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[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "cell biology" ]
Mechanical force regulates tendon extracellular matrix organization and tenocyte morphogenesis through TGFbeta signaling
elife-38069-v2
[ [ "Cells in all multicellular organisms are exposed to mechanical forces through adhesions to neighboring cells and to the extracellular matrix ( ECM ) , as well as the ebb and flow of the environment .", "Force has been shown to influence cellular processes such as cell division , survival , migration , and differentiation ( Behrndt et al . , 2012; Culver and Dickinson , 2010; Hamada , 2015; Keller et al . , 2008; Roman and Pekkan , 2012 ) .", "Cellular responses to force include the activation of cell surface receptors such as integrins ( Itgs ) , G-protein-coupled receptors ( GPCRs ) , transient receptor potential ( TRP ) ion channels , and Piezo channels ( Busch et al . , 2017; Chachisvilis et al . , 2006; Maartens and Brown , 2015; Mederos y Schnitzler et al . , 2008; Popov et al . , 2015; Wu et al . , 2017 ) .", "Despite recent insights into the nature of such responses , few in vivo studies have investigated how cells adapt to force and alter the ECM landscape to strengthen or weaken it accordingly ( Maeda et al . , 2011; Ng et al . , 2014 ) .", "The musculoskeletal system bears among the strongest forces experienced by any tissue , such as the tensional forces exerted upon tendons and ligaments ( Heinemeier et al . , 2013; Wang , 2006 ) .", "Tendons can withstand such forces due to the specialized organization of collagen ( Col ) fibers and proteoglycans within each tendon fibril .", "Tendon injuries are extremely common and debilitating , especially in athletes , the elderly , and patients with neuromuscular diseases such as muscular dystrophy ( Bönnemann , 2011; Walden et al . , 2017 ) .", "Despite their prevalence , little is known about how tendon fibroblasts ( tenocytes ) respond in vivo to tensional force at muscle attachments , or how they adapt to changes in mechanical load .", "Tendons form a variety of attachment sites - connecting muscles to cartilages and bones as well as other muscles and soft tissues .", "A myotendinous junction ( MTJ ) is a specialized ECM-rich region at the interface of muscle-tendon attachment sites that functions as the primary sources of force transmission .", "Each type of attachment bears varying levels of force , which correlates with distinct composition and organization of its tendon ECM ( Ker et al . , 2000; Wang , 2006 ) .", "While extensive research has been conducted to evaluate the effects of exercise on size and strength of muscle fibers , less is known about how it effects tendon morphology and function .", "Understanding this is key to gaining insights into the causes of tendon defects and developing new treatments for tendon injuries or atrophy .", "Previous studies in vitro have suggested that tenocytes actively respond to changes in force in their environment by modulating ECM composition and organization ( Maeda et al . , 2010; Rullman et al . , 2009 ) .", "Excised tendons stretched in collagen gels , as well as tissue samples from chronic Achilles tendonitis patients , upregulate various collagens and ECM-modulating proteins , particularly Col3 , Matrix Metalloproteinase 9 ( MMP9 ) and MMP13 ( Ireland et al . , 2001; Pingel et al . , 2014 ) .", "In addition , collagen fibril size decreases and fibril packing increases in tendinopathies , likely due to increased ECM turnover ( Pingel et al . , 2014 ) .", "These studies have suggested ECM modifications and morphological changes in tendinopathies but they have largely been limited to cultured tendons or tendon fragments .", "In vivo , several growth factor signaling pathways and transcription factors have been implicated downstream of mechanical force in tendon development and repair in mice .", "These include several members of the Transforming Growth Factor ( TGF ) superfamily , including TGFβ and Bone Morphogenetic Proteins ( BMPs ) , as well as Fibroblast Growth Factors ( FGF ) ( Gumucio et al . , 2015; Nourissat et al . , 2015 ) .", "Mice lacking the transcription factor Scleraxis ( Scx ) show severe defects in force-transmitting and load-bearing tendons , suggesting that Scx is essential for maintenance of tendon ECM in response to mechanical force ( Murchison et al . , 2007 ) .", "In addition , Scx directly regulates transcription of tendon ECM components , including Col1a1 ( Havis et al . , 2014; Subramanian and Schilling , 2015 ) .", "Our studies of the ECM protein Thrombospondin 4b ( Tsp4b ) in zebrafish have shown that it is an essential scaffolding protein for tendon ECM assembly , required to maintain muscle attachments subjected to mechanical force via muscle contraction , and able to strengthen attachments when overexpressed ( Subramanian and Schilling , 2014 ) .", "Here , we show that mechanical force causes remarkable morphological changes in tenocytes in zebrafish , which form a dense curtain of projections at MTJs , and in their surrounding ECM .", "Tenocyte projections have been reported in electron micrographs of mammalian tendon fascicles yet how they form and their functions in tendon development remain largely unexplored ( Kalson et al . , 2015; Knudsen et al . , 2015 ) .", "Our results suggest that tenocytes play an active role in sensing force and thereby regulating ECM composition and overall tendon strength .", "In addition , we show that the force of muscle contraction regulates the growth and branching of tenocyte projections via TGFβ signaling .", "Such feedback between tenocytes and ECM may be a common mechanism for force adaptation within the musculoskeletal system ." ], [ "Tenocytes in zebrafish express two Scx orthologues , scxa and scxb ( Chen and Galloway , 2014 ) .", "Using a bacterial artificial chromosome ( BAC ) transgenic line that expresses mCherry under the control of regulatory elements for scxa , Tg ( scxa:mCherry ) , we examined the morphogenesis of tenocytes during embryonic ( 20 hr post fertilization ( hpf ) to 72 hpf ) and early larval ( 72 hpf to 5 dpf ) zebrafish development .", "Expression of scxa:mCherry was first detected at 20 hpf in muscle and tendon progenitors of the somites .", "In a developing zebrafish embryo , muscles in the trunk establish attachments at bilateral , ‘chevron’ shaped somite boundaries that subdivide each muscle segment forming the vertical myoseptum ( VMS ) .", "In addition , dorsal and ventral compartments within each somite are subdivided by a horizontal myoseptum ( HMS ) , which extends laterally from the notochord ( NC ) , along which oblique myofibers attach .", "By 24 hpf , as the first myofibers differentiated , scxa:mCherry expression in muscle progenitors diminished and became progressively restricted to scattered tendon progenitors along the HMS and VMS ( ~24 cells per VMS ) ( Figure 1A , D , G ) ( Figure 1—video 1 ) .", "Cells with the highest levels of scxa:mCherry expression were located laterally , adjacent to the HMS , while more medial cells expressed lower levels ( Figure 1A’ , D’ , G’ ) .", "By 36 hpf , scxa:mCherry+ cells doubled in number ( ~44/VMS ) and became increasingly localized to the HMS and VMS at future MTJs ( Figure 1B , E , H ) ( Figure 1—video 1 ) .", "At this stage , cells with the highest scxa:mCherry expression that were located medially and in the ventral somites began to extend projections laterally along the VMS , perpendicular to the orientation of muscle fibers ( Figure 1B’ , E’ , H’ ) .", "By 48 hpf these projections extended 70–80 μm ( Figure 1C , F , I ) .", "3D-reconstructions of confocal stacks at 60 hpf revealed that this polarized network of tenocyte projections covered the entire VMS ( Figure 1C’ , F’ , I’; Figure 1—figure supplement 1 ) .", "Time-lapsed videos of Tg ( scxa:mCherry ) embryos capturing images at 20 min intervals from 48 to 60 hpf showed that tenocyte projections are dynamic and constantly changing in length and branching pattern ( Figure 1—video 2 ) .", "Tenocytes along the HMS near the NC have shorter , more convoluted projections than tenocytes along the VMS ( Figure 1I’; Figure 1—figure supplement 1B ) .", "Thus , during the period in which axial muscles in the trunk begin to contract and embryos become motile , tenocytes align along future MTJs and undergo dramatic changes in cell shape that correlate with the establishment and strengthening of muscle attachments .", "Cranial tendons also undergo dramatic morphological changes during the onset of muscle attachment and contractility .", "A cluster of scxa:mCherry+ tenocyte progenitors is first observed at 36 hpf in the ventral midline near the future attachment sites of the sternohyoideus ( SH ) and adductor mandibulae ( AM ) , which are among the earliest muscles to differentiate at 53 hpf ( Schilling and Kimmel , 1997 ) .", "By 48 hpf , three major clusters of scxa:mCherry+ tenocytes are visible ventrally , one anterior that forms ventral mandibular , hyoid , and oculomotor muscle tendons and two posterior clusters associated with each SH ( Figure 1—figure supplement 2A ) .", "The anterior cluster subdivides over 14 hr into separate attachment sites for mandibular ( IMA , IMP , AM ) and hyoid ( IH ) muscles ( Figure 1—figure supplement 2B ) .", "Double labeling with anti-MHC and anti-mCherry antibodies revealed a tight correlation between the timing of the onset of muscle contraction and tenocyte morphogenesis in each of these clusters ( Figure 1—figure supplement 2A , B , D , E , G , H ) .", "Cranial myofibers remain immotile at 48–62 hpf and their corresponding tenocytes form clusters of rounded cells at future attachment sites .", "These tenocytes then undergo compaction and elongation as contractions begin at 72 hpf ( Figure 1—figure supplement 2C , F , I ) .", "Based on the close correlation between the onset of muscle contraction and tenocyte morphogenesis , we hypothesized that mechanical force serves as a cue for tenocytes to elongate and form projections .", "To test this idea , we first injected full-length mRNA encoding codon-optimized α-bungarotoxin ( αBtx ) , a specific irreversible antagonist of acetylcholine receptors that blocks neuromuscular synapses and prevents skeletal muscle contractions ( Swinburne et al . , 2015; Westerfield et al . , 1990 ) .", "Embryos injected with αBtx mRNA at the one-cell stage were completely paralyzed until 60 hpf , after which they gradually recovered motility as αBtx activity declined .", "Depth-coded , 3D-reconstructed images of living trunk tenocytes along the MTJs of somites 16–17 at 48 hpf revealed an average reduction of 13 μm ( 18% ) in axial tenocyte projection length in αBtx-injected embryos compared to uninjected controls ( Figure 2A , B , E ) .", "Paralyzed embryos also showed reduced branching complexity in their projections ( Figure 2F ) and projection density along the VMS ( Figure 2—figure supplement 1 ) .", "To restore mechanical force , we electrically stimulated αBtx-injected embryos to induce muscle contractions , as described previously ( Subramanian and Schilling , 2014 ) .", "Stimulation at 48 hpf for 2 min at 20V caused no visible muscle damage or significant change in tenocyte projection lengths compared to controls ( Figure 2C , E ) while the same stimulation of αBtx-injected embryos rescued both tenocyte projection length and density along the VMS almost completely ( Figure 2D , E; Figure 2—figure supplement 1 ) .", "The observed reductions in projection length and density were caused by paralysis rather than any unanticipated effect of αBtx , since homozygous mutants paralyzed due to lack of a functional voltage-dependent L-type calcium channel subtype beta-1 ( Cacnb1 ) , necessary for excitation-contraction coupling in muscle , showed similar ( 10–15 μm ) reductions in projection length ( Figure 2—figure supplement", "2 ) ( Zhou et al . , 2006 ) .", "Tenocytes in cacnb1 mutant embryos fail to compact and elongate .", "Since αBtx-injected embryos recover from paralysis at 65 hpf , prior to cranial muscle contractions , we compared cranial tenocyte patterning in immunostained 4 dpf cacnb1 mutant embryos with their siblings .", "We observed both a failure of cranial tenocytes to compact and elongate , as well as reduced projections and frayed myofibers ( Figure 2—figure supplement 3 ) .", "These results indicate a strong correlation between mechanical force from muscle contraction and tenocyte morphogenesis , suggesting that force stimulates the dynamic growth and branching of tenocyte projections .", "We previously showed that Tsp4b secreted by tenocytes is essential for ECM organization at MTJs and strengthens muscle attachments ( Subramanian and Schilling , 2014 ) .", "We hypothesized that force stimulates tenocytes to secrete Tsp4b from the projections they extend into the tendon ECM .", "Consistent with this , injection of tsp4b-gfp full length mRNA into Tg ( scxa:mCherry ) embryos produced Tsp4b-GFP protein that localized to MTJs along the attachment sites at 48 hpf ( Figure 3A–C , I ) .", "This exogenous Tsp4b-GFP protein was dramatically reduced in αBtx-injected embryos , particularly around projections , and became diffuse compared to uninjected controls ( Figure 3D–F , I ) .", "Likewise , immunohistochemical staining for Tsp4b at 48 hpf in αBtx-injected embryos showed dramatic reductions along the attachment sites compared to controls ( Figure 3G , H , J ) .", "In contrast , other ECM proteins such as laminin ( Lam ) at 48 hpf and and fibronectin ( Fn ) at 24 hpf showed no significant changes at the MTJ in αBtx-injected embryos at 48 hpf ( Figure 3—figure supplement 1 ) .", "Defects in Tsp4b distribution were due to the lack of mechanical force , since restoring force in paralyzed embryos through electrical stimulation rescued both local levels and the overall area of Tsp4b protein localization along the VMS ( Figure 3—figure supplement 2 ) .", "To test the hypothesis that changes in Tsp4b localization were due to reduced tsp4b gene expression in response to lack of force , we performed real-time PCR and found a significant reduction in tsp4b expression at 48 hpf in αBtx-injected embryos , while no significant change in expression was observed at 24 hpf ( Figure 3—figure supplement 3 ) .", "These results suggest a role for mechanical force in both assembly of tendon ECM and expression of key MTJ ECM genes during development and demonstrate that muscle contractions regulate the composition and organization of the tendon ECM .", "Cellular projections in neurons , keratinocytes and pigment cells are rich in microtubules ( MTs ) and in some cases F-actin , while filopodial extensions of cells are typically more actin-based ( Eom et al . , 2015; Witte et al . , 2008 ) .", "To determine the cytoskeletal structure of tenocyte projections we injected full-length mRNA encoding eGFP-αtubulin and found that this fusion protein localized to MTs along the length of tenocyte projections  ( Figure 4A–C ) ( Rusan et al . , 2001 ) .", "Similar injections of plasmids encoding EGFP-Lifeact-7 failed to show labeled actin in the projections .", "To determine if MTs are critical for maintaining projections , we treated embryos with Nocodazole , which caused them to fragment ( Figure 4D , E ) .", "Immunohistochemical staining of Nocodazole-treated embryos for Tsp4b showed scattered Tsp4b + puncta localized at MTJs along the VMS and reduced Tsp4b protein levels in the VMS ( Figure 4F , G , H , I ) .", "These results suggest that MTs are the key structural components of tenocyte projections required to sustain the organization of tendon ECM .", "Previous studies from primary cultures of tenocytes and stretch tests on isolated tendons in vitro have proposed a mechanoresponsive role for TGFβ signaling ( Gumucio et al . , 2015; Havis et al . , 2016; Maeda et al . , 2011 ) .", "TGFβ secreted by muscles or latent in the ECM of the MTJ could be released in response to force and thereby regulate both tenocyte morphogenesis and ECM production .", "To address this hypothesis , we treated Tg ( scxa:mCherry ) embryos with a chemical inhibitor of TGFβ signaling ( SB431542 – which blocks TGFβ receptors ) for 12 hr from 24 to 36 hpf ( Chen and Galloway , 2014 ) .", "This treatment severely reduced signaling in both muscle fibers and tenocytes as confirmed by immunostaining for phosphorylated SMAD3 ( pSMAD3 ) in SB431542-treated embryos compared to controls ( Figure 5A–C , E–G , I ) .", "In addition , tenocyte projections were reduced in length by an average of ~20 μm in SB431542-treated embryos ( Figure 5D , H , J ) , similar to the effects of αBtx ( Figure 2B , E ) .", "However , unlike embryos injected with αBtx , SB431542-treated embryos continued to swim actively .", "These results suggest that TGFβsignaling acts downstream of muscle contraction to stimulate growth and branching of tenocyte projections .", "To confirm if muscle contraction is essential for activation of TGFβ signaling , we stained control and αBtx-injected , Tg ( scx:mCherry ) embryos with anti-pSMAD3 .", "While control embryos showed strong pSMAD3 localization in the nuclei of muscles and tenocytes , pSMAD3 staining was strongly reduced in the nuclei of tenocytes in αBtx-injected embryos ( Figure 6A–G ) .", "Here , in contrast to embryos treated with SB431542 ( Figure 5C , G ) , paralysis specifically reduced pSMAD3 in tenocytes and not in muscle nuclei .", "This correlated with the reduction in length of tenocyte projections ( Figure 6H ) .", "These results suggest that mechanical force from muscle contraction serves as a cue for TGFβ mediated signaling in tenocytes to control their morphogenesis and differentiation .", "To further confirm that mechanical force has a role in induction of TGF-β responses in tenocytes , we stained control and αBtx-injected embryos with or without electrical stimulation , with anti-pSMAD3 antibody to verify if localization of pSMAD3 in nuclei of tenocytes could be rescued .", "αBtx-injected embryos stimulated with mild electric current showed increased pSMAD3 localization in tenocyte nuclei strongly suggesting that mechanical force from muscle contraction can rescue TGFβ signaling in tenocytes ( Figure 6—figure supplement 1 ) .", "Previous studies have linked mechanical force with the expression of tenogenic and myogenic genes ( Chen et al . , 2012; Maeda et al . , 2011 ) .", "Our results showing similar tenocyte projection defects in Nocodazole-treated , αBtx-injected and SB431542 treated embryos suggest that they induce similar changes in expression of force-responsive genes .", "Real-time PCR analysis on cDNA prepared from 48 hpf control and αBtx-injected embryos revealed that paralysis led to an almost complete loss of expression of tsp4b , as well as TGFβ-induced protein ( tgfbip ) and , connective tissue growth factor a ( ctgfa ) , while expression levels of other tendon genes , such as scxa , were unaffected ( Figure 6—figure supplement 2A ) .", "All three genes ( tsp4b , tgfbip and ctgfa ) were restored to control levels of expression with electrical stimulation ( Figure 6—figure supplement 2A ) .", "We further validated the results with digital droplet PCR ( ddPCR ) on cDNA prepared from FACS sorted tenocytes and whole embryos respectively .", "Our ddPCR results from whole embryo cDNA preparation agreed with the real-time PCR analysis ( Figure 6—figure supplement 2B ) .", "Real-time PCR analysis of SB431542-treated embryos showed significant reductions in expression of tsp4b and tgfbip genes ( Figure 6—figure supplement 3A ) .", "Loss of tenocyte projections through destabilization of microtubules in nocodazole-treated embryos also led to reduced expression of tsp4b and tgfbip while expression of ctgfa and scxa were elevated ( Figure 6—figure supplement 3B ) .", "Taken together , these results are consistent with the hypothesis that mechanical force acts through TGFβ signaling to regulate tenocyte-specific transcription including ECM components such as Tsp4b ." ], [ "Tendons primarily experience tension from muscle contractions ( Lavagnino et al . , 2015; Wang , 2006 ) .", "In contrast , skeletal cell types ( e . g . osteocytes , osteoblasts , chondrocytes ) are exposed to compressive forces ( Klein-Nulend et al . , 2012 ) or shear forces in the case of chondrocytes in joints exposed to fluid flow ( Servin-Vences et al . , 2017 ) .", "We show that in the absence of tension during development , tenocytes reduce the extent and spread of their projections into the tendon ECM and this can be rescued by a short bout of contraction .", "Tendon defects and injuries result from dramatic changes in tension experienced either instantly or periodically over extended periods of muscle disuse or overuse ( Franchi et al . , 2013; Gaut and Duprez , 2016; Wang et al . , 2012 ) .", "Embryonic tenocyte progenitors experience muscle contractions at early stages and must continuously adapt to changes in muscle strength .", "Our results support the idea that the establishment and adaptation of MTJs occurs in response to mechanical force from muscle contraction and involves both changes in tenocyte morphogenesis and ECM production .", "We show that paralysis reduces tenocyte branching and tendon ECM , which can be rescued by restoring muscle contractions through electrical stimulation .", "Early experiments on developing chick embryos have shown that induced lack of muscle activity ( either by lack of neuronal innervation or by injecting paralysis-inducing drugs ) negatively affects the growth of associated skeletal structures , suggesting a role for force from muscle contraction as an essential cue for proper growth and differentiation of the skeleton ( Hall and Herring , 1990; Hamburger and Waugh , 1940 ) .", "During development , the skeleton is exposed to two major types of force – contractile ( tension ) force from muscles and compressional force ( e . g . gravity ) .", "A contractile force from muscles has a greater impact on the growth of bones when compared to compression , indicating a primary role for muscle function in guiding the growth of associated skeletal tissues ( Ellman et al . , 2014; Warden et al . , 2013 ) .", "Recent studies in paralyzed limbs have shown that the development of a tendon-bone attachment unit , the enthesis , is affected by lack of muscle contraction ( Schwartz et al . , 2013; Tatara et al . , 2014 ) .", "Our studies suggest that muscle contraction has a similar role in the development of tendons .", "Immobilization experiments performed on canine models have shown that mechanical force is required for repair of tendon injuries ( Gelberman et al . , 1982 ) .", "More recent studies using paralysis and restricted movement have shown that mechanical force has multiple roles in the maintenance of tenogenic gene expression , secretion of tendon ECM , and tenocyte survival ( Gaut et al . , 2016; Hettrich et al . , 2011; Maeda et al . , 2011 ) .", "Cellular filopodia and neuronal axons require either F-actin and MTs or both in the formation and maintenance of projections , and new classes of cellular projections are emerging from recent studies such as cytonemes and airinemes ( Bornschlögl , 2013; Eom et al . , 2015; Huang and Kornberg , 2015; Witte et al . , 2008 ) .", "MTs also serve as pathways for trafficking various proteins , RNA , and other intracellular components along projections .", "Tenocytes in Drosophila rely on a network of polarized MTs for the maintenance of cellular structure and function ( Subramanian et al . , 2003 ) , but similar requirements for cytoskeletal components have not been investigated in vertebrate tendons .", "Here , we show that zebrafish tenocytes are rich in MTs , which are required to maintain projections .", "Pharmacological disruption of MTs destabilized the projections without affecting tenocyte cell bodies .", "This reduced Tsp4b localization suggesting that tenocyte projections both sense force and respond to it by altering ECM organization in an MT-dependent manner .", "A caveat to this result is that treatment of embryos with Nocodazole causes global destabilization of MT in the entire embryo .", "Hence , the effects on tenocyte projections and tendon ECM organization could also arise in response to MT destabilization in neighboring muscle fibers , axons and other cells .", "Similar roles for cellular projections have been observed in pigment cells , where airinemes composed of both F-actin and MTs play a role in long-range signaling by secreting signaling ligands at the tips of their projections ( Eom et al . , 2015 ) .", "We find that loss of tenocyte projections leads to upregulated expression of scxa and ctgfa in MT-deficient embryos , suggesting that they revert to a more dedifferentiated state .", "Previous EM studies of human and rat tendons have described tenocytes projecting into the tendon matrix , but their functional significance has remained unclear ( McNeilly et al . , 1996; Pingel et al . , 2014 ) .", "3D reconstructions from EM studies suggest a role for these projections , referred to as ‘fibropositors’ in one study , in secreting collagen fibrils ( Canty et al . , 2004 ) .", "Analysis of Scx expression in chick and mouse using immunostaining and transgenic reporter lines , respectively , have shown that limb tendons elongate as the musculoskeletal system matures ( Brent et al . , 2003; Kardon , 1998; Pryce et al . , 2007 ) .", "Our results in zebrafish reveal that such elongated projections are conserved , but quite distinct in different classes of tenocytes .", "While cranial tenocytes resemble those in the limb in that they extend in parallel to the direction of force , axial tenocytes extend their projections perpendicular to the plane of muscle contraction ( with opposing directions of contractile force ) ( Figure 1 and Figure 1—figure supplement 2 ) .", "Based on our results , we propose that these distinct morphologies reflect a more structural , load-bearing role for cranial ( and limb ) tenocytes , while early larval axial tenocytes in zebrafish function as tension sensors in the myoseptum .", "Many important questions remain and form the basis of future studies , including why these cells are so polarized and how this mediolateral polarity develops .", "Consistent with the tension-sensor hypothesis , the timing of the outgrowth of tenocyte projections tightly correlates with the onset of muscle contraction .", "Tension sensing projections are observed in other musculoskeletal tissues as osteocytes extend projections into the bone matrix where they are thought to form a network of force sensors ( Cowin et al . , 1991 ) that modulate bone formation and resorption ( Klein-Nulend et al . , 2012; Schaffler et al . , 2014 ) .", "Likewise , the cues that cause osteoblasts to form these projections as they differentiate into osteocytes remain unknown ( Franz-Odendaal et al . , 2006 ) .", "Similar to our results with zebrafish tenocytes , mammalian osteocyte projections increase in density in response to force , consistent with a role as force sensors and responders in both cases .", "In both bone and tendon , the ECM undergoes dynamic changes in expression of collagens , fibronectin , laminin and MMPs , and this is also the case in the developing somites of zebrafish embryos ( Jenkins et al . , 2016; Snow and Henry , 2009 ) .", "Tenocyte projection formation also correlates with the establishment of tendon ECM .", "Previous studies have shown remodeling of MTJ ECM between 24 and 48 hpf with a progressive reduction of Fn , which is replaced by increased in levels of Lam at the MTJ ( Jenkins et al . , 2016 ) .", "Our results suggest that initial production and accumulation of Fn is independent of force at 24 hpf , when tsp4b also shows force-independent expression .", "The later force-dependent expression and localization of Tsp4b at 48 hpf indicates that dynamic regulation of tendon ECM occurs after the onset of muscle contraction , which suggests a role for mechanical force in the process .", "Mammalian tenocytes actively sense mechanical force in vitro , resulting in changes in gene expression , cytoskeletal organization and ECM secretion ( Banos et al . , 2008; Gaut et al . , 2016; Havis et al . , 2016; Maeda et al . , 2010; Maeda et al . , 2013; Maeda et al . , 2011 ) .", "This depends , at least in part , on gap junctional complexes that localize to tenocyte projections ( Maeda et al . , 2012 ) .", "Exercise induces Tenomodulin ( Tnmd ) and Col1a1 expression and tenocyte proliferation in rats ( Eliasson et al . , 2009; Zhang and Wang , 2013 ) and stress induces COL4A1 and COL6A1 expression in chick tenocytes ( Marturano et al . , 2014 ) .", "Despite these changes in gene expression , the molecular mechanisms underlying these cellular signaling responses to force are unclear .", "Embryonic tenocyte projections in zebrafish end in bouton-like structures close to the dermis ( Figure 1—figure supplement 1A , B ) , which may act as signaling beacons and ECM secreting centers .", "The strong correlation between onset of muscle function , changing myotendinous ECM and tenocyte morphogenesis suggests a model in which force is transduced through cues from the ECM that induce the formation of projections ( Figure 7A ) .", "Similar processes may underlie the projections of osteocytes and other mesenchymal cell types .", "Such feedback likely allows tendons to adapt to changing mechanical force during normal development and exercise , as well as in healing and repair of tendon injuries .", "Our results show for the first time that activation of TGFβ signaling in response to mechanical force is required for tenocyte morphogenesis , in particular the growth and branching of tenocyte projections .", "Paralyzed embryos ( αBTX-injected ) lose pSMAD3 expression in tenocytes and projections shorten , which is rescued by restoring muscle contraction .", "Similarly , pharmacological inhibition of TGFβ receptors reduces pSMAD3 expression and shortens tenocyte projections .", "Studies of mechanotransduction have identified several putative signaling pathways involved , depending on the tissue , including TGFβ , YAP/TAZ , and Integrins , as well as membrane channels such as TrpV4 and Piezo receptors ( Busch et al . , 2017; Gumbiner and Kim , 2014; Lavagnino et al . , 2015; Servin-Vences et al . , 2017 ) .", "Some of these pathways such as TGFβ and YAP/TAZ share intermediate signaling components and targets , which complicates our understanding of their role in mechanotransduction in specific tissues ( Qin et al . , 2018; Szeto et al . , 2016 ) .", "This could help explain the modest reduction in expression of tgfbip and ctgfa2 in SB431542-treated embryos , as other mechanotransduction signaling pathways may still function in these embryos to partially maintain expression levels of these genes ( Figure 6—figure supplement", "3 ) In vitro studies of tenocyte primary cultures and excised tendon tissue have shown elevated TGFβ signaling in response to mechanical load ( Heinemeier et al . , 2003; Heinemeier et al . , 2007; Maeda et al . , 2013; Maeda et al . , 2011; Yang et al . , 2004 ) and mice show elevated TGFβ signaling in muscles and tendons following exercise ( Maeda et al . , 2011 ) .", "These studies suggest that TGFβ signaling , in addition to its earlier role in tenocyte specification ( Havis et al . , 2016 ) , is involved in mechanotransduction in these cells after they differentiate .", "TGFβ signaling is activated by many factors , including integrins , BMP1 and MMPs which can act on the large latent complex ( LLC ) , to release active TGFβ ligand from the ECM ( Horiguchi et al . , 2012; Keski-Oja et al . , 2004; Todorovic et al . , 2005 ) .", "This could be the critical cue from the ECM that induces and modulates the formation of tenocyte projections .", "One candidate for initiating these events is Tsp4 , since Tsps can activate TGFβ signaling by destabilizing latency-associated peptide ( LAP ) ( Bailey Dubose et al . , 2012 ) .", "The tendon matrix is rich in MMPs and Tsps , which dynamically change in composition and activity depending on mechanical force ( Jenkins et al . , 2016; Popov et al . , 2015; Subramanian and Schilling , 2014 ) .", "The dynamic reductions in Tsp4b that we have shown in response to paralysis could fail to activate latent TGFβ in the tendon ECM .", "Furthermore , because we see reductions in Tsp4b expression in paralyzed embryos , our results support a model where force triggers TGFβ signaling leading to increased expression of Tsp4b , which in turn activates TGFβ expression , creating a positive feedback loop ( Figure 7A , B ) .", "Transection of tendons or injection of botulinum toxin ( Botox ) to induce paralysis in mice causes tenocyte death and reduced expression of tenogenic genes ( Maeda et al . , 2011 ) .", "In contrast , we observe neither cell death nor significant changes in tenogenic gene expression in paralyzed ( cacnb1 mutant ) zebrafish embryos until 5 dpf , several days after tenocyte differentiation .", "We interpret such a response as a separate response to prolonged disuse rather than an adaptation to force .", "These studies have shown a direct relationship between mechanical force and tendon development through TGFβ signaling in tenocytes .", "How do tenocyte progenitors begin the process of elongation and growth of projections ?", "What are the roles of these projections during tendon embryonic development and in adult tendons ?", "Osteocytes are known to induce repair pathways in bone when cracks or stress damage their processes ( Dooley et al . , 2014; Mulcahy et al . , 2011 ) .", "Do tenocyte projections perform a similar role in tendon repair ?", "These are some of the questions that need to be addressed in the field of tendon biology .", "Understanding the relationship between force and tendon development is essential for developing effective treatment strategies that include engineering tendons to treat tendon injuries .", "Force sensing projections that allow cells to adjust their surrounding ECM , such as those we have described in tenocytes , may also be a more general feature of cells , particularly within the musculoskeletal system ." ], [ "Tg ( scx:mCherry ) transgenics were generated by injecting a BAC construct ( CH211-251g8 ) containing mCherry ORF inserted in frame after the start codon of the scxa gene ( McGurk et al . , 2017 ) .", "A new mutant allele of cacnb1 was identified in a forward genetic screen and outcrossed with Tg ( scxa:mCherry ) to create a cacnb1;Tg ( scxa:mCherry ) line .", "All embryos were raised in embryo medium at 28 . 5°C ( Westerfield , 2007 ) , and staged as described previously ( Kimmel et al . , 1995 ) .", "Craniofacial muscles and cartilages were labeled as described previously ( Schilling and Kimmel , 1997 ) .", "Adult fish and embryos were collected and processed in accordance with approved UCI-IACUC guidelines .", "A Pmtb-t7-alpha-bungarotoxin ( αBtx ) vector ( Megason lab , Addgene , 69542 ) was used to synthesize αBtx mRNA following a previously published protocol and injected into Tg ( scx:mCherry ) embryos at the 1–2 cell stage ( Subramanian and Schilling , 2014; Swinburne et al . , 2015 ) .", "A pIRESneo-EGFP-alpha Tubulin plasmid ( Wadsworth lab , Addgene , 12298 ) was used to synthesize EGFP-α Tubulin mRNA following a previously published protocol and injected into Tg ( scx:mCherry ) embryos at the 1–2 cell stage ( Rusan et al . , 2001; Subramanian and Schilling , 2014 ) .", "A stock solution of 50 mM SB431542 ( Tocris 1614 , SID: 241182574 ) ) , a selective inhibitor of TGFβ type I receptor was prepared in DMSO ( Fisher Scientific D1281 , SID: 349996472 ) and diluted to a final working concentration of 10 μM in embryo medium .", "Embryos were incubated in 10 μM SB431542 for 12 hr .", "Treated embryos were rinsed in pre-warmed ( 28 . 5°C ) embryo medium before fixation for immunostaining or RNA extraction .", "A stock solution of 33 mM Nocodazole ( Sigma M1404 , SID: 336851328 ) , an inhibitor of tubulin polymerization , was prepared in DMSO and diluted to a final working concentration of 0 . 33 mM in embryo medium .", "Embryos were incubated in 0 . 33 mM Nocodazole for 12 hr at 28 . 5°C .", "Treated embryos were either mounted for live imaging or fixed for immunostaining .", "Whole embryo RNA was extracted from control and paralyzed embryos collected at 48 hpf according to standard protocols using Trizol ( Invitrogen 15596018 ) and Direct-zol RNA MinipPrep kits ( Zymo Research R2061 ) .", "RNA concentration was normalized between samples and used as a template for cDNA synthesis .", "cDNA was synthesized with oligodT primers using the standard protocol of ProtoScript II First Strand cDNA Synthesis Kit ( NEB E6560 ) .", "The synthesized cDNA was diluted to 1:20 and used as a template for RT-PCR using the protocol for the Luna Universal qPCR master mix ( NEB M3003S ) .", "The primers used for RT-PCR are listed in Table 1 .", "The reaction was run on a LightCycler 480 II Real time-PCR Instrument ( Roche ) and analyzed using LightCycler 480 Software .", "Each qPCR experiment was designed with triplicates of reactions for every biological sample and two biological samples were used for each analysis ( Subramanian and Schilling , 2014 ) .", "cDNA was prepared from whole embryo RNA using the standard protocol of ProtoScript II First Strand cDNA Synthesis Kit ( NEB E6560 ) .", "The cDNA concentration was determined following standard protocol and reagents from the Qubit SSDNA assay kit ( Invitrogen Q10212 ) and fluorescence was read on a Qubit 2 . 0 fluorometer ( Invitrogen Q32866 ) .", "A total concentration of 1 ng was used from each sample to prepare 20 ml of ddPCR reaction following the instructions and reagents from QX200 EvaGreen ddPCR Supermix ( Bio-Rad 186–4033 ) .", "Primers for the PCR are listed in Table 1 .", "The droplets were generated using QX200 Droplet Generation Oil for EvaGreen ( Bio-Rad 1864005 ) on a QX200 Droplet Generator ( Bio-Rad 1864002 ) .", "The PCR reaction was run on a standard thermocycler under standard cycling conditions .", "Following the PCR the droplets were analyzed using QX200 Droplet Reader ( Bio-Rad 1864003 ) .", "The data were analyzed using QuantaSoft Analysis Pro Software .", "Electrical stimulation was used to induce muscle contraction , as previously described ( Subramanian and Schilling , 2014 ) .", "Both αBtx injected and control embryos or larvae were anaesthetized with Tricaine ( ethyl 3-aminobenzoate methanesulfonate , Sigma A5040 , SID: 329770864 ) , placed on a silicone plate in embryo medium and stimulated for 2 min at 20V , 6 msec duration , 4 pulses/sec frequency and 6 msec delay between successive pulses .", "With these settings neither control nor paralyzed embryos showed any muscle detachment .", "Embryos were allowed to recover in embryo medium for 12 hr and further processed for immunostaining or RT-PCR .", "All embryos used for immunofluorescence experiments were fixed in 4% neutral pH buffered paraformaldehyde ( PFA ) for 2 hr at room temperature ( 25°C ) or overnight at 4°C .", "The embryos were washed with 1X Phosphate Buffered Saline ( PBS , CID: 24978514 ) and permeabilized with cold acetone ( Fisher Scientific A94 , SID: 349996362 ) for 15 min at −20°C .", "Following permeabilization , they were rehydrated in PBDT ( PBS with 2% DMSO and 1% Triton X-100 ( Sigma T9284 ) ) and processed according to a standard antibody staining protocol .", "Primary antibodies used: rabbit anti-Tsp4b ( 1:500 ) ( RRID: AB_2725793 ) , mouse anti-myosin heavy chain ( MHC ) ( Developmental Hybridoma - 1:250 , A1025 , RRID: AB_528356 ) , chicken anti-GFP ( Abcam – 1:1000 , ab13970 , RRID: AB_300798 ) , rat monoclonal anti-mCherry ( Molecular Probes −1:500 , M11217 , RRID: AB_2536611 ) , rabbit anti-Laminin ( Abcam – 1:200 , ab11575 , RRID: AB_298179 ) , rabbit anti-Fibronectin ( Abcam – 1:200 , ab2413 , RRID: AB_2262874 and rabbit anti-pSMAD3 ( Antibodies-online – 1:500 , ABIN1043888 , RRID: AB_2725792 ) .", "DiAmino PhenylIndole ( DAPI ) ( Invitrogen – 1:1000 , D1306 , RRID: AB_2629482 ) was used to mark cell nuclei .", "Preabsorbed secondary antibodies were all obtained from Jackson ImmunoResearch and used for indirect immunofluorescence at 1:1000 , including: Alexa Fluor 488 conjugated donkey anti-mouse IgG ( 715-546-150 , RRID: AB_2340849 ) , DyLight 549 conjugated donkey anti-rabbit IgG ( 711-506-152 , RRID: AB_2616595 ) , Alexa Fluor 488 conjugated donkey anti-rabbit IgG ( 711-545-152 , RRID: AB_231358 ) , Cy5 conjugated Goat anti-mouse IgG ( 115-176-071 ) , Alexa Fluor 594 conjugated donkey anti-rat IgG ( 712-586-153 , RRID: AB_2340691 ) , and Alexa Fluor 488 conjugated donkey anti-chicken IgY ( 703-486-155 ) .", "After staining , embryos were mounted in 1% low melt agarose in PBS and imaged .", "Embryos processed for fluorescent immunohistochemistry were imaged using a Nikon A1 confocal system with an Nikon Eclipse Ti inverted microscope using a CFI Plan Apochromat VC 60XC ( water immersion ) objective .", "Confocal stacks were analyzed using Image J software .", "The depth-coded 3D reconstructions were created using Nikon software ( NIS-Elements AR 4 . 60 . 00 64-bit ) .", "To better visualize tenocyte projections along the Z-axis , the 3D reconstructed image was rotated to about 45o .", "The length of projections was measured using the Neurite Tracer plugin on Image J . Sample size and number of data points required for each experiment were determined using a power analysis calculator ( www . powerandsamplesize . com ) .", "The embryos were collected from a single tank of fish and processed for injection and downstream stimulation together to minimize variation introduced during handling .", "Fixation and staining of embryos were also performed together for all samples in a given experiment .", "Imaging of embryos within each experiment was performed with identical parameters .", "In order to control for variation in position of tenocyte cell bodies and antibody penetrance variation , projection length , fluorescence intensity of ECM proteins and pSMAD3 were always measured in the ventral half of the VMS in somites 16–19 .", "In experiments where a normal distribution was not present , we analysed the significance using a Wilcoxon Rank Sum test .", "In datasets involving two samples of unequal variance , a t-test was used .", "In experiments with more than two experimental conditions , an ANOVA single-factor analysis was performed with posthoc multiple comparisons using Tukey method on R . Data were also quantified and analyzed separately by two of the authors to account for user bias and they obtained similar results .", "Fluorescence Intensity ( FI ) to quantify protein localization was measured as described previously ( Subramanian and Schilling , 2014 ) ." ] ]
[ "Mechanical forces between cells and extracellular matrix ( ECM ) influence cell shape and function .", "Tendons are ECM-rich tissues connecting muscles with bones that bear extreme tensional force .", "Analysis of transgenic zebrafish expressing mCherry driven by the tendon determinant scleraxis reveals that tendon fibroblasts ( tenocytes ) extend arrays of microtubule-rich projections at the onset of muscle contraction .", "In the trunk , these form a dense curtain along the myotendinous junctions at somite boundaries , perpendicular to myofibers , suggesting a role as force sensors to control ECM production and tendon strength .", "Paralysis or destabilization of microtubules reduces projection length and surrounding ECM , both of which are rescued by muscle stimulation .", "Paralysis also reduces SMAD3 phosphorylation in tenocytes and chemical inhibition of TGFβ signaling shortens tenocyte projections .", "These results suggest that TGFβ , released in response to force , acts on tenocytes to alter their morphology and ECM production , revealing a feedback mechanism by which tendons adapt to tension ." ]
[ "Tendons – the fibrous structures that attach muscles to bones – must withstand some of the strongest forces in the body .", "Little is known about how tendons develop or adapt to withstand these forces .", "Studies have shown that muscles respond actively to force , as seen during exercise .", "Do tendons respond in similar ways ?", "Tendons consist of collagen fibers surrounded by a ‘matrix’ of proteins .", "Also embedded in the matrix are specialized cells called tenocytes , which regulate the production of the different components of the tendon .", "A genetic modification allows tenocytes to be tracked using a fluorescent gene product that can be viewed using a microscope .", "Subramanian et al . have now used this technique in zebrafish to watch how the behaviors of the tenocytes change in response to forces applied to the tendon .", "Subramanian et al . show that at the start of muscle contraction , tenocytes put forth long projections from their cell bodies that extend perpendicular to the muscle fibers .", "This suggests that the projections act as force sensors .", "Consistent with this idea , paralyzing the muscle causes the projections to shrink .", "This shrinkage correlates with changes in how the tendon matrix proteins are organized .", "Further investigation reveals a force-responsive signaling pathway in the tenocytes that controls how these cells grow and produce key tendon matrix proteins .", "Subramanian et al . believe this pathway is central to how tendons adapt to the forces applied during muscle contraction .", "A better knowledge of how force affects tendon structure could ultimately help to improve treatments for tendon injuries and tendon atrophy .", "In particular , understanding how force affects how tenocytes develop could help researchers to develop new ways to regenerate and repair tendons ." ]
2018
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Roles of the membrane-reentrant β-hairpin-like loop of RseP protease in selective substrate cleavage
elife-08928-v1
[ [ "Regulated intramembrane proteolysis ( RIP ) is a common mechanism for transmembrane signalling and is a part of many important cellular processes in both prokaryotes and eukaryotes ( Ha , 2009; Wolfe , 2009; Urban , 2013 ) .", "Proteases involved in RIP are called intramembrane-cleaving proteases ( I-CLiPs ) and catalyse the proteolytic cleavage of transmembrane segments ( TMs ) of target membrane proteins within the lipid bilayer .", "Escherichia coli RseP belongs to S2P zinc metalloproteinase family of I-CLiPs .", "It plays a key role in regulating the σE extracytoplasmic stress response ( Hizukuri et al . , 2013; Kroos and Akiyama , 2013 ) , in which the dedicated sigma factor σE is activated through the successive cleavage of a single membrane-spanning anti-σE RseA in response to cell surface stresses .", "The first cleavage catalysed by DegS ( site-1 cleavage ) on the periplasmic side triggers the intramembrane cleavage by RseP ( site-2 cleavage ) ( Alba et al . , 2002; Kanehara et al . , 2002 ) .", "Recent studies suggest that in addition to being involved in RseA cleavage , RseP acts in the proteolytic removal of remnant signal peptides from the membrane ( Saito et al . , 2011 ) .", "RseP spans the membrane 4 times , with both the termini facing the periplasm .", "Its central periplasmic region contains tandem PDZ domains ( PDZ-N and PDZ-C; Figure 1A ) ( Kanehara et al . , 2001; Kinch et al . , 2006; Inaba et al . , 2008 ) .", "The S2P family proteases share a conserved core domain containing 3 TMs .", "In RseP , these correspond to TM1–TM3 ( Figure 1A , B ) ( Kinch et al . , 2006 ) .", "The first and the third TM contain HExxH and LDG active site motifs , respectively .", "Disulfide crosslinking and co-immunoprecipitation experiments suggest that TM3 plays a critical role in binding of a substrate TM ( Koide et al . , 2008 ) .", "Helix destabilisation of substrate TMs promotes their binding to and cleavage by RseP ( Akiyama et al . , 2004; Koide et al . , 2008 ) . 10 . 7554/eLife . 08928 . 003Figure 1 . Structures of RseP and the model substrates used in this study .", "( A ) Schematic representation of RseP .", "( B ) Crystal structure of mjS2P ( PDB code: 3B4R A ) .", "The secondary structure was analysed using STRIDE ( Frishman and Argos , 1995 ) , and the image was generated using PyMOL ( https://www . pymol . org ) ( Schrödinger , 2010 ) .", "In A and B , the 3 TM helices composing the core membrane domain are shown in light green; the MRE β-loop , HExxH motif and LDG motif are shown in red , yellow and orange , respectively , and the coordinated zinc atom is shown in blue .", "( C ) The structure of the mjS2P MRE β-loop .", "The polypeptide backbone is extracted from the model in Figure 1B and shown as sticks .", "C , O and N atoms are shown in pink , red and blue , respectively .", "The amino acid residues of the corresponding region of RseP are assigned on the model .", "( D ) Sequences of the MRE β-loop of RseP ( E . coli ) , SpoIVFB ( B . subtilis ) and mjS2P ( M . jannaschii ) .", "The regions predicted to form β-strands are boxed .", "The secondary structure is assigned based on the analysis of amino acid sequences by PsiPred ( http://bioinf . cs . ucl . ac . uk/psipred ) ( for RseP and SpoIVFB ) or the analysis of the crystal structure by STRIDE ( for mjS2P ) .", "The sequence alignment and secondary structure prediction suggest that this region of RseP also assumes the β-hairpin-like structure .", "The conserved PxGG motif is boldfaced .", "( E ) Schematic representation of the model substrates and their amino acid sequences .", "RseA- and LacY-TM1 ( LY1 ) -derived regions are shown as white and hatched rectangles , respectively .", "YqfG- or YoaJ-derived region is shown as a gray rectangle .", "Amino acid sequences of RseA and LY1 TMs and the entire region of YqfG and YoaJ are shown .", "The possible TMs of YqfG and YoaJ are underlined . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 003 RseP-catalysed site-2 cleavage of RseA depends on site-1 cleavage ( Alba et al . , 2002; Kanehara et al . , 2002 ) , which results in stress-dependent σE activation , because site-1 cleavage is induced by stress signals ( Walsh et al . , 2003; Kulp and Kuehn , 2011; Lima et al . , 2013 ) .", "Previous studies suggest that RseP PDZ domains are involved in the site-1 cleavage-dependence of the site-2 cleavage by RseP ( Kanehara et al . , 2003; Bohn et al . , 2004; Grigorova et al . , 2004; Inaba et al . , 2008; Hizukuri and Akiyama , 2012 ) .", "Although the crystal structure of an S2P homolog ( mjS2P ) from Methanocaldococcus jannaschii ( Feng et al . , 2007 ) has been reported , it does not have a PDZ domain , and the three-dimensional structure of an S2P homolog with PDZ domain ( s ) is not available .", "We recently reported the crystal structure of the PDZ tandem of an Aquifex aeolicus RseP homolog .", "The structure suggests that the 2 PDZ domains create a single pocket-like structure that covers the core membrane domain on the membrane surface ( Hizukuri et al . , 2014 ) .", "Structural models and biochemical and genetic results suggest that the PDZ tandem acts as a size-exclusion filter to allow only the periplasmically truncated form of RseA to enter the recessed active site in the membrane domain of RseP ( Kroos and Akiyama , 2013; Hizukuri et al . , 2014 ) .", "However , mechanisms underlying substrate proteolysis by RseP remains elusive .", "It is unclear whether the size-exclusion function of the PDZ tandem is sufficient to discriminate between substrates and non-substrates and how a substrate TM , which assumes an α-helical conformation that needs to be unwound for proteolysis , is recognised and presented to the proteolytic active site .", "The mechanism of substrate discrimination and specific cleavage in RIP is one of the major problems to be solved ( Langosch et al . , 2015 ) .", "Detailed knowledge of mechanisms underlying substrate discrimination and cleavage would help in controlling the cleavage of membrane proteins by I-CLiPs including S2P proteases .", "The structure of mjS2P indicates that it contains a region , located close to the active site , that is looped into the membrane domain from the cytoplasmic side ( Figure 1B , C ) .", "This membrane-reentrant loop is unique because it is in an extended or β-strand conformation , whereas a membrane-embedded polypeptide is generally in an α-helical conformation .", "Therefore , we have named this region membrane-reentrant β-loop ( MRE β-loop ) .", "The MRE β-loop is conserved in proteins belonging to groups I and III of S2P subfamilies , including RseP ( group I ) , Bacillus subtilis SpoIVFB ( group III ) and mjS2P ( group III; Figure 1D ) ( Ha , 2009; Kroos and Akiyama , 2013; Zhang et al . , 2013 ) .", "A recent study showed that the MRE β-loop of SpoIVFB can be crosslinked with a substrate through disulfide bonds , suggesting its possible involvement in substrate interaction ( Zhang et al . , 2013 ) .", "However , functional roles of the MRE β-loop remain largely unknown .", "Here , we analysed the function of the RseP MRE β-loop and found that it could directly bind to and promotes selective cleavage of RseP substrates .", "Therefore , we propose that the MRE β-loop stabilises substrate TMs in an extended conformation and presents them to the proteolytic active site ." ], [ "We performed systematic mutational analysis to investigate the possible role of the MRE β-loop in RseP function .", "We constructed an MRE β-loop deletion mutant ( Δloop mutant ) containing a short linker ( Gly–Gly ) in place of residues Ile-61 to Glu-75 .", "Accumulation level of the Δloop mutant of RseP-HM ( RseP with a C-terminal His-Myc bipartite tag ) was comparable to that of wild-type RseP-HM ( Figure 2 ) , suggesting that the Δloop mutation did not cause global structural changes in RseP .", "The Δloop mutant did not exhibit complementation activity against a ΔrseP mutation ( Figure 2A ) .", "Its protease activity was examined using model substrates HA-MBP-RseA148 and HA-MBP-RseA ( LY1 ) 148 ( Figure 2C , D ) .", "HA-MBP-RseA148 is a derivative of the DegS-cleaved form of RseA ( RseA148 ) and contains a maltose-binding protein ( MBP ) domain with an N-terminal haemagglutinin ( HA ) tag in place of the cytoplasmic region of RseA ( Figure 1E ) ( Hizukuri and Akiyama , 2012 ) .", "HA-MBP-RseA ( LY1 ) 148 is essentially similar to HA-MBP-RseA148 , except that it has the first TM ( LY1 ) of lactose permease ( LacY ) instead of the RseA TM ( Figure 1E ) ( Hizukuri and Akiyama , 2012 ) .", "Our previous studies showed that an N-terminally-attached tag has little effect on substrate cleavage by RseP ( Akiyama et al . , 2004; Saito et al . , 2011 ) .", "The model substrates were co-expressed with RseP-HM or its derivatives in a ΔrseA/ΔrseP strain .", "The substrates were converted from the full-length form ( FL ) to a cleaved form ( CL ) after co-expression with wild-type RseP but not with its proteolytically inactive mutant having an amino acid alteration ( E23Q ) in the H22ExxH active site motif , indicating that these substrates underwent RseP-mediated proteolysis , as described previously ( Hizukuri and Akiyama , 2012 ) .", "Co-expression of the substrates with the Δloop mutant did not convert them from FL to CL , indicating that the mutant was proteolytically inactive . 10 . 7554/eLife . 08928 . 004Figure 2 . Effects of Pro substitutions in the MRE β-loop on RseP function .", "( A and B )", "Complementation assay .", "Strain KK31 [rseP::kan/pKK6 ( Para-rseP ) ] harbouring a plasmid encoding the indicated mutant form of RseP-His6-Myc ( RseP-HM ) under the lac promoter or the corresponding vector was grown in L medium containing 0 . 02% arabinose .", "The cultures were serially diluted with saline , and 4 μl of the diluted cultures were spotted on L agar plates containing 0 . 02% arabinose or 1 mM IPTG .", "The plates were incubated at 30°C for 20 hr . ( C–F ) Immunoblotting analysis of substrate cleavage .", "KA306 ( ΔrseA/ΔrseP/ΔclpP ) cells harbouring a plasmid encoding the indicated model substrate was transformed with plasmids encoding the indicated mutant forms of RseP-HM and were grown at 30°C in M9-based medium containing 1 mM IPTG and 1 mM cAMP for 3 hr .", "Proteins were precipitated using TCA , solubilised in 1% SDS and analysed by 10% Laemmli–SDS-PAGE and immunoblotting with anti-HA or anti-Myc antibody .", "FL and CL indicate full-length and RseP-cleaved forms , respectively , of each model substrate .", "( G–I )", "Pulse-chase analysis of substrate cleavage .", "KA306 cells harbouring an appropriate combination of plasmids encoding the indicated RseP-HM mutant and the model substrate were grown in M9-based medium containing 1 mM IPTG at 30°C .", "The cells were labelled with [35S]-methionine for 30 s and were chased with unlabelled methionine as indicated .", "Proteins were immunoprecipitated using agarose-conjugated anti-HA antibody and were analysed by 10% Laemmli–SDS-PAGE .", "Cleavage ( % ) was calculated using the following equation: cleavage ( % ) = 100 × ( CL ) /[ ( FL ) + ( CL ) ] , where FL and CL are the intensities of the respective bands that were corrected for methionine content .", "Two independent experiments were performed , and mean values are shown along with standard deviations .", "See Figure 2—source data 1 for gel images and quantitated band intensities data for Figure 2G–I .", "See Figure 2—figure supplement 1 for integral association of HA-MBP-YqfG and HA-MBP-YoaJ with membrane .", "See Figure 2—figure supplement 2 for complementation and model substrate cleavage activity of the RseP derivatives with a Cys substitution in the MRE β-loop . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 00410 . 7554/eLife . 08928 . 005Figure 2—source data 1 . Zip file containing gel images and quantified band intensity data for the pulse-chase experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 00510 . 7554/eLife . 08928 . 006Figure 2—figure supplement 1 . Membrane localization of the model substrates . Strain KK374/pKH441 ( hflD ) was further transformed with the plasmids encoding the indicated model substrate .", "Cells were grown in the M9-based medium containing 1 mM IPTG and 1 mM cAMP at 30°C .", "An aliquot of the cells were sonically disrupted and fractionated into the soluble ( S ) and membrane ( P ) fractions ( ‘sonic’ samples ) .", "Another aliquot was treated with 0 . 1 N NaOH and fractionated into the supernatant ( S ) and pellet ( P ) fractions ( ‘NaOH’ samples ) .", "W indicates unfractionated whole cell samples .", "Proteins were analyzed by 12 . 5% SDS-PAGE and immunoblotting using anti-HA , anti-HflD , anti-FtsH , and anti-SecB antibodies .", "HflD , FtsH and SecB are peripheral membrane , integral membrane and cytosolic proteins , respectively , and served as controls for the fractionation .", "Because the chromosomally-encoded HflD is expressed at a very low level and hard to detect , we expressed HflD from a multicopy plasmid .", "The majority of the model substrates , HA-MBP-RseA ( LY1 ) 148 , HA-MBP-YqfG and HA-MBP-YoaJ , was associated with the membrane both after sonical disruption and after alkali extraction , although higher proportions of HA-MBP-YoaJ was recovered in the soluble fractions as compared with the other two model substrates , indicating that most of the SMP-derived model proteins are integrally associated with the membrane . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 00610 . 7554/eLife . 08928 . 007Figure 2—figure supplement 2 . Effects of the MRE β-loop Cys substitutions on the RseP function .", "( A ) Assay of substrate cleavage by immunoblotting .", "Strain KA306 ( ΔrseA/ΔrseP/ΔclpP ) harboring pYH124 ( HA-MBP-RseA ( LY1 ) 148 ) was further transformed with a plasmid encoding the indicated single Cys mutant form of RseP-HM .", "Cells were grown at 30°C in M9-based medium containing 1 mM IPTG and 1 mM cAMP for 3 hr .", "Proteins were acid-precipitated , solubilized in 1% SDS and analyzed by 10% SDS-PAGE and anti-HA or anti-Myc immunoblotting as indicated .", "( B ) Complementation assay .", "Strain KK31 ( rseP::kan/pKK6 ( Para-rseP ) ) was further transformed with a plasmid encoding the indicated mutant form of RseP-HM under the lac promoter , or the corresponding vector .", "Cells were grown in L-0 . 02% arabinose , serially diluted with saline and a 4 µl each of the diluted culture was spotted on an L-0 . 02% arabinose or L-1 mM IPTG agar plate .", "The plates were incubated at 30°C for 20 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 007 We next examined the effects of MRE β-loop mutations on the cleavage of other substrates .", "Recent studies have shown that some small membrane proteins are encoded by the E . coli genome and expressed as functional proteins ( Hemm et al . , 2008; Alix and Blanc-Potard , 2009; Fontaine et al . , 2011 ) .", "YqfG and YoaJ , small membrane proteins with unknown functions , are predicted to have type II ( NIN-COUT ) membrane orientation according to the TMHMM program ( Krogh et al . , 2001 ) .", "Membrane topology of YoaJ was confirmed experimentally ( Fontaine et al . , 2011 ) .", "We examined the cleavage of these proteins by RseP because their structural features were analogous to those of signal peptides that were recently identified as RseP substrates ( Saito et al . , 2011 ) .", "To facilitate detection , we attached an HA-MBP domain to the N-termini of YqfG and YoaJ ( Figure 1E ) and expressed them in ΔrseA/ΔrseP strain ( Figure 2E , F ) .", "Anti-HA immunoblotting showed the accumulation of a protein of the expected size ( approximately 46 kDa ) when HA-MBP-YqfG was expressed alone ( Figure 2E ) .", "The protein was converted to a slightly smaller fragment upon co-expression with wild-type RseP-HM but not with RseP-HM E23Q mutant , indicating that HA-MBP-YqfG was cleaved by RseP presumably within its TM .", "As expected , the Δloop mutant did not cleave HA-MBP-YqfG .", "In contrast , HA-MBP-YoaJ did not undergo any cleavage ( Figure 2F ) .", "Cell fractionation and alkali extraction showed that most HA-MBP-YoaJ was integrally associated with the membrane ( Figure 2—figure supplement 1 ) , suggesting that its inability to undergo cleavage was not because of defective membrane localisation .", "These results show that the MRE β-loop plays a critical role in the proteolytic function of RseP and that RseP selectively cleaves type II membrane proteins , even those with a small periplasmic domain .", "Cys-scanning mutagenesis was performed to identify functionally important residues in the MRE β-loop .", "We constructed single Cys RseP derivatives by substituting Cys residues in place of the 15 residues in the MRE β-loop of Cys-less RseP-HM , which contained Ala residues in place of the 2 original Cys residues ( Cys-33 and Cys-427 ) .", "Complementation and model substrate cleavage assays showed that all the single Cys mutants , except G68C supported normal cell growth and cleaved HA-MBP-RseA ( LY1 ) 148 ( Figure 2—figure supplement 2 ) .", "Although the G68C mutant was inactive in complementation , it cleaved the model substrate with a slightly lower but significant efficiency .", "These results indicate that no residue in the MRE β-loop is specifically required for the proteolytic function of RseP .", "It is unclear why the G68C mutant failed to complement the ΔrseP mutation despite its substantial protease activity against the model substrate .", "G68C mutation might destabilise or inactivate the mutant protein under conditions employed in the complementation assay or might impair the cleavage of certain substrates that affect cell viability ( see below ) .", "We then performed a Pro-scanning experiment to test whether the secondary or tertiary structure of the MRE β-loop affects RseP function .", "Introduction of a Pro residue would disturb the higher-order structures of the MRE β-loop because Pro lacks the amide proton unlike other amino acids .", "Because the MRE β-loop originally had 1 Pro residue at position 65 , we replaced other residues with Pro individually .", "In contrast to the results for Cys mutants , Pro substitution at positions 67 , 68 , 69 and 70 , which are thought to be located in the distal part ( Leu-66 to Glu-75 ) of the MRE β-loop , abolished complementation ( Figure 2B ) .", "Consistently , the cleavage of HA-MBP-RseA148 was impaired moderately ( G67P ) or severely ( G68P , Y69P and V70P ) by these mutations ( Figure 2C ) .", "Similar cleavage defects were observed with HA-MBP-RseA ( LY1 ) 148 for G67P , Y69P and V70P mutants ( Figure 2D ) .", "Interestingly , the G68P mutant , which was almost completely defective in cleaving HA-MBP-RseA148 , efficiently cleaved HA-MBP-RseA ( LY1 ) 148 .", "Pro mutations in the MRE β-loop exerted more profound effects on the cleavage of HA-MBP-YqfG than on the cleavage of the other 2 model substrates .", "Cleavage was severely impaired by G67P , G68P , Y69P and V70P mutations and was moderately impaired by I61P , L66P and K71P mutations .", "However , the latter 3 mutations did not inhibit the cleavage of the other 2 model substrates to detectable levels .", "Substrate cleavage kinetics was investigated by pulse-chase experiments ( Figure 2G–I and Figure 2—source data 1 ) .", "We found that the initial rates of HA-MBP-RseA ( LY1 ) 148 and HA-MBP-YqfG cleavage by co-expressed RseP-HM were slightly lower than that of HA-MBP-RseA148 cleavage ( Figure 2G ) .", "During the 15 min chase period , the G68P mutant significantly cleaved HA-MBP-RseA ( LY1 ) 148 ( Figure 2H ) while the K71P mutant efficiently cleaved HA-MBP-RseA148 and HA-MBP-RseA ( LY1 ) 148 but only slightly cleaved HA-MBP-YqfG , which was consistent with immunoblotting results ( Figure 2I ) .", "Thus , the intrinsic susceptibility of each model substrate to wild-type RseP was not correlated with its susceptibility to MRE β-loop mutants , indicating that the MRE β-loop mutations resulted in the differential cleavage of different model substrates .", "We investigated the possible role of the MRE β-loop in RseP–substrate interaction by co-immunoprecipitation experiments ( Figure 3 ) .", "Inverted membrane vesicles ( IMVs ) were prepared from ΔrseA/ΔrseP strain expressing HA-MBP-RseA and RseP-HM derivatives , solubilised with n-dodecyl-β-d-maltoside ( DDM ) and were subjected to immunoprecipitation with agarose-conjugated anti-Myc or anti-HA antibody .", "The precipitated proteins were analysed by anti-HA or anti-Myc immunoblotting .", "In all the following co-immunoprecipitation/crosslinking experiments , RseP derivatives carried a mutation ( E23Q ) in the active site motif to prevent substrate cleavage during expression and immunoprecipitation/crosslinking .", "We previously used a similar approach to show that several residues in TM3 , including Asn-389 , play important roles in substrate binding ( Koide et al . , 2008 ) .", "Consistent with the previous results , wild-type RseP-HM was co-immunoprecipitated with HA-MBP-RseA and HA-MBP-RseA was co-immunoprecipitated with RseP-HM , but no co-immunoprecipitation was observed with RseP-HM having an N389L mutation ( Figure 3A ) .", "Deletion of the MRE β-loop almost completely abolished co-immunoprecipitation , suggesting its importance in RseP–substrate interaction ( Figure 3B ) .", "We found that all the MRE β-loop Pro mutants , except D74P located in the C-terminus of the loop , significantly decreased co-immunoprecipitation efficiency , indicating that the Pro mutations compromised the interaction between RseP-HM and model substrates ( Figure 3A ) .", "The result that absence of or mutations in the MRE β-loop interfered with RseP–substrate interaction suggests that the MRE β-loop is required for stable substrate binding by RseP .", "Pro substitutions in the proximal part ( Ile-61 to Ile-64 ) of the MRE β-loop had little effect on substrate proteolysis ( Figure 2 ) and decreased co-immunoprecipitation .", "These mutants would exhibit weak but sufficient interaction with substrates to promote cleavage in a membrane-integrated state . 10 . 7554/eLife . 08928 . 008Figure 3 . Effects of MRE β-loop mutations on RseP–substrate interaction .", "( A and B )", "Co-immunoprecipitation assays of RseP–substrate interaction .", "IMVs were prepared from KK211 ( ΔrseA/ΔrseP ) cells harbouring a plasmid encoding the indicated mutant of RseP-HM and pSTD881 ( HA-MBP-RseA140 ) .", "The IMVs were solubilised with DDM and were subjected to immunoprecipitation with anti-HA or anti-Myc antibody .", "The immunoprecipitates and the DDM-solubilised proteins ( input ) were analysed by 12 . 5% Laemmli–SDS-PAGE and immunoblotting with anti-HA and anti-Myc antibodies .", "Proteins form approximately fourfold more number of cells were loaded on the gel for the immunoprecipitated samples as compared with the input samples .", "All the RseP-HM derivatives carried the E23Q mutation .", "See Figure 3—figure supplement 1 for interaction between RseP-HM and HA-MBP-YoaJ . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 00810 . 7554/eLife . 08928 . 009Figure 3—figure supplement 1 . Analysis of RseP–YoaJ interaction .", "( A ) Co-immunoprecipitation assay of RseP–YoaJ interaction .", "IMVs were prepared from cells of KK211 ( ΔrseA/ΔrseP ) carrying a plasmid encoding the indicated mutant form of RseP-HM in addition to pSTD881 ( HA-MBP-RseA140 ) or pKA210 ( HA-MBP-YoaJ ) , solubilized with DDM and were subjected to immunoprecipitation with anti-HA .", "Immunoprecipitates and the DDM-solubilized proteins ( input ) were analyzed by 12 . 5% Laemmli–SDS-PAGE and anti-HA and anti-Myc immunoblotting .", "For the immunoprecipitated samples , proteins were loaded onto the gel in an amount corresponding to approximately fourfold more cells than were used for the input samples .", "All the RseP-HM derivatives carried the E23Q mutation .", "( B ) Effect of YoaJ overproduction on cleavage of HA-RseA148 .", "Cells of AD1811 ( ΔrseA ) /pKA195 ( HA-RseA148 ) /pKA268 ( HA-MBP-YoaJ ) or KK211 ( ΔrseA/ΔrseP ) /pKA195/pKA268 were grown at 30°C in M9-based medium containing 1 mM IPTG and 1 mM cAMP for 3 hr .", "Proteins were acid-precipitated , solubilized in 1% SDS and analyzed by 10% Laemmli–SDS-PAGE and anti-HA , anti-RseA and anti-RseP immunoblotting as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 009 We then examined the interaction of RseP with YoaJ ( Figure 3—figure supplement 1 ) .", "Anti-HA co-immunoprecipitation showed that RseP-HM was pulled down with HA-MBP-YoaJ in an MRE β-loop-dependent manner , indicating that YoaJ can interact with RseP although it is not cleaved by RseP and that the MRE β-loop can affect this interaction .", "However , overexpression of HA-MBP-YoaJ little affected the cleavage of co-expressed HA-RseA148 by RseP .", "It would be possible that YoaJ interacts with the MRE β-loop with much lower affinity than RseA does in the membrane , although similar amounts of RseP-HM were co-immunoprecipitated with HA-MBP-YoaJ and HA-MBP-RseA148 after membrane solubilisation .", "Alternatively , YoaJ might bind to RseP at a site different from that for RseA and other substrate proteins .", "In this case , deletion of the MRE β-loop might have indirectly disturbed the former site .", "The nature of the RseP–YoaJ interaction should be clarified in future study .", "The above results and the presumed location of the MRE β-loop near the proteolytic active site suggest that the loop is directly involved in substrate binding by RseP .", "This was examined by site-directed in vivo photo-crosslinking experiments ( Figure 4 ) .", "Amber suppression-mediated incorporation of p-benzoylphenylalanine ( pBPA ) , a non-natural photoreactive amino acid ( Young et al . , 2010 ) , allowed the expression of RseP-HM derivatives with pBPA in the MRE β-loop .", "We examined the crosslinking of plasmid-expressed RseP-HM with chromosomally encoded RseA .", "We used ΔrseP/ΔompA/ΔompC strain as the host strain because rseP can be deleted in rseA+ background when genes encoding outer membrane proteins OmpA and OmpC are deleted ( Douchin et al . , 2006 ) .", "The host strain expressed DegS , which cleaved some amount of RseA to RseA148 .", "Expression of pBPA-containing RseP-HM proteins decreased the accumulation of RseA148 , indicating the functionality of these proteins , although some exhibited lower proteolytic activity against RseA ( Figure 4—figure supplement 1A ) .", "After the exposure of cells to UV irradiation , the proteins were analysed by SDS-PAGE and immunoblotting .", "Anti-RseA immunoblotting showed UV irradiation produced bands of approximately 71 kDa ( XL ) when pBPA was incorporated at position 69 , 71 or 74 , suggesting that these bands represented photo-crosslinked products between RseP-HM and RseA ( Figure 4A , upper panels ) .", "However , these bands were not detected by anti-Myc immunoblotting ( Figure 4A , lower panels ) .", "As the expression level of plasmid-expressed RseP-HM was much higher than that of chromosomally encoded RseA , it seemed likely that only a small portion of RseP-HM was crosslinked with RseA .", "The over-expression of RseP-HM caused an increased background , making it difficult to detect weak bands of crosslinked products by anti-Myc immunoblotting .", "To confirm whether the 71 kDa band represented an RseP–RseA crosslinked product , we conducted immunoprecipitation experiments by using total proteins obtained from UV-irradiated RseP ( Y69pBPA ) -HM-expressing cells ( Figure 4—figure supplement 1B ) .", "Anti-RseA antibody precipitated a predominant 71 kDa band that reacted with both anti-RseA and anti-RseP antibodies ( upper panels ) .", "Anti-Myc antibody also precipitated a single anti-RseA-reactive protein of 71 kDa ( lower left panel ) .", "Although no clear band of the corresponding size was obtained after anti-RseP immunoblotting ( lower right panel ) , this may be due to increased background disturbance because of the high expression of RseP-HM .", "These results further support the notion that the 71 kDa band was the RseP–RseA crosslinked product . 10 . 7554/eLife . 08928 . 010Figure 4 . In vivo photo-crosslinking between RseP and RseA .", "( A ) Analysis of the MRE β-loop/RseA crosslinking .", "KA418 ( ΔompA/ΔompC/ΔrseP ) /pEVOL-pBpF cells were transformed with a plasmid encoding an RseP derivative with an amber mutation at the indicated position .", "The cells were grown at 30°C in M9-based medium containing 0 . 02% arabinose and 1 mM pBPA for 6 hr and were irradiated with UV light for 0 or 10 min .", "Proteins were precipitated using TCA and were analysed by SDS-PAGE with a 10% wide-range gel ( Nacalai Tesque , Inc . Kyoto , Japan ) containing 1% SDS and immunoblotting with anti-RseA antibody or by SDS-PAGE with a 10% Laemmli gel and immunoblotting with anti-Myc antibody .", "XL indicates crosslinked products .", "( B ) Effect of the N389L mutation on photo-crosslinking .", "KA418/pEVOL-pBpF strain was transformed with a plasmid encoding an RseP derivative with an amber mutation at the indicated position with or without the N389L mutation .", "The cells were subjected to photo-crosslinking analysis , as indicated in A . ( C ) Effect of periplasmic truncation of RseA on photo-crosslinking .", "KA418/pEVOL-pBpF or KA438 ( KA418 , ΔdegS ) /pEVOL-pBpF cells were transformed with a plasmid encoding an RseP derivative with an amber mutation at the indicated position .", "The cells were subjected to photo-crosslinking analysis , as indicated in A . All the RseP-HM derivatives in A–C carried the E23Q mutation .", "See Figure 4—figure supplement 1 for proteolytic activity of the pBPA-containing RseP derivatives and verification of RseP–RseA photo-crosslinking . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 01010 . 7554/eLife . 08928 . 011Figure 4—figure supplement 1 . Functionality of the pBPA-containing RseP derivatives and characterization of the photo-crosslinked product .", "( A ) Proteolytic activities of the RseP-HM derivatives having pBPA in the MRE β-loop .", "Strain KA418 ( ΔrseP/ΔompA/ΔompC ) /pEVOL-pBpF was further transformed with a plasmid encoding an RseP-derivative with an amber mutation at the indicated positions .", "Cells were grown at 30°C in the M9-based medium containing 0 . 02% arabinose and 1 mM pBPA for 6 hr .", "Proteins were acid-precipitated , solubilized in SDS sample buffer and analyzed by 10% SDS-PAGE and anti-RseA or anti-Myc immunoblotting as indicated .", "( B ) Identification of the photo-crosslinked proteins by immunoprecipitation .", "The UV-irradiated , acid-precipitated proteins from cells of KA429 or KA436 carrying a plasmid encoding the indicated RseP-HM derivatives in addition to pEVOL-pBpF were solubilized in 1% SDS and subjected to immunoprecipitation with agarose-conjugated anti-RseA ( upper panels ) or anti-Myc ( lower panels ) antibodies .", "Immunocomplexes were dissolved in SDS-sample buffer and analyzed by 10% SDS-PAGE and anti-RseA or anti-Myc immunoblotting as indicated .", "For the immunoprecipitated samples , proteins were loaded onto the gel in an amount corresponding to approximately 70-fold more cells than was used for the input samples . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 011 The N389L mutation that destabilised the RseP–substrate interaction considerably decreased RseA crosslinking at the 3 positions ( Figure 4B ) , suggesting that the photo-crosslinking reflected the functional interaction between RseP and RseA .", "We examined the effect of the cleavage by DegS ( Figure 4C ) .", "Periplasmic truncation of RseA was suppressed in the absence of DegS , thus increasing the accumulation of intact RseA , although few non-specific cleavages by other cellular proteases still occurred under this condition .", "RseP ( Y69pBPA ) -HM generated a significantly lower amount of the crosslinked product in the ΔdegS strain than in the degS+ strain , indicating that the MRE β-loop mainly interacts with the DegS-cleaved form of RseA .", "To determine whether the MRE β-loop directly interact with RseA TM , we examined disulfide crosslinking between RseP-HM and HA-RseA140 mimicking the DegS-cleaved form of RseA ( Kanehara et al . , 2002 ) .", "An RseP-HM variant having a unique Cys residue at position 69 , 70 or 71 was co-expressed with HA-RseA140 , which has a unique Cys at position 109 , or with its variant HA-RseA ( A108C/C109A ) 140 , which has a unique Cys in place of Ala-108 ( Cys-109 was replaced with Ala ) , in a ΔrseA/ΔrseP/ΔdegS strain .", "Ala-108 and Cys-109 are located in the middle of the RseA TM , and RseP-catalysed cleavage occurs between these residues ( Akiyama et al . , 2004; Flynn et al . , 2004 ) .", "Cells expressing these proteins were treated with Cu2+ ( phenanthroline ) 3 to induce disulfide bond formation .", "After quenching the oxidant , proteins were acid-denatured and solubilised in SDS .", "The samples were analysed directly by SDS-PAGE or after 2-mercaptoethanol treatment to cleave the disulfide bonds .", "The oxidant treatment generated a ∼75 kDa band that reacted with both anti-HA and anti-Myc antibodies when Y69C and K71C variants but not Cys-less and V70C variants of RseP-HM were co-expressed with HA-RseA ( A108C/C109A ) 140 ( Figure 5A ) .", "This band disappeared when the samples were treated with 2-mercaptoethanol before SDS-PAGE ( Figure 5B ) .", "The same results were obtained when the single Cys RseP-HM variants were co-expressed with HA-RseA140 ( C109 ) .", "These results were consistent with those of photo-crosslinking experiments because pBPA at positions 69 and 71 but not at position 70 was crosslinked with RseA , indicating that the MRE β-loop was near the RseP cleavage site .", "Together , our results strongly suggest that the MRE β-loop can directly bind to substrate TMs . 10 . 7554/eLife . 08928 . 012Figure 5 . Disulfide crosslinking of RseP and RseA . AD1840 ( ΔrseA/ΔrseP/ΔdegS ) cells harbouring a combination of plasmids encoding Cys-less or the indicated single Cys derivative of RseP-HM and HA-RseA140 were treated with Cu2+ ( phenanthroline ( PT ) ) 3 at 37°C for 5 min .", "After quenching the oxidant , proteins were precipitated using TCA and free thiol groups were blocked by treatment with N-ethylmaleimide ( NEM ) .", "The samples were analysed by 10% Laemmli–SDS-PAGE and immunoblotting with anti-HA ( upper panels ) or anti-Myc ( lower panels ) antibody , followed by treatment without ( A ) or with ( B ) 10% 2-mercaptoethanol ( ME ) .", "All the RseP-HM derivatives carried the E23Q mutation .", "Asterisk indicates a possible dimer of RseP-HM ( Koide et al . , 2008 ) that was not characterised further . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 012 We previously showed that helix-destabilising residues in substrate TMs promote the binding and cleavage of a substrate by RseP ( Akiyama et al . , 2004; Koide et al . , 2008 ) .", "Our current results showed that several Pro substitutions in the MRE β-loop exerted opposite effects , that is , they compromised substrate binding and cleavage .", "Therefore , we examined whether destabilisation of a substrate TM helix improves its cleavage by RseP MRE β-loop mutants .", "Because the MRE β-loop was inserted halfway into the membrane from the cytoplasmic side , we replaced each residue in the N-terminal region ( Ile-8 to Phe-15 ) of the presumed YqfG TM by a Pro residue , a strong helix destabiliser , and investigated its cleavage by RseP G67P and K71P mutants , which showed severe and moderate defects , respectively , in cleaving HA-MBP-YqfG ( Figure 6 ) .", "Wild-type RseP-HM cleaved all the HA-MBP-YqfG mutants almost as efficiently as it cleaved the original HA-MBP-YqfG ( Figure 6A ) .", "L11P and L12P mutations but not other mutations appreciably increased the cleavage by RseP ( G67P ) -HM ( Figure 6B ) .", "In contrast , cleavage by RseP ( K71P ) -HM was markedly improved by mutations other than L12P and L13P .", "L12P and L13P also improved cleavage , but their effects were much lower ( Figure 6C ) .", "We focused on 2 YqfG TM mutations I8P and L12P that resulted in differential cleavage by RseP ( G67P ) -HM and RseP ( K71P ) -HM and confirmed their effects by pulse-chase experiments ( Figure 6D–F and Figure 6—source data 1 ) .", "RseP ( WT ) -HM efficiently cleaved wild-type HA-MBP-YqfG and its I8P and L12P mutants ( Figure 6D ) .", "G67P mutation in the RseP MRE β-loop severely impaired its ability to cleave wild-type HA-MBP-YqfG .", "Only some cleavage was observed after the 60 min chase period ( Figure 6E ) .", "K71P mutation , which had a weaker effect on immunoblotting , also impaired the ability of RseP to cleave wild-type HA-MBP-YqfG during the 15 min chase period ( Figure 6F ) .", "RseP ( G67P ) -HM significantly cleaved HA-MBP-YqfG ( L12P ) but not HA-MBP-YqfG ( I8P ) ( Figure 6E ) and RseP ( K71P ) -HM efficiently cleaved HA-MBP-YqfG ( I8P ) but not HA-MBP-YqfG ( L12P ) ( Figure 6F ) , which was consistent with the immunoblotting results .", "Replacement of Leu-12 in YqfG by Asn , another strong helix-destabilising amino acid , also promoted cleavage by the RseP MRE β-loop Pro mutants .", "However , replacement of the same residue by a helix-forming Trp residue resulted in no increase in cleavage ( Figure 6A–C ) , suggesting that helix destabilisation is important for improving substrate cleavage .", "The I8P or L12P mutation did not promote cleavage by the RseP Δloop variant , indicating that these mutations did not bypass the RseP MRE β-loop function for substrate cleavage ( Figure 6—figure supplement 1 ) . 10 . 7554/eLife . 08928 . 013Figure 6 . Suppression of the effect of RseP MRE β-loop mutations by destabilising substrate TM helix .", "( A–C )", "Immunoblotting analysis of YqfG cleavage .", "KA306 cells carrying a combination of plasmids encoding the indicated RseP-HM mutants and HA-MBP-YqfG mutants were grown and were subjected to immunoblotting analysis , as described in Figure 2 .", "( D–F )", "Pulse-chase analysis of YqfG cleavage .", "KA306 cells harbouring a combination of plasmids encoding the indicated RseP-HM mutants and HA-MBP-YqfG mutants were grown and subjected to pulse-chase analysis , as described in Figure 2 .", "Two independent experiments were performed , and mean values are shown along with standard deviations .", "See Figure 6—source data 1 for gel images and quantitated band intensities data for Figure 6D–F .", "( G ) Immunoblotting analysis of LY1 cleavage .", "KA306 cells carrying a combination of plasmids encoding the indicated RseP-HM mutants and HA-MBP-RseA ( LY1 ) 148 mutants were grown and were subjected to immunoblotting analysis , as above . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 01310 . 7554/eLife . 08928 . 014Figure 6—source data 1 . Zip file containing gel images and quantified band intensity data for the pulse-chase experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 01410 . 7554/eLife . 08928 . 015Figure 6—figure supplement 1 . Cleavage of the I8P and L12P mutants of HA-MBP-YqfG depends on the MRE β-loop . KA306 cells harbouring a combination of plasmids encoding the indicated RseP-HM mutants and HA-MBP-YqfG mutants were grown and total proteins were subjected to immunoblotting analysis , as described in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 015 Similar effects of a Pro substitution was also observed for LY1 , another substrate TM ( Figure 6G ) ; the F21P mutation in LY1 significantly improved the cleavage of this TM by RseP ( V70P ) -HM that was almost inactive in cleavage of wild type LY1 , but the Pro substitution of the neighbouring residue , Phe-20 , did not .", "The results that helix-destabilising mutations in substrate TMs at least partially suppress the defects in substrate cleavage induced by Pro substitutions in the RseP MRE β-loop in an allele-specific manner suggests a specific interaction between substrate TMs and the RseP MRE β-loop ." ], [ "RseP is one of the most extensively characterised proteins among the S2P family of I-CLiPs .", "However , detailed mechanisms underlying its substrate recognition and cleavage are not completely understood .", "We focused on the MRE β-loop , a conserved intramembrane β-hairpin-like structure , and investigated its role in substrate proteolysis by RseP .", "Deletion or Pro substitution in the MRE β-loop impaired substrate cleavage , indicating its critical role in the proteolytic function of RseP .", "Crosslinking and co-immunoprecipitation experiments showed that the MRE β-loop was directly involved in substrate binding .", "Several Pro substitutions differentially affected RseP-catalysed proteolysis of substrates .", "Helix-destabilising mutations in substrate TMs suppressed the defects caused by mutations in the MRE β-loop in an allele-specific manner .", "These results collectively suggest that the MRE β-loop specifically recognises substrate TMs .", "The proximal part of the MRE β-loop has a short β-strand ( Ile72-Leu-Leu ) in the reported crystal structure of mjS2P ( Feng et al . , 2007 ) .", "However , no secondary structure is assigned for the distal part of the MRE β-loop .", "Analysis of the secondary structure with STRIDE ( Frishman and Argos , 1995 ) suggested that Val80-Ala-Met in the distal part forms a β-strand ( Figure 1B–D ) .", "This region is in an extended conformation with the side chains facing the opposite sides in an alternate manner ( as expected for a β-strand ) suggesting that this region forms a β-strand .", "Sequence-based prediction suggested that the MRE β-loops of RseP and SpoIVFB also have β-strands in the corresponding regions ( Figure 1D ) , suggesting that the MRE β-loop commonly assumes a β-hairpin-like structure .", "Cys-scanning mutagenesis suggest that no specific residue in the MRE β-loop is essential for the proteolytic function of RseP because the Cys substitutions had little or only slight effect on the substrate cleavage .", "In contrast , substrate cleavage was severely impaired by Pro substitutions .", "These observations suggest that a higher-order structure of the MRE β-loop is important for the proteolytic function of RseP .", "Interestingly , some Pro substitutions in the MRE β-loop resulted in the differential cleavage of the 3 model substrates .", "Pulse-chase experiments showed that RseP ( G68P ) -HM significantly cleaved LY1 but only slightly cleaved RseA TM and YqfG TM .", "In contrast , RseP ( K71P ) -HM efficiently cleaved RseA TM and LY1 but only slightly cleaved YqfG TM .", "These 3 substrates were rapidly cleaved by wild-type RseP-HM .", "Therefore , the effects of MRE β-loop mutations cannot be simply explained by the intrinsic susceptibility of these substrates to RseP .", "In addition , some mutations in YqfG TM suppressed G67P and K71P mutations of the RseP MRE β-loop in an allele-specific manner .", "The I8P mutation improved the proteolysis by RseP ( K71P ) -HM but not by RseP ( G67P ) -HM , whereas the L12P mutation improved the proteolysis by RseP ( G67P ) -HM compared with that of RseP ( K71P ) -HM .", "Similarly the F21P , but not F20P , mutation in the LacY TM1 region ( LY1 ) partially suppressed the V70P mutation of the RseP MRE β-loop .", "These results suggest that the MRE β-loop specifically recognises substrate TMs .", "Direct evidence of MRE β-loop–substrate interaction was obtained by co-immunoprecipitation and crosslinking experiments .", "Pro substitutions in the MRE β-loop markedly decreased the co-immunoprecipitation of RseP-HM with HA-MBP-RseA after membrane solubilisation , suggesting that the integrity of the MRE β-loop is required for stable RseP–substrate interaction .", "Disulfide- and photo-crosslinking experiments showed that residues at positions 69 and 71 of the MRE β-loop contact with substrates .", "Cys residues at these positions disulfide-bonded with Cys residues on either side of the scissile bond in RseA .", "A similar result was reported recently for SpoIVFB and its substrate Pro-σK ( Zhang et al . , 2013 ) .", "A Cys residue in place of Val-70 in the MRE β-loop of SpoIVFB formed disulfide bonds with Cys residues at several positions around the cleavage site in Pro-σK , suggesting that the MRE β-loop of SpoIVFB directly interacts with Pro-σK .", "However , the role of the MRE β-loop in SpoIVFB function has not been investigated experimentally .", "Although RseP also has a Val residue at the position corresponding to Val-70 in SpoIVFB ( Figure 1D ) , we did not observe any substrate crosslinking at this position .", "This might be due to differences in the mode of enzyme–substrate interaction between RseP and SpoIVFB .", "Indeed , the substrates of these enzymes show some differences .", "In contrast to RseA , which is a single-spanning membrane protein with type II orientation , Pro-σK is suggested to be peripherally associated with the membrane ( Zhou et al . , 2013 ) .", "In addition , although helix-destabilising residues in substrate TMs are important for their efficient cleavage by RseP , these residues are not required for the cleavage of Pro-σK by SpoIVFB ( Zhou et al . , 2013 ) .", "Thus , the role of the MRE β-loop in substrate binding and cleavage might slightly differ between RseP and SpoIVFB .", "The N389L mutation in RseP TM3 , which was previously shown to weaken the RseP-substrate interaction ( Koide et al . , 2008 ) , inhibited the in vivo photo-crosslinking of the MRE β-loop with RseA , suggesting that the observed crosslinking reflected a functional RseP–substrate interaction .", "The TM4 ( corresponding to RseP TM3 ) of mjS2P that contains one of the active site residues ( corresponding to Asp-402 in RseP TM3 ) can be located in the vicinity of the proteolytic active site and the MRE β-loop ( Feng et al . , 2007 ) .", "We previously showed that Cys introduced at the positions of Pro-397 and Pro-399 in RseP TM3 can form a disulfide bond with Cys at multiple positions of RseA TM .", "In mjS2P , the residues corresponding to Pro-397 and Pro-399 of RseP reside in a loop-like structure .", "The region containing residues 397 and 399 might be flexible and act with the MRE β-loop in stable binding of a substrate .", "An α-helix is generally not amenable to proteolysis and unwinds to an extended structure to undergo cleavage ( Wolfe , 2009 ) .", "Many zinc metalloproteinases have a β-strand ( edge strand ) close to their proteolytic active site that binds to substrates in an extended conformation through β-strand addition and presents them to catalytic residues ( Stocker and Bode , 1995; Langklotz et al . , 2012 ) .", "Our results suggest that the MRE β-loop stabilises the extended conformation of a substrate by directly binding to it through β-strand addition , thus promoting substrate cleavage in a manner similar to the edge strand ( Figure 7 ) .", "Crosslinking at alternate positions in the Tyr69-Val-Lys region supports the idea that this region is in a β-strand conformation during its interaction with a substrate .", "According to this model , mutations that disrupt the secondary structure of the distal part of the MRE β-loop would compromise its interaction with a substrate .", "Introduction of a Pro residue will disturb substrate accommodation through β-strand addition or interstrand interaction because Pro residues cannot serve as hydrogen bond donors because of the absence of the amide proton .", "Moreover , if the compromised interaction of the MRE β-loop with a substrate prevents efficient conformational conversion of the substrate TM and decreases its cleavage efficiency , destabilisation of the α-helical structure of the substrate TM might improve its cleavage by RseP MRE β-loop mutants , which is consistent with our results .", "A recent structural study of E . coli GlpG that belongs to the rhomboid family of I-CLiPs revealed that the P1–P4 region of a substrate forms a β-sheet with the GlpG loop 3 located near the active site ( Zoll et al . , 2014 ) .", "Mutations in loop 3 severely compromise the GlpG activity , suggesting the interaction between the substrate and loop 3 is functionally important ( Baker and Urban , 2012 ) .", "Although the loop 3 of GlpG and the MRE β-loop of RseP differ in that the former is located near the periplasmic surface of the membrane and forms a parallel β-sheet with a substrate whereas the latter is located near the cytoplasmic surface and forms a anti-parallel β-sheet , they might have a similar role in stabilising an extended substrate structure and promoting its cleavage . 10 . 7554/eLife . 08928 . 016Figure 7 . A model describing the function of the MRE β-loop .", "( A ) The periplasmic PDZ tandem of RseP acts as a size-exclusion filter to discriminate between substrates based on the size of their periplasmic region .", "( B ) The binding of the MRE β-loop serves as an additional checkpoint for substrates with a small periplasmic region and contributes to selective substrate cleavage .", "( C ) The MRE β-loop binds to substrate TMs with some specificity because of β-strand addition , stabilises its extended conformation and presents the substrate to the recessed proteolytic active site of RseP . DOI: http://dx . doi . org/10 . 7554/eLife . 08928 . 016 Differential effects of Pro substitutions in the MRE β-loop on the cleavage of the model substrates and allele-specific suppression of these mutations by helix-destabilising mutations in substrate TMs suggest that the MRE β-loop–substrate interaction occurs with some specificity .", "MRE β-loop-assisted cleavage of a substrate may be affected by helix stability and amino acid sequence of substrate TMs .", "We recently suggested that the periplasmic PDZ tandem of RseP acts as a size-exclusion filter to prevent the cleavage of substrates with bulky periplasmic domains ( Figure 7A ) ( Hizukuri et al . , 2014 ) .", "In this study , we observed that RseP did not cleave YoaJ , a type II membrane protein with a very small periplasmic domain , suggesting the presence of additional mechanism ( s ) for substrate discrimination .", "In vivo photo-crosslinking experiments suggest that the MRE β-loop interacts with RseA after site 1 cleavage ( Figure 7B ) and that it acts as the second checkpoint for membrane proteins that passed the first check by the PDZ filter and contributes to their selective proteolysis .", "It should be noted that mjS2P does not have PDZ domains .", "Thus , the MRE β-loop could play a central role in cleaving specific substrates in this enzyme .", "Although the MRE β-loop may not interact with intact RseA , our previous chemical crosslinking and co-immunoprecipitation experiments showed that RseP directly interacted with intact RseA in the membrane ( Kanehara et al . , 2003 ) , suggesting that RseP has a separate binding site ( exosite ) for intact RseA .", "Because the PDZ filter excludes intact RseA from the intramolecular active site , the presumed exosite may exist on the external surface of the enzyme .", "Other families of I-CLiPs such as rhomboid and γ-secretase have also been suggested to have an exosite ( Kornilova et al . , 2005; Strisovsky et al . , 2009; Watanabe et al . , 2010; Arutyunova et al . , 2014 ) .", "A systematic in vivo crosslinking approach would help in identifying binding sites for intact RseA .", "This along with the structural analysis of RseP , especially in complex with a substrate , would be essential for verifying our model and for understanding molecular mechanisms underlying substrate recognition and intramembrane proteolysis by RseP .", "Further , we would like to study the physiological significance of RseP-catalysed cleavage of YqfG and identify additional substrates of RseP to elucidate all the cellular roles of RseP ." ], [ "All the bacterial strains used in this study are derivatives of E . coli K12 and are listed in Supplementary file", "1 . KA306 was constructed by transferring the clpP::cat marker derived from MC4100 clpP::cat ( provided by M . Kitagawa ) into AD2328 .", "The clpP::cat marker was introduced to stabilise the RseP-cleaved fragment of HA-MBP-RseA148 .", "KA418 was constructed as follows .", "First , ΔompA::kan from JW0940 ( Baba et al . , 2006 ) was introduced into CU141 by P1 transduction , and the kan cassette of the resulting strain was deleted using pCP20 , as described previously ( Datsenko and Wanner , 2000 ) .", "Introduction of ΔompC::kan from JW2203 ( Baba et al . , 2006 ) and deletion of the kan cassette were performed in a similar manner to yield YH426 .", "KA363 was constructed by introducing rseP::kan from KK211 into YH426 .", "We found that KA363 had spontaneously lost F′lac+ lacIq .", "We thus constructed KA418 by re-introducing F′lac+ lacIq into KA363 by conjugation .", "KA438 was constructed by transferring degS::tet from AD1839 ( Kanehara et al . , 2002 ) to KA418 by P1 transduction .", "Plasmids used in this study are listed in Supplementary file", "2 . pKA1 and pKA117 were constructed by replacing the region encoding the MRE β-loop ( Ile61 to Glu75 ) in rseP-his6-myc on pKK49 or a corresponding region in rseP ( Cys-less ) -his6-myc on pSTD892 with GGCGGT ( encoding Gly–Gly ) by site-directed mutagenesis .", "pKA19 was constructed by introducing the E23Q mutation into pSTD892 .", "pKA52 was constructed by replacing a 1 . 5 kb BamHI–SacI fragment of pKA1 with a corresponding fragment of pKK34 .", "pKA65 was constructed by cloning a 1 . 4 kb EcoRI–HindIII fragment of pYH19 in the same site of pSTD689 .", "Other plasmids were constructed by site-directed mutagenesis by using appropriate combinations of primers and template plasmids .", "Mutations were confirmed by DNA sequencing .", "pMZ14 was constructed by cloning a PCR-amplified fragment encoding YqfG into the SalI–PstI site of pSTD835 and by ligating an EcoRI–HindIII fragment of the resulting plasmid with EcoRI-/HindIII-digested pSTD689 .", "pKA195 was constructed by cloning a 0 . 6 kb EcoRI–HindIII fragment of pYH18 in the same site of pSTD689 .", "For construction of pKA210 , first , a PCR-amplified fragment encoding YoaJ was cloned in the SalI–PstI site of pSTD835 , and then an EcoRI–HindIII fragment of the resulting plasmid was ligated with EcoRI-/HindIII-digested pSTD689 .", "pKA268 was constructed by cloning a 1 . 4 kb EcoRI–HindIII fragment of pMZ14 in the same site of pUC118 .", "L medium ( 10 g/l bactotryptone , 5 g/l yeast extract and 5 g/l NaCl; pH adjusted to 7 . 2 by using NaOH ) and M9 medium ( without CaCl2 ) ( Miller , 1972 ) were used for bacterial cultivation .", "Ampicillin ( 50 μg/ml ) , chloramphenicol ( 20 μg/ml ) , spectinomycin ( 50 μg/ml ) , kanamycin ( 25 or 12 . 5 μg/ml ) and/or tetracycline ( 25 μg/ml ) were added for selecting transformants and transductants and for growing plasmid-harbouring strains .", "Cells were grown at 30°C in L medium containing 1 mM isopropyl-β-d-thiogalactopyranoside ( IPTG ) and cAMP and were suspended in 50 mM HEPES-KOH ( pH 8 . 0 ) containing 50 mM KCl , 10% glycerol and 1 mM dithiothreitol ( DTT ) .", "The cells were then fractionated after sonication or subjected to alkali extraction .", "For cell fractionation ( Kihara et al . , 2001 ) , the cells were mixed with 1/10 vol of 1 mg/ml lysozyme in 100 mM EDTA ( pH 8 . 0 ) and were incubated at 0°C for 30 min .", "The cells were then disrupted by sonication and ultracentrifuged ( 99 , 000×g for 1 hr at 4°C ) .", "The pellet was suspended in 50 mM HEPES-KOH ( pH 8 . 0 ) containing 50 mM KCl , 10% glycerol and 1 mM DTT .", "Proteins were precipitated using 5% ( final concentration ) trichloroacetic acid ( TCA ) .", "Alkali fractionation was performed as described previously ( Ito and Akiyama , 1991 ) .", "The cells were suspended in 50 mM HEPES-KOH ( pH 8 . 0 ) containing 50 mM KCl , 10% glycerol and 1 mM DTT .", "After addition of 1/10 vol of 10 mg/ml lysozyme in 50 mM EDTA ( pH 8 . 0 ) , the cells were incubated at 0°C for 5 min and were disrupted by freezing-thawing .", "The samples were then mixed with an equal volume of cold 0 . 2 N NaOH , vortexed vigorously for approximately 10 s and ultracentrifuged in a microfuge ( 99 , 000×g for 1 hr at 4°C ) .", "The supernatant was mixed with 1/10 vol of 100% ( wt/vol ) TCA .", "Acid denatured protein precipitates were collected by centrifugation , washed with acetone , and analysed by 12 . 5% SDS-PAGE and immunoblotting .", "Because the chromosomally encoded HflD was expressed at a very low level and was difficult to detect , we expressed HflD from a multicopy plasmid .", "Cells harbouring an appropriate combination of plasmids encoding an RseP derivative and a model substrate were grown at 30°C in M9 medium supplemented with 20 μg/ml of each of the 20 amino acids , 2 μg/ml thiamine , 0 . 4% glucose , 1 mM IPTG and 1 mM cAMP for 3 hr .", "A part of the culture was mixed with an equal volume of 10% TCA ( Hizukuri and Akiyama , 2012 ) .", "Protein precipitates were recovered by centrifugation , washed with acetone and dissolved in 1× SDS sample buffer .", "Proteins were analysed by 10% Laemmli–SDS-PAGE and immunoblotting with anti-Myc and anti-HA antibodies , as described previously ( Inaba et al . , 2008 ) .", "The blotted proteins were visualised and quantified using ECL or ECL Prime Western Blotting Detection Reagent ( GE Healthcare , Waukesha , WI ) and LAS-3000 Mini Lumino-Image Analyzer ( Fujifilm , Tokyo , Japan ) .", "Cells harbouring an appropriate combination of plasmids encoding an RseP derivative and a model substrate were grown at 30°C in M9 medium supplemented with 20 μg/ml of each of 18 amino acids ( except Met and Cys ) , 2 μg/ml thiamine and 0 . 4% glucose to a mid-log phase and were induced with 1 mM IPTG and 5 mM cAMP for 10 min .", "The cells were then labelled with 370 kBq/ml [35S]-methionine ( American Radiolabeled Chemicals , St . Louis , MO ) for 30 s .", "Chasing was performed using 200 μg/ml unlabelled methionine for the indicated periods .", "Proteins were precipitated using 5% ( final concentration ) TCA , washed with acetone , dissolved in 50 mM Tris–HCl ( pH 8 . 1 ) containing 1 mM EDTA and 1% SDS and immunoprecipitated with anti-HA antibody , as described previously ( Akiyama et al . , 2004 ) .", "Labelled proteins were separated by SDS-PAGE and were visualised and quantified using a phosphor imager ( BAS1800 ) ( Fujifilm ) .", "Co-immunoprecipitation experiments were performed as described previously ( Koide et al . , 2008 ) .", "Total membranes were suspended in 50 mM HEPES-KOH ( pH 7 . 5 ) containing 50 mM KCl and 20% glycerol , diluted 10-fold with 50 mM HEPES-KOH ( pH 7 . 5 ) containing 300 mM KCl and 10% glycerol and solubilised with 1% DDM on ice for 1 hr .", "After clarification , the supernatant was incubated with agarose-conjugated mouse monoclonal anti-HA ( F-7 ) or anti-Myc ( 9E10 ) antibody ( Santa Cruz Biotechnology , Inc . Santa Cruz , CA ) at 4°C for 3 . 5 hr with rotation .", "Immunocomplexes were collected , washed 3 times with 50 mM HEPES-KOH ( pH 7 . 5 ) containing 300 mM KCl , 10% glycerol and 0 . 1% DDM and dissolved in 1× SDS sample buffer .", "The samples were analysed by 12 . 5% Laemmli–SDS-PAGE and by immunoblotting with rabbit polyclonal anti-Myc and anti-HA antibodies ( Santa Cruz Biotechnology , Inc . ) .", "Cells harbouring a plasmid encoding an RseP derivative with an amber mutation in the MRE β-loop region and pEVOL-pBpF ( Addgene , Inc . , Cambridge , MA ) were grown at 30°C in M9 medium supplemented with 2 μg/ml thiamine , 0 . 4% glucose , 0 . 02% l-arabinose and 1 mM pBPA for 6 hr .", "After adding 100 μg/ml ( final concentration ) spectinomycin to halt protein synthesis , the cells were irradiated with UV light ( 365 nm ) at 4°C for 10 min , as described previously ( Narita et al . , 2013 ) .", "Proteins were precipitated by mixing the UV-irradiated cells with 1/20 vol of 100% TCA , washed with acetone , dissolved in 1× SDS sample buffer and analysed by 10% Laemmli–SDS-PAGE and immunoblotting with anti-RseA and anti-Myc antibodies .", "For verifying the in vivo crosslinked products , TCA-precipitated proteins were dissolved in 50 mM Tris–HCl ( pH 8 . 1 ) containing 1% SDS and 1 mM EDTA , diluted 33-fold with 50 mM Tris–HCl ( pH 8 . 1 ) containing 150 mM NaCl and 1% NP-40 and immunoprecipitated with TrueBlot Anti-Rabbit IgG IP Beads ( eBioscience , Inc . , San Diego , CA ) plus anti-RseA antibody or agarose-conjugated anti-Myc antibody ( Akiyama et al . , 2004 ) .", "The proteins were separated by SDS-PAGE on a 10% wide-range gel ( Nacalai Tesque , Inc . , Japan ) and by immunoblotting with anti-RseA and anti-RseP antibodies by using Can Get Signal Immunostain Enhancer Solution ( Toyobo , Co . , Ltd , Osaka , Japan ) and TrueBlot Anti-Rabbit IgG .", "Cells harbouring an appropriate combination of plasmids encoding an RseP derivative and a model substrate were grown at 30°C in L medium containing 1 mM IPTG and 1 mM cAMP for 3 . 5 hr .", "Disulfide bond formation was induced as described previously ( Koide et al . , 2008 ) .", "Briefly , the cells were treated with 0 . 1 mM Cu2+ ( phenanthroline ) 3 or 3 mM 2-phenanthroline at 37°C for 5 min .", "Oxidation was terminated by incubating the cells with 12 . 5 mM neocuproine .", "Proteins were precipitated using TCA and dissolved in 100 mM Tris–HCl ( pH 7 . 5 ) containing 1 . 5% SDS , 5 mM EDTA and 25 mM NEM .", "The samples were mixed with an equal volume of SDS sample buffer containing no or 10% 2-mercaptoethanol , boiled at 98°C for 5 min and analysed by 10% Laemmli–SDS-PAGE and immunoblotting with anti-Myc and anti-HA antibodies ." ] ]
[ "Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP , which belongs to S2P family of intramembrane-cleaving proteases , remain unclear .", "We examined the function of a conserved region looped into the membrane domain of RseP to form a β-hairpin-like structure near its active site in substrate recognition and cleavage .", "We observed that mutations disturbing the possible β-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates .", "Co-immunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates .", "Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner .", "These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site , which contributes to substrate discrimination ." ]
[ "Cells have communication systems that enable them to respond to potentially dangerous changes in their external environment .", "For example , bacteria have an enzyme called RseP that helps to activate responses to external stresses .", "This enzyme sits in the membrane that surrounds the cell and cuts a protein called RseA to release a signal molecule into the cell interior .", "This signal molecule then promotes the expression of particular genes to protect the cell from harm .", "Previous studies have identified the ‘active site’ of RseP , which is the region of the enzyme that actually cuts the target protein .", "However , it is not clear how the enzyme is able to identify and cleave RseA and its other ‘substrate’ proteins .", "The enzyme also contains a structure called a β-hairpin-like loop that is close to the active site , which is not commonly found in membrane proteins .", "Here , Akiyama , Mizuno et al . used genetic and biochemical techniques to study the role of this loop structure in the RseP enzyme of the E . coli bacterium .", "The experiments show that the loop specifically binds to a section of substrate proteins—called the transmembrane segment—that spans the cell membrane .", "Several genetic mutations that affected the loop altered the ability of RseP to bind to and cleave substrates .", "The effect of these mutations in RseP could be suppressed by introducing genetic mutations in substrates that altered the transmembrane segments .", "Akiyama , Mizuno et al . propose that the β-hairpin-like loop of the RseP enzyme binds the transmembrane segment of a substrate and presents it to the active site .", "A previous study showed that another region of RseP called the periplasmic PDZ domains can act as a filter to stop RseP cutting other membrane proteins in error .", "Akiyama , Mizuno et al . 's findings suggest that the β-hairpin-like loop serves as an additional checkpoint to identify RseA and other proteins that RseP targets .", "The next step is to carry out further experiments to test this model ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "ecology", "neuroscience" ]
Patterns of call communication between group-housed zebra finches change during the breeding cycle
elife-07770-v1
[ [ "Vocal communication plays an important role for a variety of social animals , because it is often directly linked with individual survival and successful reproduction .", "Vocal signals are especially important in group-living species , because they can be used to maintain group cohesion and coordinate common activities ( reviewed in Fichtel and Manser , 2010 ) , but also to recognise , locate and interact with specific individuals inside such groups ( Jouventin et al . , 1999a , 1999b; Aubin and Jouventin , 2002; Balsby et al . , 2012; Ter Maat et al . , 2014 ) .", "However , in songbirds , most vocalisation studies have focused on male song and its relationship with hormones and reproduction ( Nottebohm et al . , 1987; Perez et al . , 2012; Gahr , 2014 ) in solitary , territorial , temperate-zone species ( Marler , 2004 ) .", "But songbirds also produce calls in a variety of contexts ( Marler , 2004 ) , sometimes in very large numbers throughout the day ( Beckers and Gahr , 2010 ) .", "The usage and function of such calls is still unknown , mainly because it has been challenging to investigate with individual-level resolution naturally occurring vocal interactions between group members , in relevant contexts or different life-history stages .", "It has been hypothesized that zebra finches ( Taeniopygia guttata ) , which are group-living , socially monogamous and opportunistically breeding songbirds ( Zann , 1996; Perfito et al . , 2007 ) , share a different form of vocal communication with their life-long partner compared to other members of their group ( Zann , 1996 ) .", "In this species , both sexes produce diverse calls in large numbers , in various social contexts ( Zann , 1996; Beckers and Gahr , 2010; Ter Maat et al . , 2014 ) , and depending on group structure ( Elie et al . , 2011 ) .", "Some soft calls , that is , low-amplitude vocalisations used in close-range signalling ( Dabelsteen et al . , 1998 ) , have been suggested to play a role in pair communication ( Zann , 1996; Elie et al . , 2010; Ter Maat et al . , 2014 ) .", "However , until recently , it was not possible to record and reliably assign all calls of individual zebra finches in the presence of their mates and within a group ( Elie et al . , 2011 ) .", "Because calls are short and may be low in amplitude , especially when used at close range , earlier studies often resorted to strongly reduced social contexts or impoverished environments to investigate vocalisations at the individual level ( Blaich et al . , 1996; Vignal et al . , 2004; Anisimov et al . , 2014 ) .", "Hence , these studies mainly addressed mechanistic questions of vocal behaviour , but to understand the underlying meaning of calling , individual-based information in a socially relevant context is necessary .", "Our aim was thus to investigate with individual-level resolution the calling behaviour of zebra finches that were behaving freely in social groups , in a changing environment .", "We aimed at studying the dynamics of different call types and their usage—on the individual level as well as in interactions between mates and other group members—in relation to reproductive state and successful egg-laying .", "To do this , we housed groups of four females and four males previously unknown to each other together in large aviaries for about 3 weeks , provided them with nest material , and continuously recorded individual vocalisations of all group-members using microphone telemetry ( = on-bird microphone transmitters ) .", "While the birds formed pairs and passed through different stages of their breeding cycle , we recorded vocalisations , performed behavioural observations , took blood samples for hormone determination , and monitored their nests to measure reproductive performance ." ], [ "30 out of 32 birds formed pairs ( for definitions , see ‘Materials and methods’ , Tables 1 ,", "2 ) within 1 to 7 days and began occupying nest boxes within 5 . 6 ± 3 . 3 days ( mean ± standard deviation [SD] ) .", "The birds were not entirely synchronized in their reproductive stages and some nest building began even before actual nest material was provided ( using scraps of food or single threads from backpack material ) .", "But the addition of proper nest material triggered nesting activities in all birds but one , resulting in a mean onset of nest building at 7 . 3 ± 2 . 4 days .", "Two categories of reproductive status , ‘nest stage’ and the more detailed ‘breeding stage’ resulted from our behavioural observations and nest checks ( see ‘Materials and methods’ ) and are depicted in an overview of reproductive activity over the first 20 days ( Figure 2 ) .", "Hereby , ‘nest stage’ reflected large-scale changes of reproductive stages and was confirmed by correlated changes in sex steroid levels ( Figure 2—figure supplement 1 , see Appendix 1 ) .", "Zebra finches reproduce as soon as environmental conditions permit to ensure successful breeding ( see ‘Discussion’ ) .", "Therefore , birds that produced a clutch of eggs during the trials were termed ‘successful’ and those that did not as ‘unsuccessful’ at egg-laying .", "Pairs had eggs after 11 . 6 ± 5 . 8 days and began incubating them after 18 . 3 ± 5 . 1 days . 10 . 7554/eLife . 07770 . 013Table 1 . Overview and short description of different agonistic , affiliative or sexual , and neutral behaviours and whether they were measured as occurrences ( count ) or every 2 min ( duration ) DOI: http://dx . doi . org/10 . 7554/eLife . 07770 . 013BehaviourDescriptionCount/DurationTypeDisplacementFocal bird arrives at another bird's location forcing it to leaveCountAgonisticFightingFor example , bill-fight , full body fight , chasingCountClumpingBirds sit in direct physical contact with each otherDurationAffiliative or sexual behaviourAllopreeningOne bird preens another birdDurationCopulation solicitationFemale fans tail at maleCountCopulationMale mounts femaleCountEnter nest boxBirds enter the same nest box without fightingCountForagingBird is foraging on ground , feeding , drinkingDurationNeutralPreeningBird is self-preeningDurationFlyingBird flies around in aviaryCountIncubatingBird sitting inside nest box with eggsDuration10 . 7554/eLife . 07770 . 014Table 2 . Overview and short description of breeding stages and nest stagesDOI: http://dx . doi . org/10 . 7554/eLife . 07770 . 014Breeding stageDescriptionNest stageUnpairedBird does not show increased prosocial behaviour towards specific individualPre-nestingNo nestPaired but without nestTerritorialPair defending nest site without nest materialEarly nestingNest inspectionPair inspecting different nest boxesNest buildingPair bringing nest material to nest boxLater nestingLayingPair's female laying eggsIncubationPair members incubating Apart from calls related to parental behaviour ( ‘thucks’ ) , we found all vocalisation types described by Zann ( 1996 ) .", "The five most frequently used and distinct call types ( ‘distance calls’ , ‘stacks’ , ‘tets’ , ‘cackles’ and ‘whines’ , Figure", "3 ) were used for further analyses ( 182 . 752 calling events ) .", "Within these five call types , we found differences in the number of vocalisations uttered per bird and recording in relation to reproductive stage and sex ( Figure 4 , Figure 4—figure supplement 1 , Appendix 2 , Appendix 2—table 1 , see ‘Materials and methods’ for sample sizes ) .", "For distance calls , cackles and whines , the numbers of vocalisations changed for males and females in the same way ( no interaction between ‘nest stage’ and sex ) .", "Loud distance calls were produced most when birds were not yet paired or nesting , with highest levels during pre-nesting , that then decreased during the early and again during the later nest stages ( Figure 4 , Fstage = 18 . 5 , Fsex = 1 . 13 , Rmarginal2 = 0 . 20 , Rconditional2 = 0 . 37; see ‘Materials and methods’ for an explanation of F , Rmarginal2 , Rconditional2 ) .", "Cackles and whines increased at the onset of reproductive activities , with cackles showing a peak in both sexes during the early nest stage ( Fstage = 3 . 80 , Fsex = 2 . 28 , Rmarginal2 = 0 . 08 , Rconditional2 = 0 . 33; Figure 4 ) , and whines showing a peak during early and later nest stages ( Fstage = 8 . 34 , Fsex = 1 . 58 , Rmarginal2 = 0 . 12 , Rconditional2 = 0 . 24; Figure 4 ) .", "Tets did not change throughout the three nest stages for either sex ( Fstage = 0 . 49 , Fsex = 0 . 39 , Rmarginal2 = 0 . 01 , Rconditional2 = 0 . 34; Figure 4 ) .", "Stack calls showed an interaction between nest stage and sex: the amount did not change for males ( Fstage = 0 . 06 , Rmarginal2 = 0 . 001 , Rconditional2 = 0 . 42; Figure 4 ) , but in females , stacks were produced slightly more often during the early nest stage than during the pre-nesting stage ( Fstage = 2 . 37 , Rmarginal2 = 0 . 05 , Rconditional2 = 0 . 31; Figure 4 ) .", "In sum , this shows that call-type usage , that is , the repertoire composition , changed at the individual level over the breeding cycle .", "Because the recordings were gained in temporal synchrony between all group members ( see ‘Materials and methods’ , Figure 7 ) , our study also allowed investigating how individual birds used calls to interact with other individuals of the group . 10 . 7554/eLife . 07770 . 015Figure 7 . Synchronous external and on-bird recordings . Example spectrograms ( x-axis: time [ms] , y-axis: frequency [kHz] ) of synchronous recordings with ( A ) group recording from external microphone without individual information and ( B–D ) individual recordings from three ( out of eight ) on-bird microphones .", "Dark vertical lines ( in C and D ) represent wing beats that hardly show up in the noisy external recording .", "Note the higher power in the low frequencies in the on-bird microphone recordings , compared to the external recording . DOI: http://dx . doi . org/10 . 7554/eLife . 07770 . 015 These interaction-level data indicated that vocal networks were dynamic , differed in pair and group communication , and were related to the breeding stages .", "Peristimulus time histograms ( PSTHs , for details see ‘Materials and methods’ and Ter Maat et al . , 2014 ) that compare the onsets of the birds' vocalisations relative to each other revealed that calls did not occur randomly .", "Instead , in many cases , the calling behaviour of a specific bird elicited significant changes in the calling behaviour of another bird within a time frame of 0 . 5 s ( relative to each dyad's baseline ) .", "The resulting correlation indices ( see ‘Materials and methods’ for calculation and sample sizes ) were plotted in confusion matrices showing all possible combinations of birds and call types ( Figure 5A ) .", "These matrices demonstrated dynamic interaction patterns between the birds: while birds shared many ‘significant interactions’ with other birds in various call types at the beginning of the group trial , interactions decreased and became more and more specific with progressing reproductive activities ( Figure 5A ) .", "On the day that nest material was provided , the diagonal between the top left and the bottom right lit up ( Figure 5A ) , suggesting that an interaction pattern emerged that was pair specific and synchronous within each group .", "Two of the 10 pairs did not show this vocal interaction pattern ( pairs L and F in Figure 5A ) and also did not lay eggs .", "In total , out of all possible combinations between all call types from all individuals ( within-bird interactions excluded ) only 6 . 5% resulted in significant interactions ( see ‘Materials and methods’ for definition ) .", "4 . 8% showed positive values , that is , the calls of one bird led to an increase in the calls of another bird , and 1 . 7% showed negative values , that is , the calls of one bird led to a decrease in the calls of another bird .", "Within pairs ( n = 10 ) , 9 . 2% of the possible interactions was significant , with 8 . 3% being positive and 0 . 9% negative .", "Further , the data suggest that vocal activity and the amount of vocal interactions decreased with progressing breeding stages , but the ratio of vocal interactions with the partner compared to those with other group members showed a fivefold to sixfold increase ( from 0 . 74 when unpaired to 4 . 24 during incubation; Figure 5—figure supplement 1 ) .", "Within pairs , not all possible call-type combinations were used in significant positive vocal interactions; for example , distance calls were never used in combination with whines .", "The highest percentages of within-pair calling interactions took place in tets , stacks , and cackles ( Figure 5B ) .", "Same-call interactions ( bottom left to top right diagonal in Figure 5B ) were not more common than interactions between different call types .", "However , same-call interactions were more symmetrical between females and males , and changed over the breeding stages almost in the same way for both sexes .", "In contrast , different call-type interactions were less symmetrical between the sexes .", "For example , tets were more likely to be answered by stacks when the responding bird was a female , and stacks were more likely to be answered by tets or cackles when the responding bird was a male , especially at the onset of nesting activities ( Figure 5B ) .", "In this case , the asymmetries thus changed over the breeding stages , with a peak at ‘nest inspection’ .", "Breeding stage thus had an effect on different call-type combinations .", "The number of positive within-pair calling interactions was not only related to reproductive state , but also to whether or not a pair succeeded in producing a clutch of eggs ( ‘successful egg-laying’ ) .", "The number of call-type combinations with significant interactions increased over reproductive stages for pairs that laid eggs , but failed to do so for pairs that did not lay eggs ( Figure 6 ) .", "This means that pairs involving in more call-type interactions at certain stages were more likely to produce a clutch of eggs .", "Successful pairs only shared significant interactions in few call-type combinations before nesting ( 1 . 4 ± 0 . 83 call-type combinations ±SD ) and increased these interactions during the later nest stage ( 8 . 92 ± 4 . 41 ) .", "Unsuccessful pairs , on the other hand , showed decreasing numbers and also higher levels of variation throughout the breeding stages ( from 4 . 48 ± 3 . 35 to 1 . 25 ± 1 . 5 , respectively ) , suggesting a less specific usage of call types in interactions ." ], [ "In songbirds , there is increasing evidence that not only song , but also calls can play a role in reproduction ( Groth , 1993; Marler , 2004; Elie et al . , 2010; Ter Maat et al . , 2014 ) .", "However , the usage and function of call repertoires , especially in group-living species , has been unknown , so far .", "The zebra finch is one of the prime model organisms for studies on vocal communication , especially with regard to song .", "Its vocal repertoire , including song and different call types , has been described in most detail by Zann's laboratory and field observations ( Zann , 1996 ) to which we found various parallels in our data .", "For example , as suggested ( Zann , 1996 ) , loud distance calls occurred most before birds were paired or nesting , and cackles and whines increased at the onset of breeding activities ( Figure 4 , Figure 4—figure supplement 1 ) .", "However , in most previous analyses of zebra finch vocal behaviour , technical limitations constrained a reliable separation between individual sound sources when birds behaved in social contexts involving direct contact with multiple individuals ( Zann , 1996; Elie et al . , 2010 , 2011 ) .", "This was especially relevant when birds vocalised quietly and in close proximity to each other ( Elie et al . , 2010 ) .", "Although it has been suggested that the vocal output of entire zebra finch groups depends on group structure ( Elie et al . , 2011 ) and that quiet calls may play a role in pair formation ( Zann , 1996; Elie et al . , 2010; Ter Maat et al . , 2014 ) , these previous studies did not investigate calling interactions inside groups with individual-level resolution .", "For instance , it had been stated that quiet tet calls are produced almost at all times , are not directed at specific individuals , and therefore do not stimulate specific replies ( Zann , 1996 ) .", "Instead , we found that tet calls did not occur randomly , but in meaningful interactions between individual birds .", "Other studies recorded individual vocalisations , but resorted to strongly reduced social contexts and environmental enrichment , often coupled with frequent disturbances through bird handling ( e . g . , Blaich et al . , 1996; Vignal et al . , 2004; Anisimov et al . , 2014 ) .", "Therefore , despite slightly different vocalisation classification paradigms ( Vignal et al . , 2004; Elie et al . , 2011; Anisimov et al . , 2014; Ter Maat et al . , 2014 ) , between-study differences in the birds' vocal repertoire contents are most likely due to social context , as this can impact a multitude of physiological and behavioural aspects which in turn may be linked to vocalisations , for example , high rates of stack-call production in isolated birds ( Zann , 1996 ) .", "Our setup allowed combining a species-relevant context with recording techniques ( Figure 1 , Video 1 ) that ensured longer-term individual recordings of freely behaving birds with infrequent bird handling .", "This enabled us to explore new aspects of vocal communication , including functional aspects of vocal interactions , as discussed below .", "Zebra finches are opportunistic breeders , and in arid habitats , they rely on short and unpredictable periods of rainfall to successfully rear their young .", "Therefore , birds need to start breeding immediately when environmental conditions permit ( Zann et al . , 1995; Prior et al . , 2013 ) , and may do so throughout the year , in the wild as well as in captivity ( Zann , 1996; Perfito et al . , 2007; Perfito , 2010 ) .", "In our study , newly joined birds quickly formed pairs and increasingly engaged in reproductive activities which were correlated with increased concentrations of gonadal hormones ( see Appendix 1 ) .", "In parallel , shifts in reproductive stages were associated with changes in calling behaviour .", "First of all , at the individual level , the call repertoire changed , sometimes showing sex-specific patterns .", "Second , vocal networks were dynamic , and showed increasing differences between pair and group communication .", "The most synchronous and therefore most apparent changes in reproductive stages occurred at the sudden onset of nest material which was accompanied by nesting behaviours in most birds and was reflected by a pair-specific pattern lighting up in the vocal interaction matrices .", "Intriguingly , the quality of within-pair vocal interactions was associated with successful egg-laying .", "Although all pairs engaged in nesting behaviours at some point , those sharing interactions in more call types during later nest stages were more likely to succeed in producing a clutch of eggs during the 3-week trials .", "In addition , the different levels of variation , especially before the onset of breeding activities ( pre-nesting ) , suggest that successful pairs were more specific in their call-type usage in interactions , demonstrating the importance of call types in pair communication during the breeding cycle .", "Because in the wild , birds need to start breeding immediately with the unpredictable onset of rain ( see above ) , such rapid changes in fine-tuned pair communication could be essential for successful reproduction .", "Our findings thus offer an additional aspect of opportunistic breeding behaviour by showing that changes in the environment , leading to changes in reproductive state , were accompanied by transient changes in the calling behaviour inside groups , involving a shift towards dyadic pair communication .", "In zebra finches , song has been shown to be important for mate choice and pair formation but to lose significance once a stable pair bond is established ( Adkins-Regan and Tomaszycki , 2006 ) .", "Our findings not only support that calls and calling interactions between mates are important for pair formation ( Elie et al . , 2010; Ter Maat et al . , 2014 ) , but also suggest an important role for successful reproduction ( egg-laying ) .", "In this species , song is produced only by males , and thus constitutes a unilateral signal .", "Calls , on the other hand , are produced by both sexes and can be exchanged bilaterally .", "Therefore , they have the potential to be used in mutual and more complex behavioural interactions supporting pair formation and synchronisation as well as pair-bond maintenance , as suggested for other mutual behavioural displays ( reviewed by Bradbury and Vehrencamp , 2011 ) .", "Hereby , some call types are used more frequently in vocal interactions than others .", "We therefore suggest that for rapid calling exchanges especially the soft and short tet , stack , and cackle calls may be more suitable than longer and more variable calls , such as whines .", "It remains to be explored whether or how gonadal hormones affect calling behaviour across changing reproductive stages ( see Appendix 1 ) .", "Due to large differences in the temporal resolution of sampling methods for hormones and behaviour , a direct comparison between concentrations of circulating hormones and of call communication is difficult ( see Appendix 1 for a more detailed discussion of hormones and calling dynamics in groups ) .", "Next to hormones , the decision of an initiating bird to produce a certain call and of a responding bird to answer with a certain call may depend on the specific context , on the behaviour of the other group members , or on the responder's previous experience ( as suggested for social bonding in other bird species; Vignal et al . , 2004; Emery et al . , 2007 ) .", "To date , we can only speculate on how such vocal interaction patterns are established in this songbird species .", "Learning might play a role in females and males , because both sexes initiated calling interactions and responded to calls in an increasingly synchronised pattern during progressing reproductive stages .", "Also , it remains to be investigated whether call-type usage between potential mates supports assortative mating ( Moravec et al . , 2006 ) , or has a direct effect on fertility , as suggested for specific vocalisations in budgerigars ( Melopsittacus undulatus ) ( Brockway , 1965 , 1967 ) .", "Physiological and behavioural synchronisation of pair members have been suggested to play an important role in successful reproduction ( Wickler and Seibt , 1980; Cheng , 2003; Hirschenhauser et al . , 2008; Ouyang et al . , 2014 ) .", "To our knowledge , ours is the first study to demonstrate an association between intra-pair calling dynamics and successful egg-laying , thus highlighting the potential fitness relevance of calls and calling interactions in communication systems ." ], [ "Animal housing and welfare were in compliance with the European directives for the protection of animals used for scientific purposes ( 2010/63/EU ) .", "Protocols were approved by the Government of Upper Bavaria .", "Between November 2011 and August 2012 , four group trials of about 3 weeks each were carried out in succession on a total of 32 adult zebra finches ( 4 females and 4 males each ) which were fully adult offspring from our breeding colony .", "Future group members of the opposite sex had never previously met , that is , had been raised in separate rooms , and siblings were not included in the same trials .", "We also ensured that birds had not bred before .", "Birds were individually recognised by one numbered and two coloured leg bands , and were kept in a 14:10 hr light:dark cycle with ad-libitum access to water and seeds and additional greens and egg-food .", "The birds were caught from large same-sex aviaries and equipped with on-bird audio transmitter ‘backpacks’ ( Figure 1 ) .", "They were subsequently held in smaller same-sex aviaries ( 170 × 165 × 80 cm ) for habituation to the backpacks before the beginning of the trials .", "Different habituation phases have been reported in similar studies , which is likely due to differences in the weight of the backpacks applied to the birds .", "Reported backpack weights on captive zebra finches ranged from 0 . 6 g ( Adreani et al . , 2015 , in prep . ) to 3 g ( Anisimov et al . , 2014 ) , and resulted in habituation phases between a few days ( 1 day for movement , 4 days for call rates , Adreani et al . , 2015 , in prep . ) up to about 2 weeks ( Anisimov et al . , 2014 ) .", "Our backpacks weighed 1 g , and based on prior observation ( unpublished data ) , we chose a 1-week habituation period to ensure birds had fully recovered from any behavioural effects of the backpacks and any associated handling .", "Each trial began by placing the four males and the four females inside a large aviary ( Figure 1—figure supplement 1; for timeline see Figure 8 ) .", "This mixed-sex aviary ( 2 m × 2 m × 2 . 5 m ) contained four large perches , four empty nest boxes , and eight antennas protruding vertically into the top area of the cage , thus offering additional perching opportunities ( Figure 1—figure supplement 1 ) .", "1 week after the beginning of the recordings , nest material ( coconut fibres and soft white lining ) was provided and recurrently refilled .", "While the birds went through different breeding stages , they were blood-sampled for hormone analyses ( see Appendix 1 ) , and non-vocal behaviours were recorded during regular observations .", "Vocal behaviour was recorded almost continuously throughout the day by means of microphone telemetry , and exemplary morning sound recordings were analysed ( see ‘Materials and methods’ on sound analyses ) .", "Behavioural observations and handling of technical equipment were carried out from behind a large , green curtain inside the experimental room , and to control for human disturbance , the observer quietly entered the room at least 10 min before each observation period .", "Animal care , nest checks as well as any handling of birds , took place outside of recording periods ( see below ) . 10 . 7554/eLife . 07770 . 016Figure 8 . Timeline of trials . Timeline indicating housing conditions and approximate timing of backpack application , habituation phase ( minimum 7 days ) , beginning of trial and sound recordings ( ca . 3 weeks , see ‘Materials and methods’ ) , onset of nest material availability , end of trial .", "Of the continuous sound recordings , mornings ( 220 ± 20 min ) of different days ( differed between trials , not indicated in graph ) equally representing birds' breeding stages were analysed .", "Blood sampling occurred three times ( indicated by red arrows ) : before the beginning of the trials , 1 day after males and females were joined and 1 day after nest material became available . DOI: http://dx . doi . org/10 . 7554/eLife . 07770 . 016 For behaviour and hormone analyses , the data from all four trials ( 0 , I , II and III ) were used ( n = 32 ) .", "For sound analyses , only trials I , II , and III were used , because trial 0 served as a test run for the sound recordings and experimental design ." ] ]
[ "Vocal signals such as calls play a crucial role for survival and successful reproduction , especially in group-living animals .", "However , call interactions and call dynamics within groups remain largely unexplored because their relation to relevant contexts or life-history stages could not be studied with individual-level resolution .", "Using on-bird microphone transmitters , we recorded the vocalisations of individual zebra finches ( Taeniopygia guttata ) behaving freely in social groups , while females and males previously unknown to each other passed through different stages of the breeding cycle .", "As birds formed pairs and shifted their reproductive status , their call repertoire composition changed .", "The recordings revealed that calls occurred non-randomly in fine-tuned vocal interactions and decreased within groups while pair-specific patterns emerged .", "Call-type combinations of vocal interactions changed within pairs and were associated with successful egg-laying , highlighting a potential fitness relevance of calling dynamics in communication systems ." ]
[ "As the name implies , songbirds produce song , but they may also emit large numbers of shorter calls .", "Because calls are often given in social situations , they are difficult to record and to assign to the correct individual .", "Therefore , it is still unclear what information is communicated by these calls and how important they are .", "Zebra finches are highly vocal songbirds , with males singing and both females and males producing calls .", "In their natural habitat , Australia , these chatty birds pair for life and live in groups .", "To ensure successful breeding , zebra finches need to begin breeding activities as soon as the unpredictable environment allows .", "Therefore , even in captivity , they will readily breed when given nesting material .", "To find out about the role of zebra finch calls in relation to different environmental or social factors , Gill et al . brought together in groups female and male zebra finches that had not met before , and followed their individual calls during different breeding stages .", "This was done using a technique called microphone telemetry that involves placing tiny wireless microphones on the birds .", "The finches quickly formed breeding pairs , and when provided with nesting material , began building nests and laying eggs .", "While doing so , and especially when pairs began building nests , the birds changed how often they used certain calls and started using different call types; for example , they made more breeding-related ‘cackles’ .", "Calls often featured precisely timed back-and-forth calling interactions , and , over time , were directed more and more towards their partner than other members of the group .", "Pairs that performed more of these call exchanges during nesting were more likely than others to lay a clutch of eggs .", "Overall , Gill et al . show that both the timing and types of calls used in pair communication are important for successful breeding .", "Future research could investigate the role of calls in group communication in more detail—possibly even in the wild—and how calling behaviour is reflected in the brain ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "immunology and inflammation" ]
Method development and characterisation of the low-molecular-weight peptidome of human wound fluids
elife-66876-v1
[ [ "Wound infections after surgery and in relation to burns , as well as in non-healing wounds , are significant medical and societal problems ( Sen et al . , 2009 ) .", "Surgical site infections ( SSIs ) are leading nosocomial infections in developing countries and the second most frequent nosocomial infections in Europe and the United States ( Allegranzi et al . , 2011 ) .", "For example , in European hospitals , the overall rates of SSI range between 3% and 4% of patients undergoing surgery ( Saleh and Schmidtchen , 2015 ) .", "In some procedures , such as when using skin grafts , or performing hernia surgery using biomaterials , the infection risk is higher and may exceed 5–10% ( Saleh and Schmidtchen , 2015; Futoryan and Grande , 1995; Falagas and Kasiakou , 2005 ) .", "The economic burden of failing skin repair is therefore extensive ( Lindholm and Searle , 2016 ) .", "Currently , the costs of treating wounds are estimated to be over 3% of the total health care budgets of Western countries ( Guest et al . , 2017; Phillips et al . , 2016 ) and these expenses are projected to grow with the increasing development of antimicrobial resistance , as well as ageing of the population and the rising incidence and prevalence of diseases such as obesity and diabetes ( MedMarket Diligence , 2021 ) .", "All these factors contribute not only to an increased risk for SSI , but also to an increase in the incidence of non-healing wounds in elderly patients with circulatory insufficiencies .", "Besides the enormous economic burden , wound infections in acute and non-healing wounds lead to increased risk of invasive infections and sepsis , dysfunctional wound closure , and risk for unaesthetic scarring , as well as a reduced quality of life ( Consensus I , 2012 ) .", "Given the above , there is an unmet need for improved methods to measure and predict wound healing status and infection risk in different types of wounds .", "Furthermore , detailed characterisation of wounds may enhance our understanding of the causes that lead to failure and possibly reveal novel therapeutic targets .", "A non-invasive way of investigating wound healing is through analysis of wound exudates .", "These contain proteins , peptides and other biological components , such as metabolites , which can be characterised to various degrees .", "In agreement , several studies have reported the use of proteomics for the analysis of wound fluids ( Eming et al . , 2010; Kalkhof et al . , 2014; Escalante et al . , 2009 ) .", "However , although a powerful methodology , mass spectrometry analyses on samples after trypsin digestion of the proteins mainly report on the presence of proteins and their high-molecular-weight fragments , whereas information about the endogenous fragmentation and resulting peptides is lost .", "In wounds , however , endogenous protein degradation is of high relevance for the understanding of healing as balanced proteolytic activity is essential for progression of healing , whereas aberrant protease activity , such as seen in patients with infected acute wounds ( Trengove et al . , 1999; Saleh et al . , 2019 ) and non-healing ulcers ( Trengove et al . , 1999; Grinnell and Zhu , 1996 ) , may lead to deteriorated wound healing .", "Various endogenous proteases , such as neutrophil elastase , matrix metalloproteases , and collagenases , play important roles in both functional and impaired healing ( Trengove et al . , 1999; Agren , 2000; Yager and Nwomeh , 1999; Herrick et al . , 1997 ) .", "Additionally , exogenous proteases secreted by colonising or infecting bacteria may influence healing as well ( McCarty and Percival , 2013; Suleman , 2016 ) .", "These different proteases may degrade endogenous proteins into peptides with sequences that are enzyme specific , and this may result in peptide patterns that reflect the nature and level of protein degradation occurring in a wound .", "This subject has been addressed to some extend by Sabino et al . who applied quantitative proteomics strategies to assess the wound proteome and the activity of distinct protease groups along the healing process , thus mapping proteolytic pathways ( Sabino et al . , 2018; Sabino et al . , 2015 ) .", "However , given the dynamics of wound healing and the highly proteolytic environment , the generation of endogenous peptides in wounds , their structures , and possible utilisation as biomarkers for wound healing still remains to be explored .", "The large-scale analysis of endogenous peptides has , since its introduction in 2001 ( Schrader and Schulz-Knappe , 2001; Bergquist and Ekman , 2001; Schulz-Knappe et al . , 2001 ) , resulted in a new omics field , that is peptidomics or peptidome research .", "Peptidomics investigations have been conducted for a number of different biological samples , including plasma ( Tammen et al . , 2005 ) , cerebrospinal fluid ( Hölttä et al . , 2012 ) , saliva ( Vitorino et al . , 2012 ) , tears ( Hayakawa et al . , 2013 ) , and brain tissues ( Secher et al . , 2016 ) .", "As the low-molecular-weight peptidome of wounds could act as downstream reporters of the proteolytic action of endogenous and exogenous proteases they could serve as a promising tool for diagnosis and/or prognosis of wound healing .", "In a recent study , we showed that peptides from a selected protein , human thrombin , are detected and could be attributed to proteolytic actions ( Saravanan et al . , 2017 ) .", "Specific thrombin-derived peptide sequences were identified in wound fluids from acute and non-healing ulcers , respectively .", "The result , although focusing on one single protein , demonstrated a proof-of-concept pointing at the possibility of defining peptide biomarkers for improved diagnosis of wound healing and infection .", "In the present study , we aimed at developing a robust peptidomics method for the characterisation of the peptidome of wound fluids .", "Using this method , we here compare acute non-infected wound fluids with plasma samples and find significantly higher protein and peptide numbers in wound fluids compared with plasma , which typically were also smaller in size as compared to plasma-derived peptides .", "Finally , we analyse wound fluids collected from dressings after facial surgery and compare three uninfected and normally healing surgical wounds with three inflamed and Staphylococcus aureus -infected wounds .", "We further demonstrate the utility of peptidomics in wound fluid analysis , showing peptide profiles of various selected proteins .", "Together , the work defines a workflow for analysis of peptides derived from human wound fluids and demonstrate a proof-of-concept that such wound fluids can be used for analysis of subtle qualitative differences in peptide patterns derived from individual patient samples .", "Moreover , as recently also demonstrated ( Hartman et al . , 2021 ) , the uploaded data sets derived from the present report were further mined and processed using global bioinformatic approaches exploring quantitative differences in the identified peptidomes , demonstrating the applicability of the peptidome data generated in this study ." ], [ "To determine the most optimal method for extraction of peptides from wound fluids , acute wound fluids were mixed with 6 M ( final concentration ) urea in the absence or presence of 0 . 05% RapiGest or 0 . 1% TFA followed by 30 kDa filtration as indicated in Figure 1A .", "SDS–PAGE analysis of the filtrate showed high-molecular-weight proteins before filtration ( BF ) and low-molecular-weight peptides after filtration ( Figure 1B ) .", "Urea alone ( U ) did only result in a few peptide bands , whereas both RapiGest ( U + R ) and TFA ( U + T ) resulted in more abundant peptide patterns .", "To further analyse and optimise peptide extraction , various volumes of wound fluids were filtrated as described above , followed by desalting and concentrating of defrosted samples using StageTips and finally liquid chromatography–tandem mass spectrometry ( LC–MS/MS ) analysis of peptides between 700 and 6400 dalton ( the workflow is depicted in Figure 1A ) was performed .", "The results showed a sample volume-dependent increase in the number of identified peptides and corresponding proteins for all used buffers ( Figure 1C ) .", "In agreement with the SDS–PAGE results , urea alone resulted in lower numbers of identified proteins and unique peptides than the other two buffers .", "Interestingly , similar numbers of proteins were identified when using urea and TFA or urea and RapiGest ( 180 versus 173 ) for 100 µL of WF , whereas more unique peptides were found using the latter buffer ( 2931 versus 3543 ) .", "Based on these results , 100 µL of wound fluids in 6 M urea supplemented with 0 . 05% RapiGest was selected for further studies .", "To determine the robustness of the selected sample preparation method , each step of the workflow was investigated .", "The results showed no difference in peptide patterns on SDS–PAGE after dividing one sample preparation over two 30 kDa cut-off filters ( Figure 2A ) .", "Nevertheless , LC–MS/MS analyses of two injections per sample showed that 62% ( 170 ) of the peptides came from the same proteins ( Venn diagrams , Figure 2A ) , whereas 58% ( 3363 ) of all unique peptides were found in both samples , indicating that the filters do interfere with peptide recovery .", "Next , we investigated the reproducibility of sample injection using one preparation injected three times on the same day .", "As shown in Figure 2B , we found similar heatmap patterns for the protein scores for the three injections .", "Furthermore , similar patterns were also found for the number of unique peptides identified and the percentage coverage of the protein by these peptides .", "Moreover , comparison of one preparation injected twice on two different days resulted in clear correlations in protein score , number of unique peptides , and protein coverage ( Figure 2C ) , indicating that sample storage at 4°C does not significantly influence the results .", "Finally , we tested the reproducibility of the entire sample preparation method by comparing two independently generated samples of the same wound fluid and found a good correlation for all three measured parameters ( Figure 2D ) .", "Taken together , the above results show that the selected sample preparation method and storage is robust and can be used for peptidome comparison of various donor fluids .", "Whereas proteases are activated and/or released in the wound environment during inflammation , plasma from healthy donors should not contain activated proteases and therefore far less peptides .", "Indeed , a clear difference between three wound fluids and three plasma samples could be observed on SDS–PAGE , the latter samples containing fewer distinct bands and of a higher molecular weight ( Figure 3A ) .", "In agreement , LC–MS/MS analysis showed substantially more peptides in wound fluids as compared to plasma ( Figure 3B ) .", "When pooling the results of the three individuals for each fluid type , over five times as many proteins and 6 . 8 times as many peptides were observed in wound fluids as compared to plasma samples ( Figure 3C ) .", "Moreover , only 9 . 6% ( 30 ) of all proteins and 2% ( 121 ) of all peptides were detected in both types of fluids , although not necessarily in all three fluids in each group .", "Interestingly , we found relatively more small peptides ( 700–1500 Da ) in wound fluids , whereas peptides larger than 1700 Da were relatively more prevalent in plasma ( Figure 3D , right panel ) .", "Finally , heatmaps of the proteins that were common either for the three plasma samples or for the three wound fluid samples were generated , ordered on function or localisation , showing clear differences between these two types of samples in the protein score , number of unique peptides , and coverage ( Figure 3E ) .", "As expected , no peptides derived from proteases were detected in plasma .", "Besides the number of identified peptides , another difference between plasma and wound fluids is the degree and type of post-translational modifications ( a summary of mass spectrometry results for all sample types is shown in Supplementary file 1 ) .", "For each wound fluid , we found a higher degree of peptide modifications than in plasma .", "In plasma , the average deamidation of the peptides was 3 . 1% as compared to 6 . 4% for aWF sample , whereas the degree of oxidation of methionine was 21 . 7% in wound fluids , while only 4 . 5% in CP samples .", "All peptides identified in the plasma and wound fluid samples are listed in Supplementary Datasets 1 and", "2 . To investigate similarities of sterile acute wound fluids , five wound fluids from different donors were processed and analysed with LC–MS/MS .", "The data of four injections per sample ( two injections a day at two different days ) were merged and then subjected to a database search .", "For all wound fluids , high numbers of peptides were detected ( ranging from 2649 to 4271 , Supplementary file 2 ) and these peptides corresponds to a total of 373 proteins ( Supplementary file 1 ) .", "As illustrated in Figure 4A , 74 proteins were detected in all five wound fluid samples .", "Within this group of 74 proteins , there were 783 identical peptides ( Figure 4A , second Venn diagram ) , which amounts to 15% of the total peptides for the common proteins .", "Notably , the slightly higher number of common peptides in the Venn diagram for all detected proteins ( Figure 4A , third Venn diagram ) as compared to that found for the common proteins can be explained by the presence of 14 peptides that were not unique for one of the 74 common proteins , but were also assigned to other proteins that were not included in the common group of proteins .", "Interestingly , we found a difference in the average peptide length , ranging from 12 . 3 to 13 . 7 amino acids ( AA ) , of the identified peptides from the five different wound fluids ( Supplementary file 2 ) , suggesting that the samples have been exposed to different levels of proteolytic activity .", "Finally , heatmaps of the proteins that were common for all five samples were generated showing resemblances in protein score , numbers of unique peptides , and protein coverage ( Figure 4B ) , which further proves the usefulness of peptidomics in wound fluid analysis .", "Finally , and as a proof-of-concept , we selected six patients from a previously published clinical study ( Saleh et al . , 2019 ) , who had undergone facial full-thickness skin grafting , and either had healed well at the 7 day follow-up after surgery ( Figure 5A , low inflammation group ) or had an inflamed wound infected by amongst others S . aureus ( high inflammation group ) .", "Cytokine analysis of extracts made of the wound dressings indeed showed increased levels of IL-1β , IL-6 , IL-8 , and TNF-α in the high inflammation as compared to the low inflammation group ( Figure 5B ) .", "Further analyses of the dressing extracts using SDS–PAGE ( Figure 5C ) and zymograms ( Figure 5D ) showed a positive correlation between protein degradation and enzymatic activity , both more apparent in the high inflammation group .", "Next , two independently generated sample preparations were made of each extract , using the sample preparation method above , and subjected to LC–MS/MS .", "The data of four injections per sample ( two injections for each of the duplicate extracts ) were merged and then subjected to a database search .", "All identified peptides are listed in Supplementary Dataset 3 , while a summary of the total numbers of identified unique peptides , proteins , and the average length and number of amino acids ( AA ) is given in Supplementary file", "3 . Interestingly , when pooling the results of the three patients per group , we found that 163 proteins ( 2666 peptides ) were found in both groups , although not necessarily in each single patient , whereas 156 proteins ( 5273 peptides ) were only found in the low inflammation group versus 90 ( 4661 peptides ) in the high inflammation group ( Figure 5E ) .", "These results suggest a higher degree of proteolysis in the high inflammation group , as per identified protein an average of 52 unique peptides was found as compared to 34 in the low inflammation group .", "Indeed , this reflects the expected increase of endogenous proteases in the infected and inflamed wounds due to the influx of immune cells , such as neutrophils and macrophages , combined with the presence of bacterial proteases .", "Furthermore , the individual samples within the high inflammation group show a higher degree of variability as only 23% of the proteins ( 10% of all unique peptides ) were found in all three patient dressing extracts versus 34% ( 16% of the peptides ) for the low inflammation group ( Figure 5—figure supplement 1 ) .", "This variability is further visualised in heatmaps , generated of the proteins that were found in all three extracts of either the low inflammation group or the high inflammation group ( Figure 5F ) .", "As expected , some proteins , such as the serum proteins albumin ( ALBU ) and hemoglobin ( HBA/HBB/HBD ) , are identified in all wound fluids and dressing extracts ( heatmaps , Figures 2–5 ) , whereas complement component C9 ( CO9 ) , which is a constituent of the membrane attack complex that plays a key role in the innate immune response against bacteria , is only found in fluids from infected wounds .", "Interestingly , the antimicrobial protein dermcidin ( DCD ) , constitutively expressed by eccrine sweat glands , was detected in the low inflammation group , whereas the protein was absent in the high inflammation group .", "This may be explained by the reported proteolytic degradation of dermcidin by staphylococci ( Lai et al . , 2007 ) , to such an extent that the remaining peptides can no longer be detected by LC–MS/MS .", "Contrastingly , the antimicrobial protein lactoferrin ( TRFL ) was only detected in the high inflammation group .", "As lactoferrin is secreted by neutrophils upon degranulation , and influx of neutrophils is extensive in inflamed wounds , these results are as expected .", "To further investigate protein fragmentation , peptide profiles and peptide alignment maps were generated in order to visualise the qualitative changes at the peptide level .", "Figures 6–8 show selected peptide profiles representing proteins of relevance for fibrin formation , wound healing and inflammation , as well as antimicrobial defence .", "Notably , these profiles do not contain peptides that are post translationally modified .", "Furthermore , the depicted peptide alignment maps are only showing a selection of the sequences detected for the protein areas that are highlighted by blue boxes .", "As expected , peptides derived from the three different fibrinogen chains alpha , beta , and gamma were identified ( Figure 6 ) .", "Peptides similar to clinically relevant fibrin degradation products , composed of fibrinopeptides , were found .", "Furthermore , the antimicrobial and anti-inflammatory peptide GHR28 from the beta chain ( Påhlman et al . , 2013 ) was identified in the three non-inflamed wounds , whereas only smaller fragments were detected in the inflamed and infected wounds .", "Prothrombin is a major protease activated during wound healing , and it was notable that peptides from the thrombin light chain were observed in all three wound samples from non-infected normally healing wounds , whereas they were absent in two of the infected wound fluids .", "Furthermore , fragments derived from thrombin-derived C-terminal peptides ( TCPs ) , previously found to exert antimicrobial and immunomodulatory effects in wounds ( Saravanan et al . , 2017; Papareddy et al . , 2010; van der Plas et al . , 2016; Hansen et al . , 2017 ) , were identified .", "Whereas thrombin is a major liver-derived enzyme , others such as cathepsin G and elastase are secreted from neutrophils during inflammation and infection .", "In agreement , peptide fragments from these two enzymes were particularly observed in samples from infected wounds .", "For cathepsin G , peptides were identified from two antimicrobial regions .", "Whereas the first region was discovered using synthetic peptides ( Shafer et al . , 1993 ) , the second region corresponds to the previously described antimicrobial peptide CG-1 , which was identified in granule extracts from neutrophils ( Miyasaki et al . , 1995 ) .", "Interestingly , a similar C-terminal fragmentation pattern was identified in neutrophil elastase .", "Taken together , these results indicate that specific degradation patterns of major plasma and neutrophil proteases can be detected using wound extracts derived from dressings after surgery .", "High-molecular-weight kininogen ( HMWK ) is a multifunctional 120 kDa glycoprotein found in plasma and in granules of platelets ( Schmaier et al . , 1983 ) .", "The protein is composed of six domains , each having different properties and specific ligands ( Colman and Schmaier , 1997 ) .", "Whereas domains 2 and 3 have cysteine protease inhibitor activities , the D4 domain contains the bradykinin sequence , which is released by plasma kallikreins during contact activation and actions of proteases such as neutrophil elastase ( Kozik et al . , 1998 ) .", "Thus , limited proteolysis of HMWK generates highly vasoactive and pro-inflammatory peptides , which are formed at sites of tissue injury and inflammation .", "The cell-binding D5 from HMWK contains regions dominated by histidine , glycine , and in certain parts , interspersed lysine residues , which also can mediate antimicrobial effects ( Nordahl et al . , 2005 ) .", "As seen in Figure 7 , these peptide fragments were indeed observed in the postoperative wounds .", "Further evidence for the usefulness of the methodology is provided by the detection of fragmentation patterns from additional protease inhibitors such as alpha-1-antitrypsin , antiplasmin , as well as inter-alpha-trypsin inhibitor heavy chain H4 , all yielding peptide sequences from distinct regions of these inhibitors , thereby reflecting the involvement of these inhibitors in direct and specific protease control ( Janciauskiene et al . , 2018; Lijnen , 2006; Lord et al . , 2020 ) , their sensitivity to endogenous and bacterial proteases , and the resulting peptide fragments respective metabolism and turnover in the complex wound environment .", "Cleavage of A1AT occurs at the reactive centre loop by proteases such as neutrophil elastase , although MMPs and S . aureus enzymes can also cleave the antiproteinase ( Nelson et al . , 1998; Potempa et al . , 1986 ) .", "Hence A1AT may be reporting wound-derived protease activity .", "Moreover , cleavage between Phe376-Leu377 generates a carboxy-terminal 42-residue peptide , and variants of this fragment , such as a C-terminal 36 aa peptide , have been identified in several human tissues , where they may exert various immunomodulatory functions ( Blaurock et al . , 2016; Subramaniyam et al . , 2006 ) .", "Notably , such fragments were also identified in the wound fluids from surgical wounds .", "Interestingly , C-terminal regions of antiplasmin were only observed in normally healing wounds , and it is notable that this region of plasmin contains bioactive epitopes that can modulate urokinase activity ( Lee et al . , 2002 ) and interact with endothelial cells ( Thomas et al . , 2007 ) .", "Recently , C-terminal fragments generated in vivo were indeed identified ( Abdul et al . , 2020 ) .", "Inter-alpha-trypsin inhibitor heavy chain H4 is a 120 kDa serum glycoprotein secreted primarily by the liver .", "Peptides derived from the proline-rich region may be biomarkers for a variety of disease states including breast cancer ( van den Broek et al . , 2010 ) , further illustrating that surgical wounds contain regions of biological and diagnostic importance .", "Notably , peptides from residues 488–504 were only identified in normally healing wounds ( Figure 7 , orange arrow ) .", "In addition to the proteases and protease inhibitors above , the complement cascade is another fundamental defence system activated during wounding and infection .", "Factor B is part of the alternate pathway of the complement system is cleaved by factor D into two fragments: Ba and Bb .", "Bb , a serine protease , then combines with complement factor 3b to generate the C3 or C5 convertase .", "Inspection of the peptide profile for this factor showed that fragments from particularly the S1 peptidase domain were present in the infected wound ( Figure 8 ) .", "For the major complement component 3 ( CO3 ) , a series of similar fragmentation patterns were identified , with peptides originating in different regions of CO3 .", "Importantly , N-terminals of the well-known anaphylatoxin C3a , which harbours antimicrobial activity ( Nordahl et al . , 2004 ) , as well as the opsonin C3b were identified .", "Besides common fragments found for all six patients , two peptide sequences were only found in normally healing wounds ( orange arrows ) .", "As mentioned , complement component 9 , which is part of the membrane attack complex that plays a key role in the immune response against bacteria , was only detected in fluids from infected wounds .", "Further evidence for distinct proteolytic events affecting selected proteins is seen in the peptigrams representing serum amyloid A-1 ( SAA1 ) peptides , as well as dermcidin ( DCD ) -derived fragments , which both were only found in non-infected wounds ( Figure 8 , lower panels ) .", "SAA1 can upregulate inflammatory cytokines , whereas SAA1 peptides suppress inflammation ( Zhou et al . , 2017 ) .", "Moreover , the C-terminal SAA ( 86–104 ) binds and inhibits human cystatin and carboxy-terminal fragments are involved in amyloid diseases ( Maszota et al . , 2015 ) .", "Interestingly , similar C-terminal fragments were found in non-infected wounds , indicating that SAA is proteolyzed in wounds , suggesting a yet undisclosed physiological role for such fragments during normal wound healing .", "DCD is an antimicrobial peptide found in skin ( Schittek et al . , 2001 ) .", "Proteolytic processing of DCD gives rise to several truncated peptides ( Steffen et al . , 2006 ) .", "Interestingly , variants of the peptide DCD-1L having the N-terminal SSLLEK ( Rieg et al . , 2005 ) were indeed identified in normally healing surgical wounds .", "A proteolytically processed form of fetuin-A , also denoted α2-HS glycoprotein , lacking a segment of 40 amino acid residues bridging its heavy and light chain portions ( ‘connecting peptide’ ) has been described , suggesting that this peptide is released by post-translational processing to fulfil biological role ( s ) of fetuin-A .", "The connecting peptide region , which is highly susceptible to proteolytic breakdown in vitro and in vivo ( Nawratil et al . , 1996 ) , was present in the surgical wound fluids .", "Interestingly , peptides starting with the N-terminal LPPAGS were only detected in the three inflamed wounds ( purple arrow ) , whereas those starting with LLAAPP were unique for the three non-inflamed wounds ( orange arrow ) .", "Finally , to show the validity and robustness of the method , the peptide profile of serum albumin is presented , showing a high similarity between the fragmentation patterns in the individual wounds ." ], [ "Endogenous peptides serve as biomarkers of disease progression for several different diseases , and the here presented strategy and methodology identified the wound fluid peptidome as a source for assessment of wound fluid dynamics , as these fragments represent proteolytic events that are the result of basic physiological processes involving coagulation and complement activation , but also additional proteolytic activities by endogenous and bacterial proteases .", "Degradation of proteins occurs due to the action of set of a proteases and biological processes that results in a low-molecular-weight fraction of endogenous peptides with specific cleavage points .", "For the analysis of the wound fluid peptidome by mass spectrometry , it is necessary to extract and enrich the peptides , thereby excluding proteins and other interfering molecules .", "Several different protocols and methods are available for extraction of peptides from biofluids prior to the mass spectrometry measurement .", "In our work , we have chosen to investigate the peptidome of wound fluids by a combination of low-molecular-weight filtration , solid phase purification , and enrichment followed by mass spectrometry detection and database search .", "Although solubility of the peptides and the binding of peptides to proteins will affect the outcome of the filtration procedure , previously it was found that solubilisation using urea was efficient for the extraction recovery of endogenous peptides ( Secher et al . , 2016 ) .", "The LC–MS/MS analysis was performed using data-dependent analysis , where the instrument is set to sequence as many peptides as possible for each sample and the same sample was injected in duplicates .", "The chosen method allowed us to characterise more than 7800 peptides in acute wound fluids derived from the degradation of over 370 proteins .", "We have focused our subsequent analysis on a subset of peptides corresponding from the degradation of the most abundant proteins that could be measured in all of the examined acute wound fluids .", "The definition of the wound peptidome of acute wounds in well-defined sterile wound fluids validated the approach , identifying multiple peptides derived from several protein families .", "The following analyses on dressing extracts from skin-grafted surgical wounds provided a proof-of-principle , showing the possibility of analysis of selected peptide regions as potential biomarkers for inflammation and wound healing .", "This is of high relevance from a clinical perspective , as skin grafting surgery is normally associated with a higher risk of SSI ( Saleh and Schmidtchen , 2015; Saleh et al . , 2019 ) , motivating the use of such wound fluids from this type of dermatologic surgery .", "As briefly mentioned in the introduction , classical proteomics approaches have been employed in the study of different wound types .", "Eming et al . , 2010 studied the distribution of tissue repair proteins in exudates of healing acute and non-healing venous wounds on the legs .", "Subsequent studies have added to the categorisation of proteins identified in various wounds such as diabetic and pressure ulcers ( Broadbent et al . , 2010; Krisp et al . , 2013; Wyffels et al . , 2010; Steinsträßer et al . , 2010; Edsberg et al . , 2012 ) .", "Auf dem Keller and colleagues applied quantitative proteomics strategies to dissect proteolytic pathways during wounding and the activity of distinct protease groups along the healing process .", "Moreover , they also mapped proteolytic pathways in vivo and established protease–substrate relationships that will help to better understand protease action in cutaneous wound repair and other inflammatory conditions in skin ( Sabino et al . , 2018; Sabino et al . , 2015; Sabino , 2017; Savickas et al . , 2020 ) .", "However , it is of note that methods for detection of downstream products reflecting inflammation are rare .", "Although Auf dem Keller’s work involves studies on such protease patterns , their method focuses on ‘N-terminomics’ , while low-molecular-weight peptides , such as the ones studied here , are mainly lost during the preparation steps .", "Interestingly , at the proteomic level , in their studies on healing and non-healing wounds on the legs , Eming et al . , 2010 showed that the serine protease thrombin , as well as the antimicrobial DCD , was exclusively detected in healing wounds .", "In contrast , the neutrophil-derived lactoferrin was increased in non-healing wounds .", "Moreover , the three major fibrinogen chains alpha , beta , and gamma were dominating in the wound fluids from all wound types .", "Although the wounds types are obviously different with respect to localisation and pathogenesis , the clinical grading into non-healing and healing is the same for both studies .", "It is therefore notable that corresponding observations on the above proteins were indeed made in this study at the peptidome level as well , lending further support for the diagnostic potential of the here described methodology .", "The significance of the methodology is further underscored by recent bioinformatics applications based on the present datasets ( Hartman et al . , 2021 ) .", "Thus , in a global approach , algorithms in Python as well as open source softwares such Deep-AmPEP30 ( Yan et al . , 2020 ) and Proteasix ( Klein et al . , 2013 ) were employed .", "Analyses of distribution of amino acids at N- and C-terminal positions , the corresponding four amino acid long terminal segments , and in the complete sequence were conducted .", "Wound fluids derived from dressings were found to contain larger proportion of acidic residues in the P1 position .", "Moreover , specific sequences like HKYH , LERM , and SKYR were especially prevalent at the C-terminal in infected wounds .", "In silico prediction using Proteasix showed that cleavages by proteases such as MMP 7 , MMP 9 , and neutrophil elastase were most prevalent , and in particular neutrophil elastase was particularly up regulated in infected wound samples ( Hartman et al . , 2021 ) , serving as a possible indicator of wound infection .", "DeepAMP identified novel antimicrobial peptides in hemoglobin , and moreover , peptides derived from LPS-binding regions of hemoglobin were identified in infected surgical wounds .", "Apart from demonstrating the power of data-driven approaches to wound fluid peptidomics , the results also highlight the usefulness of novel bioinformatic tools for analysis of the vast datasets obtained using the methodology disclosed in this report .", "Today , the current method for characterisation of infection and related wound complications in the clinic is first to assess wound status according to clinical experience combined with bacterial detection .", "There are many obvious signs of advanced infection including redness , heat , swelling , purulent exudate , smell , and pain , but the challenge is to translate these observations to objective and sensitive methods that correctly evaluate wound status and , in particular , the associated inflammation , before wounds even reach this dysfunctional state .", "Moreover , the presence of bacteria in wounds does not correlate with wound inflammation and infection .", "For example , non-healing ulcers are frequently colonised by staphylococci , even without infection , and post-surgical wounds can also harbour S . aureus without signs of clinical infection ( Saleh et al . , 2019; Grice and Segre , 2012; Johnson et al . , 2018 ) .", "Therefore , given that mere bacterial presence is not discriminatory for wound infection , there is a clear need for objective methods that enable detection of dysfunctional wound healing to better guide the use of treatment .", "Approaches under development today to evaluate bacteria and the concomitant inflammation are use of biological or chemical sensors of wound exudates to detect bacterial antigens , monitor pH , temperature , oxygen , and enzymes .", "Spectroscopic and imaging techniques are also possible as future advanced wound monitoring techniques ( Dargaville et al . , 2013; World Union of Wound Healing Societies ( WUWHS ) , 2008; Li et al . , 2021; Lindley et al . , 2016 ) .", "Methods that measure host responses as one way to assess wound healing , and particularly inflammation , have also been developed .", "For example , the major enzymes from neutrophils , human neutrophil elastase ( HNE ) , and cathepsin G have been reported as early-stage warning markers for non-healing ulcers .", "In the clinic , detection of HNE or MMPs is used by a device developed by Systagenix , measuring elevated protease activity through their Woundcheck Protease Status diagnostic .", "In a recent study , it was identified that wounds that had a clinical appearance of being more inflamed indeed had higher levels of proteases , as demonstrated for the major MMPs , MMP-2 and −9 .", "These wounds had increased cytokine levels relative to the wound surface area , particularly observed for IL-1β , but also for TNF-α ( Saleh et al . , 2019 ) .", "Moreover , these wounds also had significantly higher overall bacterial loads , a factor of importance in the development of SSIs .", "As demonstrated in this report , apart from the cytokines IL-1β , IL-6 , IL-8 , and TNF-α , a vast number of potential new peptide sequences could be detected in these wounds , using a qualitative approach focusing on major protein families involved in coagulation , complement activation , and protease control .", "Furthermore , subsequent mining of the peptidome data sets with bioinformatics methods identified additional subtle sequence differences ( Hartman et al . , 2021 ) .", "Together , these results provide a proof-of-principle for utilisation of wound fluid peptidomics in future biomarker-oriented studies on larger well-defined patient groups .", "Combining the use of such new peptides as biomarkers along with classical analyses of cytokines and MMPs could yield higher diagnostic sensitivity and specificity with respect to wound status and infection risk .", "Moreover , the development of sensors , based on antibodies or aptamers that target selected peptides , could make this diagnostic approach more attractive in the clinics , enabling fast readouts and thus facilitate the needed clinical studies .", "Finally , the peptidomics data generated here provides previously undisclosed data on proteolytic fragmentation patterns during wounding , which may aid in the discovery of novel bioactive peptides and elucidation of their biological roles during wound healing ." ], [ "This study was carried out in accordance with the recommendations of the Ethics Committee at Lund University , Lund , Sweden , with written informed consent from all subjects in accordance with the Declaration of Helsinki .", "The protocols for the use of human blood ( permit no . 657–2008 ) and human wound materials ( 708–01 , 509–01 , and 762–2013 ) were approved by the Ethics Committee at Lund University .", "Plasma was collected from citrated venous blood from three healthy donors by centrifugation at 2000 g .", "Sterile acute wound fluids , obtained from surgical drainages after mastectomy from five donors , were collected for 24 hr , 24–48 hr after surgery followed by centrifugation as described previously ( Lundqvist et al . , 2004 ) .", "Wound fluids from six patients that underwent facial full-thickness skin grafting were extracted from Mepilex ( Mölnlycke Health Care , Sweden ) tie-over wound dressings , which had been on the wound for 1 week , as described ( Saleh et al . , 2019 ) .", "In short , wound exudate was extracted from dressings after incubation in 10 mM Tris ( pH 7 . 4 ) for 1 hr at 8°C while shaking .", "All samples were stored at −20°C before use .", "Frozen plasma and/or sterile acute wound fluids were defrosted and mixed with three parts freshly made 8 M urea ( in 10 mM Tris , pH 7 . 4 , yielding a final urea concentration of 6 M ) alone or supplemented with RapiGest SF ( 0 . 05% final concentration; Waters , USA ) or trifluoroacetic acid ( 0 . 1% final concentration; Sigma-Aldrich , Germany ) and incubated for 30 min at room temperature ( RT ) followed by size exclusion .", "For this purpose , centrifugal filters with 30 kDa cut-off ( Microcon 30 , regenerated cellulose , Millipore , Ireland ) were first rinsed with 100 µL buffer and centrifuged at 14 , 000 g for 15 min at RT , then loaded with the incubated sample mixtures , followed by 30 min centrifugation ( 14 , 000 g at RT ) and a final filter washing step with 100 μL of extraction buffer ( 5 min , 14 , 000 g ) .", "Due to large variations in protein content of the dressing extracts ( between 2 . 8 and 16 . 5 mg/mL ) , which was measured using the Pierce BCA Protein Assay Kit ( Thermo Scientific , USA ) according to manufacturer’s instructions , protein concentrations were standardised .", "For this purpose , 10 mM Tris was added to 280 µg of each fluid to obtain a sample volume of 100 µL in total , which was then incubated with 300 µL 8 M urea supplemented with RapiGest SF for 30 min , followed by the size exclusion steps as described above , with the modification that the filters were centrifuged at 10 , 000 g .", "For all samples , the two filtrates containing the peptides were pooled and analysed by SDS–gel electrophoresis directly or stored at −20°C before analysis by LC–MS/MS .", "Extracted peptide samples or 20 µg of each dressing extract was denatured at 85°C for 5 min in 1× SDS sample buffer followed by separation on 10–20% Tris–Tricine mini gels in 1× Tris–Tricine SDS running buffer for 90 min at 125 V . Gels and buffers were derived from Invitrogen ( USA ) .", "Gels were stained using GelCode Blue Safe Protein Stain ( Thermo Scientific , USA ) or the SilverQuest Silver Staining Kit ( Invitrogen ) according to manufacturer’s instructions , and patterns were visualised using a Gel Doc Imager ( Bio-Rad Laboratories , USA ) .", "Gels were prepared consisting of a separation gel ( 0 . 1% [w/v] gelatine , 0 . 1% [w/v] SDS , 10% acrylamide in 375 mM Tris buffer [pH 8 . 8] , 0 . 05% [v/v] TEMED , and 0 . 05% [w/v] APS ) and a stacking gel ( 0 . 1% [w/v] SDS , 4% acrylamide in 125 mM buffer [pH 6 . 8] , 0 . 1% [v/v] TEMED , and 0 . 05% [w/v] APS ) .", "Dressing extracts ( 5 µg ) were mixed with sample buffer ( 20% [v/v] glycerol , 5% [w/v] SDS , 0 . 03% [w/v] bromophenol blue , 0 . 4 M Tris–HCl pH 6 . 8 ) in a 1:1 ratio , transferred to the slots , and gels were run in electrophoresis buffer ( 25 mM Tris , 0 . 2 M glycine , and 0 . 5% [w/v] SDS in H2O at pH 8 . 7 ) for 60 min at 150 V . Next , gels were washed in H2O , incubated for 1 hr in 2 . 5% Triton X-100 , washed again , and placed in enzyme buffer ( 5 mM CaCl2 , 1 µM ZnCl2 , 200 mM NaCl , and 50 mM Tris–HCl pH 7 . 5 ) .", "After overnight incubation at 37°C while shaking ( 50 rpm ) , gels were washed and stained using Coomassie brilliant Blue .", "Enzymatic activity was observed after destaining the gels with a solution consisting of 10% EtOH and 14% acetic acid in H2O .", "Cytokine levels in the dressing extracts were determined using ELISA kits ( R and D systems , USA ) according to manufacturer’s instructions .", "Results , adjusted to the total protein concentrations of the extracts , are mean values of triplicate measurements .", "Prior to LC–MS/MS analyses , 80µL of defrosted peptide extracts were acidified with 5 µL of 10% formic acid and then trapped and enriched on StageTip columns ( Rappsilber et al . , 2007 ) .", "Next , peptides were extracted with 70% ACN and 0 . 1% TFA and dried down before reconstitution in 20 µL of 2% ACN and 0 . 1% TFA .", "For each run , 2 µL of reconstituted sample was injected , which corresponded to 1 . 6 µL of the original wound fluid or plasma sample .", "For the evaluation of the sample preparation method , LC–MS/MS analyses were carried out on an Orbitrap Fusion Tribrid MS ( Thermo Scientific ) as described earlier ( Mehmeti et al . , 2019 ) , with the following modifications .", "During the elution steps , the percentage of solvent B increased from 5% to 22% in the first 20 min , then increased to 20% in 85 min , then to 30% in 20 min , and to 90% in a further 5 min , where it was kept for 5 min .", "Subsequent analysis of patient samples was performed using an HFX Orbitrap MS system ( Thermo Scientific ) equipped with a Dionnex 3000 Ultimate HPLC ( Thermo Fisher , USA ) .", "Injected peptides were trapped on an Acclaim PepMap C18 column ( 3 µm particle size , 75 µm inner diameter × 20 mm length ) .", "After trapping , gradient elution of peptides was performed on an Acclaim PepMap C18 column ( 100 Å 2 μm , 150 mm , 75 μm ) .", "The outlet of the analytical column was coupled directly with the mass spectrometer using a Nano Easy source .", "The mobile phases for LC separation were 0 . 1% ( v/v ) formic acid in LC–MS grade water ( solvent A ) and 0 . 1% ( v/v ) formic acid in acetonitrile ( solvent B ) .", "Peptides were first loaded onto the trapping column and then eluted to the analytical column with a gradient .", "The percentage of solvent B increased from 2% to 27% in the first 107 min , then increased to 32% in 10 min , then to 90% in 15 min where it was kept for 5 min .", "The peptides were introduced into the mass spectrometer via a Stainless steel emitter 40 mm ( Thermo Fisher ) , and a spray voltage of 1 . 9 kV was applied .", "The capillary temperature was set at 275°C .", "Data acquisition was carried out using a top 20 based data-dependent method .", "MS was conducted in the range of 350–1350 m/z at a resolution of 120 , 000 FWHM .", "The filling time was set at a maximum of 100 ms with limitation of 3 × 106 ions .", "MSMS was acquired with a filling time maximum 300 ms with limitation of 5 × 104 ions , a precursor ion isolation width of 2 . 0 m/z , and resolution of 15 , 000 FWHM .", "Normalised collision energy was set to 28% .", "Only multiply charged ( 2+ to 5+ ) precursor ions were selected for MS2 .", "The dynamic exclusion list was set to 30 s .", "MS/MS spectra were searched with PEAKS software .", "UniProt Human , including 20 , 413 protein sequences , was used with nonspecific cleavage; 5 ppm precursor tolerance and 0 . 5 Da fragment tolerance were used for the experiments conducted with the Fusion instrument and 0 . 02 Da for the fragments when the HFX was used .", "Oxidation ( M ) and deamidation ( NQ ) were treated as dynamic modification .", "Search results were filtered by using 1% FDR for the wound fluids , or a 23 score for the plasma samples , and at least two unique peptides for each protein .", "The mass spectrometry peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE ( Perez-Riverol et al . , 2019 ) partner repository with the dataset identifiers PXD023884 and PXD023244 .", "For data visualisation , graphs and heatmaps were generated using Graphpad Prism , Venn diagrams were made in VennDis ( Ignatchenko et al . , 2015 ) , while peptide profiles and peptide alignment maps were made using the web-based application Peptigram ( Manguy et al . , 2017 ) ." ] ]
[ "The normal wound healing process is characterised by proteolytic events , whereas infection results in dysfunctional activations by endogenous and bacterial proteases .", "Peptides , downstream reporters of these proteolytic actions , could therefore serve as a promising tool for diagnosis of wounds .", "Using mass-spectrometry analyses , we here for the first time characterise the peptidome of human wound fluids .", "Sterile post-surgical wound fluids were found to contain a high degree of peptides in comparison to human plasma .", "Analyses of the peptidome from uninfected healing wounds and Staphylococcus aureus -infected wounds identify unique peptide patterns of various proteins , including coagulation and complement factors , proteases , and antiproteinases .", "Together , the work defines a workflow for analysis of peptides derived from wound fluids and demonstrates a proof-of-concept that such fluids can be used for analysis of qualitative differences of peptide patterns from larger patient cohorts , providing potential biomarkers for wound healing and infection ." ]
[ "Infected wounds and burns represent a serious risk to patients: they can delay healing and , if left untreated , can lead to generalised infection or sepsis , organ failure and death .", "Wounds and burns get infected when harmful micro-organisms , such as bacteria , enter the wound .", "Predicting the risk of infections , and detecting them early , could reduce their impact and make treating them easier .", "A way to distinguish between healing and infected wounds is to study how proteins are broken down in each situation .", "Proteases are the enzymes that break down proteins , and they are different in healing wounds and infected wounds that are failing to heal .", "This is because , while the body produces proteases , the bacteria that cause infection do so too .", "Each protease breaks down proteins in a specific way , resulting in a different set of protein fragments , known as peptides .", "Together , all the peptides in a wound are referred to as the wound’s ‘peptidome’ .", "Studying the peptidome of a wound could show whether it is infected , and even what type of bacteria might be responsible , which could help identify suitable treatments .", "Van der Plas et al . used a technique called mass spectrometry to study the peptidome of wounds after surgery .", "Sterile post-surgical wounds showed high levels of peptides compared to plasma , the liquid component of blood , with up to 4 , 300 different peptides .", "Comparing healing wounds to ones infected with the bacterium Staphylococcus aureus revealed that infected wounds contained peptides from about 150 proteins not found in uninfected wounds , while peptides from 90 proteins were unique to uninfected wounds .", "The peptides exclusive to uninfected wounds included some linked to antimicrobial activity and immune system activity .", "Van der Plas et al . ’s results suggest that analysing the peptidome may be an approach to tracking the healing status of wounds , making it easier to detect infection before symptoms are apparent .", "The next step will be to study more wounds and identify the reliable peptide markers to use them for diagnostic tests ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience", "tools and resources", "genetics and genomics" ]
A genetic, genomic, and computational resource for exploring neural circuit function
elife-50901-v2
[ [ "The anatomy of neural circuits is being characterized with increasing resolution and throughput , in part following a dramatic increase in the size of circuits amenable to detailed electron microscopy reconstruction ( Swanson and Lichtman , 2016 ) and the development of genetic tools to access individual cell types ( Luo et al . , 2018 ) .", "These efforts reveal anatomy at unprecedented detail , but not the molecular properties of cells .", "In principle , the genes expressed in each cell of a neural circuit should serve as a molecular proxy for cell physiology .", "However , most genomic efforts have focused on surveying neuronal diversity rather than characterizing circuit function ( Ecker et al . , 2017 ) .", "To develop a resource exploring molecular correlates of circuit function , here we use an approach that genetically targets cell types within a well-characterized brain region to measure high-quality transcriptomes that can be integrated with connectomes .", "Drosophila affords an ideal system to study neural circuits in detail , as both excellent genetic tools and high resolution connectomes are available .", "Here we focus on the repeating columnar circuits of the visual system , found in the optic lobes , a widely used model for studying circuit development and function with an extensive genetic toolbox and well-described anatomy ( Figure 1A; Nériec and Desplan , 2016; Silies et al . , 2014; Apitz and Salecker , 2014 ) .", "This network begins with photoreceptor neurons and contains several layers of connected neurons which process incoming luminance signals into multiple parallel streams of visual information ( Figure 1B ) .", "Many of its cellular components have been described by light microscopy , including classical Golgi studies ( Fischbach and Dittrich , 1989 ) and recent analyses using genetic methods ( Morante and Desplan , 2008; Otsuna and Ito , 2006; Nern et al . , 2015; Wu et al . , 2016 ) .", "Electron microscopy reconstruction work has characterized the synaptic connections of many optic lobe neurons ( Meinertzhagen and O'Neil , 1991; Meinertzhagen and Sorra , 2001; Rivera-Alba et al . , 2011; Takemura et al . , 2013; Takemura et al . , 2015; Takemura et al . , 2017; Shinomiya et al . , 2019 ) .", "Comparative studies have also explored the evolution of this ancient brain structure ( Strausfeld , 2009 ) .", "Despite this wealth of information , many of its fundamental properties remain unknown , including the neurotransmitters used at many of its synapses .", "Measuring the genes expressed in specific cells of the brain is challenging due to its compact and complex organization .", "RNA sequencing ( RNA-seq ) addresses this challenge by profiling either single cells or genetically labeled populations of cells ( Ecker et al . , 2017 ) .", "The latter approach requires genetic tools to access individual cell types but provides more direct access to cells of interests than sampling of unmarked single cells , especially for sparse cell types .", "Profiling identified cell types provides a direct link to previous work on the anatomy and physiology of those cell types .", "Cell type-specific drivers also facilitate follow-up experiments , for example evaluating the role of individual genes in individual cells .", "In Drosophila , large collections of GAL4 driver lines ( Jenett et al . , 2012; Tirian and Dickson , 2017 ) and the possibility to further refine these patterns with intersectional methods such as split-GAL4 ( Luan et al . , 2006; Dionne et al . , 2018 ) enable genetic access to many neuronal populations ( see , for example , Tuthill et al . , 2013; Aso et al . , 2014; Wu et al . , 2016 ) .", "We therefore chose the genetic , rather than single cell , approach to build a genomics resource to explore circuit function .", "We previously developed an Isolation of Nuclei Tagged in a specific Cell Type ( INTACT ) method ( Deal and Henikoff , 2010 ) to measure transcriptomes and epigenomes of genetically-marked neuronal populations in Drosophila ( Henry et al . , 2012 ) and mouse ( Mo et al . , 2015 ) .", "Here , we develop a tandem affinity purification of INTACT nuclei ( TAPIN ) method with increased specificity , sensitivity , and throughput .", "By combining this method with an extensive set of new driver lines with predominant expression in specific cell types and a new probabilistic method to interpret transcript abundance , we build a resource of high-quality transcriptomes for one hundred driver lines .", "We selected drivers that expressed in cell types constituting the lamina ( Fischbach and Dittrich , 1989; Tuthill et al . , 2013; Edwards et al . , 2012 ) as well as the major cell types of the circuits that compute the direction of visual motion ( Mauss et al . , 2017; Figure 1C ) .", "We further included neuronal populations in two central brain regions , the mushroom body and central complex , primarily to serve as informative outgroups .", "By profiling these driver lines , we develop an expression catalog for 67 Drosophila cell types as well as several broader cell populations .", "Through validation experiments and comparisons to the literature we demonstrate that this resource is useful both for identifying individual genes expressed in specific cell types and for revealing broader patterns such as the expression of all members of a gene family across many cell types .", "As an example , we describe the expression of neurotransmitters and their receptors and use this information to interpret synaptic connectivity .", "For example , we unexpectedly found that the R8 photoreceptors express acetylcholine in addition to histamine and show that this apparent co-transmitter phenotype is further supported by differential expression of neurotransmitter receptors in R8 postsynaptic partners .", "Our results demonstrate that combining expression and connectomes leads to specific testable hypotheses about circuit mechanisms that are inaccessible to either approach alone ." ], [ "To enable transcriptome analyses of defined cell populations , we first assembled a collection of genetic drivers to access them .", "For this study , we combined drivers from existing collections for cell types in the lamina ( Tuthill et al . , 2013 ) , the mushroom body ( Aso et al . , 2014 ) , and the lobula ( Wu et al . , 2016 ) with new driver lines for many additional optic lobe cell types and also some neurons of the central complex ( Wolff and Rubin , 2018 ) , T . Wolff , personal communication ) .", "Nearly all of these drivers were generated using an intersectional method , split-GAL4 , to refine expression patterns of GAL4 driver lines .", "To characterize new driver lines , we imaged expression patterns across the entire fly brain to determine overall driver specificity ( Figure 1D , Figure 1—figure supplement 1 ) and examined anatomical features such as layer patterns in higher resolution images to identify specific cell types ( Supplementary file 1A , Figure 1—figure supplement 2 ) .", "For most lines , we further confirmed the identity of labeled cells by examining the morphology of individual cells using stochastic labeling ( Figure 1—figure supplement 2 ) .", "Although we noted that a few patterns also include some additional contaminating cells ( Supplementary file 1A ) , these driver lines are the most specific tools currently available to access individual cell types in the optic lobe .", "Next , we employed an improved INTACT method to measure nuclear transcriptomes in genetically defined cell populations ( Henry et al . , 2012 ) , and we also developed a new variant of the method that permits higher throughput with increased purity and sensitivity .", "In both approaches , nuclei are purified using a nuclear tag whose expression is driven in a cell population of interest by either a standard or split GAL4 driver ( Figure 2A ) .", "The new variant protocol , tandem affinity purification of INTACT nuclei ( TAPIN ) , uses a bacterial protease ( IdeZ ) to specifically cleave antibodies in the hinge region separating their Fc and antigen binding F ( ab’ ) 2 fragments ( Figure 2B , Figure 2—figure supplement 1B ) .", "Treating protein A magnetic bead-bound nuclei with this protease generates both nucleus-F ( ab’ ) 2 and bead-Fc complexes .", "Soluble nucleus-F ( ab’ ) 2 is then recaptured on protein G magnetic beads , removing non-specifically bound material from the first capture .", "INTACT successfully profiled many of the abundant cell types in the optic lobe ( >1000 cells per brain ) , but failed for sparser cell types and those whose nuclei were difficult to purify by differential centrifugation ( photoreceptors , glia , T4 , T5 ) .", "We solved these problems with TAPIN , which does not purify nuclei prior to bead capture .", "The greatest advantage of TAPIN is its ability to purify nuclei from sparse cell types ( <50 cells/brain ) ( Supplementary file 1A ) .", "INTACT is not suitable for these lines because of loss during differential centrifugation .", "This difficulty cannot be overcome by processing more brains per experiment because differential centrifugation is difficult to scale .", "TAPIN solves this problem by running a first capture on crude extracts generated from hundreds to thousands of fly heads .", "The substantial background in this first capture is reduced 5- to 6- fold in a second capture with only a modest decline in both the yield of nuclei and amplified cDNA ( Figure 2C ) .", "We applied INTACT and TAPIN to the cell populations defined by the genetic drivers we described above ( Supplementary file 1B ) .", "Most drivers express in a single anatomically defined cell type or a small group of related cell types .", "We note that a few of our cell types are strictly groups of related cell types ( for example , the muscle cells or , at a different level of a cell type hierarchy , the T4 and T5 cells , with four subtypes each ) .", "Additional drivers target more heterogeneous cell populations sharing a common property ( e . g . , driver lines aimed at recapitulating the expression of a neurotransmitter marker ) .", "Altogether , we built 250 RNA-seq libraries from 242 samples of purified nuclei ( 46 using INTACT and 196 using TAPIN ) and eight manually dissected samples ( Supplementary file 1B ) .", "We estimated relative transcript abundance in each library using kallisto ( Bray et al . , 2016 ) .", "Libraries built from more nuclei yielded more cDNA ( Figure 2D ) , allowed more genes to be detected ( Figure 2E ) , had more estimated transcripts ( Figure 2—figure supplement 1C ) , more reproducible transcript abundance ( Figure 2F ) , and less bias in coverage across gene bodies ( Figure 2—figure supplement 1D , E ) .", "We focused on 203 libraries that had at least 8500 genes detected , 3µg cDNA yield , and 0 . 85 Pearson’s correlation of transcript abundances in two biological replicates .", "These 203 libraries consist of at least two biological replicates built from 100 drivers that covered 67 cell types ( 53 visual system , 7 mushroom body , 5 central complex , 2 muscle ) , 6 broader cell populations ( ChAT , Gad1 , VGlut , Kdm2 , Crz , and NPF ) , and 2 manually dissected tissues ( the lamina and remainder of the optic lobe ) ( Materials and methods ) .", "We provide the read and abundance data for the remaining sub-optimal libraries ( 47 libraries covering 24 cell types ) in the event they may be informative , but we do not consider these to be of sufficient quality and do not consider them further here .", "We did not sort the sex of flies when preparing TAPIN-seq libraries , as we did not observe large differences in male and female expression profiles ( Figure 2—figure supplement 1F ) .", "We were encouraged by the clear enrichment of previously identified markers in cell types where they were expected .", "For example , we recovered transcription factors ( TFs ) previously found in the developing monopolar interneurons and inner photoreceptors ( Tan et al . , 2015; Figure 2G ) .", "We further confirmed our measurements by comparing TAPIN-seq results for twelve cell types that were also recently profiled by FACS-seq ( Konstantinides et al . , 2018; Figure 2—figure supplement 2A , B ) and found concordant expression of cell type-enriched genes .", "This concordance also argues against major differences between nuclear and cytoplasmic transcriptomes .", "In combination with the technical quality of our libraries , this confirmation by independent gene expression measurements validated our approach , and also motivated us to explore how to best interpret a large dataset of relative abundances .", "To study the relation between cell types , we built a dendrogram based on the expression states we inferred for the whole transcriptome and estimated the support for each branch point with bootstrap resampling ( Figure 4A ) .", "The broad groupings were well supported and mostly intuitive: muscle were outgroups , followed by a mushroom body cell type ( PAM_4 ) , the glia , the photoreceptors , and the remaining neurons .", "Several fine groupings of anatomically closely related neurons were also well supported ( e . g . , Kenyon cells; C2 , C3; Lawf1 , Lawf2; T4 , T5; LPLC1 , LPLC2 ) .", "However , mid-level branchings were not well supported , indicating the lack of a simple hierarchical relationship .", "Neurons were generally grouped by region: central complex , mushroom body , and optic lobe .", "One surprise was the grouping of Tm20 and Dm1 , away from all other optic lobe cell types .", "Upon closer examination , the identity of genes expressed exclusively in these two lines ( lz , Pdh , bw ) suggest that this grouping is driven by shared pigment cell contamination in the GAL4-tagged patterns of these driver lines .", "Similarly , the unusual position of PAM_4 is likely due to some unidentified non-neuronal cells in the driver .", "These are examples of imperfections in the GAL4 driver lines .", "While they can lead to some false positives for the main target cell types , they can also provide additional information .", "For example , analyzing the overlap between Dm1 and Tm20 allowed us to infer marker genes expressed in the pigment cell population .", "We next identified genes that marked cell groups in the tree , using three criteria: genes that expressed in all the cells within a group , at most two cells outside this group , and with transcript abundance higher than all cells outside the group ( For simplicity , we will hereon refer to cell type as just cell . This is not meant to exclude the possibility of heterogeneity within the individual cells of a profiled population . ) .", "We used these criteria to identify markers for photoreceptors ( n = 108 ) , glia ( n = 60 ) , and muscle ( n = 76 ) ( Figure 4B , Supplementary file 1D ) .", "These genes included many known as well as new markers .", "For example , genes enriched in photoreceptors include signaling components ( Arr2 , Galphaq ) and transporters ( trpl , Eaat2 ) with known physiological roles as well as uncharacterized orphan transporters ( e . g . , CG8468 ) .", "We also identified 18 markers for pigment cells using the Tm20 and Dm1 profiles .", "In addition to the three types of lamina glia we profiled , several other glia types are present in both the lamina and the medulla .", "Genes expressed exclusively in the dissected samples ( lamina , remainder of optic lobe ) and not in the TAPIN libraries identified marker genes for optic lobe cells that we did not directly profile , such as medulla glia .", "Indeed , the genes identified in this way included several known markers for astrocytes ( alrm , wun2 , Obp44a ) ( Huang et al . , 2015 ) .", "We examined the breadth of expression of different functional groups of genes , as defined by FlyBase gene group curation .", "HOX-like homeobox TFs were among the most specifically expressed group , while groups of core cellular machinery ( e . g . , beta importins , mitochondrial complexes ) were among the most broadly expressed groups ( Figure 4C ) .", "Some groups included both broadly and very specifically expressed genes .", "For example , among cell adhesion molecules , we noted an interesting distribution for three gene groups proposed to be involved in protein-protein interactions that underlie synaptic connectivity ( Özkan et al . , 2013; Tan et al . , 2015; Carrillo et al . , 2015 ) .", "While 11 DPR-interacting proteins ( DIP ) were among the most specifically expressed genes ( expressed in a median of 6 cells ) , beat ( median , 25 . 5 cells ) and DPR ( median , 51 cells ) genes were more broadly expressed ( Figure 4—figure supplement 1A–D ) .", "As physical interactions among these and other extracellular proteins have been systematically characterized ( Özkan et al . , 2013 ) , we combined their expression and interaction patterns to estimate the number of potential interaction between cells in the lamina ( Figure 4—figure supplement 1E ) , many of which are in actual contact ( Figure 4—figure supplement 1F ) .", "Except for a clear paucity of transcripts encoding interacting protein pairs expressed by glia , these global expression-based patterns did not correlate well with connectivity in the lamina .", "However , we found that every pair of lamina cells expressed mRNAs for tens of interacting protein pairs , highlighting the broad potential for cell-cell interactions not only in the developing ( Tan et al . , 2015 ) but also adult optic lobe .", "Single cell RNA-seq ( scRNA-seq ) was recently used to map the optic lobe ( Konstantinides et al . , 2018 ) and brain ( Davie et al . , 2018 ) .", "Despite its routine use , interpreting scRNA-seq measurements – clustering single cell transcriptomes and labeling these clusters as known cell types – remains challenging .", "For example , the 52 single cell clusters found in the optic lobe ( 23 labeled as known cell types , including 7 types of glia; Konstantinides et al . , 2018 ) and the 87 clusters found in the whole brain ( 41 labeled , also including seven glia; Davie et al . , 2018 ) far under-estimate the expected diversity of cell types – over one hundred anatomically distinct neuronal cell types have been described in the optic lobe alone ( Fischbach and Dittrich , 1989; Morante and Desplan , 2008; Otsuna and Ito , 2006; Nern et al . , 2015; Wu et al . , 2016 ) .", "Furthermore , comparing the number of single cells in each optic lobe cluster to the true abundance of each cell type ( as established by neuroanatomical studies ) reveals that the single cell map does not proportionally represent abundance ( ranging from ~5 times fewer Dm8/Tm5c cells to ~7 times more Dm12 cells than expected in the optic lobe map; Figure 5A ) .", "The whole brain map , measured using the more sensitive 10X scRNA-seq platform rather than Drop-seq used for the optic lobe map ( Svensson et al . , 2017 ) , showed similar cell type abundances ( Figure 5B ) .", "Without detailed neuroanatomy to serve as ground-truth , this similarity could be interpreted as reproducibility across platforms .", "Instead , our results suggest caution when interpreting cell type frequencies from scRNA-seq maps , as they can be skewed by experimental artifacts such as cell type-specific differences in RNA isolation yields , computational over-clustering , or inaccurate cell type labeling .", "Given the known number of cell types and their frequencies , it is clear that interpreting single cell measurements is challenging .", "Comparison with our data reveals additional challenges in mapping scRNA-seq clusters to known cell types .", "To compare our data to the brain map , we used non-negative least squares regression to model each TAPIN-seq transcriptome as a linear weighted sum of single cell cluster transcriptomes , assuming that large regression coefficients reflect matching cell types ( Davie et al . , 2018; Cao et al . , 2019 ) .", "We interpreted the results of this comparison against an ideal scenario where single cell clusters were perfectly resolved and accurately labeled , and assuming that the driver lines used for TAPIN-seq profiling had minimal expression outside of the main target cell type .", "In this scenario , we would expect a unique cluster matching each of our TAPIN-seq profiles of cell type-specific driver lines , as well as many unmatched clusters , reflecting cell types that we did not profile .", "However , we observed few one-to-one matching clusters for our TAPIN-seq profiles ( e . g . , T1 , Tm1 , Dm8 , Dm9 , Pm3 ) , several one-to-many matches ( e . g . , photoreceptor cluster #53 matching our R1-6 , R7 , R8-Rh5 , and R8-Rh6 profiles; also , L1-5 cluster #20 , Lawf1/2 cluster #58 , and T4/T5 cluster #24 ) , clusters with no TAPIN-seq matches ( e . g . , clusters #7 , 15 , 23 ) , as well as TAPIN-seq cell types without a matching cluster ( e . g . , Dm4 , Dm11 , Mi4 , Tm2 , Tm20 , LPLC1 ) ( Figure 5C ) .", "The matches confirmed several clusters labeled as single cell types ( e . g . , Tm1 , Mi1 ) or multiple cell types ( e . g . , photoreceptors , L1-5 , Lawf1/2 , T4/5 , Kenyon cells ) and also suggested possible labels for previously unlabeled clusters ( e . g . , Dm8 cluster #52 , Dm9 cluster #74 , pigment cell cluster #76 ) and alternative labels for previously labeled clusters ( e . g . , TmY5a TAPIN matches the TmY14 cluster #11; Lai TAPIN matches Dm8/Tm5c cluster #39 ) .", "We observed similar results when analyzing the optic lobe map , with few apparent single cell – TAPIN-seq matches ( Figure 5—figure supplement 1 ) .", "Although this result could arise from major errors in our TAPIN-seq profiles , this possibility is unlikely given our earlier validation results and the concordance between our TAPIN-seq profiles and cell type-enriched genes identified from independent FACS-seq measurements ( Figure 2—figure supplement 2 ) .", "As a separate comparison of the bulk and single cell profiles , we examined the bulk expression of genes marking each single cell cluster ( Figure 5—figure supplement 2 ) .", "Confirming the regression results , this analysis also found few one-to-one matches in which single cell cluster markers were enriched in only a single TAPIN-seq profile .", "Instead , most cluster markers were either enriched in multiple bulk cell types ( over-clustering ) , or were not enriched in our data ( cell types we did not profile by TAPIN-seq ) .", "As before , many TAPIN-seq profiles were not enriched for cluster markers , reflecting cell types that were either missing or clustered with other cell types in the single cell map .", "We further explored specific examples where the TAPIN-seq data offered new insight into the single cell maps by suggesting alternative labels or labeling unannotated clusters .", "The single cell clusters labeled as TmY14 matched the TmY5a TAPIN profile ( Figure 5C ) .", "The cluster was originally labeled based on the expression of a single transcription factor , knot ( kn ) , as determined using a kn-GAL4 reporter .", "We also observed kn expression in our TmY5a measurements and further confirmed its expression in both TmY14 and TmY5a cells using the kn-GAL4 reporter line , suggesting that the cluster likely includes not only TmY14 cells , but also TmY5a and other kn-expressing cells ( Figure 5—figure supplement 3 ) .", "Similarly , we found that the Dm2 cluster ( optic lobe cluster #55 ) , which was labeled based on a Dm2 FACS-seq profile , matched our Mi15 profile ( Figure 5—figure supplement 2A ) .", "This observation is concordant with previous reports that the line used to FACS sort Dm2 also expresses in Mi15 ( Supplementary Figure 2 in Takemura et al . , 2013 , Supplementary file 1 Table S4 in Nern et al . , 2015 ) .", "Finally , we found that the Dm8/Tm5c cluster ( brain cluster #39 ) matches our Lai TAPIN-seq profile , while unlabeled clusters match our Dm8 and Dm9 TAPIN-seq profiles ( Figure 5D ) .", "Our measurements also suggest labels for other previously unannotated clusters , such as brain cluster #76 which likely reflects pigment cell , as demonstrated by enrichment of its marker genes in both Dm1 and Tm20 profiles – both measured with lines that also express in pigment cells ( Figure 5—figure supplement 2B ) .", "As expected , the genes marking this cluster include known pigment cell markers ( e . g . , Pdh , rdhB ) .", "Altogether , our results demonstrate that cell type-identified data , such as bulk transcriptomes , can help interpret single cell RNA-seq measurements .", "The proteins that synthesize and transport neurotransmitters are well known , enabling us to use their expression to predict neurotransmitter phenotype .", "We used histamine decarboxylase ( Hdc ) , glutamate decarboxylase ( Gad1 ) , the vesicular acetylcholine transporter ( VAChT ) , and the vesicular glutamate transporter ( VGlut ) to identify potential histaminergic , GABAergic , cholinergic , and glutamatergic cell types , respectively ( Figure 6A ) .", "Our model unambiguously inferred expression states for these genes and indicated a single transmitter ( from this group ) for nearly all neurons we profiled .", "A second cholinergic marker , choline acetyltransferase ( ChaT ) , matched VAChT expression almost perfectly ( the two genes also share an exon ) .", "The sole exception , apparent expression of ChAT but not VAChT in R7 photoreceptors , likely results from a subset of dorsal rim R8 cells labeled by the R7 driver line ( further discussed below , also see Supplementary file 1A ) .", "Besides these four neurotransmitters that we identified by one or two marker genes , we also identified candidate dopaminergic neurons based on the combined expression of tyrosine hydroxylase ( ple ) , dopa decarboxylase ( ddc ) , vesicular monoamine transporter ( Vmat ) and dopamine transporter ( DAT ) .", "While DAT , ple , and ddc were also expressed individually in several cell types that did not express Vmat , only known dopaminergic cell types and one medulla neuron ( Mi15 ) expressed this combination ( Figure 6A ) .", "One neuronal cell type , T1 , expressed none of the neurotransmitter markers VGlut , VAChT , Vmat , and Gad1 ( Figure 6A ) .", "Although T1 does express most pan-neuronal genes , it does not express bruchpilot ( brp ) , a key component of presynaptic active zones .", "Consistent with this result , EM reconstruction has identified very few T1 presynaptic specializations ( Takemura et al . , 2008 ) .", "Transmitters for nearly half of our cell types have been previously proposed and generally agree with our results .", "For example , VAChT/ChAT expression in Kenyon cells supports recent reports showing they are cholinergic ( Barnstedt et al . , 2016; Crocker et al . , 2016 ) .", "Fluorescence in situ hybridization and immunolabeling guided by our measurements confirmed the expression of ChAT , Gad1 , and VGlut in Mi1 , Mi4 , and Mi9 , respectively ( Long et al . , 2017; Takemura et al . , 2017 ) .", "However , we see considerable differences between our assignments and some previous work that used reporter transgenes ( Raghu and Borst , 2011; Varija Raghu et al . , 2011; Raghu et al . , 2013 ) , which we generally attribute to unfaithful transgene expression patterns .", "We believe our assignments to be more reliable , however they are not without problems .", "For example , one assignment inferred by our model that seems unlikely and is not supported by other available data is the presence of Gad1 in Mi9 , which was not detected in the FISH or antibody experiments mentioned above .", "Given the presence of some contaminating Mi4 cells in at least one Mi9 driver and the lower Gad1 abundance ( mean 276 TPM in Mi9; 2165 TPM in Mi4; 1870 mean TPM in predicted GABAergic cells ) , we attribute the Mi9 Gad1 signal to contaminating contributions from other GABAergic cells such as Mi4 .", "A principal goal of our work is to provide a foundation for combining molecular data such as neurotransmitter and receptor expression patterns with anatomical or functional connectivity data .", "One application of expression information is to constrain mechanistic models of neural circuits such as the extensively studied motion detection circuit in the fly eye ( reviewed in Mauss et al . , 2017; Figure 7—figure supplement 1A ) .", "The combined availability of expression and connectomics data for many cell types in a brain region also makes it possible to systematically identify and further explore unusual patterns of receptor or transmitter expression; for example , cell types in which an otherwise widely expressed receptor is absent or cells with unusual combinations of receptor subunits .", "Below we discuss three examples , focused on potential signs of synaptic transmission , of how such patterns can lead to specific and unexpected hypotheses about circuit function .", "As we illustrate , combining expression data with synapse-level anatomy permits analyses which are inaccessible to either approach alone ." ], [ "We present an approach to characterize the function of neural circuits by combining genetic tools to access their component cells , TAPIN-seq to measure their transcriptomes , and a probabilistic model to interpret these measurements ( Figures 1 , 2 and 3 ) .", "We used this approach to establish an extensive resource of the genes expressed in 67 Drosophila cell types , including 53 in the visual system , covering photoreceptors , lamina , and components of the motion detection circuit ( Figure 4 ) and systematically compare our results to single cell RNA-seq ( Figure 5 ) .", "Our approach enables an extensive analysis of neurotransmission in the Drosophila visual system , including the neurotransmitters sent and received across the network as well as transcription factors that potentially regulate neurotransmitter identity ( Figures 6 and 7 ) .", "We also provide specific examples of integrating transcriptomes and connectomes to illuminate circuit function ( Figures 8 and 9 ) .", "Many recent studies have explored gene expression in neurons .", "However , only a few of these were aimed at neurons in genetically tractable organisms and brain regions for which detailed anatomical data , especially at the level of synaptic connections , are available .", "Previous work in the mouse retina has used both genetic ( Siegert et al . , 2012 ) and single cell approaches ( Macosko et al . , 2015 ) to characterize transcriptional regulators as well as classify cell types .", "More recent work in Drosophila used single cell RNA-sequencing to characterize heterogeneity in olfactory projection neurons ( Li et al . , 2017 ) , the midbrain ( Croset et al . , 2018 ) , the optic lobe ( Konstantinides et al . , 2018 ) , and the whole brain ( Davie et al . , 2018 ) .", "The expression patterns of many genes have also been mapped in C . elegans neurons , whose connectivity has long been known , although these studies typically focus on individual genes rather than genome-wide catalogs ( Hobert , 2016 ) .", "The unique combination of an extensive genetic toolbox to access individual cell types in the Drosophila visual system and systematic efforts to map its connectivity , make it well suited for exploring whether a comprehensive catalog of gene expression is useful for understanding circuit function .", "Towards this end , we profiled a diverse array of cell types including all of the neuronal cell types that populate the lamina and a subset of cell types in the medulla and lobula complex including those known to play a central role in the detection of motion .", "We also analyzed a number of cell types residing in deeper brain structures such as the mushroom body and central complex .", "Our approach requires genetic driver lines to obtain transcriptomes of specific cell populations .", "The recent availability of large collections of reagents for split-GAL4 intersections ( Dionne et al . , 2018; Tirian and Dickson , 2017 ) make it possible to obtain such lines for virtually any cell type of interest .", "This expanding genetic toolbox works well with our TAPIN-seq method to profile transcriptomes .", "In some cases , available driver lines , including some used in this study ( see Supplementary file 1A ) , may label some additional cell types .", "In addition to the presence of different , anatomically distinct cell types in a driver pattern , heterogeneity could also result from as yet unrecognized subpopulations within a seemingly uniform group of cells .", "For example , R7 and R8 photoreceptor neurons each include two major subtypes ( pale and yellow ) with different rhodopsin expression but very similar , if not identical , cell morphology ( Wernet and Desplan , 2004 ) .", "While drivers with even higher specificity could be obtained through testing of additional split-GAL4 intersections or perhaps triple intersections ( Dolan et al . , 2017 ) , we did not find the contributions of small numbers of ‘off-target’ cells to be a major limitation for many applications of expression data .", "In general , the transcriptomes support the high specificity of the intersectional lines we used to access visual system cells ( Figure 1 ) .", "For example , we found specific expression of known marker genes ( Figures 2G and 4B ) and also that most neurons only express genes for a single neurotransmitter type ( Figure 6A ) .", "In general , the expression patterns observed in our validation experiments ( for example , Figure 3G and Figure 3—figure supplement 2 ) were highly consistent within a cell type .", "This suggests that individual cells of many if not all of the anatomical cell types we profile do indeed share specific molecular signatures , even if subdivisions within some types might exist .", "The availability of specific driver lines makes such validation at cellular resolution possible in a way that is otherwise difficult , for example in single cell RNA-seq studies .", "Driver lines also permit repeated access to the same cell type in multiple animals at defined time points , enabling the study of behavioral or circadian conditions in individual cell types without having to sequence the whole brain or dissected brain regions .", "Modifying the one-step affinity capture in the original INTACT method to a two-step capture in TAPIN-seq increased its specificity , sensitivity , and throughput without the need for time-consuming and labor-intensive centrifugation steps ( Figure 1 ) .", "We initially tried improving the original INTACT method by using density gradient centrifugation to purify nuclei prior to the bead capture step , but this was cumbersome , low throughput , and ineffective for cell types with few nuclei per brain .", "In addition , for reasons that remain unclear , both photoreceptors and T4 cells consistently yielded few nuclei with this approach .", "Even with TAPIN , the libraries obtained with some sparser driver lines did not meet the quality control standards we applied .", "We suspect that the quality of these sub-optimal libraries can be improved by starting with more flies , which is simplified by TAPIN-seq’s ability to use frozen material , enabling the collection of many flies on multiple days at defined time points .", "In contrast , manual or FACS sorting of dissociated cells is more challenging to scale up , because these more labor-intensive tissue procurement schemes cannot be simplified in the same way .", "It is also worth noting that our tandem affinity purification approach can improve the specificity of any immunopurification method that uses a capture antibody that is cleavable by IdeZ ( all IgG subclasses ) , without requiring expression of a traditional TAP tag ( Rigaut et al . , 1999 ) .", "TAPIN-seq complements single-cell RNA-seq studies of neurons in several ways ( Ecker et al . , 2017; Konstantinides et al . , 2018 ) .", "First , our high-resolution transcriptomes will serve as a reference for interpreting single-cell measurements .", "In particular , comparing our expression catalog to recent single cell maps of the optic lobe and whole brain highlights the challenges in interpreting single cell measurements .", "Several cell types that we profiled don’t appear as clusters in the single cell map , while others are grouped into the same cluster .", "The well-established neuroanatomy of the optic lobe makes it an ideal setting to evaluate the accuracy of single cell RNA-seq measurements and raises a broader caution when interpreting scRNA-seq surveys of less well-characterized tissues: the composition of cell types ( or states or clusters ) observed by scRNA-seq can deviate significantly from their true abundance and requires validation with independent methods .", "While we analyze fly neurons here , the cautions may also be worth considering in other tissues and species ( e . g . , the Human Cell Atlas effort; Regev et al . , 2017 ) .", "Having both deep bulk transcriptomes and single cell maps of the same tissue also provides an opportunity for developing new analytical tools that can harness available cell type-identified information while clustering single cell data .", "Second , combining our approach with single-cell profiling could more efficiently profile heterogeneity within a brain region or genetically defined cell population .", "Such a combined approach could also help to characterize known cell types for which specific driver lines are not yet available or help to identify and exclude contributions from ‘off-target’ cells to a bulk profile .", "Finally , the complementarity between bulk and single-cell measurements extends to other genomic features that can be measured in TAPIN-seq purified nuclei , including accessible chromatin and modified histones .", "We expect this combination of genomic tools to help decipher the transcriptional and epigenetic regulation of neuronal expression programs .", "Transcriptome measurements can be of limited utility because it is challenging to interpret relative transcript abundance .", "In this study we developed a probabilistic mixture modeling approach to classify relative abundances into binary on and off states .", "This model was a useful guide for interpreting our measurements; most genes are readily described with the two-state model , although the expression of some genes is not ( e . g . , Rab11; Figure 3—figure supplement 1L ) .", "Even for specific genes whose expression is more continuous than bimodal , the results still offer a useful family-wide summary of expression patterns .", "For example , DPR family members are more broadly expressed than DIP genes ( Figure 4C; Figure 4—figure supplement 1D ) , an observation supported by two recent studies using different methods ( Cosmanescu et al . , 2018; Venkatasubramanian et al . , 2019 ) .", "Despite our model’s utility , it is important to remember the many potential sources of error ( minor cell types in driver line patterns , transcript carry over during TAPIN , biases in RNA-seq library construction and sequencing , etc . ) that can affect measurements of relative transcript abundance and the resulting model inferences .", "Having observed most discrepancies between our modeling results and protein-level expression near the boundary between on and off states , it is prudent to treat these cases more carefully .", "Our resource provides additional foundation for systematic functional and molecular studies of the Drosophila visual system .", "We illustrated how the resource can characterize neurotransmission in the network , particularly when combined with connectome information detailing connectivity between cell types as well as the grouping of post-synaptic partner cell types .", "We predict neurotransmitters used by every cell we profiled and found two likely cases of co-transmission ( Figure 6A ) .", "The expression patterns of the major fast-acting transmitters histamine , acetylcholine , glutamate and GABA were comparatively simple: Nearly all neuronal cell types in our catalogue appear to express exactly one of these four transmitters .", "However , the transcriptomes suggest that many cells also have the potential to release specific neuropeptides , other chemical messengers such as nitric oxide , or form gap junctions with other cells .", "While selected transmitter markers ( e . g . , Gad1 or VGlut ) could also be assigned to cell types using methods such as immunolabeling or FISH , these approaches are not practical for comprehensive sampling of markers across these different modes of cell-cell communication .", "This is particularly clear when the expression patterns of neurotransmitter receptors are also considered ( Figure 7A ) .", "Our results suggest that , for canonical small molecule transmitters , neurotransmitter output space is tightly tuned while input space is not: neurons typically send just one type of signal but can receive many ( Figures 6 and 7 ) .", "The expression patterns of neurotransmitter receptors provide further context for determining circuit mechanisms ( Figure 7—figure supplement 1 , Figures 8 and 9 ) .", "Our results also implicate transcription factors involved in regulating neurotransmitter phenotype , including several that appear to have conserved roles in specifying neuronal identity in other species ( Figure 6—figure supplement 1 ) .", "The availability of connectivity data for many neurons in the visual system allowed us to interpret neurotransmitter use and receptor distribution in the context of circuit architecture ( Takemura et al . , 2013; Takemura et al . , 2015; Rivera-Alba et al . , 2011 ) .", "For example , our data show expression of both histaminergic and cholinergic markers in R8 photoreceptors .", "We further find that some major synaptic targets of R8 cells , as identified by electron microscopy , do not express known receptors for histamine ( Figure 8F ) .", "Given that R7 and R8 cells were previously thought to be exclusively histaminergic both results are unexpected and individually might appear difficult to explain ( Gao et al . , 2008 ) .", "However , in combination , these findings make a strong case for a dual histaminergic and cholinergic transmitter phenotype of R8 cells .", "In contrast , R7 only expresses the histaminergic marker Hdc , and all of its targets express a histamine receptor ( Figure 8G ) .", "Finally , our approach especially complements ongoing efforts to map circuit connectivity , which is complete for C . elegans , and is becoming accessible on a whole brain level for Drosophila ( Zheng et al . , 2018 ) , and for portions of the mouse brain such as the retina .", "Methods to obtain and interpret serial electron micrographs , array tomography and other methods for mapping connectivity are rapidly progressing ( Swanson and Lichtman , 2016; Micheva and Smith , 2007; Kebschull et al . , 2016 ) .", "All told , we are entering a period in neuroscience where connectomics will become pivotal .", "We expect that genomic approaches , such as the methods for data collection and analysis that we describe here , will enhance these efforts by using transcriptomes to provide , at high-throughput , a molecular proxy for physiological features that are otherwise inaccessible to connectomic methods ." ], [ "Flies were reared on standard cornmeal/molasses food at 25°C .", "For profiling experiments adults , 4–7 days of age , were entrained to a 12:12 light:dark cycle and anesthetized by CO2 at ZT8 - ZT12 .", "Samples can be stored indefinitely at −80°C after flash freezing in liquid N2 .", "We used female flies for all anatomical characterizations .", "All raw and processed transcriptome data is available from NCBI GEO ( accession GSE116969 ) .", "The shell scripts used to process the raw RNA-seq data , and the R and Stan programs that implement the mixture model as well as generate all figures and tables in this paper are available at github ( http://github . com/fredpdavis/opticlobe ) .", "The cell type-level expression table can be explored interactively at http://www . opticlobe . com ." ] ]
[ "The anatomy of many neural circuits is being characterized with increasing resolution , but their molecular properties remain mostly unknown .", "Here , we characterize gene expression patterns in distinct neural cell types of the Drosophila visual system using genetic lines to access individual cell types , the TAPIN-seq method to measure their transcriptomes , and a probabilistic method to interpret these measurements .", "We used these tools to build a resource of high-resolution transcriptomes for 100 driver lines covering 67 cell types , available at http://www . opticlobe . com .", "Combining these transcriptomes with recently reported connectomes helps characterize how information is transmitted and processed across a range of scales , from individual synapses to circuit pathways .", "We describe examples that include identifying neurotransmitters , including cases of apparent co-release , generating functional hypotheses based on receptor expression , as well as identifying strong commonalities between different cell types ." ]
[ "In the brain , large numbers of different types of neurons connect with each other to form complex networks .", "In recent years , researchers have made great progress in mapping all the connections between these cells , creating ‘wiring diagrams’ known as connectomes .", "However , charting the connections between neurons does not give all the answers as to how the brain works; for example , it does not necessarily reveal the nature of the information two connected cells exchange .", "Assessing which genes are switched on in different neurons can give insight into neuronal properties that are not obvious from physical connections alone .", "To fill that knowledge gap , Davis , Nern et al . aimed to measure the genes expressed in a well-characterized network of neurons in the fruit fly visual system .", "First , 100 fly strains were established , each carrying a single type of neuron colored with a fluorescent marker .", "Then , a biochemical approach was developed to extract the part of the cell that contains the genetic code from the neurons with the marker .", "Finally , a statistical tool was used to assess which genes were on in each type of neurons .", "This led to the creation of a database that shows whether 15 , 000 genes in each neuron type across 100 fly strains were switched on .", "Combining this information with previous knowledge about the flies’ visual system revealed new information: for example , it helped to understand which chemicals the neurons use to communicate , and whether certain cells activate or inhibit each other .", "The work by Davis , Nern et al . demonstrates how genetic approaches can complement other methods , and it offers a new tool for other scientists to use in their work .", "With more advanced genetic methods , it may one day become possible to better grasp how complex brains in other organisms are organized , and how they are disrupted in disease ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "structural biology and molecular biophysics" ]
Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors
elife-00745-v1
[ [ "Nucleocytoplasmic transport across the nuclear envelope is controlled and facilitated by nuclear pore complexes ( NPCs ) , very large , modular protein assemblies ( Brohawn et al . , 2009; Capelson et al . , 2010; Aitchison and Rout , 2012 ) .", "NPCs are positioned in circular openings within the nuclear envelope , generated by the fusion of inner and outer nuclear membrane .", "About 30 different proteins , collectively called nucleoporins or nups , make up an NPC .", "These nups are arranged around a central eightfold rotational symmetry axis along the central pore opening .", "Cryo-electronmicroscopic ( EM ) studies have shown that the principal architecture of the NPC is conserved among all eukaryotes , however metazoan NPCs are more elaborate and have additional features compared to single cell organisms , explaining in part substantially different size estimates for the assembly ( 60–120 MDa vs 40–50 MDa ) ( Reichelt et al . , 1990; Alber et al . , 2007; Brohawn et al . , 2009; Grossman et al . , 2012 ) .", "The entire structure is built around a central , donut-shaped scaffold , with prominent extensions that distinguish both the cytoplasmic and the nucleoplasmic faces of the NPC .", "The center of the NPC is thought to be filled by unstructured , highly repetitive protein extensions characterized by phenylalanine-glycine dipeptides ( FG repeats ) , which are found in ∼1/3 of all nups ( Rout et al . , 2000; Cronshaw et al . , 2002 ) .", "FG repeats are critical for NPC function and allow for selective passage of transport cargos that are bound to soluble nuclear transport receptors ( NTRs ) ( Görlich and Kutay , 1999; Weis , 2003 ) .", "The mechanistic details of how FG repeats contribute to NPC selectivity remain somewhat controversial , however , it has been firmly established that all soluble NTRs specifically bind to FG-repeat domains and that interactions between NTRs and FG repeats are critical for nucleocytoplasmic transport .", "The central scaffold of the NPC donut structure is formed by approximately 15–20 nups that are organized into four major subcomplexes .", "These are the Y- ( or Nup84 ) complex , the Ndc1 complex , the Nsp1 complex , and the Nic96 complex .", "The Y-complex is essential for NPC biogenesis and has been well characterized .", "It contains seven universally conserved proteins that form a highly branched , Y-shaped structure ( Lutzmann et al . , 2002; Kampmann and Blobel , 2009 ) .", "Currently ∼90% of the complex has been elucidated in crystallographic detail ( Berke et al . , 2004; Brohawn et al . , 2008; Brohawn and Schwartz , 2009; Leksa et al . , 2009; Whittle and Schwartz , 2009 ) .", "From this analysis it is evident that the structural elements , and also their interactions are shared with vesicle coats , specifically the outer coat of COPII vesicles ( Devos et al . , 2004; Brohawn et al . , 2008; Onischenko and Weis , 2011 ) .", "The Ndc1 complex contains proteins thought to anchor the NPC in the highly-curved pore membrane ( Onischenko et al . , 2009 ) .", "The Nsp1 complex is built from three coiled-coil proteins , each with prominent FG-repeats .", "The Nsp1 complex is centrally located in the NPC , with the FG-repeats contributing to the selective transport barrier ( Hülsmann et al . , 2012 ) , and the coiled-coil moiety functioning as a bridge to the other NPC scaffold units ( Grandi et al . , 1995; Bailer et al . , 2001 ) .", "The Nic96 complex organizes four architectural proteins , Nic96 , Nup157/170 , Nup188 , and Nup192 , as well as the more disordered protein Nup53/59 ( Nehrbass et al . , 1996; Marelli et al . , 1998; Theerthagiri et al . , 2010; Amlacher et al . , 2011 ) .", "Structures for large fragments of Nic96 and Nup170 are available and show distinct stacked helical arrangements ( Jeudy and Schwartz , 2007; Schrader et al . , 2008; Whittle and Schwartz , 2009 ) .", "Nic96 interacts directly with the Nsp1 complex ( Bailer et al . , 2001; Schrader et al . , 2008 ) , while Nup53/59 binds directly to the pore membrane and interacts with other Ndc1 complex components ( Onischenko et al . , 2009 ) .", "This suggests that the Nic96 complex functions to bridge the pore membrane to the central Nsp1 complex .", "Intriguingly , Nic96 and its vertebrate ortholog Nup93 were shown to bind FG-repeats in vitro ( Schrader et al . , 2008; Xu and Powers , 2013 ) .", "Nup188 and Nup192 are the last two large nucleoporins whose atomic structures are unknown .", "To obtain structural information for these two related proteins , we set out to crystallize Nup188 .", "We succeeded in solving the structures of two fragments , together covering 85% of the 202 kDa full-length protein .", "Nup188 is a stacked helical protein with intrinsic flexibility .", "Interestingly , a structural comparison reveals similarity between Nup188 and NTRs .", "Moreover , we can show that both Nup188 and Nup192 specifically bind to FG-repeats and translocate across NPCs by facilitated diffusion .", "These findings blur the line between the strict separation of soluble NTRs and stationary nups , question some of the current models of FG-repeat organization , and suggest that the NPC and the soluble transport machinery are evolutionary related ." ], [ "To determine the structure of Nup188 we first expressed full-length protein from Saccharomyces cerevisiae ( Sc ) and the thermophilic fungus Myceliophthora thermophila ( Mt ) ( Berka et al . , 2011 ) .", "Although the Mt homolog behaved better in vitro , it did not yield crystals of the full-length protein .", "Using a series of truncation constructs , generated through a combination of limited proteolysis , phylogenetic analysis and structure prediction , we were able to obtain crystals of two major parts of MtNup188 .", "The N-terminal fragment ( Nup188N ) includes residues 1–1160 and its structure was solved to 2 . 65 Å resolution ( Figure 1 and Table 1 ) .", "The C-terminal fragment ( Nup188C ) includes residues 1445–1827 , and we solved its structure to 3 . 0 Å resolution ( Figure 2 and Table 1 ) .", "Each structure was solved using single-anomalous dispersion ( SAD ) experimental phases derived from crystals grown from selenomethionine-labeled protein .", "Nup188N crystallized in space group C21 with one copy per asymmetric unit .", "With a Wilson B-factor of 45 Å2 the structure is generally well defined .", "Nup188C crystallized in space group P212121 with two copies per asymmetric unit .", "Compared to Nup188N , it is not quite as well defined ( Wilson B-factor is 75 Å2 ) , likely because those crystals have a higher solvent content and are more loosely packed . 10 . 7554/eLife . 00745 . 003Figure 1 . Crystal structures of Nup188N .", "( A ) Schematic diagram of full-length Myceliophthora thermophila Nup188N with details about the subdomain arrangement .", "The crystallized fragment is boxed and gradient-colored in red with helical HEAT- and ARM-repeats indicated .", "( B ) The crystal structure of Nup188N is shown in cartoon representation .", "The helices are gradient-colored as in ( A ) .", "The inner helical ring is in pale-yellow to highlight the superhelical organization of the protein .", "The SH3-like domain insert is shown in green .", "( C ) Close-up of the ring-closing latch .", "The clamping helices α11 and α12 ( red ) contact a substantial surface area formed by helices 44 , 46 , and 47 .", "( D ) The SH3-like domain of Nup188 compared to the canonical , peptide-bound SH3 domain of Sem-5 from C . elegans ( PDB 1SEM ) .", "Conserved residues important for peptide interaction are labeled .", "Note that these residues are not conserved in the SH3-like domain of Nup188 .", "In addition , the SH3-like domain is a circular permutation of the canonical SH3-domain . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 00310 . 7554/eLife . 00745 . 004Table 1 . X-Ray data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 004MtNup188N ( 1–1160 ) MtNup188N ( 1–1160 ) MtNup188C ( 1445–1827 ) PDB code4KF74KF8Data collectionData setNativeSelenomethionineSelenomethionineSpace groupC2C2P212121a , b , c ( Å ) 169 . 0 , 94 . 8 , 91 . 6168 . 3 , 94 . 6 , 91 . 464 . 4 , 66 . 7 , 162 . 5α , β , γ ( ° ) 90 , 98 . 9 , 9090 , 98 . 4 , 9090 , 90 , 90Wavelength ( Å ) 0 . 97920 . 97920 . 9792Resolution range ( Å ) 66 . 8–2 . 65 ( 2 . 72–2 . 65 ) 90 . 4–2 . 90 ( 3 . 02–2 . 90 ) 81 . 1–3 . 00 ( 3 . 11–3 . 00 ) Total reflections170 , 859209 , 69550 , 886Unique reflections41 , 67358 , 24914 , 539Completeness ( % ) 100 ( 99 . 9 ) 99 . 9 ( 98 . 8 ) 99 . 9 ( 99 . 7 ) Redundancy4 . 1 ( 3 . 8 ) 3 . 6 ( 3 . 1 ) 3 . 5 ( 3 . 1 ) Rmerge ( % ) 3 . 1 ( 77 . 2 ) 3 . 0 ( 32 . 8 ) 2 . 7 ( 56 . 1 ) Rp . i . m . ( % ) 2 . 0 ( 51 . 0 ) 4 . 1 ( 26 . 3 ) 1 . 6 ( 27 . 0 ) I /σ ( I ) 31 . 3 ( 1 . 8 ) 9 . 6 ( 1 . 2 ) 21 . 4 ( 4 . 8 ) Wilson B factor ( Å2 ) 51 . 650 . 075 . 0RefinementResolution range ( Å ) 66 . 8–2 . 6581 . 1–3 . 00Rwork ( % ) 18 . 127 . 5Rfree ( % ) 22 . 529 . 4Number of reflectionsTotal41 , 66114 , 536Rfree reflections19191139Number of atomsProtein85164366Water1280B factors ( Å2 ) Protein64 . 492 . 3Water44 . 9RMSDBond length ( Å ) 0 . 0040 . 003Bond angles ( ° ) 0 . 9040 . 769Ramachandran plotFavored ( % ) 95 . 092 . 2Allowed ( % ) 4 . 16 . 8Outliers ( % ) 0 . 91 . 0The highest resolution shell is in parenthesis .", "Rmerge is the merging R factor .", "Rp . i . m . is the precision-indicating merging R factor .", "For definitions , see Weiss ( 2001 ) . 10 . 7554/eLife . 00745 . 005Figure 2 . Crystal structures of Nup188C .", "( A ) Schematic diagram of full-length Myceliophthora thermophila Nup188C with details about the subdomain arrangement .", "The crystallized fragments is boxed and gradient-colored in blue with helical HEAT- and ARM-repeats indicated .", "( B ) Crystal structure of the C-terminal part of Nup188 , gradient-colored as in ( A ) , with the inner helices in pale-yellow .", "( C ) Superposition of chains A ( blue ) and B ( gray ) of Nup188C shows the flexibility seen in the crystal , in which the outer helices of chain B move by 3–4 Å relative to the helices in chain A . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 005 Nup188N is built from 52 stacked helices .", "They fold into a large right-handed superhelical ring that resembles a lock washer ( Figure 1B ) .", "Although the overall structure has a general repeat pattern and is built around 2-helix ( HEAT repeat ) , and 3-helix ( ARM-repeat ) elements , several distinct details specific to Nup188 make it a rather unique protein .", "We can discern an inner ring of helices forming the concave surface and an outer ring of helices forming the convex surface creating three distinct subdomains ( Figure 1A , B ) .", "The first subdomain consists of 15 helices that are arranged in irregular orientations without an obvious repeat pattern .", "Next , helices α16–25 make up subdomain II and are organized around three HEAT repeats ( α16/17 , α18/19 , α22-23 ) .", "Helix α23 protrudes from the molecule and is sandwiched between helices α24 and α25 , such that they are splayed apart at a 90° angle .", "In consequence , the helical stack pattern is interrupted .", "Subdomain III includes helices α26 to α52 and is structured around the second helical stack element , pivoted against subdomain II .", "It has 10 helical repeats of both the HEAT and the ARM type and forms two curved layers of helices .", "Between helix α32 and α33 , we find a β-stranded insertion with structural homology to Src-homology ( SH3 ) domains ( see Figure 1D ) .", "The Nup188N ring structure is closed by four helices ( α10–α13 ) from subdomain I that latch onto a complementary surface created by helices α44 , α46 and α47 .", "The interface is mixed in character , with van der Waals contacts as well as polar and ionic interactions .", "In total , the latch covers an 1511 Å2 interface ( Figure 1C ) .", "As noted , Nup188 contains an inserted β-stranded-domain , which has structural similarity to the SH3 domain superfamily ( Li , 2005 ) .", "The domain is arranged in five anti-parallel β-strands that fold into a β-barrel , characteristic for the SH3 protein family ( Figure 1D ) .", "Sequence alignment and secondary structure prediction of Nup188 from a broadly diverged range of species indicate that all Nup188 orthologs contain this SH3-like domain and that the position within the protein is conserved ( data not shown ) .", "In contrast to canonical SH3 domains , the SH3-like domain in Nup188 has a different β-strand topology , which represents a circular permutation ( Figure 1D ) .", "SH3 domains are protein interaction domains known to predominantly bind proline-rich peptides ( Ren et al . , 1993 ) , although non-consensus ligands have also been found in recent years ( Saksela and Permi , 2012 ) .", "The surface residues typically involved in peptide binding in previously characterized SH3 domains are not present in Nup188 .", "Instead Nup188 has a different set of residues that are conserved between Nup188 orthologs .", "This conservation suggests that Nup188 also binds a specific protein or peptide motif , but it most likely does not interact with known SH3 targets .", "The C-terminal fragment of Nup188 , Nup188C , is a right-handed arc-shaped superhelical structure built from 19 helices that form 6 helical repeats , which are stacked in regular order ( Figure 2 ) .", "The first helical pair ( α1 and α2 ) forms a HEAT repeat followed by 5 ARM repeats .", "The inner face of the structure is built from helices α2 , α5 , α8 , α12 , α15 , and α19 .", "The C-terminal helix α19 caps the structure .", "At the N terminus , helices α1 and α2 expose a hydrophobic surface , which strongly suggests that the full-length protein continues in a stacked pattern .", "This hydrophobic surface engages in a twofold symmetric crystal packing contact with a neighboring Nup188C molecule resulting in a horseshoe-shaped dimer .", "Because this contact is generated by a protein truncation , we suggest that it is a crystal artifact and not physiologically relevant .", "Similar packing contacts are frequently observed with truncated , stacked helical proteins .", "Interestingly , the two Nup188C copies in the asymmetrical unit have a different curvature , likely reflecting the intrinsic flexibility of the molecule ( Figure 2C ) .", "Since we were unable to obtain crystals for full-length Nup188 , we attempted to model the missing 283-residue segment ( Figure 3A ) , taking various information into consideration .", "To start , secondary structure prediction suggests that the segment folds into 10 helices .", "Furthermore , the adjacent ends of both crystallized fragments , Nup188N and Nup188C , are regular repeats , therefore we reasoned that the missing segment very likely forms five helical pairs connecting the two parts into one continuous superhelical element ( Figure 3B ) .", "This assumption is supported by 3D comparisons of the crystallized fragments with published structures of other stacked helical proteins .", "For both fragments we find superposable helical proteins in the PDB that extend in a repeat pattern further into the space where we expect the missing residues of Nup188 to be positioned .", "The assumption of a continuous helical structure is also supported by limited proteolysis experiments .", "Although MtNup188 is cut in numerous positions , the resulting fragments still behave like the full-length protein in a gel filtration experiment ( data not shown ) . 10 . 7554/eLife . 00745 . 006Figure 3 . Structural details of Nup188 and comparison to other proteins .", "( A ) Schematic diagram of full-length Myceliophthora thermophila Nup188 with details about the subdomain arrangement for the entire protein .", "( B ) Full-length Nup188 gradient-colored in red-gray-blue .", "The modeled region and the overall dimensions are indicated .", "( C ) .", "Single-particle negative stain EM class averages of full-length MtNup188 , ScNup188 and MtNup192 .", "Classes are roughly grouped and are ordered from more ‘closed’ ( left ) to ‘open’ conformations ( right ) .", "The three proteins are evidently structurally related .", "( D ) Structural similarity between Nup188 and importin-α and -β .", "The aligned areas of Nup188N , Nup188C , importin-α ( 3L3Q ) and importin-β ( 1QGR ) are gradient-colored in orange from N to C terminus .", "Non-aligned helices are in gray and the repeat numbering is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 006 To experimentally support our model of full-length MtNup188 we performed single particle EM analysis .", "We obtained 2D class averages from negatively stained particles ( Figure 3C ) demonstrating that MtNup188 can adopt multiple conformations .", "Several 2D classes are consistent with the crystal structures and show a ring-like structure with a bent , elongated extension .", "The overall dimensions are consistent with our full-length model measuring ∼170 Å in length and ∼85 Å across the N-terminal ring .", "Other 2D classes show a twisted S-shaped molecule , where the ring is apparently opened .", "Thus , the protein can adopt various conformations in solution , consistent with the flexibility observed for many stacked helical domains .", "Like many other architectural nucleoporins , Nup188 is rather poorly conserved in sequence across diverse eukaryotes ( Neumann et al . , 2010 ) .", "To test whether Nup188 is conserved in structure , we analyzed Nup188 from S . cerevisiae by single-particle negative stain EM and compared it to the thermophilic ortholog .", "The two fungi are separated by ∼800 million years in evolution ( Hedges et al . , 2006 ) and the Nup188 proteins share 15% sequence identity .", "As expected , MtNup188 and ScNup188 are very similar in structure , at least at the resolution obtained by EM analysis ( Figure 3C ) .", "Both proteins show similar overall dimensions and share flexibility , evident in distinct 2D classes .", "Nup188 and Nup192 are most likely paralogs that originate from an early gene duplication event ( Mans et al . , 2004 ) .", "The two proteins are weakly conserved with an approximate sequence identity of 15% .", "The size range of 1600–2000 residues is also conserved among Nup188 and Nup192 ( Nup205 in vertebrates ) .", "Binding data using the Xenopus laevis homologs Nup188 and Nup205 suggest that both proteins share some similar functions in the NPC as both compete for the same binding site on the nucleoporin Nup93 ( Nic96 in yeast ) ( Theerthagiri et al . , 2010 ) .", "To analyze the structural similarity between Nup188 and Nup192 , we next performed single particle negative stain EM of MtNup192 ( Figure 3C ) .", "We found that the overall morphology of MtNup192 is very similar to Nup188 , again with a ring structure of ∼90 Å in diameter and a length of ∼185 Å .", "Our data is consistent with the recent EM analysis of Nup192 from Chaetomium thermophilum ( Amlacher et al . , 2011 ) and like Nup188 , Nup192 is also a flexible molecule in solution , with coexisting ‘open’ and ‘closed’ conformations .", "Sequence analysis and comparison between Nup188 and Nup192 do not reveal a SH3-like domain insert in Nup192 .", "No sequence conservation in this area can be observed between Nup188 and Nup192 and secondary structure predictions do not predict the presence of β-sheets in Nup192 .", "We therefore conclude that the SH3-like domain is a specific feature of Nup188 that cannot be detected in Nup192 .", "Stacked helical proteins are abundantly found in the eukaryotic proteome , serving multiple biological functions ( Aravind et al . , 2006 ) .", "To identify structures more closely related to Nup188 we used 3D similarity searches using our experimental structures as templates .", "The most regular part of Nup188N is subdomain III , which was found to be structurally similar to nuclear transport receptors ( NTRs ) ( Figure 3D ) .", "A shared core of helical repeats superimposes with an RMSD of 3 . 6 Å to importin-α ( 276 C-α positions ) and 4 . 5 Å to importin-β ( 263 C-α positions ) , respectively .", "Similarly we found that Nup188C is structurally similar to NTRs and superimposes with an RMSD of 3 . 2 Å to importin-α ( 208 C-α positions ) and 3 . 7 Å to importin-β ( 212 C-α positions ) , respectively .", "Both Nup188 and NTRs form right-handed superhelices with different degrees of curvature .", "The RMSD values show that NTRs and Nup188 are generally related , as has been speculated previously based on EM- and 3D prediction methods ( Flemming et al . , 2012 ) .", "The intriguing structural similarity between Nup188 , Nup192 and NTRs prompted us to test whether they also share other properties .", "We first set out to examine whether Nup188 and Nup192 share the unifying feature of all NTRs and interact with nucleoporin FG-repeats .", "MtNup188 , MtNup192 , importin β from S . cerevisiae ( ScKap95 ) or 3xGFP were incubated with a selection of immobilized budding yeast FG-repeat domains and the interactions were monitored in pulldown experiments .", "To control for binding specificity all proteins were pre-mixed with excess bacterial extract proteins ( Figure 4—figure supplement 1A ) .", "The panel of FG-repeat regions included Nup116 ( 348–458 ) , Nup100 ( 1–310 ) and Nup100 ( 1–610 ) representing GLFG-type repeats , and Nsp1 ( 1–603 ) representing FXFG-type repeats ( Yamada et al . , 2010 ) .", "In addition , we included the Nup116 ( 348–458 ) 10xFA mutant , which lacks the key phenylalanine residues necessary for binding to NTRs ( Bayliss et al . , 2000 , 2002; Patel et al . , 2007 ) .", "As expected , ScKap95 was firmly enriched ( 10-fold to 40-fold over bulk bacterial extract proteins ) on both types of wild-type FG-repeat regions .", "This interaction was highly specific since ScKap95 did not bind to the Nup116 ( 348–458 ) 10xFA mutant and no interaction between FG-coated beads and 3xGFP could be detected ( Figure 4A , B ) .", "Of note , ScKap95 was efficiently eluted with 1M NaCl from all the FG-coated beads consistent with the relatively weak interaction between ScKap95 and FG-repeat regions ( Pyhtila and Rexach , 2003 ) ( Figure 4A , Figure 4—figure supplement 1B ) .", "Intriguingly , both MtNup188 and MtNup192 were strongly enriched on all wild-type FG-repeat regions and did not show significant binding to the Nup116 ( 348–458 ) 10xFA variant .", "We therefore conclude that both these nucleoporins can specifically bind to the FG-repeats in vitro and behave like the NTR ScKap95 in this assay ( Figure 4A , B ) .", "The only notable deviation from the FG-binding pattern was observed with MtNup188 , which was relatively inefficiently eluted with 1M NaCl from Nup100 ( 1–310 ) and Nup100 ( 1–610 ) , suggesting that MtNup188 either interacts with these FG-repeat regions more tightly than ScKap95 or that these interactions are more hydrophobic in nature ( Figure 4—figure supplement 1B ) . 10 . 7554/eLife . 00745 . 007Figure 4 . Biochemical properties of Nup188 and Nup192 . ( A and B ) Binding of MtNup188 and MtNup192 to various FG-coated beads monitored by in vitro pulldown assays .", "( A ) Standard volumes of the soluble protein mixtures ( pulldown input ) , separate components of the mixtures ( protein alone , bacterial extract alone ) and the 1M NaCl pulldown eluates form various FG-coated beads ( 1M NaCl pulldown elates ) were precipitated by methanol-chloroform and re-solubilized in a standard volume of SDS-sample buffer .", "The samples were separated by SDS-PAGE and the gels were stained with SYPRO-Ruby .", "( B ) The diagram representing relative enrichments of ScKap95 , MtNup188 , MtNup192 and 3xGFP in various FG-pulldown eluates , as compared to bulk bacterial extract proteins .", "The enrichment values ( ratios between the eluted and input protein amounts ) were computed using protein band intensities from the SYPRO Ruby stained gels shown in ( A ) .", "( C ) HsRanQ69L alone or an equimolar mixture of HsRanQ69L with ScKap95 were subjected to gel filtration using a Superdex 200 column , and the eluted fractions were separated by SDS-PAGE .", "Ran was visualized by Western blotting against the ZZ-tag , and ScKap95 was visualized by SYPRO Ruby staining .", "( D ) Superdex 200 elution profiles of ZZ-HsRanQ69L ( ZZ-tag Western blot ) with or without the addition of MtNup188 or MtNup192 ( SYPRO Ruby ) analyzed as described in ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 00710 . 7554/eLife . 00745 . 008Figure 4—figure supplement 1 . Characterization of the FG-repeat interactions in pulldown assays .", "( A ) The specificity of the FG-pulldown assay conditions were examined by subjecting bacterial extract proteins alone to pulldown assays with various FG- coated beads .", "Bound proteins were eluted with 1M NaCl sufficient to disrupt the expected transient interactions ( left panel ) , followed by SDS sample buffer ( right panel ) .", "The input bacterial extract sample and 1M NaCl eluates were precipitated by methanol-chloroform and re-solubilized in SDS-sample buffer prior to separation by SDS-PAGE .", "The gels were stained with SYPRO Ruby .", "( B ) Proteins resistant to 1M NaCl elution in the experiment described in Figure 4A were stripped-off by SDS-sample buffer and separated by SDS-PAGE followed by SYPRO Ruby gel staining .", "The ‘protein sample’ lanes are the same as ‘protein alone’ in Figure 4A , and are included as references for the protein amounts remaining after 1 M NaCl elution . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 008 NTRs of the importin-β/karyopherin family can also form direct complexes with the small GTPase Ran in its GTP-bound form ( Floer and Blobel , 1996; Chook and Blobel , 1999; Cook et al . , 2007 ) .", "Sequence analysis did not detect any obvious RanGTP binding surface in either Nup188 or Nup192 .", "Consistent with this , we were unable to detect binding between RanGTP and either MtNup188 or MtNup192 by gel filtration analysis ( Figure 4D ) whereas the NTR ScKap95 bound robustly to RanGTP ( Figure 4C ) .", "We conclude that in contrast to NTRs neither MtNup188 nor MtNup192 can form a stable complex with RanGTP in our binding assay conditions .", "A defining functional property of NTRs is their ability to mediate transport across the NPC .", "The structural similarity between MtNup188 , MtNup192 and NTRs and their common ability to bind FG-repeats led us to ask whether MtNup188 and MtNup192 could also efficiently translocate through intact NPCs .", "To this end MtNup188 and MtNup192 were tagged with YFP , and their nuclear translocation was monitored by quantitative fluorescence microscopy using the standard in vitro nuclear transport assay in digitonin-permeabilized HeLa cells ( Adam et al . , 1990 ) .", "YFP-HsImportin-β and 3xGFP were used as positive and negative controls , respectively .", "To select only intact nuclei for our analyses , each reaction was supplemented with 155 kDa TRITC-labeled dextran , and only nuclei that excluded TRITC-dextran were analyzed ( see Figure 5—figure supplement 1 and materials and methods section for details ) .", "As expected , YFP-importin-β efficiently translocated into nuclei independent of the presence of a nuclear transport mixture ( Xenopus cytosol plus energy regeneration system ) .", "At the same time no significant nuclear translocation of 85 kDa 3xGFP was observed in any of the tested conditions , consistent with the approximately 40–60 kDa diffusion permeability cutoff of intact NPCs ( Figure 5A , B ) ( Mohr et al . , 2009 ) .", "Strikingly , both YFP-MtNup188 and YFP-MtNup192 mimicked the behavior of YFP-HsImportin-β and accumulated in intact nuclei even in the absence of a nuclear transport mixture .", "Nuclear uptake was completely blocked in the presence of WGA , a broad inhibitor of NPC-mediated transport ( Finlay et al . , 1987; Mohr et al . , 2009 ) ( Figure 5A , B ) .", "Furthermore , translocation of both nucleoporins , and of YFP-HsImportin-β , was strongly inhibited upon addition of an unlabeled importin-β ( Figure 5C , D ) .", "Together these results demonstrate that the translocation of YFP-MtNup188 and YFP-MtNup192 occurs through NPCs .", "Since the sizes of YFP-MtNup188 and YFP-MtNup192 ( ∼220 kDa ) are far above the diffusion permeability cutoff of the NPCs this efficient nuclear translocation cannot be explained by simple passive diffusion . 10 . 7554/eLife . 00745 . 009Figure 5 . Nuclear translocation of Nup188 and Nup192 in digitonin-permeabilized HeLa cells .", "( A and B )", "The nuclear translocation of YFP-MtNup188 and YFP-MtNup192 ( green ) was monitored in digitonin-permeabilized HeLa cells in transport buffer alone ( −cytosol ) , in transport buffer containing Xenopus cytosol and an energy regenerating mix ( +cytosol ) , or in transport buffer after pre-treatment of nuclei with WGA ( +WGA ) ( A ) .", "Similar assays were performed in transport buffer with or without the addition of unlabeled HsImportin-β ( B ) .", "155kD TRITC-dextran ( red ) was used to check nuclear integrity .", "YFP-HsImportin-β and 3xGFP were used as positive and negative controls for facilitated NPC translocation , respectively .", "( C and D )", "Quantitative analysis of the protein translocation experiments described in ( A and B ) , respectively .", "The intranuclear fluorescence intensities of the proteins were quantified and normalized against the background ( extranuclear ) fluorescence .", "Bar graphs represent mean values ± standard deviations .", "Asterisks ( * ) indicate a significant difference in median value using the Mann-Whitney U test ( p<0 . 01; >50 nuclei for each condition ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 00910 . 7554/eLife . 00745 . 010Figure 5—figure supplement 1 . Outline of the nuclear translocation assay .", "( A ) Illustration of the protocol used for the nuclear translocation assays .", "( B ) An example of two-channel confocal images used to simultaneously monitor the nuclear translocation of fluorescent protein fusions ( YFP channel ) and 155 kDa TRITC-dextran ( TRITC channel ) .", "( C ) Schematic representation of the procedure used to quantify the nuclear translocation of fluorescent protein fusions into intact nuclei .", "Fluorescence intensities inside and outside the nuclei were quantified using custom written code in MATLAB .", "The normalized intranuclear fluorescence intensity for each nucleus was calculated by taking the ratio of the fluorescence inside to outside the nucleus .", "All nuclei whose normalized TRITC fluorescence exceeded 0 . 3 were considered ‘non-intact’ and omitted from the analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 010 To examine more directly if YFP-MtNup188 and YFP-MtNup192 cross the NPC by facilitated diffusion we measured the rates of their NPC translocation and compared them to the inert 3xGFP , which is almost threefold smaller compared to the model transport receptor YFP-HsImportin-β .", "We followed the nuclear accumulation kinetics of each protein and calculated initial nuclear translocation rates by fitting each set of kinetic data to a single exponential decay function ( Figure 6 , see also ‘Materials and methods’ for details ) .", "While both nucleoporins translocated slower than YFP-HsImportin-β , their translocation rates exceeded the values of 3xGFP by at least 30-fold indicating that both proteins indeed cross the NPCs in a facilitated manner ( Figure 6 ) .", "The digitonin permeabilization protocol does not remove all endogenous NTRs ( Nachury and Weis , 1999 ) .", "To test whether the nuclear translocation rates of MtNup188 or MtNup192 can be enhanced by the removal of endogenous NTRs that remain tightly associated with NPCs we performed similar analyses with nuclei that were pre-treated with RanGTP ( Nachury and Weis , 1999 ) .", "While we saw an increase in the YFP-MtNup188 translocation rate , no effect on YFP-MtNup192 was observed .", "Under these conditions the translocation kinetics of MtNup188 and MtNup192 were approximately equal , but still about fourfold below the values for YFP-HsImportin-β ( Figure 6 and Figure 6—figure supplement 1 ) .", "This suggests that the slower nuclear translocation rates of MtNup188 and MtNup192 are intrinsic properties of these two proteins .", "Interestingly , both MtNup188 and MtNup192 , unlike HsImportin-β accumulated within nuclei in the absence of exogenous energy without reaching notable saturation during the time frame of our experiments ( Figure 6 ) .", "Furthermore , both proteins continued to accumulate in nuclei even after subsequent 10-fold dilution , suggesting that they have strong intranuclear binding sites ( Figure 6—figure supplement 2 ) .", "In summary , these results demonstrate that MtNup188 and MtNup192 share functional characteristics with NTRs and translocate across NPCs by facilitated diffusion without a requirement for other cytosolic factors . 10 . 7554/eLife . 00745 . 011Figure 6 . Kinetics of Nup188 and Nup192 nuclear translocation .", "( A ) The nuclear accumulation kinetics of the scaffold nucleoporins in transport buffer as compared to YFP-HsImportin-β and 3xGFP .", "Nuclear translocation reactions were imaged immediately after the addition of the fluorescent proteins and TRITC-dextran .", "Non-intact nuclei permeable to TRITC-dextran were excluded from the analysis .", "The graph represents mean values ± standard deviations .", "( B ) Quantitative analysis of the initial nuclear translocation rates ( equal to the total protein translocation rate through NPCs ) for the experiments described in ( A ) .", "The nuclear accumulation dataset for each nucleus ( >30 for each condition ) was separately fitted to a single-exponential curve , and the derivative at time t = 0 representing the initial nuclear translocation rate were computed .", "Asterisks ( * ) denote a significant difference in median value from the 3xGFP rate using the Mann-Whitney U test ( p<0 . 01 ) .", "Box plots show the median , first and third quartiles , and non-outlier extrema .", "Values greater than six standard deviations from the mean are marked as outliers . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 01110 . 7554/eLife . 00745 . 012Figure 6—figure supplement 1 . Effect of RanGTP on the nuclear translocation kinetics of Nup188 and Nup192 . ( A ) Nuclear translocation kinetics of the scaffold nucleoporins in permeabilized HeLa cells pre-incubated with Ran and an energy-regenerating system were obtained as described in Figure 6A .", "The kinetic plots for MtNup188 and MtNup192 from Figure 6A obtained without a Ran pre-wash are shown as a reference .", "( B ) Quantitative analysis of the initial nuclear translocation rates for the experiments described in ( A ) .", "The analysis was performed as described in Figure 6B .", "The asterisk ( * ) denotes a significant difference in median values in YFP-MtNup188 translocation rates between the no Ran pre-wash and Ran pre-wash conditions using the Mann-Whitney U test ( p<0 . 01 ) .", "Box plots show the median , first and third quartiles , and non-outlier extrema . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 01210 . 7554/eLife . 00745 . 013Figure 6—figure supplement 2 . Nuclear accumulation of Nup188 and Nup192 upon 10-fold dilution . YFP-MtNup188 , YFP-MtNup192 , YFP-HsImportin-β and 3xGFP were allowed to translocate into the nuclei of permeabilized HeLa cells for 15 min , and then their nuclear fluorescence was followed after a 10-fold dilution with transport buffer as described in Figure 6A .", "Note that unlike YFP-HsImportin-β and 3xGFP , YFP-MtNup188 and YFP-MtNup192 continue to accumulate even after the 10-fold dilution . DOI: http://dx . doi . org/10 . 7554/eLife . 00745 . 013" ], [ "The analysis of the largest universally conserved nucleoporin Nup188 , which we present here , fills the last gap in the inventory of architectural nucleoporin structures .", "We find that the topology of Nup188 is distinctly different from all other scaffold nups , except for the related paralog Nup192 , in that it appears to be inherently flexible .", "Negative stain EM analysis ( Figure 3C ) reveals that the stacked helical domain can adopt a ‘closed’ conformation seen in the crystal , but that the protein can also ‘open’ up .", "A similar degree of flexibility was recently also observed for Nup192 by negative stain EM analysis ( Flemming et al . , 2012 ) .", "Other , mainly helical scaffold nucleoporins contain elements that prevent such flexibility .", "For instance , the ACE1 class of nucleoporins ( Nic96 , Nup84 , Nup85 , Nup145C ) share a distinct foldback architecture where a string of helices latches onto the core helical stack , thereby prohibiting the bending of neighboring helical elements ( Brohawn et al . , 2008 , 2009 ) .", "Three conserved scaffold nups ( Nup120 , Nup133 , Nup170 ) share a topology where an N-terminal β-propeller is linked to a C-terminal helical stack domain .", "For each of these three nucleoporins there is structural evidence that stretches within the helical elements are somewhat flexible , but likely not to the extent that is observed for Nup188 and Nup192 ( Whittle and Schwartz , 2009; Bilokapic and Schwartz , 2012 ) .", "How does this exceptional flexibility shape the functional role of Nup188 and Nup192 ?", "Based on current data , we can only speculate .", "It is conceivable that structural changes within these large nups are critical during the assembly and biosynthesis of the NPC .", "Alternatively , plasticity of Nup188 , Nup192 , and the Nic96 complex as a whole , may be important for the nuclear transport function of the NPC , for example , by accommodating large soluble cargos or regulating the translocation of inner nuclear membrane proteins .", "Although Nup188 and Nup192 are paralogous proteins with very similar structural arrangements , they have acquired distinct functions during evolution .", "For instance , the depletion of Nup188 was shown to increase the size limit for the transport of inner nuclear membrane proteins , but this was not observed upon removal of Nup205 ( the Nup192 homolog in vertebrates ) ( Theerthagiri et al . , 2010 ) .", "Our structure unexpectedly shows that Nup188 contains an SH3-like domain , but a similar domain cannot be detected in Nup192 .", "Whether this SH3-like domain contributes to the functional diversification of these two large nups in transmembrane transport is currently unknown and is an interesting question for future studies .", "SH3 domains are generally involved in protein-protein interactions but the typical residues needed for the interaction with known SH3 substrates , are not conserved in Nup188 .", "Therefore , it will be important to identify the binding partners of the SH3-like domain in Nup188 and to directly test whether this domain functions , for example , in the transport of transmembrane cargos .", "So far , soluble NTRs and scaffold nucleoporins were considered to be functionally and evolutionarily separated groups of proteins that were already present in the last eukaryotic common ancestor each very likely originating from relatively few ancestor proteins through multiple gene duplication events ( Mans et al . , 2004 ) .", "Intriguingly , our crystallographic analysis revealed that Nup188 is structurally related to NTRs .", "Moreover , single particle EM analysis showed that both Nup188 and its paralog , Nup192 , resemble the overall shapes of NTRs and display exceptional flexibility characteristic for NTRs ( Figure 3 ) ( Conti et al . , 2006 ) .", "However , the structural similarity is rather general and not specific to a particular NTR .", "Following the entire 202 kDa structure , one finds a patchwork of 10–40 kDa substructures that are closer related to one NTR vs another .", "Based on the structure alone it is not possible to conclude a common phylogenetic origin; too many proteins involved in diverse protein–protein interactions adopt stacked helical domains and these domains likely have evolved multiple times throughout evolution ( Aravind et al . , 2006 ) .", "We therefore tested whether Nup188 and Nup192 have functional similarities with NTRs .", "All known NTRs bind FG-repeats , typically exhibiting various binding sites dispersed over their surface ( Bednenko et al . , 2003 ) .", "Intriguingly , both Nup188 and the related Nup192 directly and specifically bind to FG-repeats ( Figure 4A , B ) .", "Furthermore , both Nup188 as well as Nup192 can translocate across intact NPCs in vitro , again recapitulating the behavior of NTRs ( Figure 5 ) .", "By contrast , we do not observe RanGTP binding ( Figure 4C , D ) , another characteristic of the importin–β family of NTRs ( Floer and Blobel , 1996; Chook and Blobel , 1999; Cook et al . , 2007 ) .", "Unlike the FG-binding pockets , the RanGTP interaction site is much larger and sequence-conserved , thus easier to recognize .", "Neither Nup188 nor Nup192 show surface conservation consistent with Ran binding ( Cook et al . , 2007 ) .", "Yet , the combination of shared FG-binding activity , common NPC translocation properties combined and general structural similarity make it tempting to speculate that Nup188 , Nup192 and NTRs , indeed , might have a common evolutionary origin .", "This blurs the line of a strict functional separation between nucleoporins and NTRs , and allows for a number of interesting hypotheses .", "Could it be that the ancestral NPC had only a few core nucleoporins , from which NTRs evolved to become the separate , ‘soluble phase’ ?", "Or alternatively , could some soluble NTRs or shuttling cargos have evolved to bind tightly to the original NPC membrane coat and become part of the architectural scaffold ?", "The finding that architectural nucleoporins bind FG-repeats and display NTR-like properties , raises the question of the biological function of these properties .", "Once incorporated into NPCs Nup188 and Nup192 are unlikely to shuttle across the nuclear envelope .", "The very low mobility of their common binding partner , Nup93 in vertebrate NPCs and an exceptionally slow protein turnover suggest that both proteins are very stable NPC components ( Rabut et al . , 2004; Savas et al . , 2012 ) .", "However , the NTR-like activity of Nup188 and Nup192 could be critical for de novo NPC biosynthesis during interphase nuclear growth in higher eukaryotes or in organisms , which undergo a closed mitosis when new NPCs assemble de novo into the intact nuclear envelope ( D’Angelo et al . , 2006; Dultz and Ellenberg , 2010; Menendez-Benito et al . , 2013 ) .", "Interphase NPC assembly seems to rely on steps that occur on both the cytoplasmic and the nucleoplasmic side of the nuclear envelope and thus nuclear translocation of NPC components through already existing pores might be critical for new NPC assembly in intact nuclei .", "Another possibility is that FG-scaffold interactions are important for the structural integrity of the NPC by enabling connections between subcomplexes .", "Interactions within specific subcomplexes have been studied extensively and are rather well characterized .", "In contrast , the interaction between subcomplexes is much more poorly understood , and it remains unclear how subcomplexes come together to assemble into the oligomeric NPC ring structure .", "Interestingly , the ability to interact with FG-repeats may extend to other scaffold nucleoporins as well .", "Nic96 and its vertebrate ortholog , Nup93 bind GLFG-type domains in vitro ( Schrader et al . , 2008; Xu and Powers , 2013 ) .", "Furthermore , budding yeast scaffold nucleoporins can be biochemically purified together with NTRs from yeast extracts using FG-repeat regions as a bait ( Allen et al . , 2001 ) .", "Therefore , the interactions between FG-repeats and scaffold nups could possibly be part of the glue that holds the NPC scaffold together .", "Finally , the FG-binding properties that we have discovered here might also play a role in the correct establishment of the NPC selectivity barrier for soluble or transmembrane cargos by organizing the FG meshwork within the NPC channel .", "FG domain interactions with scaffold Nups could connect FG-repeat regions to the wall of the NPC questioning models in which FG-repeats function exclusively within the central channel of the NPC to mediate nuclear transport .", "Future experiments will be needed to test these models and to characterize the role of the FG-scaffold interactions in NPC biogenesis , structure or function ." ], [ "Expression constructs were PCR amplified from genomic DNA of the thermophillic fungus Myceliophthora thermophila ( Mt ) .", "Two introns were removed using inverse PCR .", "Nup188 ( residue 1–1827 ) , Nup188N ( residue 1–1160 ) and Nup188C ( residue 1445–1827 ) were cloned into a pETDuet-1 vector from EMD Millipore ( Billerica , MA ) and N-terminally fused with a 3C protease cleavable 10xHis-7xArg-SUMO tag .", "The full ORF of S . cerevisiae ( Sc ) Nup188 was PCR amplified and inserted into pETDuet-1 .", "It was fused to an N-terminal 3C cleavable 10xHis-7xArg-SUMO tag and a C-terminal 6xHis tag .", "Full-length MtNup192 was amplified from genomic DNA and its three introns removed by inverse PCR .", "Nup192 ( residue 1–1734 ) , Nup192 ( residue 1–570 ) and Nup192 ( residue 1–1034 ) were cloned into pETDuet-1 , with an N-terminal 3C cleavable 10xHis-7xArg-SUMO tag and a C-terminal 3C cleavable 9xArg-7xHis tag .", "YFP-MtNup188 ( residue 1–1827 ) and YFP-MtNup192 ( residue 1–1734 ) expression constructs were derived from the respective non-labeled expression constructs ( see above ) , by in-frame insertion of the YFP coding sequence after the respective N-terminal affinity tags .", "The ScKap95 expression plasmid was constructed by insertion of PCR-amplified ScKap95 ORF into pET30-a ( + ) His-tag expression vector .", "The bacterial expression plasmids for GST-ScNSP1 ( 1–603 ) , GST-ScNup100 ( 1–307 ) and were made by insertion of PCR-amplified coding sequences of the respective FG-repeat segments into the pGEX-2T vector .", "All Mt proteins were expressed at 18°C in Escherichia coli BL21 ( DE3 ) RIL cells and first purified on Ni-affinity resin .", "When used for structure determination the eluted proteins were loaded on a HiTrapS FF column from GE Healthcare ( Piscataway , NJ ) and eluted with a linear NaCl gradient .", "Affinity tags were removed with 3C protease , followed by a second HiTrapS FF column purification step .", "Finally , the proteins were purified on a Superdex S200 10/300 column from GE Healthcare ( Piscataway , NJ ) in gel filtration buffer ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl and 1 mM DTT ) .", "Selenomethionine-derivatized protein was produced as described ( Brohawn et al . , 2008 ) and purified identical to the native protein , except that reducing agent was kept at 5 mM in all buffers .", "For FG-pulldowns , Ran-binding assays and protein translocation assays , the Mt proteins were purified using the same protocol that was used for structure determination , except for omitting the first HiTrapS FF column step .", "HsImportin-β versions and ScKap95 were expressed and purified on Ni-affinity resin , followed by dialysis against the gel filtration buffer .", "ZZ-HsRanQ69L was produced essentially as described in ( Nachury et al . , 2001 ) .", "Purification of human Ran and nucleotide loading was performed as described in ( Lowe et al . , 2010 ) .", "All GST-fusions of budding yeast FG-repeat regions were expressed in BL21 ( DE3 ) cells and purified on GSH-beads from GE Healthcare ( Piscataway , NJ ) in PBS pH 7 . 4 supplemented with 10 mM DTT , 0 . 1 mM PMSF and 0 . 5% Triton X-100 .", "The beads were washed with PBS and proteins were eluted with glutathione and dialyzed against gel filtration buffer .", "Small needles of Nup188N grew in hanging drops containing 1 . 5 μl of protein at 4–6 mg/ml and 1 . 5 μl of precipitant ( 0 . 1 M MES pH 6 . 5 , 4 . 5–7% ( w/v ) PEG 4000 , 150 mM ammonium sulfate and 1 mM DTT ) at 16°C .", "Large ( 300 × 50 × 50 μm ) diffraction quality crystals were obtained after micro-seeding and the addition of 1–2 . 5% ( v/v ) tert-butanol .", "Crystals were cryoprotected by briefly soaking in precipitant supplemented with 20% ( v/v ) PEG 200 and were flash-frozen in liquid nitrogen .", "Crystal of Nup188C grew in hanging drops containing 1 . 5 μl of protein at 3–5 mg/ml and 1 . 5 μl of precipitant ( 18–23% ( w/v ) PEG 3350 , 150 mM tri-ammonium citrate and 1 mM DTT ) at 16°C .", "Crystals were cryoprotected by dialysis into precipitant with 35% ( w/v ) PEG 3350 and flash-frozen in liquid nitrogen .", "Data were collected at the NE-CAT beamlines 24ID-C at Argonne National Laboratory .", "HKL2000 ( Otwinowski and Minor , 1997 ) was used to reduce data .", "The structure of Nup188N was solved using SAD phases ( usable to 3 Å ) from a selenomethionine-substituted crystal .", "Out of 23 possible sites SHELXC/D/E ( Sheldrick , 2010 ) found 22 selenium sites , which were refined in Phaser-EP ( McCoy et al . , 2007 ) .", "An interpretable map was obtained after density modification using Parrot in the CCP4 suite ( Winn et al . , 2011 ) .", "After most of the model was built , native data was used for the final rounds of model building and refinement using Coot ( Emsley et al . , 2010 ) and Phenix ( Adams et al . , 2010 ) .", "Five loops ( residue 85–91 , 447–459 , 673–682 , 886–902 , 963–970 ) and the very C-terminal 11 residues could not be built , due to poorly defined electron density .", "The structure of Nup188C was also solved using SAD phases from a selenomethionine-substituted crystal .", "All three possible sites of both molecules in the asymmetric unit were found using phenix . autosol .", "In the initial solvent-flattened electron density map several helices of both Nup188C molecules were well defined and could be built .", "In general , one copy of Nup188C is better defined then the other , presumably because of tighter crystal packing .", "Phases were improved by combining SAD phases with model phases using Phaser-EP .", "Even though the overall structure is similar , both Nup188C molecules were built independently , without using non-crystallographic symmetry restraints .", "The conformation between both molecules is different enough , that NCS was not beneficial even in the early rounds of refinement .", "The structure is complete except for three loops ( residues 1495–1509 , 1534–1538 , 1705–1720 ) and the very C-terminal four residues , all of which are poorly defined in the electron density map .", "Structure figures were made using PyMol ( Schrödinger LLC ) and structural superposition carried out using the Dali server ( Holm and Rosenström , 2010 ) and Coot .", "Recombinantly expressed and purified full-length MtNup188 , ScNup188 and MtNup192 at ∼1 . 0 mg/ml were diluted with the gel filtration buffer ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl , 1 mM DTT ) to 5 µg/ml and negatively stained with 2% uranyl acetate on continuous carbon-film grids .", "Single-particle CCD images of these three specimens were recorded on an FEI Tecnai Spirit electron microscope at 80 keV , 1–2 μm defocus and 60 , 000x magnification with a pixel size of 3 . 6 Å .", "A total of 1252 particles of MtNup188 , 3000 particles of ScNup188 , and 1408 particles of MtNup192 were boxed into separate stacks and were then each subjected to 2D classification using the single-particle data analysis package PARTICLE ( www . sbgrid . org/software/title/PARTICLE ) .", "The ‘direct particle classification’ method implemented in PARTICLE does not require image pre-alignment , therefore the result is objective and free of any alignment error or reference bias .", "All FG-pulldown assays were performed in pulldown buffer ( 10 mM HEPES pH 7 . 5 , 160 mM KOAc , 1 mM MgOAc2 , 1 mM DTT , 0 . 01% Triton X-100 ) .", "Bound proteins were eluted with 1 M NaCl elution buffer ( 10 mM Tris-HCl pH 8 . 0 , 1 M NaCl , 0 . 1% Tween-20 , 5 mM BME ) , followed by SDS-sample buffer .", "All the procedures except otherwise mentioned were performed at room temperature .", "The GST-FG fusions were pre-bound to GSH beads in the following amounts per pulldown reaction: 25 μl GSH resin; 10 nmol GST-Nup116 ( 358–458 ) ; 10 nmol GST-Nup116 ( 358–458 ) 10xFA; 5 . 0 nmol GST-Nup100 ( 1–307 ) ; 2 . 5 nmol GST-Nup100 ( 1–610 ) ; 3 . 0 nmol GST-NSP1 ( 1–603 ) .", "The beads were washed twice with the elution buffer re-equilibrated with pulldown buffer and aliquoted into separate siliconized tubes .", "The pulldown input proteins were pre-mixed with bacterial extract proteins in pulldown buffer to yield the following amounts per reaction: 0 . 5 ml pulldown buffer containing either MtNup188 ( 100 pmol ) , MtNup192 ( 100 pmol ) , ScKap95 ( 200 pmol ) or 3xGFP ( 500 pmol ) plus 200 μg bacterial extract proteins .", "The pulldown reactions were gently agitated for 20 min , quickly washed twice with 1 ml ice-cold pulldown buffer and bound proteins were eluted by 100 μl of the elution buffer followed by 100 μl of SDS-sample buffer .", "Salt eluates ( 1M NaCl ) were precipitated with methanol-chloroform and re-solubilized in 50 μl SDS-sample buffer .", "The pulldown input samples and their components were prepared for SDS-PAGE essentially as described for the salt eluates .", "The gels were stained with SYPRO Ruby ( Invitrogen ) and imaged with a UV-transilluminator equipped with a CCD-camera .", "The digital images were analyzed and prepared using ImageJ software .", "ZZ-HsRanQ69L was pre-mixed with either Ran binding buffer alone ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl , 2 mM MgOAc2 , 1 mM DTT ) , ScKap95 , MtNup188FL or MtNup192FL to yield 2 μM concentration of each protein .", "The binding reactions were incubated on ice for 20 min , passed through 0 . 45 μm filter unit and subjected to gel filtration using Superdex 200 10/300 ( GE Healthcare ) in the Ran binding buffer .", "0 . 5 ml elution fractions were collected , separated by SDS-PAGE and stained with SYPRO Ruby to visualize ScKap95 , MtNup188FL or MtNup192FL .", "ZZ-HsRanQ69L was visualized by Western Blotting using rabbit IgG followed by fluorophore conjugated secondary antibodies .", "HeLa cells were cultured in DMEM media ( +10% FBS ) and plated on glass-bottomed poly-lysine coated chambers ( MatTek , Ashland , MA ) at a seeding concentration of 2 . 5 × 105 cells/ml the day prior to use .", "The cell permeabilization protocol is based on that of ( Adam et al . , 1990 ) Briefly , cells were washed with PBS ( 137 mM NaCl , 2 . 7 mM KCl , 8 mM Na2HPO4 , 2 mM KH2PO4 , pH 7 . 4 ) followed by a wash with permeabilization buffer ( 50 mM HEPES pH7 . 3 , 50 mM KOAc , 8 mM MgCl2 ) .", "The cells were then treated with 50 μg/ml digitonin in permeabilization buffer for 5 min followed by three washes with transport buffer ( 20 mM HEPES pH 7 . 3 , 110 mM KOAc , 5 mM NaOAc , 2 mM MgOAc ) .", "After the final wash , the buffer was removed and 100 μl of transport buffer containing the experimental protein mixtures was added to the cells ( see Figure 5—figure supplement 1 ) .", "Concentrations used were: 1 μM YFP-MtNup188 /192 , 1 μM YFP- HsImportin -β , 1 μM HsImportin-β , 1 μM 3xGFP , 200 μg/mL 155 kDa TRITC-dextran ( Sigma-Aldrich , St . Louis , MO ) , 2 mM DTT , 100 μg/mL WGA ( Sigma-Aldrich ) , and 5% v/v cytosolic extract .", "Cytosolic extract from Xenopus laevis oocytes was prepared as described in ( Levy and Heald , 2010 ) .", "Experiments using the cytosolic extract were supplemented with an energy regenerating system consisting of 2 mM GTP ( Roche , Indianapolis , IN ) , 100 µM ATP ( Roche ) , 4 mM creatine phosphate ( Roche ) , and 20 U/ml creatine kinase ( units of specific activity given by Roche ) .", "For WGA pre-treatment , 100 μg/mL WGA was first incubated with the nuclei for 10 min , removed , and then the experimental mix was added to the nuclei .", "For experiments involving a Ran wash , 5 µM RanGDP and the energy regenerating system were added to the nuclei for 10 min followed by three washes with transport buffer , and then the experimental mix was added to the nuclei .", "After the transport reaction was allowed to run for 15 min at room temperature , images were acquired on a Zeiss LSM 700 laser scanning confocal microscope ( 63× oil objective; EYFP , EGFP , & TRITC channels; 1 airy unit pinhole; 4 line averaging ) using the Zen 2011 imaging software package ( Carl Zeiss , Inc . ) .", "For the time-lapse experiments the confocal imaging was started at the same time as the transport mix was added to the cells ( 63× oil objective; EYFP , EGFP , & TRITC channels; 1 airy unit pinhole; no line averaging; 15 min run time; 20 s intervals ) .", "Fluorescence quantification and data analysis were performed using custom written code in MATLAB ( The MathWorks , Inc . ) .", "The fluorescence intensity inside each nucleus and the average background fluorescence intensity were determined for both the YFP/GFP and TRITC channels .", "The intranuclear to extranuclear ratios were then calculated giving normalized fluorescence intensities for both the protein of interest and the dextran .", "All nuclei with normalized dextran fluorescence ratio greater than 0 . 3 were considered to be ‘non-intact’ ( permeable to the dextran ) and rejected .", "Only ‘intact’ nuclei were subject to further analysis ( see Figure 5—figure supplement 1 ) .", "For time-lapse experiments , the time-dependent normalized fluorescence intensity of the protein of interest was determined for each nucleus and then fit to a single exponential curve , y ( t ) = A* ( 1−exp[k*t] ) +C , where A , k , and C are fit parameters , and t is time .", "The initial rate of nuclear accumulation is given by the time derivative at t = 0 ( i . e . , −Ak ) ." ] ]
[ "Nucleocytoplasmic transport is mediated by nuclear pore complexes ( NPCs ) embedded in the nuclear envelope .", "About 30 different proteins ( nucleoporins , nups ) arrange around a central eightfold rotational axis to build the modular NPC .", "Nup188 and Nup192 are related and evolutionary conserved , large nucleoporins that are part of the NPC scaffold .", "Here we determine the structure of Nup188 .", "The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring , while the C-terminal region is a more regular , superhelical structure .", "Overall , the structure has distant similarity with flexible S-shaped nuclear transport receptors ( NTRs ) .", "Intriguingly , like NTRs , both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion .", "This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery ." ]
[ "The nucleus of a cell is surrounded by a two-layered membrane that controls the flow of molecules from the cytoplasm into the nucleus and vice versa .", "The molecular traffic between the cytoplasm and nucleus is essentially controlled by nuclear pore complexes—large , multi-protein structures that are embedded in the membrane .", "Each nuclear pore complex contains about 30 different proteins called nucleoporins or nups , which combine to form a structure with a central pore that allows the molecules to enter and leave the nucleus .", "The centre of the nuclear pore complex is thought to be filled with protein filaments that contain a large number of so-called FG repeats ( where F and G are the amino acids phenylalanine and glycine ) .", "Specialized molecules called soluble nuclear transport receptors , which carry various cargoes between the cytoplasm and nucleus , can bind to these FG repeats , and the interaction between the receptors and the FG repeats is crucial for the selective transport of molecules between the cytoplasm and the nucleus .", "The large size of the nuclear pore complex has hindered efforts to work out its structure , but in recent years researchers have been able to obtain structures for many individual nups and their subcomplexes .", "Now , Andersen et al . have determined the structure of one of the largest nups , Nup188 .", "This has led to the discovery that it and a related nup , Nup192 , share unexpected features with soluble nuclear transport receptors .", "In general the first step when attempting to determine the structure of a biomolecule is to form a crystal .", "Since full-length Nup188 did not crystallize , Andersen et al . instead crystallized two large fragments of Nup188 , determined the structures of these fragments , and then combined these to produce the likely structure of the full-length protein .", "They found that Nup188 has a structure that consists of stacked helices and is more flexible than other nups .", "Moreover , its structure was very similar to those of soluble nuclear transport receptors , and this led Andersen et al . to investigate whether Nup188 also had similar functional features .", "Surprisingly , they discovered that both Nup188 and Nup192 could bind FG repeats , just like nuclear transport receptors .", "What is more , this binding allowed both nups to travel through nuclear pore complexes in in vitro transport reactions .", "These findings have implications for the understanding of the organization and function of FG-repeats and suggest that the stationary elements of the nuclear pore complex and soluble nuclear transport receptors are evolutionarily related ." ]
2013
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "physics of living systems", "microbiology and infectious disease" ]
Mechanism of bidirectional thermotaxis in Escherichia coli
elife-26607-v2
[ [ "For many organisms temperature is one of the crucial environmental factors that determine growth and fitness .", "Thus , it is not surprising that organisms developed sophisticated systems for sensing and responding to temperature ( Sengupta and Garrity , 2013 ) .", "Indeed , the capability to detect and follow environmental temperature gradients – thermotaxis – is inherent to many organisms , from animals to bacteria .", "Although in eukaryotes temperature is usually sensed by specific thermal sensors , behavioral controls by temperature and chemical stimuli are tightly intertwined in the well-studied examples of Caenorhabditis elegans ( Kimata et al . , 2012 ) and Drosophila melanogaster ( Montell , 2013; Ni et al . , 2013 ) .", "Such integration of behavioral responses is even more pronounced in bacteria .", "In Escherichia coli the thermotactic and chemotactic responses are mediated by the same pathway ( Maeda and Imae , 1979; Maeda et al . , 1976 ) .", "The tactic behavior of E . coli generally relies on the control of flagellar motors by a signaling pathway that decreases the rate of ‘tumbles’ ( reorientations ) upon an increase in the levels of attractants or upon a decrease in the levels of repellents .", "As a result cells make longer runs in the preferred direction , resulting in a net propagation up attractant gradients ( Brown and Berg , 1974; Macnab and Koshland , 1972 ) .", "Stimulus recognition relies on signaling complexes that consist of transmembrane receptors , the scaffold protein CheW , and the histidine kinase CheA ( Gegner et al . , 1992; Hazelbauer et al . , 2008; Sourjik , 2004 ) .", "Conformational changes that are induced by binding of chemical ligands in the periplasm are transmitted to the cytoplasmic part of the receptor and control the activity of CheA , which is inhibited by attractants and stimulated by repellents ( Falke and Hazelbauer , 2001; Hazelbauer et al . , 2008 ) .", "CheA-mediated phosphorylation of the response regulator CheY stimulates its binding to the flagellar motor and induces tumbling , whereas dephosphorylation of CheY by the phosphatase CheZ promotes smooth swimming .", "Adaptation to persistent stimuli in the chemotaxis system is mediated by the methyltransferase CheR and the methylesterase CheB , which adjust the level of receptor methylation and thereby receptor activity dependent on the background stimulation ( Goy et al . , 1977; Springer et al . , 1979 ) .", "The system functions as an integral negative feedback circuit , whereby CheR preferentially methylates inactive receptors , thus increasing their activity , whereas CheB preferentially demethylates active receptors ( Barkai and Leibler , 1997; Shapiro et al . , 1995; Terwilliger et al . , 1986; Yi et al , 2000 ) .", "The most abundant receptors in E . coli are Tsr and Tar , which respectively sense the amino acids serine and aspartate but can also detect other stimuli ( Adler et al . , 1973; Greer-Phillips et al . , 2003; Kondoh et al . , 1979; Maeda et al . , 1976; Mesibov and Adler , 1972; Slonczewski et al . , 1982; Springer et al . , 1979 ) .", "In the cell , all chemoreceptors form large mixed clusters in the inner membrane , where cooperative interactions between multiple receptors serve to amplify chemotactic stimuli ( Ames et al . , 2002; Briegel et al . , 2012; Sourjik and Berg , 2004; Studdert and Parkinson , 2004; Zhang et al . , 2007 ) .", "The coupling of neighboring receptors within clusters also allows integration of signals perceived by different types of receptors , so that the net response of a cooperative signaling unit is determined by the net of the free-energy changes due to stimulation of individual receptors ( Keymer et al . , 2006; Mello and Tu , 2005; Neumann et al . , 2010 ) .", "Notably , although the activities of different receptors are tightly coupled , adaptation to stimuli results in preferential methylation of the stimulus-specific receptor ( Lan et al . , 2011 ) .", "Chemotaxis is typically assumed to enable bacteria to find conditions that are optimal for growth , and correlation between chemotactic and metabolic preferences has indeed been observed for E . coli ( Yang et al . , 2015 ) .", "Consistent with that , E . coli’s response to chemical ligands is usually unidirectional , that is cells either follow gradients only upwards ( attractant ) or only downwards ( repellent ) .", "In contrast , gradients of other stimuli that are sensed by the chemotaxis pathway , such as pH ( Yang and Sourjik , 2012 ) , osmolarity ( Adler et al . , 1988 ) , or temperature ( Maeda et al . , 1976 ) , might need to be followed bidirectionally in order to find optimal growth conditions .", "However , while the unidirectional chemotactic response of E . coli is comparatively well understood , mechanisms of bidirectional taxis in bacteria remain to be established .", "E . coli is well known to follow temperature gradients and to react to temporal changes in temperature , similar to the tactic response to chemical effectors ( Maeda et al . , 1976 ) .", "This tactic response to temperature is mainly mediated by Tar and Tsr ( Imae et al . , 1984; Lee et al . , 1988; Maeda and Imae , 1979; Mizuno and Imae , 1984 ) , although minor receptors Trg , Aer , and Tap might also be temperature-sensitive ( Nara et al . , 1991; Nishiyama et al . , 2010 ) .", "The thermotactic behavior of E . coli is thus primarily determined by the interplay of the responses mediated by Tar and Tsr ( Yoney and Salman , 2015 ) .", "At low temperatures , E . coli is normally thermophilic ( heat-seeking ) , with an increase in temperature causing an attractant-like response ( Maeda et al . , 1976 ) .", "However , the thermotactic response of E . coli to higher temperatures , 36°C to 42°C , remained ambiguous , with different studies showing either loss of response or its inversion to cryophilic ( Maeda et al . , 1976; Paster and Ryu , 2008; Yoney and Salman , 2015 ) .", "Adaptation to a combination of attractants sensed by Tsr ( serine ) and Tar ( aspartate or its non-metabolizable analogue α-methyl-DL-aspartate , MeAsp ) can clearly invert the thermotactic response ( Imae et al . , 1984; Salman and Libchaber , 2007 ) , which is likely due to increased methylation of adapted receptors since amino acid replacement of methylation sites also altered thermotactic responses of Tar and Tsr ( Nara et al . , 1996; Nishiyama et al . , 1999a; Nishiyama et al . , 1999b; Oleksiuk et al . , 2011 ) .", "Several studies showed that Tar functions as a warm sensor in low methylation states but as a cold sensor in high methylation states , whereas Tsr was suggested to similarly function as a warm sensor in low methylation states but to lose its temperature sensitivity in high methylation states ( Imae et al . , 1984; Salman and Libchaber , 2007; Yoney and Salman , 2015 ) .", "In contrast , other work showed that both receptors mediate thermophilic response in the low-modification states ( zero or one methyl groups per receptor monomer ) and cryophilic response in the high-modification states ( Oleksiuk et al . , 2011 ) .", "Despite this clear evidence that the thermotactic behavior of E . coli depends on both the ambient temperature and chemotactic stimuli , the interpretation of E . coli behavior in gradients of temperature is complicated by the fact that , besides the signaling pathway , temperature also affects cell swimming , respiration , and metabolism ( Demir et al . , 2011; Maeda et al . , 1976; Salman et al . , 2006 ) .", "Hence , the key questions whether E . coli is capable of accumulation at a specific intermediate temperature solely by means of thermotaxis , and the mechanism of such accumulation , remain unanswered .", "Importantly , a standard mathematical model of the chemotactic network , developed by Barkai and Leibler ( Barkai and Leibler , 1997 ) , and generalized to allow for coupled teams of receptors ( Keymer et al . , 2006; Mello and Tu , 2005; Sourjik and Berg , 2004 ) , cannot explain the accumulation temperature .", "Although a model that can account for the bidirectional taxis ( termed ‘precision sensing’ ) was proposed ( Jiang et al . , 2009 ) , this model critically relies on an ad hoc assumption about the temperature dependence of the pathway activity that was not experimentally verified .", "In this study we investigated the thermotactic response of E . coli at the level of the intracellular pathway activity , thus independently of any direct temperature effects on motility .", "Additionally , we analyzed cell behavior using microfluidic devices that were designed to minimize the time cells spend in temperature gradients , thus reducing secondary effects on cell physiology .", "The results obtained with both assays were consistent , demonstrating that thermotactic behavior of E . coli can indeed be explained solely by specific receptor-mediated responses .", "We showed that in the absence of chemical attractants the response of E . coli is always thermophilic although it is weakened with increasing ambient temperature .", "By contrast , inversion of the pathway response from thermophilic to cryophilic at intermediate temperature ( inversion temperature ) and bidirectional cell accumulation towards intermediate temperature ( accumulation temperature ) were observed when the cells were adapted to ligands sensed by either Tar or Tsr , but not when both ligands were present at similar levels .", "Our results are consistent with the hypothesis that the mechanism of bidirectional thermotaxis relies on the interplay between Tar and Tsr receptors in different methylation states , and we employed a mathematical analysis to elucidate the details of the underlying mechanism .", "Finally , we demonstrate that the preferred accumulation temperature observed in the presence of serine roughly corresponds to the optimal growth temperature , suggesting that the thermotactic behavior of E . coli could indeed be explained by growth-rate optimization ." ], [ "To investigate the thermotactic response of E . coli at the level of the pathway activity , we utilized an in-vivo assay based on Förster ( fluorescence ) resonance energy transfer ( FRET ) ( Neumann et al . , 2012; Sourjik and Berg , 2002a; Sourjik et al . , 2007 ) ( Figure 1—figure supplement 1A ) .", "The FRET assay relies on phosphorylation-dependent interactions between CheY and CheZ , which are fused to yellow and cyan fluorescent proteins , respectively .", "The formation of the CheY-YFP/CheZ-CFP complex , which is proportional to the kinase activity of CheA , leads to an increase in the ratio of YFP to CFP fluorescence due to energy transfer from CFP to YFP .", "In our FRET experiments , ΔcheY-cheZ cells expressing the CheY-YFP/CheZ-CFP FRET pair were exposed to rapid stepwise changes in temperature while under a constant flow of buffer ( Figure 1—figure supplement 1A ) .", "Note that to facilitate the measurements we used a strain deleted for flgM , the negative regulator of flagellar and chemotaxis gene expression , that elevates in proper proportion the levels of chemotaxis proteins and thereby enhances the chemotactic response ( Kollmann et al . , 2005; Steuer et al . , 2011 ) .", "Similar to the response observed upon stimulation with a chemical attractant in the same setup ( Figure 1—figure supplement 1C ) , the FRET response to an increase in temperature revealed a rapid transient decrease of the YFP/CFP ratio , reflecting a decrease in the kinase activity , that is a thermophilic response ( Figure 1A and Figure 1—figure supplement 1D ) .", "This transient response was specific because it was not observed in a ΔcheA strain ( Figure 1—figure supplement 1E ) .", "A decrease in temperature resulted in an opposite response , that is , a transient increase in FRET , similar to the removal of attractant ( Figure 1A and Figure 1—figure supplement 1C ) or the addition of repellent .", "The time course of subsequent adaptation in the presence of persistent stimulation was also similar for thermal and chemical stimuli , indicating that the adaptation to temperature similarly relies on the CheR/CheB receptor methylation system .", "This was directly confirmed by measuring the methylation profile of Tsr and Tar , both of which shifted towards higher-methylated states at higher temperature ( Figure 1—figure supplement 2 ) .", "The only major noticeable difference between thermal and chemical stimulation in the FRET experiments was an increase in the basal YFP/CFP ratio at higher temperature , which was also seen in the ΔcheA strain and is caused by temperature dependence of YFP and CFP fluorescence ( Figure 1—figure supplement 1E ) ( Kumar and Sourjik , 2012; Oleksiuk et al . , 2011 ) .", "We subsequently used FRET to measure the response to 3°C incremental steps of temperature in the range from 21°C to 42°C ( Figure 1B ) .", "We observed that the thermophilic response of wild-type cells persisted at a similar level up to 30°C , but decreased rapidly at higher ambient temperatures .", "Nevertheless , even at the highest tested temperature ( jump from 39°C to 42°C ) , the response remained weakly thermophilic .", "Hence , for cells that were adapted in the buffer ( in the absence of chemoattractants ) we did not observe any inversion of the pathway response to temperature .", "These results were generally consistent with the behavioral response of motile cells in a thermal gradient established across a microfluidic channel ( Figure 1C and Figure 1—figure supplement 1B and Figure 1—figure supplement 3 ) .", "The design of the experiment was such that cells in the sample volume experienced only a brief exposure to a temperature gradient , thus minimizing secondary effects of temperature that might have complicated the interpretation of previous studies .", "Here , when adapted in the buffer , cells accumulated towards the warmer side of the gradient in the channel , consistent with thermophilic behavior ( Figure 1C ) .", "We quantified this behavior using a thermal migration coefficient ( TMC ) ( Figure 1C Inset , see Materials and methods for details ) .", "In line with the FRET-based pathway-activity analysis , this thermophilic behavior weakened in the range of higher temperatures but never inverted .", "Thus , the results of both FRET and microfluidics assays clearly show that in the absence of chemotactic stimuli , E . coli has an exclusively thermophilic response that decreases at high ambient temperatures , but it does not actively avoid high temperature .", "We next systematically investigated previously reported inversion of the thermotactic response from thermophilic to cryophilic upon adaptation to high concentrations of serine and aspartate ( or the non-metabolizable analogue of aspartate , MeAsp ) ( Imae et al . , 1984; Paster and Ryu , 2008 ) .", "Firstly , we measured the pathway response to temperature changes in cells that were stimulated with a combination of MeAsp and serine ( Figure 2A ) .", "These two attractants were kept at a fixed ratio of 10:1 , which reflects an approximately tenfold lower chemoattractant efficiency of MeAsp compared to serine ( Neumann et al . , 2010 ) .", "Indeed , steady pre-stimulation with high levels of both attractants inverted the response to cryophilic over the entire range of temperatures , with the response again becoming weaker at high temperatures .", "At low concentrations of attractants the response remained thermophilic and no avoidance of high temperature was observed but the response amplitude was reduced compared to buffer-adapted cells .", "Adaptation to intermediate levels of serine and MeAsp completely abolished the thermotactic response over the entire range of tested temperatures , meaning that the pathway becomes temperature-insensitive when both major chemoreceptors are stimulated at an approximately equal intermediate level .", "As a next step , we investigated the effects of adaptation to different levels of either MeAsp or serine alone .", "In contrast to previous reports ( Imae et al . , 1984; Salman and Libchaber , 2007; Yoney and Salman , 2015 ) , we observed that adaptation to MeAsp ( Figure 2B ) or serine ( Figure 2C ) had nearly identical effect on the thermotactic response .", "As in the case of stimulation with a mixture of serine and MeAsp , we observed that adaptation to either attractant weakened the thermophilic response in a dose-dependent manner and could eventually invert it to cryophilic .", "However , the pattern of this inversion by individual attractants was clearly different .", "Whereas combined stimulation either inverted or abolished the thermotactic response over the entire temperature range ( Figure 2A ) , cells adapted to individual attractants showed cryophilic response at high temperature but retained thermophilic response at low temperatures ( Figure 2B , C ) .", "Such an inversion ( ‘cross-over’ ) temperature , where the FRET response changes from thermophilic to cryophilic , implies that E . coli can indeed bidirectionally accumulate towards a preferred temperature using the chemotaxis pathway .", "However , the inversion and accumulation temperatures may not be exactly identical due to weak direct effects of temperature on E . coli motility ( Oleksiuk et al . , 2011 ) .", "These conclusions were confirmed by microfluidic experiments , where a combination of serine and MeAsp changed the response to cryophilic ( Figure 2D ) and where , in the presence of individual attractants , the response turned from thermophilic at low temperatures to cryophilic at high temperatures ( Figure 2—figure supplement 1 ) .", "This latter inversion of the thermotactic response dependent on the ambient temperature implies that in a thermal gradient and in presence of either one of the major chemoattractants cells should accumulate at some transition temperature ( which is expected to be close to the inversion temperature in the FRET assay ) , being attracted to it from both lower and higher temperatures .", "Such accumulation was indeed observed in the presence of serine in a gradient that spanned a range of temperatures with both thermophilic and cryophilic responses ( Figure 2E and Figure 2—figure supplement 2 ) .", "Notably , no cell accumulation was observed in the same thermal gradient in the absence of attractant stimulation ( Figure 2—figure supplement 2A ) , meaning that it is not simply due to the wider temperature range used in these experiments .", "Our observation that chemotactic stimulation of Tar or Tsr individually – but not together – creates an accumulation temperature indicates that the effect might be related to the interplay between the two receptors .", "To test this conclusion , we investigated the thermosensing properties of cells that express only one type of receptor , either Tar ( Figure 3A ) or Tsr ( Figure 3B ) using the FRET assay .", "Similar to wild-type cells , in the absence of chemoattractants both Tar- and Tsr-only cells exhibited thermophilic responses that decreased with ambient temperature .", "Adaptation to their respective attractants also decreased and inverted the thermophilic response in a dose-dependent manner , which was again similar for both receptors .", "Notably , the inversion to cryophilic response occurred over the entire temperature range and no significant cross-over from thermophilic to cryophilic response occurred with the change of temperature at any given concentration of serine or MeAsp .", "The responses of the Tar and Tsr-only cells were thus similar to the response of wild-type cells adapted to combinations of serine and MeAsp ( Figure 2A ) , but different from adaptation to only one of these chemoattractants ( Figure 2B , C ) .", "These observations confirm that chemotactic stimulation of Tar or Tsr can either inhibit or invert their thermosensing properties .", "The results also clearly demonstrate that in the presence of only one receptor type there is no temperature-dependent response inversion at any given level of chemotactic stimulation , and thus no accumulation temperature .", "The observed dependence of the thermotactic response on ambient temperature and chemotactic stimulation is likely to be explained by the known effects of methylation on receptor thermosensing ( Imae et al . , 1984; Nara et al . , 1996; Nishiyama et al . , 1997; Paster and Ryu , 2008; Salman and Libchaber , 2007 ) .", "Our results are consistent with a previous observation suggesting that the interplay between methylation and thermosensing is similar for Tar and Tsr ( Oleksiuk et al . , 2011 ) , which both mediate thermophilic response in low-methylation states but cryophilic response in high-methylation states .", "For the buffer adapted cells , where the level of receptor modification is low ( Endres et al . , 2008 ) , the response of both receptors is thus thermophilic .", "However , because adaptation to positive ( attractant-like ) temperature stimuli is mediated by increased methylation of receptors ( Figure 3C , Figure 1—figure supplement 2 and Figure 3—figure supplement 1 ) , the thermophilic response becomes weaker at higher temperature .", "In contrast , for cells of single-receptor strains adapted at high concentration of the respective chemoattractant , the methylation level is high and the response is thus cryophilic .", "Adaptation to this repellent-like response leads to increased demethylation of receptors at higher temperature ( Figure 3C ) , thus weakening the cryophilic response .", "At intermediate levels of chemotactic stimulation that correspond to zero thermotactic response , there are no associated changes in receptor methylation and therefore no switch from thermophilic to cryophilic behavior .", "As mentioned above , our results for Tsr are apparently in contrast with some previous studies .", "Specifically , Yoney and Salman ( Yoney and Salman , 2015 ) reported gradual dose-dependent inhibition of the thermophilic response but no response inversion to cryophilic response when cells that express Tsr as the only major receptor were stimulated with glycine , a Tsr-specific attractant .", "This discrepancy can be explained by the much lower apparent affinity of glycine to Tsr compared to serine ( Yang et al . , 2015 ) .", "Indeed , in FRET experiments we found that at glycine concentrations of up to 1 mM , the temperature response of the Tsr-only strain is either thermophilic or non-detectable ( Figure 3—figure supplement 2A , B ) .", "The response of the Tsr-only strain became weakly cryophilic only when the concentration of glycine was increased to a very high level of 30 mM ( Figure 3—figure supplement 2C ) .", "Moreover , Yoney and Salman reported accumulation of the wild-type E . coli cells in a thermal gradient that was established in a complex medium that contained a mixture serine and aspartate ( Yoney and Salman , 2015 ) .", "Notably , in these experiments the exposure of bacterial culture to a thermal gradient was preceded by a prolonged incubation , which most likely resulted in substantial depletion of serine from the medium as it is rapidly consumed by E . coli ( Yang et al . , 2015 ) .", "Such depletion could indeed be confirmed ( Figure 3—figure supplement 3 and Supplementary material ) , suggesting that during the thermotaxis assays , cells were primarily stimulated by only one major chemoattractant , aspartate .", "The observed behavior is thus consistent with our conclusion that asymmetric stimulation of either Tsr or Tar is required for E . coli accumulation at an intermediate temperature .", "Our results strongly suggest that an accumulation temperature arises from the interplay between Tar and Tsr when only one type is strongly stimulated by its ligand .", "This suggests that the level of chemotactic stimulation that leads to the response inversion as well as the accumulation temperature might be affected by the relative expression levels of Tar and Tsr .", "Because these levels are known to vary with the growth phase of an E . coli culture , with Tsr being more abundant during the early to mid-exponential phase ( Kalinin et al . , 2010; Yang and Sourjik , 2012 ) , we tested the thermotactic response in cultures grown to different optical densities ( Figure 3—figure supplement 4 ) .", "Indeed , we observed that cells adapted to high concentrations of serine showed earlier response inversion ( i . e . , inverted at lower temperature ) when grown to low optical density , and did not invert at all when grown to high optical density .", "This observation may explain why no inversion of the thermal response upon adaptation to serine was observed in a previous study ( Imae et al . , 1984 ) hat used E . coli culture grown to high density .", "The opposite dependence on the growth phase was observed for MeAsp-stimulated cells , which inverted at higher temperature when grown to low optical density .", "Here we only considered the interplay between Tar and Tsr in defining the overall thermotactic response of wild-type cells .", "While other receptors might also be temperature sensitive ( Nara et al . , 1991; Nishiyama et al . , 2010 ) , their low abundance in E . coli as compared to the abundances of Tar and Tsr ( Li and Hazelbauer , 2004 ) makes it unlikely that they significantly contribute to the thermotactic response of wild-type cells .", "Indeed , adaptation to the ligands of Trg ( glucose , galactose ) or Tap ( dipeptides ) had no noticeable effect on the thermotactic response ( Figure 3—figure supplement 5 ) .", "How can we understand the experimental observation of an accumulation temperature ?", "As mentioned above , the standard model of the chemotactic network cannot explain the observed accumulation of E . coli towards a specific temperature when both Tsr and Tar receptors are present with one type stimulated by attractant .", "To explain this accumulation , we therefore developed a minimal model for the activity of chemotaxis receptors , based on the one detailed in Meir et al . ( Meir et al . , 2010 ) ( see Source code file 1 - Modelling ) .", "The aim of the model is to provide insight into the origin of an accumulation temperature , not to quantitatively account for the experimental data which depends on many unknown temperature-dependent parameters .", "The basic elements of the model are", "( i ) a free-energy model for the probability that a team of chemoreceptors will be active , depending on the methylation level of the receptors and the concentration of ligand and", "( ii ) a kinetic model for the rate of change of the receptor methylation level due to the enzymes CheR and CheB .", "Importantly , this model incorporates the failure of precise adaptation when the number of available methylation or demethylation sites becomes small ( Meir et al . , 2010 ) , which we believe is essential to explain the accumulation temperature .", "Some indication of the importance of imprecise adaptation is already apparent for the single-receptor cells ( Figure 3A , B ) – the response in the presence of high ligand concentration is flatter as a function of temperature , consistent with the assumption of slowing down of methylation near saturation .", "Consistent with our experimental results and with previous work ( Oleksiuk et al . , 2011 ) , we assume for simplicity identical behavior of Tar and Tsr , except for their different ligand specificities .", "To most simply illustrate the physical origin of the accumulation temperature for wild-type cells , we chose the basic signaling unit ( team ) of allosterically interacting receptors to be a trimer , where Tar and Tsr are randomly mixed so that each of the three receptors can be either Tsr or Tar with probability reflecting relative expression level ( Ames et al . , 2002; Hansen et al . , 2010 ) ( see Materials and methods ) .", "As clear from the comparison of the data ( Figure 2B , C and Figure 3A ) to the results of the mathematical model , the model captures correctly the complete inversion of the thermal response form thermophilic to cryophilic with methylation level or ligand concentration ( Figure 4A ) for a single type of receptor , as well as cross-over ( i . e . , temperature-dependent inversion ) of the response for mixed receptors ( Figure 4B ) .", "Within the model , these qualitative features can be understood as follows ( for a more detailed discussion see Supplementary material ) : As discussed above , for a single receptor type the thermal response has either one sign or the other over all temperatures , with the sign determined by methylation level ( which depends on the attractant concentration ) .", "Changes of temperature alone can never change the sign of the thermal response because if the thermal response approaches zero , as necessary for a sign change , so necessarily does the adaptive change in methylation .", "Since methylation level determines the sign of the thermal response , no change in methylation means no change in the sign of the thermal response , and thus no inversion of the response at a certain temperature ( Figure 4A and Figure 4—figure supplement 1A ) .", "This conclusion also holds for a mixture of different receptors provided adaptation is perfect and thermosensing properties of receptors are identical; as the net thermal response approaches zero , so does the net change in methylation level .", "Even if one type of receptor becomes more methylated , in the standard model this is exactly compensated by the other type becoming less methylated , and as a consequence the response remains either thermophilic or cryophilic over the entire temperature range ( Figure 4—figure supplement 2 ) .", "However , an inversion of the response at a certain temperature becomes possible if changes of temperature lead to a net change in receptor methylation .", "This is exactly what happens in our mathematical model: when receptors of one type are near the saturation level of their methylation , they cannot adapt perfectly , that is their methylation level changes only weakly upon temperature stimulation , and thus cannot compensate for the response of receptor of the other type , which undergo an opposite and larger change in their methylation level ( Figure 4—figure supplement 1B , C ) .", "The resulting net change in total methylation of the receptor system can readily produce a change in the sign of the overall thermal response , leading to an inversion of the response at a certain temperature .", "The actual value of the inversion temperature depends on receptor methylation levels and is thus a function of attractant concentrations .", "Note that our model for the inversion temperature relies on exactly the same slowing of methylation rates near saturation previously introduced to explain the failure of precise adaptation in chemotaxis ( Meir et al . , 2010 ) , and it requires no further ad-hoc assumptions about differences between thermosensing properties of Tar and Tsr or asymmetry between thermophilic and cryophilic responses .", "This contrasts with the model of Jiang et al . ( Jiang et al . , 2009 ) , which attributes an accumulation temperature to temperature-dependent adapted activity , but which does not explain why an inversion temperature only occurs when both Tar and Tsr are present and then in the presence of ligand for only one of the two receptor types .", "What is the physiological significance of the accumulation temperature observed upon stimulation with amino acid attractants ?", "A previous study proposed that a cryophilic response in the presence of amino acids might be a form of quorum sensing behavior , whereby amino acid secretion at high density would cause cell accumulation at lower temperatures thus slowing metabolism ( Salman and Libchaber , 2007 ) .", "However , the benefit of such behavior is not obvious and under normal growth conditions E . coli does not secrete chemoattractive amino acids – instead these amino acids are the first to be consumed from the medium ( Prüss et al . , 1994; Selvarasu et al . , 2009; Yang et al . , 2015 ) .", "At the same time , high concentrations of several amino acids , most notably of serine , were shown to have a growth-inhibitory effect ( Amos and Cohen , 1954; Neumann et al . , 2014; Rowley , 1953; Yang et al . , 2015 ) .", "We thus investigated this toxicity of serine for E . coli MG1655 in M9 glycerol minimal medium as a function of growth temperature .", "We observed that the effect of serine on E . coli growth was indeed temperature-dependent: whereas at low temperature ( 24°C ) serine is growth-promoting ( Figure 5A ) , it becomes inhibitory at high temperature ( 39°C ) ( Figure 5B ) .", "This temperature dependence could be seen both for the growth delay ( Figure 5C ) , where the addition of serine led to a prolonged phase of slower growth at higher temperature , and for the maximal growth rate ( Figure 5—figure supplement 1 ) .", "By comparison , the effect of aspartate on growth was much weaker .", "Interestingly , the observed delay in growth in the presence of serine was minimal at 30°C ( Figure 5C ) .", "These results indicate that at least for cells adapted in the presence of serine , the emergence of an accumulation temperature might have a simple physiological meaning , being an adaptive mechanism that enables E . coli to optimize its growth in the presence of this amino acid ." ], [ "Temperature critically affects growth , metabolism , and other biological processes , and many organisms are capable of following gradients of temperature in their environment .", "In this work , we clarify the mechanism that enables E . coli , which is commonly used as a model of bacterial behavior , to accumulate at a preferred ambient temperature using bidirectional thermotaxis .", "Our results show that the entire complexity of the thermotactic behavior of E . coli can be accounted for by the interplay between the effects of temperature on the activity of the major chemoreceptors Tar and Tsr and the dependence of receptor methylation on both ambient temperature and chemoeffector stimulation:", "( i ) When cells are adapted in the buffer or in the presence of low levels of Tar and Tsr chemoattractants , both major receptors mediate thermophilic responses , leading to an overall thermophilic response .", "This response becomes weaker at higher temperature , because the increased levels of methylation make both receptors less thermophilic , consistent with previous analyses ( Nishiyama et al . , 1999a; Nishiyama et al . , 1997 ) .", "However , the response in this case cannot invert if the temperature is the sole stimulus , since weaker response also leads to smaller increase in methylation , meaning that there is no driving force to increase receptor methylation beyond the level that makes receptors temperature-insensitive .", "( ii ) When Tar and Tsr are both stimulated with intermediate levels of their respective attractants , their intermediate methylation state makes them – and therefore also wild-type cells – temperature insensitive .", "Since there is no change in methylation in the absence of a thermotactic response , no inversion from thermophilic to cryophilic response ( or vice versa ) occurs .", "( iii ) Adaptation to high levels of both Tar and Tsr ligands makes both receptors and hence the wild-type response cryophilic .", "Adaptation to high temperature in the cryophilic regime results in the reduction of methylation and thus weakens the cryophilic response .", "Such symmetric regulation of the thermal response for Tar and Tsr is in contrast to previous studies that suggested that only Tar can function both as a warm and cold sensor with Tsr acting either as a warm sensor or not responding ( Imae et al . , 1984; Salman and Libchaber , 2007 ) .", "Notably , the cryophilic response observed at high levels of ligand stimulation shows less dependence on ambient temperature than the thermophilic response .", "( iv ) Finally , upon adaptation to either one of Tar or Tsr ligands , only the cognate receptor is preferentially methylated ( Lan et al . , 2011 ) and thus inverted in its thermosensing .", "Although in nature E . coli is unlikely to be stimulated by a single amino acid , the mechanism proposed here applies as long as the concentrations of serine and aspartate in the medium are strongly different .", "Such a scenario is consistent with a recent study suggesting that E . coli chemotaxis has evolved to primarily follow gradients of individual amino acids rather than of amino acid mixtures ( Yang et al . , 2015 ) .", "We further demonstrate that this thermotactic behavior can be accounted for by a mathematical model that describes clusters of mixed receptors as a dynamical and partially decoupled lattice ( Hansen et al . , 2008; Yang and Sourjik , 2012 ) .", "Our analysis suggests that the cross-over from thermophilic to cryophilic response relies on the previously characterized failure of precise adaptation in bacterial chemotaxis ( Meir et al . , 2010; Neumann et al . , 2014 ) .", "This failure occurs at high levels of receptor methylation , which lowers the rate of further methylation .", "Because of this , positive and negative changes in methylation of the attractant-stimulated Tar and unstimulated Tsr ( or vice versa ) upon a change of temperature are not precisely compensatory , which enables receptor teams to cross the zero point of the thermotactic response .", "Importantly , our model does not require any ad-hoc assumptions of differences between the thermosensing behavior of Tar and Tsr or of the temperature dependence of other pathway parameters , and it can naturally explain the apparent difference between the thermophilic and cryophilic responses as a function of ambient temperature .", "Consistent with this model of the interplay between Tar and Tsr and with previous observations ( Yoney and Salman , 2015 ) , we demonstrate that the inversion of the thermotactic response by individual attractants strongly depends on the ratio between these two receptors , which itself is a function of the growth phase of the cell culture ( Kalinin et al . , 2010; Salman and Libchaber , 2007; Yang and Sourjik , 2012 ) .", "This work thus elucidates the mechanism of bacterial thermotaxis and its control by chemical stimuli .", "Although largely consistent with previous studies on thermotaxis , including a recent work that emphasized the importance of the interplay between Tar and Tsr ( Yoney and Salman , 2015 ) , surprisingly our results demonstrate that bidirectional thermotaxis requires asymmetric stimulation of either one of the two major receptors , Tar or Tsr .", "This finding clearly contrasts with previous studies that did not observe inversion of the Tsr response to temperature ( Imae et al . , 1984; Salman and Libchaber , 2007; Yoney and Salman , 2015 ) .", "These differences are likely to be explained first by the very low signaling strength of glycine ( Yang et al . , 2015 ) , the Tsr attractant used in two of these studies ( Salman and Libchaber , 2007; Yoney and Salman , 2015 ) , and second by the weaker cryophilic response mediated by Tsr that apparently eluded detection by Mizuno and Imae ( Imae et al . , 1984 ) .", "It was also proposed that both receptors need to be stimulated with attractants to enable accumulation at a specific temperature ( Yoney and Salman , 2015 ) .", "However , this conclusion relied on experiments that were performed upon prolonged incubation of the bacterial suspension in the growth medium containing a mixture of amino acids .", "Because under these conditions serine is rapidly depleted ( Yang et al . , 2015 ) ( Figure 3—figure supplement 3 ) and glycine is only a weak attractant , these bacteria were likely asymmetrically stimulated by aspartate .", "This finding can reconcile the previously observed accumulation behavior with the mechanism proposed in our study , further emphasizing the importance of precise control of medium composition in thermotaxis experiments .", "Secretion of attractants into the medium during long ( up to 3 . 5 hr ) pre-stimulus incubation may also explain the inversion of the thermotactic response at high temperature observed by Paster and Ryu ( Paster and Ryu , 2008 ) .", "Finally , our growth experiments also indicate that the observed thermotactic behavior could be at least partly interpreted as a strategy to achieve optimal growth .", "The optimal growth temperature for E . coli is normally assumed to be ~39°C , and we indeed observed that the growth rate in minimal medium with glycerol or glucose as a carbon source steadily increases up to this temperature .", "Therefore , the thermophilic drift up to this temperature in a thermal gradient would promote cell growth .", "Nevertheless , this thermophilic drift is gradually slowed down , apparently preventing cell movement to even higher temperatures that would elicit a heat-shock response , although we did not observe active avoidance of high temperature by E . coli under these conditions .", "However , temperature dependence of cell growth was also affected by addition of amino acids .", "While the addition of serine , and to some extent also of aspartate , was beneficial for growth at 24°C , serine had a strong inhibitory effect on growth at higher temperatures .", "This inhibitory effect was previously described ( Neumann et al . , 2014 ) , and it might be caused by isoleucine or SAM starvation ( Hama et al . , 1991; Zhang et al . , 2010; Zhang and Newman , 2008 ) .", "However , the temperature dependence of this inhibition had not yet been demonstrated , and we propose that this dependence might explain the avoidance of high temperature by E . coli in the presence of high levels of serine .", "Interestingly , in our experiments the minimal growth delay in the presence of serine was achieved around 30°C , roughly matching the preferred accumulation temperature under these conditions .", "Thus , the bidirectional taxis might enable E . coli to optimize its growth rate in a thermal gradient .", "The mechanism of bidirectional thermotaxis described here for E . coli is likely to be relevant not only for closely related species such as Salmonella that also possess Tar and Tsr but also for other bacteria and even archaea .", "Although no other bacterial thermotactic responses have been characterized so far , conservation of receptor sequences across prokaryotic chemotaxis systems makes it likely that many of them can respond to temperature and act on their temperature preference .", "Moreover , similar principles of bidirectional sensing might be used by bacteria to locate optimal points in gradients of other physical or chemical stimuli ." ], [ "All Escherichia coli K12 strains and plasmids used in this study are listed in Table 1 and Table 2 , respectively .", "Strains used for FRET analyses were derived from RP437 ( Parkinson and Houts , 1982 ) .", "VS223 ( ΔcheY-cheZ , ΔflgM ) , in this study referred to as wildtype , was transformed with pVS88 ( cheZ-ecfp/cheYeyfp ) - coexpressing cheZ-ecfp/cheYeyfp from a bicistronic mRNA; VS181 ( Δ ( cheY cheZ ) Δtsr Δ ( tar tap ) Δtrg Δaer ) transformed with pVS88 and pVS121 ( tar[EEEE] ) or pVS362 ( tsr[EEEE] ) for receptor production and referred to as Tar-only and Tsr-only strain , respectively .", "Alternatively , for protein quantification VH1 ( Δ ( cheR cheB cheY cheZ ) Δtsr Δ ( tar tap ) Δtrg Δaer ) was transformed with pVS88 and receptors in different modification states ( see Table 1 ) .", "For behavioral analysis E . coli AW405 ( HCB1 ) ( Armstrong et al . , 1967 ) was transformed with pCMW1 ( Girgis et al . , 2007 ) for gfp expression .", "Growth analysis was performed using E . coli MG1655 .", "All strains used for FRET and microfluidics were grown aerobically in tryptone broth ( TB; 1% tryptone , 0 . 5% NaCl , pH 7 . 0 ) at 275 rpm at 34°C as described previously ( Neumann et al . , 2010 ) .", "Briefly , cells diluted 1:100 in TB from an overnight culture , supplemented with appropriate antibiotics ( 100 µg/ml ampicillin , 17 µg/ml chloramphenicol or 50 µg/ml kanamycin ) and inducers ( isopropyl β-D-thiogalactoside ( IPTG ) or sodium salicylate; see Table 2 ) were grown to an optical density OD600 of 0 . 6 for FRET and protein quantification or OD600 of 0 . 35 for microfluidics , unless indicated otherwise .", "For analysis of growth at different temperatures , an overnight culture grown at the respective temperatures in M9 minimal medium ( 47 mM Na2HPO4 , 22 mM KH2PO4 , 8 mM NaCl2 , 2 mM MgSO4 , 100 μM CaCl2 , 1x trace metals ( TEKnova , Hollister , CA ) and 0 . 2% glycerol , pH7 ) was diluted 1:200 and incubated with shaking at 217 rpm in a total volume of 110 µl in a 96-well plate ( Greiner Bio-One , Frickenhausen , Germany ) and OD600 was measured using a plate reader ( M200 Absorbance , Tecan , Männedorf , Switzerland ) .", "Where indicated , M9 medium was supplemented with amino acids .", "Growth was analyzed calculating the growth delay ( time from inoculation to the half-maximal OD600 ) or the growth rate using a custom-written MATLAB ( MathWorks , Natick , MA ) script ( see Source code file 3- Growth ) .", "Cell preparation and FRET measurements were done as prescribed previously for chemotactic stimulation ( Neumann et al . , 2010; Sourjik and Berg , 2002a; Sourjik et al . , 2007 ) .", "Briefly , cells harvested at mid-exponential growth phase were washed with tethering buffer ( 10 mM KPO4 , 0 . 1 mM EDTA , 1 μM methionine , 10 mM lactic acid , pH 7 ) and stored at 4°C for 20 min to inhibit protein synthesis .", "Cells were 100-fold concentrated , attached to a polylysine-coated coverslip and placed in a flow chamber under constant flow ( 500 µl/min ) for the entire experiment .", "Temperature in the flow cell was controlled using a Peltier element ( Figure 1—figure supplement 1A ) ( Oleksiuk et al . , 2011 ) .", "Cells were adapted in tethering buffer ( with or without attractants ) for at least 25 min at a constant flow and at 21°C before stimulation with indicated steps of temperature .", "The rate of temperature change could be readily monitored due to the direct and immediate effect of temperature on the fluorescence of CFP and YFP ( Figure 1—figure supplement 1E Inset ) .", "Fluorescence of 300–500 cells was continuously recorded in the yellow ( HQ535/30 ) and cyan ( D485/40 ) channels using photon counters ( Hamamatsu , Hamamatsu City , Japan ) with 1 . 0 s integration time , using a custom-modified Zeiss Axio Imager . Z1 fluorescence microscope .", "FRET response was measured as the change in the ratio of yellow to cyan fluorescence due to energy transfer and normalized to the response of buffer-adapted cells to saturating stimulation with chemical attractant , either α-methyl-DL-aspartate ( MeAsp; Sigma Aldrich , Taufkirchen , Germany ) or L-serine ( Acros Organics – Thermo Fisher Scienitifc , Nidderau , Germany ) , at 21°C .", "Cell preparation was done using a filtration method as described previously by Berg and Turner at room temperature ( Berg and Turner , 1990 ) .", "Cells were washed in tethering buffer and within 30 min of cell harvesting a diluted cell suspension ( OD600 = 0 . 002 ) was loaded into the microfluidics device made of polydimethylsiloxane ( PDMS ) .", "The device consists of a central assay channel with a width of 500 µm , flanked on either side by water circulation channels , with cold water circulated through the channel on the left side and the warm circulated through the channel on the right side ( Figure 1—figure supplement 1B ) , resulting in a near-linear temperature gradient between these last two channels ( Figure 1—figure supplement 3 ) .", "Prior to loading with washed cells , the channel of the device was flushed with 0 . 1% BSA ( BioLabs B9001S , New England BioLabs , Ipswitch , MA ) and EtOH for one hour , followed by tethering buffer for 4 hr .", "For adaptation MeAsp and L-serine were added to the buffer as stated in the text .", "The profile of the thermal gradient was measured and calibrated as a function of the spectral shifts of 50 µM pH-sensitive 2′ , 7′ bis ( two carboxyethyl ) 5 ( and 6 ) carboxyfluorescein ( BCECF ) and 50 mM Tris buffer ( pH 7 . 1 ) .", "To that end fluorescence intensities were measured at two excitation wavelengths , 490 nm , and 440 nm , with fixed emission at 535 nm .", "The ratio of these two fluorescent intensities was used for calibration .", "Cells were imaged ~500 µm from the end of the assay channel and , 15 µm above the cover slip , at a flow rate of 30 µm/second .", "Time-lapsed images were taken at 12 frames/second for 30 min using an inverted microscope ( Nikon Eclipse Ti-U , 20x objective , EGFP/mRFP-1 filter cube , EXFO X-Cite 120Q lamp ) coupled with a CCD camera ( Stingray F145B ) , and custom software written in LabVIEW ( National Instruments , Austin , TX ) .", "Under used settings , each pixel of the movie corresponds to 0 . 3 µm .", "Data analysis was performed using custom scripts written in MATLAB ( see Source code file 2 – Microfluidics ) .", "Cells were identified using standard machines vision techniques , first by image thresholding for particle identification and then by filtering out particles less than five pixels , or with an eccentricity greater than 0 . 95 .", "Trajectories of cells were compiled from their 2D centroid position using nearest neighbor criteria to concatenate these positions .", "To remove trajectories from non-motile cells , we filtered them based on their ratio of standard deviation in the X and Y directions , since non-motile cells moved very little in the X direction .", "Cells below the empirical threshold std ( Y ) /std ( X ) of 18 were defined as motile .", "Furthermore , all trajectories with a path length shorter than 10 pixels were eliminated from further analysis .", "Examples for typical trajectories are shown in Figure 1—figure supplement 1B .", "The X-positions from all viable trajectories were binned and normalized by the number of measurements to produce the thermotaxis histograms .", "This plot shows the relative frequency of occurrence of various X-positions of swimming cells at the downstream end of the microfluidic channel .", "The thermotaxis migration coefficient ( TMC ) was used as a metric of cell tendency to migrate toward the warm ( +1 ) or cold ( −1 ) side of the microfluidics channel .", "TMC was defined asTMC=−2*[ ( mean ( X ) −Xmin ) / ( Xmax−Xmin ) − 0 . 5] where X is the array of all X-positions of viable paths , Xmin is left-side cutoff in the microfluidics channel ( expressed in pixels ) , and Xmax is the right-side cutoff .", "The extent of receptor methylation was determined using quantitative immune blotting as described in ( Neumann et al . , 2010 ) .", "Cells were treated as described above for FRET and concentrated to OD600 of approx .", "13 , adapted for 30 min with or without attractant at 21°C , and subsequently incubated for 20 min at indicated temperature .", "The reaction was stopped by addition of 4 × 95°C Laemmli buffer , samples were boiled and separated using 40 cm long 8% SDS-PAGE .", "Proteins were transferred to a 0 . 2 mm Hybond ECL nitrocellulose membrane by tank blotting and detected using an α-Tar antibody as primary antibody and a IRDyes 800-conjugated secondary antibody ( Rockland , Limerick , PA ) using an Odyssey Imager ( LI-COR , Bad Homburg , Germany ) .", "Protein bands were quantified using the line-scan tool in ImageJ ( http://rsbweb . nih . gov/ij ) .", "In the free-energy model for receptor activity , the probability A that a team of receptors will be active is A=1/ ( 1+eF ) , where F=∑ifi is the sum of free-energy differences between active and inactive states of each of the receptors participating in the team , expressed in units of the thermal energy kBT .", "Following Jiang et al . ( Jiang et al . , 2009 ) and Oleksiuk et al . ( Oleksiuk et al . , 2011 ) , the free-energy difference for a receptor is given by ( 1 ) fi=f0 ( [L] ) + ( T−T0 ) f1−[g0+ ( T−T0 ) g1]m , where fi has been expanded to first order in the temperature difference from a reference temperature T0 ( with the index i suppressed on the RHS ) ( Jiang et al . , 2009; Oleksiuk et al . , 2011 ) .", "Here , f0 ( [ L ] ) =f0 ( 0 ) +log[ ( 1+[ L ]Koff ) / ( 1+[ L ]Kon ) ] , where f0 ( 0 ) is the free-energy difference in the absence of ligand , [ L ] is the ligand concentration specific to the receptor type , and Kon/off are the receptor-ligand dissociation constants in the active ( on ) and inactive ( off ) states .", "For a chemoattractant , Koff< Kon .", "For simplicity , the free-energy difference is assumed to depend linearly on the methylation level of the receptor dimer , m=0 , …mtot ( =8 ) , with a coefficient g=[ g0+ ( T−T0 ) g1 ]>0 ( Endres et al . , 2007; Shimizu et al . , 2010 ) .", "In principle , all these parameters could be different between the Tar and Tsr receptors , but here we take them to be identical , except for the fact that the free energy of the Tar receptor is only affected by the presence of MeAsp , while that of Tsr is affected only by serine .", "For simplicity , we assumed the basic allosteric signaling unit to be a trimer of receptors with randomly mixed Tar and Tsr ( Ames et al . , 2002; Hansen et al . , 2010 ) .", "In such a trimer , each of the three receptors can be either Tsr or Tar with probability reflecting their relative expression levels ( 1:1 in our case ) .", "Following the model of Meir et al . ( Meir et al . , 2010 ) for slowing down of methylation due to the scarcity of methylation sites , the kinetics of methylation is determined by ( 2 ) dm ( t ) dt=γRmtot−m ( t ) mtot−m ( t ) +N0[1−A ( t ) ]−γBm ( t ) m ( t ) +N0A ( t ) , where N0 is the parameter that determines how abruptly methylation and demethylation slow down as the receptors approach the saturated limits m=8 and m=0 , respectively .", "The adapted steady-state methylation level mss obeys ( 3 ) mtot−mssmtot−mss+N0 1−A1−A0=mssmss+N0AA0 where A0 is the adapted activity in the absence of slowing down of methylation/demethylation ( N0=0 , 0≤m≤8 ) .", "Here we assume that all receptor dimers in the trimer have the same adapted methylation level m , which is obtained by solving Equation ( 3 ) for a given ligand concentration and a given temperature , in view of the above dependence of the probability A on m , [ L ] , and T . The average activity A¯ is given by averaging this probability over all trimers .", "For the case of mixed receptors , we assume equal fractions of Tar and Tsr with trimer probabilities reflecting random mixing .", "Unless indicated otherwise , the parameters used for simulations were A0=1/3 ( a typical wild-type adapted activity; ( Neumann et al . , 2012; Sourjik and Berg , 2002b; Sourjik et al . , 2007 ) , N0=2 ( which corresponds to the saturation parameter previously obtained in Meir et al . ( Meir et al . , 2010 ) for T = 34°C in the current model ) , T0 = 24°C ( which is a convenient arbitrary reference temperature ) , f1=1 . 2 , g0=0 , and g1=0 . 2 ( where these f and g parameters are chosen to yield an accumulation temperature near the range observed experimentally ) .", "For additional model details see Supplementary material .", "The thermotactic response at temperature T is given by [ A¯ ( T+3 ) −A¯ ( T ) ]/ΔA¯ , where the response , as in the experiment , is normalized by the change of activity ΔA at the lowest temperature upon saturation by ligand ." ] ]
[ "In bacteria various tactic responses are mediated by the same cellular pathway , but sensing of physical stimuli remains poorly understood .", "Here , we combine an in-vivo analysis of the pathway activity with a microfluidic taxis assay and mathematical modeling to investigate the thermotactic response of Escherichia coli .", "We show that in the absence of chemical attractants E . coli exhibits a steady thermophilic response , the magnitude of which decreases at higher temperatures .", "Adaptation of wild-type cells to high levels of chemoattractants sensed by only one of the major chemoreceptors leads to inversion of the thermotactic response at intermediate temperatures and bidirectional cell accumulation in a thermal gradient .", "A mathematical model can explain this behavior based on the saturation-dependent kinetics of adaptive receptor methylation .", "Lastly , we find that the preferred accumulation temperature corresponds to optimal growth in the presence of the chemoattractant serine , pointing to a physiological relevance of the observed thermotactic behavior ." ]
[ "Many bacteria can move towards or away from chemicals , heat and other stimuli in their environment .", "The ability of bacteria to move in response to nutrients and other chemicals , known as chemotaxis , is the best understood of these phenomena .", "Bacteria generally swim in a fairly random way and frequently change direction .", "During chemotaxis , however , the bacteria sense changes in the concentrations of a chemical in their surroundings and this biases the direction in which they swim so that they spend more time swimming towards or away from the source of the chemical .", "The bacteria have various receptor proteins that can detect different chemicals .", "For example , the Tar and Tsr receptors can recognize chemicals called aspartate and serine , respectively , which are – amongst other things – nutrients that are used to build proteins .", "Tar and Tsr are also involved in the response to temperature , referred to as thermotaxis .", "At low temperatures , a bacterium Escherichia coli will move towards sources of heat .", "Yet when the bacteria detect both serine and aspartate they may reverse the response and move towards colder areas instead .", "However , it was not clear why the bacteria do this , and what roles Tar and Tsr play in this response .", "Paulick et al . have now combined approaches that directly visualise signalling inside living bacteria and that track the movements of individual bacterial cellswith mathematical modelling to investigate thermotaxis in E . coli .", "The experiments show that the bacteria’s behaviour could be explained by interplay between the responses mediated by Tar and Tsr .", "In the absence of both serine and aspartate , both receptors stimulate heat-seeking responses , causing the bacteria to move towards hotter areas .", "When only aspartate is present , Tsr continues to stimulate the heat-seeking response , but the aspartate causes Tar to switch to promoting a cold-seeking response instead .", "This leads to the bacteria accumulating in areas of intermediate temperature .", "In the presence of serine only , the bacteria behave in a similar way because the receptors swap roles so that Tsr stimulates the cold-seeking response , while Tar promotes the heat-seeking one .", "The intermediate temperature at which the bacteria accumulate in response to serine is also around the optimal temperature for E . coli growth in presence of this chemical , suggesting that thermotaxis might play an important role in allowing bacteria to survive and grow in many different environments , including in the human body .", "Thus , understanding how chemotaxis and thermotaxis are regulated may lead to new ways to control how bacteria behave in patients and natural environments ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Large-scale filament formation inhibits the activity of CTP synthetase
elife-03638-v2
[ [ "Many enzymes form small-scale oligomers with well-defined subunit numbers , typically ranging from 2 to 12 subunits per oligomer .", "Recent studies suggest that some enzymes can also form large , higher-order polymers in which dozens to hundreds of subunits assemble into filaments ( Barry and Gitai , 2011 ) .", "For most of these structures , we lack an understanding of both the regulation and functional significance of their polymerization .", "To address these questions , we focused on the assembly of CTP synthetase ( CtpS ) , an essential and universally conserved metabolic enzyme .", "CtpS forms large , micron-scale filaments in a wide variety of bacterial and eukaryotic species ( Ingerson-Mahar et al . , 2010; Liu , 2010; Noree et al . , 2010 ) , but the structure of these polymers , what triggers their formation , and the relationship between CtpS polymerization and enzymatic activity were unknown until now .", "Cellular CTP levels are subject to exquisitely tight homeostatic control , and CtpS is one of the most regulated enzymes in the cell .", "In both prokaryotes and eukaryotes , CtpS activity is regulated by allosteric control and feedback-inhibition of enzymatic activity , and CtpS levels are regulated by transcriptional and post-translational control ( Long and Pardee , 1967; Levitzki and Koshland , 1972b; Yang et al . , 1996; Meng et al . , 2004 ) .", "Cells in all kingdoms of life synthesize CTP using CtpS ( Long and Pardee , 1967 ) , and its essentiality makes CtpS an attractive chemotherapeutic and antiparasitic target ( Williams et al . , 1978; Hofer et al . , 2001 ) .", "The CtpS enzyme has two domains connected by an elongated linker: a glutaminase ( GATase ) domain that deaminates glutamine and a synthetase ( ALase ) domain that aminates UTP in an ATP-dependent manner to form CTP .", "CtpS has binding sites for substrates ( glutamine , ATP , and UTP ) , product ( CTP ) , and a proposed binding site for an allosteric modulator ( GTP ) ( Levitzki and Koshland , 1972b ) .", "CtpS tetramerization is necessary for its catalytic activity and is controlled by nucleotide availability; ATP , UTP , or CTP can favor tetramer formation ( Figure 1A; Levitzki and Koshland , 1972a; Anderson , 1983; Pappas et al . , 1998; Endrizzi et al . , 2004 ) .", "Of critical regulatory importance , CtpS activity is also inhibited by CTP ( Long and Pardee , 1967 ) . 10 . 7554/eLife . 03638 . 003Figure 1 . CtpS polymerization and enzymatic activity are inversely related .", "( A ) A model of oligomeric regulation of CtpS .", "Tetramer formation from CtpS dimers is favored by a combination of enzyme concentration as well as nucleotide ( substrates ATP and UTP or product CTP ) and Mg2+ binding .", "( B ) CtpS was incubated in activity buffer containing all substrates for CTP production .", "As the enzyme concentration increases , CtpS shows assembly by light scattering and the kcat value ( Vobs/[CtpS] ) decreases .", "Error bars = standard error ( SE ) , n = 3–5 .", "( C ) Negative stain image of CtpS filaments assembled after CTP synthesis reaction .", "Smaller particles in the background resemble the X-shaped CtpS tetramer .", "A single filament is shown at bottom .", "( D ) CtpS polymers formed in activity buffer were ultracentrifuged to pellet polymers .", "The pellet fraction was resuspended and CTP production was recorded .", "( E ) CtpS assembly and activity were assayed after CtpS was first polymerized , followed by addition of saturating amounts of substrate after 600 s . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00310 . 7554/eLife . 03638 . 004Figure 1—figure supplement 1 . Determination of threshold concentration for CtpS polymerization in activity buffer .", "( A ) Below 1 µM , the changes in light scattering ( blue ) are very low .", "At low [CtpS] the net changes in light scattering were both above and below zero , so the absolute values are plotted to allow comparison on a logarithmic plot ( only the values for 0 . 05 µM and 0 . 075 µM CtpS were above zero ) .", "CtpS activity ( red ) is plotted on a logarithmic plot to compare the concentration at which polymerization and activity decreases begin .", "( B ) The logarithmic plot of polymerization shows an inflection point at 1 µM .", "This predicts an approximate threshold concentration for polymerization between 1 µM and 2 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00410 . 7554/eLife . 03638 . 005Figure 1—figure supplement 2 . Representative examples of raw data from three different concentrations of CtpS incubated in activity buffer included in Figure 1B . Light scattering ( polymerization ) is shown in blue and transmittance data ( CTP accumulation ) is shown in red .", "( A ) 100 nM CtpS ( below polymerization threshold ) .", "( B ) 2 µM CtpS ( at polymerization threshold ) .", "( C ) 5 µM CtpS ( above polymerization threshold ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00510 . 7554/eLife . 03638 . 006Figure 1—figure supplement 3 . Calculation of intracellular CtpS in minimal media . Levels of native CtpS protein in wild-type E . coli NCM3722 were compared to dilutions of the purified protein of known concentration ( 9 mg/ml ) .", "Band intensities were quantified using ImageJ .", "Amount of cells in lysate was approximated to be 8 × 108/OD600 unit .", "Volume of an E . coli cell was approximated to be 1 µm3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00610 . 7554/eLife . 03638 . 007Figure 1—figure supplement 4 . CtpS activity is not sensitive to incubation on ice . Due to concerns that placing resuspended polymers on ice may affect enzymatic activity significantly , we compared the activity level of CtpS reactions under typical conditions ( t = 0 min ) vs incubation on ice .", "For later time points , 10 µl of cold CtpS-containing buffer was added to room temperature activity buffer to match the conditions of the ultracentrifuguation assay ( Figure 1C ) .", "Overall activity does not change over the course of the experiment at the lower temperature . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00710 . 7554/eLife . 03638 . 008Figure 1—figure supplement 5 . CtpS higher order structures disassemble over time after centrifugation . Light scattering values from the pellet fraction of polymerized CtpS decrease over time after centrifugation from a baseline initial value .", "Pellet fraction stored on ice and compared to initial light scattering value immediately after resuspension . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00810 . 7554/eLife . 03638 . 009Figure 1—figure supplement 6 . CtpS polymer disassembly is not caused by mechanical disruption of polymers . Addition of an equivalent volume of water to the volume contributed by substrates added in Figure 1E to polymerized CtpS does not change light scattering values .", "Error bars in B are the standard deviation of light scattering values over the time course shown in A . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 00910 . 7554/eLife . 03638 . 010Figure 1—figure supplement 7 . Correction of kcat values between initial Princeton and UC Davis data sets . Linear correction was performed as described in ‘Materials and methods’ to generate overlapping data sets . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 010 Here , we determine the function and mechanism of CtpS polymerization .", "We demonstrate that CtpS polymerization negatively regulates CtpS activity when its CTP product accumulates .", "We also present the structure of the CtpS polymers and the resulting implications for CtpS inhibition .", "We confirm the physiological significance of CtpS assembly by demonstrating that polymerization-mediated regulation is essential for the proper growth and metabolism of Escherichia coli .", "Together , these findings establish CtpS as a model for understanding enzymatic regulation by large-scale polymerization .", "Finally , we model how coupling CtpS activity to its large-scale assembly can enable cooperative regulation and discuss the implications of polymerization-based regulation for ultrasensitive metabolic control and cytoskeletal evolution ." ], [ "Because CtpS filament formation is conserved between divergent organisms , we hypothesized that CtpS polymerization may regulate its conserved enzymatic function .", "We therefore designed a system to simultaneously monitor the assembly and activity of purified E . coli CtpS .", "We used a fluorometer to assay CtpS assembly by right-angle light scattering and CtpS activity by the specific absorbance of its CTP product .", "CtpS assembly and activity were assayed across a range of enzyme concentrations in activity buffer containing saturating amounts of substrates ( UTP , ATP , and glutamine ) as well as GTP and Mg2+ ( referred to as ‘activity buffer’ throughout the text ) ( Figure 1B ) .", "CtpS protein was first pre-incubated in an incomplete activity buffer without glutamine to favor active tetramer formation .", "CTP production was then initiated by the addition of glutamine to form a complete activity buffer .", "The formation of well-ordered filaments was confirmed by negative stain electron microscopy ( EM ) ( Figure 1C ) .", "Interestingly , at CtpS levels where robust changes in light scattering are observed ( above approximately 1–2 μM ) , CtpS activity ( determined by the rate of CTP production per enzyme ) sharply decreases ( Figure 1B , Figure 1—figure supplements 1 and 2 ) .", "This abrupt transition in activity state supports the hypothesis that there is a threshold for polymerization and that polymerization is inhibitory .", "Noise and nonlinearity in the light scattering data make it difficult to determine an exact critical concentration value .", "However , based on correlation between light scattering and CTP production changes , we predict the assembly threshold of CtpS to be approximately 1–2 μM .", "The cellular level of CtpS protein in E . coli grown in minimal media was measured at 2 . 3 μM ( Figure 1—figure supplement 3 ) , indicating that the CtpS polymerization observed in vitro may be physiologically favorable .", "To determine if polymerization indeed inhibits CtpS activity , we assayed the activity of polymers purified by ultracentrifugation .", "The polymer-containing pellet was least enzymatically active immediately after centrifugation and CtpS activity increased as the polymers in the pellet disassembled ( Figure 1D , Figure 1—figure supplements 4 and 5 ) .", "CtpS polymers are thus inactive or much less than maximally active and polymerization is readily reversible .", "We directly demonstrated the reversibility of CtpS assembly and inactivation by first allowing CtpS to polymerize in activity buffer ( with all substrates present ) and then adding 1 mM UTP and ATP .", "Upon addition of these substrate nucleotides , we observed a sharp decrease in light scattering that corresponded to a sharp increase in CtpS activity .", "This transition was followed by a gradual increase in light scattering and corresponding decrease in activity back to the initial residual level ( Figure 1E ) .", "Control experiments confirmed that the decrease in CtpS polymerization was not due to mechanical disruption by substrate addition ( Figure 1—figure supplement 6 ) .", "The correlation between the decrease in light scattering and the initiation of CTP production at the time of substrate addition indicates that substrate addition leads to rapid depolymerization and subsequent enzyme reactivation .", "Immediately after this point , we observed an increase in both CTP levels and polymerization .", "We therefore conclude that polymerized CtpS enzymes are inactive and must disassociate from the polymer to resume normal enzymatic activity .", "Despite the fact that polymerization occurs in a buffer containing substrates , polymerization only occurs with CTP production , suggesting that polymerization is triggered not by the initial substrates , but rather by the accumulation of CTP product .", "In order to identify the factors that control CtpS inhibition by assembly , we first confirmed that none of the substrates alone induced polymerization ( Figure 2—figure supplement 1 ) .", "We then directly tested our hypothesis that CtpS's product , CTP , a known inhibitor of CtpS activity , stimulates CtpS polymerization .", "In the absence of substrates ( UTP , ATP , and glutamine ) , incubation with CTP caused CtpS to polymerize ( Figure 2A ) .", "The threshold concentration for robust changes in light scattering by CtpS with saturating CTP ( 1–2 μM CtpS; Figure 2—figure supplement", "2 ) agrees with the threshold concentration in the presence of substrates ( 1–2 μM CtpS; Figure 1—figure supplement 1 ) .", "This result suggests that CTP alone is sufficient to influence polymerization and that the substrates and any other products of the enzymatic reaction are not necessary .", "To confirm that CTP stimulates CtpS assembly , we used ultracentrifugation as an independent assembly assay .", "Titrating with increasing amounts of CTP caused an increase in the amount of CtpS found in the pellet with respect to the 0 mM CTP condition ( Figure 2B , Figure 2—figure supplement 3 ) . 10 . 7554/eLife . 03638 . 011Figure 2 . CTP is sufficient and necessary to stimulate CtpS polymerization .", "( A ) CtpS levels were titrated in buffer containing 1 mM CTP ( with no substrates present ) .", "Polymerization was observed in the same range of protein concentrations as in activity buffer .", "Error bars = SE , n = 3 . ( B ) CtpS was allowed to polymerize at different CTP concentrations ( with no substrates present ) .", "The polymers were collected by ultracentrifugation and changes in CtpS pellet abundance were quantified by immunoblot .", "Error bars = SE , n = 2 . ( C ) Purified CtpSE155K , which is defective in CTP binding , showed no obvious changes in light scattering during the normal conditions of wild-type polymer assembly in activity buffer .", "Initial light scattering values were normalized to 1 to place wild-type CtpS and CtpSE155K on the same scale .", "Error bars = SE , n = 3 . ( D ) CtpS Filaments of wild-type and mutants by negative stain electron microscopy .", "There were very few filaments observed in the absence of CTP ( top row ) .", "Upon the addition of nucleotide and MgCl2 , filaments were only observed in the wild-type sample ( first column ) .", "Micrographs were all taken at 55 , 000X magnification .", "( E ) CtpS was incubated in the inhibitor DON and 1 mM CTP and allowed to polymerize .", "Addition of ATP and UTP depolymerized the sample .", "Polymers did not reform . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01110 . 7554/eLife . 03638 . 012Figure 2—figure supplement 1 . CtpS enzymatic activity or CTP addition is required for CtpS polymerization . Addition of various factors ( shown in table ) to 10 µM CtpS indicates that glutamine binding is dispensable for CtpS polymerization .", "Full activity buffer ( containing 1 mM ATP , 1 mM UTP , and 10 mM glutamine ) or CTP are required for polymerization .", "Omission of a substrate from activity buffer or addition of the inhibitor 1 mM DON inhibits polymerization .", "1 mM DON has no effect on CTP-induced polymerization .", "Error bars = SE ( n = 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01210 . 7554/eLife . 03638 . 013Figure 2—figure supplement 2 . Determination of threshold concentration for CtpS polymerization in 1 mM CTP . Threshold concentration was calculated as in Figure 1—figure supplement 1 for CtpS incubated in buffer with 1 mM CTP .", "The graph shows an inflection point at 1–2 µM .", "Error bars = SE ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01310 . 7554/eLife . 03638 . 014Figure 2—figure supplement 3 . Immunoblot of CtpS pelleted by ultracentrifugation . CtpS was incubated in titrating amounts of CTP as described in ‘Materials and methods’ .", "Pellet fraction band density was calculated by ImageJ to determine fold change . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01410 . 7554/eLife . 03638 . 015Figure 2—figure supplement 4 . DON-treated CtpS is enzymatically inactive . Samples of CtpS were allowed to polymerize in activity buffer .", "Then 10 mM DON was added to stop enzymatic activity .", "( A and B )", "Two independent representative experiments are shown .", "Light scattering ( polymerization ) and transmittance ( CTP production ) are shown on the same axis in arbitrary units .", "( C ) Polymerization and activity before and after DON addition are compared .", "Overall amplitude of light scattering and transmittance data is affected by absorption by DON at the wavelengths of light used for the assay .", "Therefore , the slopes of each condition are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01510 . 7554/eLife . 03638 . 016Figure 2—figure supplement 5 . DON inhibition of activity does not inhibit polymerization upon CTP addition . In the converse experiment of Figure 2E , DON-inhibited CtpS incubated in substrates does not polymerize , but addition of 1 mM CTP stimulates polymerization . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 016 We further demonstrated that CTP binding is necessary for polymerization by showing that a CtpSE155K mutant defective for CTP-binding feedback inhibition ( reviewed in Endrizzi et al . , 2005 ) ( Trudel et al . , 1984; Ostrander et al . , 1998 ) fails to polymerize under the same CTP-producing conditions in which wild-type enzyme polymerizes ( Figure 2C ) .", "Furthermore , electron microscopy confirmed that , unlike wild-type CtpS , CtpSE155K does not polymerize in the presence of CTP ( Figure 2D ) .", "Together , our data indicate that within our studied range of enzyme concentrations , CtpS's product , CTP , is both necessary and sufficient to induce CtpS polymerization .", "The CtpS crystal structure suggests that the enzyme's UTP and CTP binding sites partially overlap ( Endrizzi et al . , 2005 ) , raising the question of whether CtpS assembly is controlled by the absolute level of CTP or the relative product/substrate levels .", "6-Diazo-5-oxo-L-norleucine ( DON ) is a glutamine analog that covalently binds glutaminase active sites and irreversibly inactivates enzymatic activity ( Chakraborty and Hurlbert , 1961 ) .", "When added to activity buffer , DON abolishes both CTP production and CtpS polymerization ( Figure 2—figure supplement 4 ) .", "However , DON-treated CtpS can still polymerize when CTP is added to the solution ( Figure 2E ) .", "Polymers formed in the presence of CTP and DON disassemble upon the addition of substrates but do not reform after substrate addition ( Figure 2E ) , presumably because the DON-inhibited CtpS cannot produce additional CTP .", "DON treatment has no effect on CtpS polymerization when the enzyme is incubated with saturating CTP ( Figure 2—figure supplements 1 and 5 ) .", "These results suggest that competition between substrate ( UTP ) and product ( CTP ) binding controls the polymerization equilibrium of CtpS .", "The dependence of polymerization on CTP levels may explain why DON treatment abolishes in vivo CtpS assembly in some cellular contexts ( Ingerson-Mahar et al . , 2010 ) but not others ( Chen et al . , 2011 ) .", "To better understand the mechanism of enzymatic inhibition by polymerization , we determined the structure of the CtpS filament by cryo-electron microscopy at 8 . 4 Å resolution ( Figure 3—figure supplement 1 ) .", "The repeating subunits of the filament are X-shaped CtpS tetramers ( Figure 3A ) .", "The helical symmetry of the filament results in CtpS tetramers stacked atop one another with the arms of the adjacent Xs interdigitated .", "The 222 point group symmetry of the tetramer is maintained within the filament , resulting in overall twofold symmetry both along and perpendicular to the helical axis .", "A significant effect of this unusual symmetry is that , unlike many biological polymers , CtpS filaments are apolar . 10 . 7554/eLife . 03638 . 017Figure 3 . Cryo-EM structure of CtpS filaments at 8 . 4 Å resolution .", "( A ) A segment of the reconstructed filament , colored by helical subunit .", "( B ) The E . coli CtpS crystal structure monomer fit into the cryo-EM density .", "Each domain was fit as a separate rigid body .", "( C ) Novel filament assembly contacts between the linker domains .", "( D ) Novel assembly contacts between the GATase domains . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01710 . 7554/eLife . 03638 . 018Figure 3—figure supplement 1 . Cryo-EM reconstruction of CtpS filaments .", "( A ) A field of CtpS filaments in cryo-EM .", "( B ) Cryo-EM image of a single CtpS filament .", "( C ) The reconstruction of CtpS filament shown at 8 . 4 Å resolution .", "In addition to the refined helical symmetry , local 2-2-2 point group symmetry was imposed on each helical subunit .", "( D ) The resolution of the final reconstruction was estimated in two ways: the standard even-odd half volume test ( blue ) , and a comparison of the cryo-EM structure to the atomic model ( red ) .", "For both measures , the resolution is estimated at 10 . 4 Å by the 0 . 5 cutoff criterion , and 8 . 4 Å by the 0 . 143 criterion . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 01810 . 7554/eLife . 03638 . 019Figure 3—figure supplement 2 . The CtpS monomer in the filament is in a similar conformation to crystallographic structures , and ADP and CTP are present .", "( A ) The fit CtpS monomer structure ( orange ) is overlaid with the available crystal structures of full-length CtpS ( gray ) , aligned on the N-terminal ALase domain .", "( B ) A difference map ( blue mesh ) was calculated between a model of the CtpS filament calculated from the fit crystal structure and the EM structure .", "Strong density ( here rendered at 8σ ) is observed for CTP in its binding site , while the ALase active site ( red residues ) remains empty .", "( C ) Similarly strong density is found in the difference map in the positions of bound ADP .", "( D ) Very weak density is observed for glutamine in the glutaminase active site ( orange sticks ) , and no density associated with the proposed GTP binding site ( red ) , suggesting weak or no binding of glutamine or GTP . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 019 To create an atomic model of the CtpS filament , we fit a monomer of the E . coli CtpS crystal structure into the cryo-EM structure as three rigid bodies ( ALase domain , GATase domain , and the linker region ) ( Figure 3B ) .", "There is a slight rotation between the GATase and ALase domains , similar to the variation seen across crystal structures of full length CtpS ( Figure 3—figure supplement 2A ) .", "There is a strong density for CTP bound at the inhibitory site , and no density in the predicted UTP active site ( Figure 3—figure supplement 2B ) , confirming the biochemical data that CTP binding favors assembly .", "Weaker density is also observed for ADP , but there is no density in the predicted GTP allosteric regulatory site ( Figure 3—figure supplement 2C , D ) .", "There is a minor rearrangement of the tetramerization interface in the filament relative to the crystal structure that results in a compression of the tetramer by about 3 Å along the length of the filament axis ( Figure 4 ) . 10 . 7554/eLife . 03638 . 020Figure 4 . Rearrangement of the CtpS tetramerization interface within the filament .", "( A ) Superposition of the E . coli crystallographic tetramer ( gray ) with the atomic model from the cryo-EM structure ( color ) , shows a rearrangement of the tetramerization contacts , primarily a compression of the tetramer along the filament axis .", "( B ) Rearrangements of the tetramerization contacts shift the relative positions of helices near bound CTP ( gray: crystal structure; color cryo-EM structure ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 020 The cryo-EM structure of the CtpS filament offers insight into the mechanism of enzymatic regulation .", "All of the enzyme active sites are solvent accessible , suggesting that UTP , ATP , and glutamine can freely diffuse into the filament ( Figure 5A ) .", "This observation rules out occlusion of active sites as a regulatory mechanism .", "An alternative mechanism of CtpS inhibition is blocking the transfer of ammonia between the GATase and ALase active sites , which are separated by ∼25 Å .", "The detailed mechanism of ammonia transfer is unknown , but likely involves a conformational rearrangement in the vicinity of a putative channel that connects the two domains ( Endrizzi et al . , 2004; Goto et al . , 2004 ) .", "One prediction is that a conformational change , induced by UTP and ATP binding , rotates the GATase domain toward the ALase domain to create a shorter channel between the active sites ( Goto et al . , 2004 ) .", "Such a large-scale rotation would be unattainable in the steric environment of the filament , as it would lead to clashing of the moving GATase domain with an adjacent CtpS tetramer ( Figure 5B , C ) .", "Regardless of the specific changes involved , quaternary constraints imposed by the filament structure likely provide the mechanism for inhibition of the synthesis reaction . 10 . 7554/eLife . 03638 . 021Figure 5 . Implications of the CtpS filament structure for the mechanism of enzyme inhibition .", "( A ) The binding sites for ATP , CTP , and glutamine are all solvent accessible in the filament , suggesting that they are freely exchangeable in the filament form .", "( B ) The approximate direction of the putative rotation of the glutaminase domain toward the amidoligase domain ( arrow ) , which is predicted to create a shorter channel for ammonia diffusion .", "( C ) In the filament structure , such a conformational change would be sterically hindered by contacts with adjacent filament subunits . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 021 To validate the filament structure and its mechanistic implications , we generated structure-guided mutants in the CtpS polymerization interface .", "Two discrete segments constitute the novel filament assembly contacts: the linker region α-helix 274–284 , and the short α-helix 330–336 of the GATase domain ( Figure 3D , E ) .", "Though the exact amino acid sequences at the inter-tetramer assembly interfaces are not well conserved , relative to the rest of CtpS , both sites feature many charged or hydrophobic residues available for potential polymerization stabilization across species ( Figure 6—figure supplement 1 ) .", "We previously demonstrated that in E . coli , an mCherry-CtpS fusion faithfully reproduces the filamentous localization of native CtpS ( as assayed by immunofluorescence ) ( Ingerson-Mahar et al . , 2010 ) .", "As an initial screen for CtpS assembly , we therefore introduced four mutations in the linker region α-helix and surrounding residues ( E277R , F281R , N285D , and E289R ) into mCherry-CtpS ( Figure 6A ) .", "All four polymerization interface mutants disrupted mCherry-CtpS localization , exhibiting a diffuse localization pattern rather than linear filaments ( Figure 6B ) . 10 . 7554/eLife . 03638 . 022Figure 6 . Linker helix residues form a polymerization interface .", "( A ) The positions of the four polymerization mutants in the model of the linker–linker filament assembly interface .", "( B ) Point mutants were engineered into an mCherry-CtpS fusion and imaged upon expression in E . coli .", "Scale bar = 3 microns .", "Wild type mCherry-CtpS forms filaments while mutant mCherry-CtpSs show diffuse localizations . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 02210 . 7554/eLife . 03638 . 023Figure 6—figure supplement 1 . Sequence alignment of several CtpS primary sequences . Note that the linker region is comprised of residues 274–284 in the E . coli sequence .", "The primary sequence of this region is not strongly conserved across species , however , there are several potential electrostatic ( blue , purple ) or hydrophobic ( red ) pi-stacking interactions between residues of adjacent tetramers .", "The linker region and nearby residues where site-mutations were engineered into E . coli CtpS is boxed in black . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 023 The loss of filamentous mCherry-CtpS localization does not exclude the possibility that the polymerization interface mutants form small filaments that cannot be resolved by light microscopy .", "Consequently , to determine if the diffuse localization in vivo reflected a polymerization defect , we purified one of the linker region helix mutants , CtpSE277R , and examined its polymerization by light scattering and EM .", "CtpSE277R did not significantly polymerize in activity buffer , and no filaments could be detected by EM ( Figure 7B , Figure 7—figure supplement 1 ) , confirming that CtpSE277R cannot properly polymerize .", "We attribute the slight linear increase in light scattering with increasing concentration of CtpSE277R to the increase in protein abundance . 10 . 7554/eLife . 03638 . 024Figure 7 . Linker helix mutations disrupt polymerization and cause a growth defect .", "( A ) The CTP production activity of titrated levels of CtpSE227R exhibited a small decrease in enzymatic activity as enzyme concentration increases when compared to wild-type protein .", "Error bars = SD , n = 3–6 .", "( B ) Purified CtpSE277R does not polymerize in the presence of CTP .", "For both wild-type and E277R CtpS , there were very few filaments observed in the absence of CTP ( top row ) .", "Upon the addition of nucleotide and MgCl2 , filaments were only observed in the wild-type sample ( first column ) .", "( C ) Growth curve comparing wild-type and CtpSE277R cells in LB media .", "CtpSE277R exhibits defective growth when compared to cells with wild-type CtpS .", "Both strains were grown overnight and subcultured into LB media .", "Growth curve comparing wild type to the defective growth of CtpSE277R mutant E . coli in minimal media .", "CtpSE277R mutants exhibit defective growth .", "Error bars = SE , n = 18 . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 02410 . 7554/eLife . 03638 . 025Figure 7—figure supplement 1 . CtpSE277R does not polymerize in vitro . Right angle light scattering by CtpSE277R in activity buffer .", "Error bars = SE , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 02510 . 7554/eLife . 03638 . 026Figure 7—figure supplement 2 . Polymerization enhances the inhibition of CtpS activity by CTP . At a CtpS concentration below the threshold concentration , ( 200 nM , red circles ) , the CTP IC50 value is 330 μM .", "At concentrations that favor polymerization ( 4 μM CtpS , green squares ) , CTP binds with higher apparent affinity with an IC50 of 170 μM .", "Abolishing polymerization with E277R mutation reduced apparent CTP activity inhibtion ( IC50 = 833 μM at 200 nM CtpSE277R ) ( purple open diamonds ) .", "The CTP synthesis vo values before normalization were 1 . 24 , 18 . 5 , and 0 . 82 μM/s for 200 nM CtpS , 4 μM CtpS , and 200 nM CtpSE277R , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 02610 . 7554/eLife . 03638 . 027Figure 7—figure supplement 3 . Growth curve comparing wild type to the defective growth of CtpSE277R mutant E . coli in minimal media . CtpSE277R mutants exhibit defective growth .", "Error bars = SE , n = 36 . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 02710 . 7554/eLife . 03638 . 028Figure 7—figure supplement 4 . CtpS protein levels are not depleted in the CtpSE277R mutant .", "( A ) Immunoblot probing CtpS and Crl ( loading control ) levels in NCM3722 kanR and CtpSE277R cells after the addition of 200 µg/ml .", "( B ) Relative intensity of CtpS normalized to Crl levels . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 028 We next determined the impact of the E277R polymerization interface mutation on CtpS activity .", "At the lowest protein concentration tested , CtpSE277R exhibited slightly reduced CTP production ( 71% of wild type maximal activity ) compared to the wild type protein ( Figure 7A ) .", "To determine if the polymerization defect of CtpSE277R was due to impaired large-scale assembly or reduced CTP production , we used EM to examine its polymerization in the presence of saturating CTP levels .", "CtpSE277R did not polymerize in the presence of high levels of CTP ( Figure 7B ) .", "We thus conclude that CtpSE277R impairs polymerization independently of its effect on activity .", "Whereas CtpSE277R was slightly impaired in its activity at low enzyme concentrations , CtpSE277R exhibited a much higher concentration at which kcat is one half of its maximum due to polymerization ( the [CtpS]0 . 5 value ) compared to wild-type CtpS ( [CtpSE277R]0 . 5 = 7 . 1 μM vs [CtpS]0 . 5 = 3 . 3 μM ) .", "Furthermore , the concentration dependence of CtpSE277R kcat was less steep than wild type , with CtpSE277R retaining 48% of its maximal activity at the highest enzyme concentration tested ( 8 μM ) ( Figure 7A ) .", "This behavior was in stark contrast to wild-type CtpS , whose activity plummeted to 4% of its maximum .", "Thus , at low enzyme concentrations , CtpSE277R exhibited slightly lower activity than wild type while at high enzyme concentrations CtpSE277R activity was significantly greater than that of wild type .", "One explanation for the comparatively modest decrease in CtpSE277R activity as a function of enzyme concentration is that CtpSE277R produces CTP , which at high CtpS concentrations can accumulate and competitively inhibit CtpS activity , resulting in a slight activity decrease .", "However , this mutant lacks the dramatic reduction in CtpS activity mediated by large-scale assembly into filaments .", "As predicted from thermodynamic linkage , the inability to polymerize also leads CtpSE77R to bind CTP less tightly , with a higher IC50 value than the wild-type enzyme ( 830 μM vs 360 μM at 200 nM enzyme , Figure 7—figure supplement 2 ) .", "These data are thus consistent with the model that CtpS is negatively regulated in two ways: CTP competitively inhibits UTP binding , and large-scale assembly sterically hinders a conformational change required for CtpS activity .", "The quantitative differences between wild type and CtpSE277R activity suggest that large-scale assembly mediates rapid and efficient inhibition of enzymatic activity .", "To determine the impact of CtpSE277R on cell physiology , we replaced wild-type CtpS with CtpSE277R at its native locus in E . coli .", "This strain exhibited defective growth compared to wild type in rich ( Figure 7C ) and minimal media ( Figure 7—figure supplement 3 ) .", "Wild type doubling time was 51 min ± 1 . 5 min , while the CtpSE277R doubling time was 130 min ± 11 min in rich media .", "Immunoblotting confirmed that CtpSE277R was expressed at similar levels to wild-type CtpS ( Figure 7—figure supplement 4 ) .", "One possible explanation for the growth impairment is that CtpSE277R could not produce enough CTP to support robust growth .", "However , CTP levels , as measured by mass spectrometry , are not reduced in the CtpSE277R strain ( Figure 8—figure supplement 1 ) .", "In fact , CTP levels are modestly higher in the mutant than in wild type cells ( 1 . 6 ± 0 . 3-fold higher ) .", "Because average CTP levels are higher in these cells , CtpSE277R likely does not impair growth due to reduced CTP production .", "Rather , the elevated CTP levels and the observation that growth became particularly affected at mid-log phase support the hypothesis that the CtpSE277R mutant is defective in regulating CTP levels when adapting to changes in the cellular environment .", "Replacing wild-type CtpS with CtpSE277R also affected levels of other nucleotides and their precursors or byproducts ( Figure 8A , Figure 8—figure supplement 1 ) .", "For example , the amount of the pyrimidine precursor orotate was 2 . 3 ± 0 . 5-fold reduced in the mutant , consistent with the idea that CtpSE277R is hyperactive and increases CTP production at the expense of its precursors .", "Together , these data indicate that disrupting the CtpS polymerization interface does not deplete CtpS or CTP .", "Instead , we hypothesize that CtpSE277R perturbs E . coli growth by disregulating nucleotide metabolism in a manner consistent with hyperactivating CtpS by disrupting a negative regulatory mechanism .", "These data are consistent with the observation that at the cellular concentration of CtpS , CtpSE277R is more active than the wild-type enzyme . 10 . 7554/eLife . 03638 . 029Figure 8 . Mutation of polymerization interface disrupts CTP homeostasis in vivo .", "( A ) Metabolic profiling of wild type and CtpSE277R mutant cells after addition of cytidine to minimal media .", "Nucleotide biosynthesis molecules are shown .", "( B ) Incorporation of C13-label into CTP pool in wild-type and CtpSE277R mutant cells .", "Incorporation occurs at similar levels in both strains .", "Error bars = SE , n = 3 . ( C ) The proportion of unlabeled ( C12 ) CTP in wild-type and CtpSE277R mutant cells .", "The ratio of C12-CTP to total CTP is higher in the CtpSE277R strain .", "Error bars = SE , n = 3 . ( D ) Model of the fraction of active ( nonpolymerized and UTP-bound ) CtpS , plotted vs CTP concentration .", "Comparison is shown between competitive inhibition with polymerization , noncompetitive inhibition with polymerization , competitive-nonpolymerizing , and noncompetitive-nonpolymerizing mechanisms .", "In all cases , we chose a fixed UTP concentration equal to Kcp , the dissociation constant of CTP and polymerized CtpS ( see Supplementary file 1 for details ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 02910 . 7554/eLife . 03638 . 030Figure 8—figure supplement 1 . Metabolomic analysis of wild-type and CtpSE277R E . coli after addition of 200 µg/ml C13-cytidine . Fold changes of metabolite levels of NCM3722 kanR E . coli and CtpSE277R E . coli were compared to wild type levels at 0 min .", "Hierarchical clustering of metabolites is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 03010 . 7554/eLife . 03638 . 031Figure 8—figure supplement 2 . CTP levels probed by mass spectrometry after addition of C13-labeled cytidine to the media . Unlabeled C12-CTP population represents the proportion of CTP synthesized by CtpS from cellular pools of UTP .", "The CtpSE277R has a higher intracellular C12-CTP pool both at the initial time point as well as at the end of the time course , where C12-CTP is almost twice as high as in the wild-type strain . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 03110 . 7554/eLife . 03638 . 032Figure 8—figure supplement 3 . CTP binding enhances polymerization with a sharp response . The concentration required to reduce CtpS-specific activity ( kcat ) to 50% of its maximum value , [CtpS]0 . 5 , is inversely related to the affinity of the polymer for Ctps tetramers .", "In the absence of CTP , the [CtpS]0 . 5 value is 3 . 3 μM ( red circles ) .", "At a CTP concentration near the IC50 value ( 400 μM ) , the [CtpS]0 . 5 value is slightly reduced ( 2 . 8 μM , green squares ) , while at 800 μM , the [CtpS]0 . 5 value significantly shifted towards polymerization ( [CtpS]0 . 5 = 1 . 4 μM , blue open diamonds ) .", "The maximum kcat values before normalization were 6 . 7 , 3 . 5 , and 1 . 04 s−1 for experiments using 0 , 400 , and 800 μM CTP , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03638 . 032 Steady-state measurements of metabolite levels cannot establish whether the observed increase in CTP levels corresponds to a defect in feedback inhibition of CtpS ( as predicted by our model ) or by stimulating CtpS activity in some other way .", "To directly assess feedback inhibition in vivo , we supplemented wild-type CtpS or CtpSE277R with C13-labeled cytidine , which is converted into C13-CTP by the nucleotide salvage pathway that functions independently of CtpS ( Ayengar et al . , 1956; Valentin-Hansen , 1978; Fricke et al . , 1995 ) .", "We note that nucleotide triphosphates cannot be imported into the cell such that we could not supplement with CTP itself .", "Furthermore , the use of C13-cytidine enabled us to use mass spectrometry to distinguish the CTP produced by nucleotide salvage ( C13-CTP ) from the CTP produced de novo by CtpS ( C12-CTP ) .", "We hypothesized that if disruption of CtpS polymerization disrupts negative feedback , then CtpSE277R should maintain high CtpS activity despite the accumulation of C13-CTP from supplementation with C13-cytidine .", "As predicted based on the independence of nucleoside import from nucleotide biosynthesis , the incorporation of C13-label into the CTP pool was similar in the wild type and CtpSE277R strains , indicating that both take up labeled cytidine and convert it into CTP at approximately the same rate ( Figure 8B ) .", "In wild type cells , as the C13-CTP pool increased , the fraction of C12-CTP sharply decreased ( Figure 8C ) .", "Thus , feedback regulation mechanisms compensate for the increased CTP production from cytidine by reducing de novo CTP production by CtpS .", "The decrease in the fraction of unlabeled CTP was less pronounced in the CtpSE277R mutant and by the end of the period assayed , unlabeled CTP levels were almost twofold higher in the CtpSE377R strain than in wild type ( Figure 8—figure supplement 2 ) .", "This result supports our conclusion that CtpSE277R hyperactivates CtpS by disrupting its negative feedback regulation and that this hyperactivation more than compensates for its reduced enzymatic activity .", "Since disruption of just one interaction in the proposed polymerization interface weakened the ability of CtpS to control CTP production even when all other forms of CtpS regulation are unaltered , we predict that any disruption of regions of inter-tetrameric contact , either by changes to the protein sequence or by chemical perturbation , would cause this deleterious regulatory defect .", "What is the benefit of using polymerization as a negative-feedback regulation strategy ?", "To quantitatively assess the impact of polymerization-mediated enzymatic inhibition , we developed a simple mathematical model of CtpS inhibition by CTP-dependent polymerization ( see Supplementary file 1 for details ) .", "A key point of the model is that the concentration of CtpS needed for polymerization depends on the free energy of polymerization , which in turn depends on the UTP and CTP concentrations .", "One mechanism for how CTP induces reversible polymerization is by CTP binding more favorably to the filament than to the free tetramer .", "This model leads to two predictions dictated by thermodynamic linkage: ( 1 ) CTP should be a more effective inhibitor at CtpS concentrations that favor polymer formation , and ( 2 ) the presence of CTP should enhance polymer formation and the reduction in CtpS specific activity ( kcat ) as CtpS concentration increases .", "Indeed , at 4 μM CtpS , near the concentration at which CtpS kcat is one half of its maximum due to polymerization ( [CtpS]0 . 5 , 3 . 3 μM , Figure 7A ) , the CTP IC50 value is reduced to 170 μM , compared to 360 μM at 200 nM enzyme ( Figure 7—figure supplement 2 ) .", "Conversely , in the presence of 800 μM CTP , the [CtpS0 . 5] value is 1 . 4 μM , reduced by more than half compared to that with no CTP ( Figure 7—figure supplement 3 ) .", "Interestingly , the presence of 400 μM CTP has only a small effect ( [CtpS]0 . 5 = 2 . 8 μM ) , suggesting an ultrasensitive response of polymerization to CTP levels .", "Another result of this polymerization-based mechanism is that the cooperativity of CTP-mediated inhibition increases as a function of the nucleation barrier to polymerization .", "Experimentally , the abundance of long polymers in vitro ( Figure 1B ) and the small number of polymers per cell in vivo ( Ingerson-Mahar et al . , 2010 ) suggest that CtpS polymerization exhibits a significant nucleation barrier .", "The conformational differences between the free and filament forms of CtpS ( Figure 4 ) may play a role in establishing this barrier .", "This barrier could result from the free energy change required to take the CtpS tetramer from a flexible ‘free’ state to more rigid ‘filament’ state upon the first assembly step of the polymer .", "Alternatively , dimerization of ‘free’ CtpS tetramers could allosterically influence one another to adopt the ‘filament’ conformation in a manner similar to one proposed for the cooperative polymerization of FtsZ ( Miraldi et al . , 2008 ) .", "Our mathematical model enables us to estimate this nucleation barrier from the average polymer length , yielding a value of order 9 kBT , where kBT is the thermal energy .", "Moreover , it demonstrates that coupling activity to polymerization with such a significant nucleation barrier represents a mechanism for generating extremely sharp transitions in enzyme activity .", "We compared the sharpness of enzyme inhibition in our novel polymerization-based mechanism to that of previously characterized mechanisms of enzyme inhibition such as competitive and allosteric inhibition ( Figure 8D; Supplementary file 1 ) .", "We found that , among the mechanisms examined , the ones involving polymerization-based negative feedback yield the sharpest decrease in enzyme activity when CTP levels are increased , thereby enabling tight regulation of CTP production by CTP levels .", "Our estimate based on average CtpS filament length of the value of the nucleation energy yields extremely sharp transitions ( see Figure 8D , where this estimate was used , and our discussion of response coefficients in Supplementary file 1 ) .", "This sharpness is apparent in comparing the concentration dependences of CtpS specific activity in the presence of CTP .", "The CTPS0 . 5 value at 400 μM CTP is slightly shifted compared to no CTP .", "At 800 μM , the CtpS0 . 5 value is substantially decreased and the curvature more concave ( Figure 8—figure supplement 3 ) .", "Because the onset of the decrease of activity can become arbitrarily sharp as the nucleation energy is increased , polymerization-mediated regulation is fundamentally different from the case of fixed stoichiometry enzyme oligomers , such as hemoglobin , that cooperatively bind an inhibitor .", "Another crucial difference with respect to such simple cooperative inhibition is that the polymerization-based mechanism also mediates negative feedback on CtpS activity from CtpS levels ( Supplementary file 1 ) .", "Hence , this mechanism uniquely enables ultrasensitive regulation of CtpS activity by both CTP and CtpS concentrations .", "Additionally , sequestering CtpS tetramers into the inactive filament ensures the availability of a CtpS pool that can be rapidly reactivated , limited only by the polymer disassembly rate .", "Our biochemical data confirm that depolymerization and subsequent repolymerization can occur within seconds ( Figure 1E ) , while investigation of the in vivo kinetics of CtpS filament assembly and disassembly presents an interesting subject for future study ." ], [ "With so many regulatory strategies in place , why add another ?", "First , layering multiple levels of regulation results in robust regulatory control with a series of fail-safes that protects the cell from disregulated nucleotide levels .", "CtpS is a key node in nucleotide metabolism because it binds ATP , UTP , CTP , and GTP .", "We propose that strict regulation of nucleotide levels is so critical to controlled growth and division that CtpS evolved as a master switch to integrate information about nucleotide abundances and maintain their proper levels and proportions .", "Nucleotide biosynthesis is both energetically costly and controls the availability of raw materials for replication , transcription , and other biosynthetic pathways .", "Thus , coordinating biomass accumulation and cellular proliferation requires extremely tight control of nucleotide levels via CtpS that no one regulatory mechanism could achieve on its own .", "The need for such tight regulation could also explain recent observations that small CtpS polymers can combine to form higher-order larger structures ( Gou et al . , 2014 ) and can co-localize with other proteins involved in nucleotide metabolism ( reviewed in Carcamo et al . , 2014 ) .", "The second advantage of employing multiple types of regulation is that each regulatory strategy has distinct kinetics that together enables regulation over a wide range of potential conditions .", "For example , transcriptional regulation is slow in comparison to regulation by ligand binding .", "Competitive or allosteric regulation by ligand binding can be cooperative if the enzymes form oligomers , as in the case of hemoglobin ( Perutz , 1989 ) .", "However , the amassed activity of such oligomers is strictly linear with respect to protein concentration .", "By contrast , our modeling indicates that coupling activity to ligand-induced polymerization is a simple mechanism for promoting cooperativity with respect to protein concentration , while at the same time maintaining cooperativity with respect to ligand binding .", "An added benefit of polymerization-mediated inhibition is that it enables cells to sequester CtpS in an activity-primed tetramer state such that CtpS can be rapidly reactivated in a manner limited only by enzyme depolymerization ( Figure 9 ) .", "Previous models for enzyme sequestration have relied on the idea of preventing substrate binding ( e . g . , [Jackson-Fisher et al . , 1999; Michaelis and Gitai , 2010] ) .", "Here , we propose an alternate mechanism for sequestration where the active sites can readily access substrates but conformational changes required for activity are restricted .", "While our data are consistent with the model of cooperative regulation by assembly , experimental noise and nonlinearities limit the current ability to measure the extent of that cooperativity , raising the possibility that there are yet more undiscovered features of CtpS regulation .", "As methods for manipulating and monitoring nucleotide levels become more available , it will also be interesting to determine the kinetics of the various CtpS regulatory mechanisms in vivo .", "Though we have only tested the E . coli CtpS enzyme , we hypothesize that other prokaryotic and eukaryotic CtpS proteins may be subject to inhibition by polymerization .", "Caulobacter crescentus CtpS disassembles in the presence of DON while Saccharomyces cerevisiae CtpS shows longer filaments when cells were exposed to additional CTP ( Ingerson-Mahar et al . , 2010; Noree et al . , 2010 ) .", "The linker region implicated in E . coli CtpS polymerization is also mutated in three independent human lung carcinoma samples ( Forbes et al . , 2008 ) , suggesting that metabolic regulation by CtpS polymerization is important for limiting human cell proliferation .", "In the future , it will be interesting to determine if other enzymes employ polymerization-mediated regulatory strategies .", "In particular , we predict that enzymes that function at key metabolic nodes would most benefit from the ultrasensitive regulation provided by polymerization .", "Such cooperative assembly can coordinate the mobilization or sequestration of functional units , thereby dynamically altering the level of active enzyme without altering the overall enzyme concentration .", "The ultrasensitive kinetics of this transition would allow cells to rapidly respond to short-term changes in their environment or metabolic needs .", "For example , immediately following cell division , daughter cells could depolymerize any CtpS filaments inherited to compensate for reduced CtpS concentrations ( perhaps from unequal partitioning ) faster than translating and folding new proteins .", "The rapid kinetics of polymerization could sequester CtpS when CTP is plentiful to prevent futile biosynthesis .", "A handful of other metabolic enzymes have been shown to form filamentous or large scale structures in vitro and in vivo ( Barry and Gitai , 2011 ) .", "CtpS may thus emerge as a model for a larger class of enzymes that are regulated by higher-order assembly to achieve cooperative enzyme activation or inactivation .", "Large-scale polymers such as cytoskeletal filaments play an essential role in organizing the cell .", "But how did such cytoskeletal polymers evolve ?", "Our findings suggest that the selective benefit conferred by improving enzymatic regulation may have led to the evolution of large-scale filaments .", "Once present , these enzymatic polymers could then be appropriated for the structural functions commonly associated with the cytoskeleton .", "Finally , gene duplication and divergence would enable uncoupling and specialization of the enzymatic and structural properties of these proteins ( Barry and Gitai , 2011 ) .", "The observation that CtpS polymerization is conserved among diverse prokaryotes and eukaryotes supports the hypothesis that CtpS polymerization arose in an early common ancestor and is a key feature of CtpS regulation .", "An example of appropriating an enzymatic polymer for structural functions comes from C . crescentus , where CtpS filaments regulate cell shape in a manner that can be uncoupled from their enzymatic activity ( Ingerson-Mahar et al . , 2010 ) .", "While the enzymatic activity and polymerization capacity of CtpS is universally conserved , its cell shape function appears to be species specific .", "Thus , polymerization appears to have evolved early to regulate enzymatic activity while CtpS polymers were only later adapted for a structural role .", "A similar evolutionary path could explain the structural similarity between hexokinase enzymes and the actin family of cytoskeletal elements ( Holm and Sander , 1993; van den Ent et al . , 2001 ) .", "Specifically , we hypothesize that actin and hexokinase may have shared a common ancestor that , like CtpS , evolved polymerization as a regulatory mechanism .", "Gene duplication and divergence may have subsequently enabled actin to specialize as a structural element , while additional layers of enzymatic regulation may have obviated the need for hexokinase assembly ( mammalian hexokinase does not polymerize ) .", "In this way , CtpS assembly and regulation may provide insight into the origins of the intracellular structural network that became the modern cytoskeleton ." ], [ "StrainDescriptionReferenceZG247NCM3722 ( Soupene et al . , 2003 ) ZG1075pyrG-His in BL21 * ( DE3 ) ( Ingerson-Mahar et al . , 2010 ) ZG1076pyrGE155K-His in BL21 * ( DE3 ) This study . ZG1077pyrGE277R-His in BL21 * ( DE3 ) This study . ZG1082mCherry-CtpS in NCM3722 ( Ingerson-Mahar et al . , 2010 ) ZG1083mCherry-CtpSE277R in NCM3722This study . ZG1084mCherry-CtpSF281R in NCM3722This study . ZG1085mCherry-CtpSN285D in NCM3722This study . ZG1086mCherry-CtpSE289R in NCM3722This study . ZG1168CtpSE277R-kanR chromosomal integrant in NCM3722This study . ZG1169WT-kanR chromosomal integrant in NCM3722This study .", "Wild-type CtpS was purified as described previously ( Ingerson-Mahar et al . , 2010 ) .", "CtpS-E155K and CtpS-E227R were purified as described previously with the exception that the 6XHis affinity tag was not cleaved in these cases .", "Similar treatment of the wild-type protein proved indistinguishable from the cleaved sample .", "Purified CtpS protein was incubated at 37°C for 20 min in 50 mM Tris HCl ( pH 7 . 8 ) , 10 mM MgCl2 , 1 mM UTP , 1 mM ATP , and 0 . 2 mM GTP to allow tetramer formation .", "CTP production was initiated by the addition of 10 mM glutamine to create a full activity buffer ( referred to in text at ‘activity buffer’ ) ( Ingerson-Mahar et al . , 2010 ) immediately prior to recording of sample measurements .", "Time between glutamine addition and initiation of sample recording averaged 5 s was based on the amount of time required to load the sample .", "Reaction was monitored at 37°C for 5 min in a QuantaMaster 40 Fluorometer ( Photon Technology International , Birmingham , NJ ) equipped with photo multiplier tubes for both scattering and transmittance .", "Right angle light scattering at 405 nm with a 1 mM slit width detected polymerization , and transmittance at 291 nm with a 0 . 25-mM slit width detected CTP production with both values reported in arbitrary units .", "Reactions were performed in 150 μl samples .", "Polymerization was monitored for 3 min unless otherwise noted .", "Detection of light scattering and transmittance alternated with an integration time of 1 s .", "CTP production velocity ( kcat , μmol/s ) was determined for the first 30 s of the reaction .", "CTP production was normalized by the concentration of CtpS enzyme in each sample .", "Due to the fluorometer assay's use of transmittance and a photon multiplier , we compared data collected to data collected over the same concentration range on a more traditional spectrophotometer setup in the Baldwin lab .", "Comparison yielded the presence of a scaling factor to be applied to the fluorometer data set to yield kcat ranges consistent with published data .", "Data were scaled to yield the same maximal kcat value for both data sets .", "The fold-change in activity over the concentrations was similar between the data sets .", "Overlay of the data are shown in Figure 1—figure supplement 7 .", "Quantification of polymerization was calculated using the difference between the average initial and final values of light scattering for each sample ( n = 5 for average ) in Figures 1B and 2A and Figure 1—figure supplement 1 , Figure 2—figure supplements 1 and 2 , and Figure 7—figure supplement 1 .", "All other light scattering values are the actual values of light scattering recorded ( in arbitrary units ) , except where noted in the figure legends .", "Enzyme concentration was determined using the extinction coefficient for CtpS , 0 . 055 μM/A280 unit .", "Concentrated enzyme ( 40–80 μM ) was annealed at room temperature for 3 min at 21°C in 10 mM MgCl2 , 60 mM HEPES pH 8 . 0 , then mixed with 1 . 5 mM ATP and 600 μM UTP and incubated for 20 min at 37°C .", "4-min incubations with substrates gave equivalent results .", "When CTP was present , it was included in the ATP/UTP mixture .", "The reactions were initiated by mixing with 10 mM final glutamine and the absorbance at 291 nm measured .", "It was not possible to measure the rates of 277R above 8000 nM ( 19 µM/s ) because the rate could not be reliably measured considering the dead time of the instrument and the procedure ( ∼5 s ) .", "The final reactions contain 0 . 1–25 mM NaCl from the enzyme storage stocks , but these concentrations of NaCl do not have noticeable effects on enzyme rate .", "The annealing step is critical for the highest specific activities from stocks stored frozen or at 4°C and is optimal at concentrations greater than 2 μM .", "From CTP inhibition experiments , the CTP IC50 value at 200 nM CtpSWT , 600 µM UTP , and 1 . 5 mM ATP was 360 μM ( Figure 7—figure supplement 2 ) .", "The concentration-dependences were complex and yielded curved Hill plots .", "IC50 values were obtained by linear extrapolation using points flanking vi = 1/2vo .", "Graphical data points represent the averaged values of 2–6 experiments with error bars indicating the standard error or standard deviation of each measurement .", "Purified CtpS protein was incubated at 37°C for 20 min in 50 mM Tris HCl ( pH 7 . 8 ) and 10 mM MgCl2 .", "1 mM CTP ( Epicentre . Madison , WI ) was added immediately before the sample was loaded into the fluorometer .", "Time between CTP addition and initiation of sample recording averaged 5 s .", "Measurements were taken as described for the activity/polymerization assay .", "Purified CtpS protein was incubated in the activity buffer or CTP buffer ( 1 mM CTP , 10 mM MgCl2 , 50 mM Tris–HCl [pH 7 . 8] ) at 37°C for 1 hr .", "Samples were centrifuged at 116 , 000×g for 15 min at 4°C using an Optima TLA 100 rotor ( Beckman , Indianapolis , IN ) .", "After centrifugation , the supernatant was removed .", "For activity assays , the pellet was resuspended in 100 μl ice cold buffer containing 50 mM Tris HCL ( pH 7 . 8 ) and 10 mM MgCl2 .", "10 μl of this CtpS pellet solution was added to complete activity buffer containing 50 mM Tris HCl ( pH 7 . 8 ) , 10 mM MgCl2 , 1 mM UTP , 1 mM ATP , 0 . 2 mM GTP , and 10 mM glutamine to monitor initial activity .", "Wild-type NCM3722 was grown to early exponential phase in M9 minimal media plus 0 . 04% glucose ( M9G ) .", "Native levels of CtpS were quantified based on a standard curve of purified CtpS and normalized based on the OD600 of the culture .", "Calculations assume 1 OD unit = 8 × 108 cells and cellular volume = 1 μm3 .", "Samples were loaded on a 10% Tris-glycine SDS PAGE gel .", "Membrane was probed with 1:15 , 000 rabbit anti-CtpS .", "Band intensities were compared using ImageJ .", "For quantification of CtpS pelleting in variable CTP , 130 μg CtpS was incubated in 500 μl appropriate concentrations of CTP buffer ( 4 . 3 μM CtpS ) .", "200 μl samples were spun at 116 , 000×g on a Beckman TLA-100 rotor for 30 min at 4°C .", "The pellet fraction was resuspended in 50 µl SDS-PAGE sample buffer .", "Samples were loaded on a 10% Tris-glycine SDS PAGE gel .", "Membrane was probed with 1:15 , 000 rabbit anti-CtpS .", "Band intensities were compared using ImageJ .", "Defocus parameters for each micrograph were determined with CTFFIND ( Mindell and Grigorieff , 2003 ) .", "CTF correction was achieved by applying a Wiener filter to the entire micrograph .", "Lengths of helix were defined in the boxer program of the EMAN software suite ( Ludtke et al . , 1999 ) .", "Overlapping segments were extracted from the CTF-corrected micrographs along the length of each helix .", "In total , 12 , 465 overlapping segments were extracted in 510 × 510 Å boxes , representing approximately 56 , 000 unique CtpS monomers .", "Segments were binned twofold prior to reconstruction , at a final pixel size of 1 . 64 Å .", "Iterative helical real space reconstruction ( IHRSR ) was performed essentially as described by Egelman ( 2007 ) and Sachse et al . ( 2007 ) , using SPIDER ( Frank , 1996 ) for projection matching and back projection , and hsearch_lorentz ( Egelman , 2000 ) for refinement of helical symmetry parameters .", "A cylinder was used as the initial reference volume , and 30 rounds of iterative refinement were carried out at increasingly smaller angular increments ( 1 . 5° in the final round ) .", "A preliminary reconstruction was performed imposing only helical symmetry , from which it was clear that the repeating helical subunit was the CtpS tetramer; in subsequent runs of IHRSR the local 2-2-2 point group symmetry of the CtpS tetramer was also enforced .", "Visualization of the cryo-EM reconstructions and rigid body fitting of the CtpS crystal structure into the EM map were performed in Chimera ( Pettersen et al . , 2004 ) .", "The CtpS crystal structure monomer was initially fit as a single rigid body into the EM map , followed by local refinement of the fit treating the two domains and linker region as three separate rigid bodies .", "The final EM map was amplitude corrected using amplitudes from the atomic model .", "Site-directed mutagenesis was performed using the QuickChange ( Agilent , Santa Clara , CA ) system with minor modifications to enable using KOD polymerase ( Millipore , Billerica , MA ) or GXL polymerase ( Takara , Mountain View , CA ) .", "Strains were grown overnight in LB with 50 µg/ml carbenicillin , subcultured , and grown until early exponential phase .", "Fluorescent protein expression was induced with 0 . 01 mM IPTG for 2–3 hr .", "Cells were immobilized on 1% agarose in water pads containing 0 . 01 mM IPTG .", "Imaging was performed using a Nikon ( Melville , NY ) TI-E microscope using a 100X Nikon Plan Apo objective ( NA = 1 . 4 ) , Chroma ET572/35X ( excitation ) and ET622/60M ( emission ) , Prior Lumen 200 Pro illumination , and 89014VS dichroic mirro .", "Images were acquired with an Andor Clara camera using NIS-Elements software .", "PCR fragments of the region from mazG to ygcG either containing a wild-type pyrG or pyrGE277R coding region and a kanamycin resistance cassette between eno and ygcG were integrated into the NCM3722 chromosome by Lamda red recombination .", "Recombineered cells were recovered on LB agar with 50 µg/ml kanamycin and 200 µg/ml cytidine .", "Strains were grown overnight in LB containing 30 µg/ml kanamycin and 200 µg/ml cytidine .", "Then cells were diluted to the same OD in 100 µl LB plus kanamycin or M9G plus kanamycin ( as noted ) in a 96-well format .", "OD600 was recorded using a BioTek ( Winooski , VT ) microplate reader at 37°C with continuous shaking .", "Strains were grown in M9 minimal media to early exponential phase .", "Media were supplemented with 13C5-ribose-labeled cytidine ( Cambridge Isotopes , Tewksbury , MA ) to a final concentration of 200 µg/ml , and cell growth was continued at 37°C .", "Sample preparations were modified based on Lu et al . ( 2007 ) .", "Specifically , 24 milliliters of bacterial cultures were harvested by centrifugation at room temperature at five time points following cytidine addition: 0 min , 5 min , 20 min , 60 min , and 120 min .", "The pellet was resuspended in 1 ml 40:40:20 methanol:acetonitrile:water quenching buffer and allowed to sit on dry ice for 15 min .", "Sample was spun at maximum speed in a microcentrifuge for 5 min at 4°C .", "Then the resulting pellet was resuspended again in 0 . 6 ml fresh 40:40:20 solution for 15 min on dry ice and then spun as before to quench and extract metabolites a second time .", "Quenching buffer supernatants were combined and concentrated threefold for mass spectrometry as in Xu et al . ( 2012 ) .", "The cryo-EM map of the CtpS filament has been deposited with the Electron Microscopy Data Bank [EMDB] accession number EMD-2700 ." ] ]
[ "CTP Synthetase ( CtpS ) is a universally conserved and essential metabolic enzyme .", "While many enzymes form small oligomers , CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes .", "By simultaneously monitoring CtpS polymerization and enzymatic activity , we show that polymerization inhibits activity , and CtpS's product , CTP , induces assembly .", "To understand how assembly inhibits activity , we used electron microscopy to define the structure of CtpS polymers .", "This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity .", "Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation .", "This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E . coli growth and metabolic regulation without reducing CTP levels .", "We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable ." ]
[ "Enzymes are proteins that perform reactions that can convert one or more chemicals ( the substrates ) into others ( the products ) .", "The rate at which an enzyme produces its product is often carefully regulated .", "Some molecules slow or stop an enzyme by binding to and blocking the site where its substrates normally bind: its ‘active site’ .", "Other molecules can also bind to sites other than the active site , which can cause the enzyme to become either more or less active .", "Almost all living things have an enzyme called CTP synthetase that makes one of the building blocks that is used to build DNA and a similar molecule called RNA .", "This enzyme converts a molecule called uridine triphosphate ( or UTP ) into another called cytidine triphosphate ( CTP ) : a reaction that is powered by breaking down molecules of adenosine triphosphate ( ATP ) .", "The amount of CTP synthetase made by a cell is carefully controlled .", "The enzyme's activity is also regulated by the levels of UTP and CTP , and by another molecule ( called GTP ) that binds to a site outside of its active site .", "Four copies of the CTP synthetase protein must work together before this enzyme can turn UTP into CTP .", "The enzyme also forms much larger aggregates , or polymers; however , it is not clear what causes these polymers to form , or what they do in a cell .", "Barry et al . have now discovered that CTP synthetase is almost completely inactivated when these polymers are formed .", "Furthermore , CTP encourages the polymers to form , whilst UTP and ATP cause them to disassemble .", "Therefore , this enzyme is least active when there is excess product in the cell , and most active when its substrates are plentiful .", "By determining the three-dimensional structure of a CTP synthetase polymer , Barry et al . reveal that although CTP is bound to the enzymes , their active sites are still freely accessible .", "However , the enzymes in the polymer appear to be locked into a shape that makes them unable to carry out their function .", "When Barry et al . then mutated the enzyme so that it was unable to form polymers it was also no longer inactivated in the same way by CTP .", "Bacterial cells with only these mutant versions of CTP synthetase are unable to properly control their levels of CTP .", "This suggests that polymer formation is important for regulating this enzyme in response to a build up of its product .", "Further work is needed to see whether the regulation of CTP synthetase activity by forming polymers is specific to this enzyme or a widespread mechanism that is used to control other enzymes too ." ]
2014
[ "Introduction", "Results and discussion", "Materials and methods" ]
[ "ecology" ]
Sequestration and activation of plant toxins protect the western corn rootworm from enemies at multiple trophic levels
elife-29307-v1
[ [ "The growth and reproduction of herbivores is constrained by both plant quality and predation by higher trophic levels ( Hunter et al . , 1997; Ode , 2006 ) .", "Certain herbivores have found a way out of this quandary by redirecting plant defenses against their own predators: By ingesting and accumulating plant toxins , a phenomenon referred to as sequestration , herbivores may make themselves unattractive or toxic to natural enemies ( Nishida , 2002; Petschenka and Agrawal , 2016 ) .", "The resulting transfer of plant toxins across three trophic levels is increasingly recognized as a powerful force that shapes the distribution and abundance of plants , herbivores and predators in natural and agricultural ecosystems ( Kumar et al . , 2014; Hopkins et al . , 2009 ) .", "Many plants store non-toxic forms of plant defenses ( so called protoxins ) separately from the enzymes that activate them and only form the toxins upon tissue disruption when protoxin and activating enzyme come together ( Hopkins et al . , 2009; Wouters et al . , 2016 ) .", "Some sequestering herbivores in turn have evolved the ability to phenocopy these two-component defense systems by stabilizing and sequestering the protoxins and producing their own activating enzymes to release the toxins in a controlled fashion ( Jones et al . , 2001; Beran et al . , 2014; Zagrobelny and Møller , 2011; Kazana et al . , 2007; Francis et al . , 2002; Opitz and Müller , 2009; Müller et al . , 2001; Pontoppidan et al . , 2001; Jones et al . , 2002 ) .", "Although much progress has been made in the identification of the molecular mechanisms involved in phenocopying such two-component defense systems ( Beran et al . , 2014; Opitz and Müller , 2009; Bridges et al . , 2002; Hartmann et al . , 1997 ) , we know surprisingly little about their actual function in defense .", "It remains for instance unclear whether re-activation is required for herbivore protection and which predators are targeted ( Beran et al . , 2014; Zagrobelny and Møller , 2011; Kazana et al . , 2007; Bridges et al . , 2002; Hartmann et al . , 1997; Zagrobelny et al . , 2007; Pratt et al . , 2008; Beran et al . , 2011; Agrawal et al . , 2012 ) .", "Testing whether two-component systems indeed protect herbivores against predators is necessary to place them in an adequate ecological and evolutionary context .", "After being taken up by herbivores , plant defense metabolites may not only influence herbivore predators and parasitoids ( Ode , 2006 ) , but also higher trophic levels ( van Nouhuys et al . , 2012; Harvey et al . , 2007 ) .", "Effects extending to four trophic levels may be particularly likely in systems where the third and the fourth trophic level are intimately linked .", "Entomopathogenic nematodes ( EPNs ) for instance feed on insect-killing symbiotic bacteria which they inject into their herbivore hosts ( Kaya and Gaugler , 1993 ) .", "So far , the specific effects of sequestered plant defenses on the fourth trophic level remain unknown .", "In contrast to aboveground herbivores , very little is known about plant toxin sequestration and the prevalence of two-component defense systems in belowground herbivores .", "Root feeders are among the most important agricultural pests and can have significant impacts on the abundance and distribution of other species , including leaf feeders ( Blossey and Hunt-Joshi , 2003 ) .", "The two milkweed beetle larvae Tetraopes tetraophthalmus and T . texanus were recently found to accumulate cardenolides from their host plants .", "Sequestered cardenolide concentrations did not correlate with resistance to EPNs ( Ali and Agrawal , 2017 ) .", "Furthermore , the larvae and adults of the spotted cucumber beetle Diabrotica undecimpunctata accumulate cucurbitacin triterpenes from their host plants ( Tallamy et al . , 1998 ) .", "Cucurbitacins are passed on to the eggs and can protect the latter against pathogenic fungi ( Tallamy et al . , 1998 ) .", "Whether cucurbitacins protect D . undecimpunctata larvae against predators ( Gould and Massey , 1984 ) and EPNs ( Barbercheck et al . , 1995 ) is unclear .", "In this study , we investigated how the western corn rootworm Diabrotica virgifera virgifera ( D . virgifera ) deals with the major defensive metabolites of maize .", "Diabrotica virgifera is among the most damaging pest insects on this planet .", "Its larvae develop exclusively on maize roots and cause over $2 billion worth of damage every year in the United States alone ( Gray et al . , 2009 ) .", "Earlier studies found that the hemolymph of the larvae is repellent to a wide variety of predators ( Lundgren et al . , 2009; Welch and Lundgren , 2014 ) , which may contribute to the limited success of biological control programs against D . virgifera ( Gray et al . , 2009 ) .", "Our own work revealed that the larvae of the western corn rootworm are fully tolerant to benzoxazinoids ( BXs ) ( Robert et al . , 2012a ) , a dominant class of secondary metabolites in maize that provides broad spectrum resistance against a variety of other pests and diseases ( Wouters et al . , 2016 ) .", "We also found that the larvae are attracted by BXs and use them to navigate the rhizosphere and locate the most nutritious maize roots ( Robert et al . , 2012a ) .", "Based on these observations , we hypothesized that D . virgifera may be able to sequester BXs , and that these compounds may be the elusive repellent factor that renders D . virgifera repellent to predators .", "BXs function as classical two-component defenses , with the pro-toxins being stored in glycosylated form in the vacuoles of maize cells , the activating β-glucosidases being present in the cytosol and BX hydrolysis occurring upon tissue disruption ( Jonczyk et al . , 2008 ) .", "We therefore also focused on understanding if and how the western corn rootworm stabilizes and re-activates these compounds for self-defense .", "EPNs are among the major natural enemies of western corn rootworm larvae and have been proposed as promising biocontrol agents to control the pest ( Kurtz et al . , 2007 ) .", "Because of the intricate relationship between EPNs and their symbiotic bacteria ( Kaya and Gaugler , 1993 ) , we studied the impact of BX accumulation on both organisms ." ], [ "Metabolite analysis revealed that maize-fed D . virgifera larvae accumulated significant amounts of BXs in their body ( Figure 1A ) .", "The highest concentrations were found for the glucosides HDMBOA-Glc ( >100 µg/g FM ) and MBOA-Glc ( >25 µg/g FM ) .", "In contrast to D . virgifera , two generalist root feeders of the same genus , D . balteata and D . undecimpunctata , accumulated lower amounts of BXs ( Figure 1A ) .", "In North and Central America , maize is attacked by many different Diabrotica species ( Szalanski et al . , 2000 ) .", "Yet , only D . virgifera is fully specialized on maize and causes substantial yield losses ( Chiang , 1973 ) .", "Our experiments suggest that this specialization might be associated with the selective accumulation of BXs .", "BX screening of different larval tissues showed that D . virgifera accumulates HDMBOA-Glc and MBOA-Glc predominantly in the hemolymph ( Figure 1B ) .", "We also detected HDMBOA-Glc and MBOA-Glc on the exoskeleton , with estimated release rates of 3–6 ng/hr and larvae ( Figure 1B ) .", "MBOA-Glc was predominant in the frass , at an average concentration of 11 . 4 mg*g FM−1 .", "Interestingly , the concentration of sequestered HDMBOA-Glc and MBOA-Glc varied between experiments , suggesting a possible impact of environmental conditions and/or small differences in larval age on sequestration patterns .", "A comparison of BX concentrations in maize roots and D . virgifera larvae that fed on these roots from hatching until third instar revealed that most larval BX levels mirror plant BX levels , with the exception of HDMBOA-Glc and MBOA-Glc .", "Although the BX abundance in the larvae were reduced by approximately 95% , HDMBOA-Glc levels were reduced by only 50% and MBOA-Glc was exclusively found in D . virgifera ( Figure 1C ) .", "Feeding D . virgifera larvae on BX-free igl bx1 double mutant plants ( Ahmad et al . , 2011 ) led to the complete absence of BXs in the larvae ( Figure 1—figure supplement 1 ) , including MBOA-Glc , demonstrating that BXs in D . virgifera are plant-derived .", "By contrast , feeding on bx1 mutant plants which show a 90% reduction in BX levels ( Maag et al . , 2016 ) still resulted in a significant , albeit lower accumulation of HDMBOA-Glc in D . virgifera larvae ( Figure 1—figure supplement 2 ) .", "HDMBOA-Glc release from the exoskeleton did not differ between wild type ( WT ) B73 and bx1 mutant fed larvae , while MBOA-Glc release was significantly reduced ( Figure 1—figure supplement 3 ) .", "The observed accumulation patterns suggest a high degree of structural selectivity regarding uptake and sequestration of BXs by D . virgifera .", "We next investigated the processes which enable D . virgifera to sequester its two main BXs .", "MBOA , a benzoxazolinone-type BX , is a common product of DIMBOA and HDMBOA degradation in insect guts that has negative consequences for insect herbivores ( Wouters et al . , 2016; Glauser et al . , 2011; Maag et al . , 2014; Wouters et al . , 2014 ) .", "When D . virgifera gut extracts were incubated with MBOA , MBOA-Glc was readily formed ( Figure 2A ) , suggesting that D . virgifera is capable of this N-glycosylation reaction as leaf feeding caterpillars are ( Maag et al . , 2016 ) .", "Since MBOA-Glc was not deglycosylated by a root extract ( Figure 2—figure supplement 1 ) , it may represent a stable form of BX that can be absorbed from the gut by D . virgifera without toxic consequences .", "In contrast to MBOA-Glc , HDMBOA-Glc is a common plant BX that is rapidly hydrolyzed by plant-derived β-glucosidases ( Glauser et al . , 2011 ) , and the resulting aglycone is highly unstable ( Nishida , 1994 ) .", "To date , no other maize-feeding herbivore apart from D . virgifera is known to be able to accumulate HDMBOA-Glc ( Wouters et al . , 2016; Glauser et al . , 2011; Maag et al . , 2014; Wouters et al . , 2014 ) .", "To determine if D . virgifera larvae can inhibit HDMBOA-Glc hydrolysis , we incubated gut extracts with HDMBOA-Glc and β-glucosidase and analyzed aliquots of the extracts over time ( 5 , 60 and 180 min ) for BXs .", "HDMBOA-Glc was hydrolyzed both in presence and absence of the gut extracts ( Figure 2B ) .", "To determine if D . virgifera can reglycosylate free HDMBOA , we incubated gut extracts with HDMBOA-Glc , β-glucosidase , and [13C6] UDP-glucose and analyzed aliquots of the extracts over time ( 5 , 60 , 180 min ) .", "No [13C]-labeled HDMBOA-Glc was detected within the 3 hr .", "Thus , our in vitro experiments suggest that HDMBOA-Glc accumulation does not proceed via inhibition of HDMBOA-Glc hydrolysis in the D . virgifera gut nor by reglycosylation of free HDMBOA .", "Alternative strategies for HDMBOA-Glc accumulation in the hemolymph may include rapid transport ( Abdalsamee et al . , 2014 ) or transient stabilization through other chemical modifications ( Wang et al . , 2012 ) .", "Once sequestered , BXs can be of defensive value to herbivores if they can be reactivated .", "When we simulated predator attack by crushing the larvae with forceps , HDMBOA-Glc was rapidly broken down , and the final catabolite MBOA ( Maresh et al . , 2006 ) accumulated at concentrations of >25 µg/g FM ( Figure 2C ) .", "No reduction in MBOA-Glc levels was observed ( Figure 2C ) .", "When D . virgifera larvae were exposed to the EPN Heterorhabditis bacteriophora , MBOA concentrations increased as well , albeit at lower levels ( Figure 2D ) .", "Together , these results show that D . virgifera produces MBOA via HDMBOA-Glc degradation upon predator and EPN attack .", "To understand whether BX glucosides may be activated by EPNs and their endobionts directly , we incubated them with purified HDMBOA-Glc , MBOA-Glc and MBOA .", "Heterorhabditis bacteriophora infective juveniles degraded HDMBOA-Glc to MBOA ( Figure 2E ) , but not MBOA-Glc .", "MBOA was not further converted by H . bacteriophora ( Figure 2E ) .", "The entomopathogenic bacterium , Photorhabdus luminescens , a symbiont of H . bacteriophora , catabolized both HDMBOA-Glc and MBOA-Glc , albeit at very low efficiency ( Figure 2F ) .", "This work shows that the activation of sequestered plant toxins may occur upon contact with predator-derived factors , which may represent an additional route by which stabilized plant toxins can be used as anti-predator defenses by herbivores .", "To investigate whether BX uptake increases D . virgifera resistance to EPNs , larvae that had been feeding on B73 wild type ( WT ) or bx1 mutant plants were exposed to H . bacteriophora infective juveniles .", "Infectivity by EPNs was around 15% on WT-fed D . virgifera .", "On bx1-fed D . virgifera , infectivity increased to 40% ( Figure 3A ) .", "Experiments with BX-deficient bx1 and bx2 mutants in the genetic background W22 ( Tzin et al . , 2015 ) confirmed that WT-fed D . virgifera are significantly more resistant to EPNs than larvae fed on BX-deficient mutants ( Figure 3A ) .", "To further explore the potential of BXs to suppress EPN infectivity , we conducted a series of experiments with H . bacteriophora and its endobiont P . luminescens using pure BXs .", "At physiological doses , pre-exposure to HDMBOA-Glc suppressed H . bacteriophora infectivity toward the non-sequestering D . balteata by 50% ( Figure 3B ) .", "MBOA-Glc and MBOA pre-exposure did not have any significant effect .", "HDMBOA-Glc and MBOA increased H . bacteriophora mortality in vitro by 10% and 20% , while MBOA-Glc had no significant effect ( Figure 3C ) .", "Photorhabdus luminescens growth was inhibited by MBOA starting at concentrations of 25 µg/g and HDMBOA-Glc starting at concentrations of 100 µg/g ( Figure 3D , Figure 3—figure supplement 1 ) .", "Again , MBOA-Glc did not have any effect .", "These experiments show that BX-dependent resistance of D . virgifera against EPNs is associated with strong toxicity of HDMBOA-Glc and MBOA against both the nematode and its endobiontic bacterium .", "Together with the mutant experiments , these data show that sequestered and reactivated BXs protect D . virgifera against predation by the third and the fourth trophic levels .", "As BXs also accumulate on the exoskeleton of D . virgifera ( Figure 1B ) , we hypothesized that they may interfere with EPN host location and preference .", "In choice tests , H . bacteriophora infective juveniles were significantly more attracted to bx1-fed D . virgifera larvae than larvae fed on WT plants ( Figure 4A ) .", "The same pattern was found when aqueous surface extracts of bx1- and WT-fed larvae were compared ( Figure 4A ) .", "Complementing surface extracts of BX-free D . virgifera with MBOA-Glc or a mixture of MBOA-Glc and HDMBOA-Glc at physiological doses significantly reduced their attractiveness for H . bacteriophora ( Figure 4B ) .", "HDMBOA-Glc alone had no effect on EPN attraction .", "Thus , MBOA-Glc reduces the attractiveness of D . virgifera to EPNs .", "Together , the results above show that D . virgifera stores BXs , which it stabilizes and re-activates to disrupt EPN infection at different levels ( Figure 5 ) .", "First , the relatively stable MBOA-Glc is released in the frass and on the exoskeleton as a repellent for host-searching infective juvenile nematodes .", "Second , HDMBOA-Glc is activated to produce MBOA , which reduces the growth of the symbiotic bacteria injected into the haemocoel by EPNs to kill and pre-digest the larvae .", "Third , HDMBOA-Glc and its reactivation products kill EPNs directly and thereby likely reduce the infectiveness of the next generation of emerging infective juveniles .", "By interfering with these different processes , D . virgifera larvae become highly resistant to EPNs .", "D . virgifera larvae are gregarious , and larvae from the same batch of eggs often feed together on the same host plant ( Robert et al . , 2012b ) .", "Reducing the build-up of high EPN densities within the rhizosphere may therefore also protect siblings from infection .", "The capacity to control the toxicity of plant secondary metabolites is crucial to the success of herbivores .", "Storing plant toxins for self-defense may be particularly advantageous , as it allows herbivores to escape bottom-up and top-down controls at the same time ( Kumar et al . , 2014; Erb and Robert , 2016 ) .", "Our work illustrates how a specialized and highly destructive maize pest has evolved the ability to utilize the major toxins of its host plant to escape predation by soil-borne natural enemies .", "This protective effect is achieved through selective stabilization and reactivation of the toxins , which allows the herbivore to target different stages of the infection process of entomopathogenic nematodes and their endobiotic bacteria and thereby suppresses infection rates by over 50% .", "These results advance our basic understanding on the impact of plant chemistry on trophic cascades and provide an explanation for the limited success of biological control programs targeting the western corn rootworm ( Gray et al . , 2009 ) ." ], [ "Maize seeds ( Zea mays L . ) of the variety B73 were provided by Delley Semences et Plantes SA ( Delley , CHE ) .", "The near-isogenic bx1 mutant line in a B73 background was obtained by backcrossing the original bx1 mutant five times into B73 ( Maag et al . , 2016 ) .", "The wild-type W22 , and the two Ds insertion mutant lines bx1::Ds ( gene identifier GRMZM2G085381; Ds , B . W06 . 0775 ) and bx2::Ds ( gene identifier GRMZM2G085661; Ds , I . S07 . 3472 ) ( Tzin et al . , 2015 ) were kindly provided by Georg Jander ( Cornell University , Ithaca , NY; USA ) .", "The igl . bx1 double mutant line 32R was obtained by crossing and backcrossing the two corresponding single mutant lines as described ( Ahmad et al . , 2011 ) .", "Plants were grown in 120-mL plastic pots ( Semadeni , Ostermundigen , CHE ) filled with moist washed sand ( 1–4 mm , Landi Schweiz AG , Dotzigen , CHE ) and a layer of 2 cm commercial soil ( Selmaterra , Bigler Samen AG , Thun , CHE ) .", "Seedlings were grown in greenhouse conditions ( 23 ± 2°C , 60% relative humidity , 16:8 h L/D , and 250 µmol*m−2*s−1 additional light supplied by sodium lamps ) .", "MioPlant Vegetable and Herbal Fertilizer ( Migros , Zürich , Switzerland ) was added every 2 days after plant emergence .", "Twelve-day-old plants were used for the experiments .", "Diabrotica virgifera virgifera ( LeConte ) eggs were generously supplied by USDA-ARS-NCARL , Brookings , SD .", "Diabrotica balteata ( LeConte ) eggs were kindly furnished by Syngenta ( Syngenta Crop Protection AG , CHE ) .", "Diabrotica undecimpunctata howardii ( Barber ) eggs were bought from Crop Characteristics ( Crop Characteristics Inc . , Farmington , MN ) .", "Entomopathogenic nematodes Heterorhabditis bacteriophora were bought from Andermatt Biocontrol ( Grossdietwil , CHE ) .", "The endosymbontic bacterium Photorhabdus luminescens EN01 was kindly provided by Carlos Molina ( E-Nema Gesellschaft für Biotechnologie und Biologische Pflanzenschutz GmbH , Schwentinental , DE ) .", "All samples were randomly allocated to the different treatments .", "Whenever possible , data collection were made blindly .", "Stabilization of MBOA in D . virgifera guts was evaluated in vitro as follows: Third instar larval guts were collected and rinsed with distilled water .", "Five guts were pooled in 50 µL protein buffer containing 50 mM MOPSO ( Acros , BE ) , 5 mM ascorbic acid ( Sigma Aldrich Chemie GmbH , Schnelldorf , DE ) , 5 mM dithiothreitol ( Sigma Aldrich Chemie GmbH ) , 10% ( v/v ) glycerol ( Fluka Chemie GmbH , Buchs , CHE ) , 4% ( w/v ) polyvinylpyrrolidone ( Sigma Aldrich Chemie GmbH ) , 0 , 1% ( v/v ) Tween 20 ( Fluka Chemie GmbH ) at pH 7 to which 0 . 25 mM MBOA ( Sigma Aldrich Chemie ) and 5 mM UDP-Glucose ( Sigma Aldrich Chemie ) were added ( n = 3 ) .", "Controls included", "( i ) boiled guts ( 10 min at 100°C; n = 3 ) ,", "( ii ) no gut ( n = 3 ) or", "( iii ) no MBOA ( n = 3 ) in the buffer .", "Each pool of guts represented one biological replicate .", "All reactions were left at ambient temperature .", "After 10 min , 8 hr and 24 hr , 15 µL reaction solution was aliquoted and immediately mixed with 15 µL 100% MeOH ( Fisher Scientific UK Ltd , Loughborough , UK ) .", "All extracts were vortexed and centrifuged at 14 , 000 rpm for 10 min at 4°C .", "Supernatants were collected for HPLC-MS analyses as described below .", "The potential hydrolysis of HDMBOA-Glc was evaluated by adding purified HDMBOA-Glc to gut extracts .", "Gut extracts were prepared as described above .", "The protein buffer contained 3 µg/mL HDMBOA-Glc and 2 units of almond β-glucosidase ( Sigma Aldrich Chemie ) ( n = 6 ) .", "Controls included", "( i ) HDMBOA-Glc and glucosidase in absence of gut ( n = 11 ) and", "( ii ) HDMBOA-Glc and gut only ( n = 2 ) .", "All reactions were left at ambient temperature .", "After 1 min , 1 hr and 3 hr , 15 µL reaction solution was aliquoted and immediately mixed with 15 µL 100% MeOH ( Fisher Scientific UK Ltd ) .", "All extracts were vortexed and centrifuged at 14 , 000 rpm for 10 min at 4° C . Supernatants were collected for HPLC-MS analyses as described below .", "The proportion of initial HDMBOA-Glc remaining in the extract was calculated for each time point .", "MBOA-Glc and HDMBOA-Glc reactivation by D . virgifera larvae was evaluated in two experiments .", "Firstly , ten third instar D . virgifera larvae were collected and ground in the protein buffer described above ( 100 µL buffer per mg of collected larval tissue; n = 8 ) .", "Aliquots were collected after 0 , 2 , 10 , 30 , 60 and 120 min and mixed with 100% MeOH ( v/v ) .", "The resulting samples were vortexed , centrifuged at 14 , 000 rpm for 10 min at 4°C , and supernatants were used for HPLC-MS analyses .", "Secondly , BX reactivation was evaluated upon EPN infection in vivo .", "Fifty D . virgifera larvae were placed in a petri dish containing moist filter paper and 10 , 000 EPNs for 24 hr .", "Control larvae were placed in similar conditions , but without EPNs .", "Diabrotica .", "virgifera larvae were collected 40 hr after EPN exposure as a preliminary experiment had shown that the EPN endobiont starts growing at around that time .", "Three larvae of the same treatment were pooled together and ground in MeOH: H2O: FA ( 50:50:0 . 5%; 100 µL buffer per mg of collected larval tissue; n control=8 , n EPNexposed=13 ) .", "The obtained extracts were vortexed and centrifuged at 14 , 000 rpm for 10 min at 4°C .", "Supernatants were collected for HPLC-MS analyses as described below .", "A thousand EPNs were placed in 1 mL tap water containing MBOA-Glc ( 2 µg/mL , ncontrol = 10 , nMBOA = 9 ) , HDMBOA-Glc ( 20 µg/mL , ncontrol = 6 , nHDMBOA-Glc = 6 ) or MBOA ( 10 µg/mL , ncontrol = 6 , nMBOA-Glc = 6 ) .", "Controls were in tap water only .", "After 24 hr , 1 mL of MeOH: FA ( 99:1% ) was added to all samples .", "EPN samples were ground using a pellet pestle motor ( Kimble Kontes , Sigma Aldrich Chemie ) for 30 s , vortexed and centrifuged at 14 , 000 rpm for 10 min at 4°C .", "Supernatants were collected for HPLC-MS analyses as described below .", "Standardized inoculums of growing P . luminescens bacteria were placed in 1 mL tap water containing MBOA-Glc ( 15 µg/mL , ncontrol = 3 , nMBOA = 6 ) , HDMBOA-Glc ( 10 µg/mL , ncontrol = 3 , nHDMBOA-Glc = 6 ) or MBOA ( 10 µg/mL , ncontrol = 3 , nMBOA-Glc = 6 ) .", "Controls were in tap water only .", "After 24 hr , 1 mL of MeOH: FA ( 99:1% ) was added to all samples .", "resulting extracts were ground using a pellet pestle motor for 30 s , vortexed and centrifuged at 14 , 000 rpm for 10 min at 4°C .", "Supernatants were collected for HPLC-MS analyses as described below .", "The effects of BX exposure on EPN survival were evaluated by incubating 1000 live EPNs in 1 mL of tap water containing either 50 µg MBOA-Glc , 150 µg HDMBOA-Glc or 25 µg MBOA ( n = 10 per treatment , each biological replicate being the average of three technical replicates ) , and counting dead and living EPNs after 7 days .", "BX exposure effects on EPN infectivity were assessed by collecting living EPNs from the above solutions and placing them with non-sequestering D . balteata larvae .", "Briefly , a hundred EPNs in 500 µL tap water were added into petri dishes ( 9 cm diameter , Greiner Bio-One GmbH , Frickenhausen , DE ) containing a filter paper and five third instar D . balteata larvae ( n = 6 per treatment ) .", "All petri dishes were sealed with parafilm ( Bemis Company Inc . , Oshkosh , WI ) to prevent the larvae from escaping .", "After 24 hr , D . balteata larvae were collected and placed into solo cups containing fresh crown root pieces .", "Five days later , all larvae were collected and dissected to determine their infection status .", "In vivo BX-mediated resistance to EPN infection was tested by exposing WT- and mutant fed D . virgifera larvae to EPNs in petri dishes as described above .", "The added roots after EPN exposure corresponded to the genotype the larvae had fed on prior to the experiment ( mutant or WT ) .", "In a first experiment , D . virgifera larvae were grown on the bx1 mutant ( n = 9 ) and the near isogenic WT ( B73; n = 8 ) .", "In a second experiment , D . virgifera larvae were grown on the bx1 ( n = 9 ) and bx2 ( n = 7 ) Ds insertion mutants and their corresponding WT ( W22; n = 8 ) .", "Inoculums of P . luminescens ( initial optical density at 600 nm: OD600 = 0 . 01 ) were grown in 70 µL of Luria Broth ( LB ) media ( Carl Roth , Karlsruhe , DE ) containing either water , MBOA , MBOA-Glc or HDMBOA-Glc at concentrations that ranged from 13 to 360 µg/mL ( n = 4 ) .", "Bacteria samples were incubated at 27 ± 0 . 02°C and analyzed over 30 hr using a Tecan Infinite M200 multimode microplate reader equipped with monochromator optics ( Tecan Group Ltd . , Männedorf , Switzerland ) .", "During incubation , the plate was shaken using orbital shaking ( 4 . 5 mm amplitude and 5 s shaking cycles ) and the OD660nm was measured every 30 min .", "Data were analyzed using the Excel add-in DMfit ( Baranyi and Roberts , 1994 ) .", "To characterize the foraging behavior of EPNs , we designed a two choice assay in petri dishes .", "A 5 mm layer of 1% agar ( Frontier Scientific Inc . ) was poured in petri dishes ( Greiner Bio-One ) .", "In a first experiment , one larva fed on B73 and one larva fed on the bx1 mutant were pinned with a needle ( Prym , DE ) on each side of the dish ( n = 10 ) .", "Pinning the last segment of their abdomen did not kill the larvae but allowed them to move around the needle .", "In a second experiment , exudates of third instar D . virgifera larvae fed on B73 and on bx1 mutant were collected by rinsing the larvae with 50 µL tap water of which 45 µL were added into two 5 mm diameter holes on each side of the petri dishes ( n = 23 ) .", "In a third experiment , the effect of BXs on EPN foraging behavior was tested by offering BX-complemented and control exudate extracts to the petri dishes .", "Exudates of larvae fed on the double bx1-igl mutant were collected as described above .", "HDMBOA-Glc ( 3 . 3 µg/mL; n = 23 ) , MBOA-Glc ( 6 . 6 µg/mL; n = 18 ) or a mix of both ( 3 . 3 and 6 . 6 µg/mL HDMBOA-Glc and MBOA-Glc , respectively; n = 26 ) were added to 45 µL larval exudates .", "The final concentrations corresponded to natural concentrations found on third instar WT larval skin .", "Tap water was added to the exudates .", "A hundred H . bacteriophora in 50 µL tap water were added in a 5 mm diameter hole in the center of the agar plate .", "The number of EPNs in the four quarters of the plates were counted after 24 hr .", "EPNs located in the quarters containing the treatment hole in their center were counted as choosing EPNs .", "Plates were no nematode moved from the center were excluded from the analysis .", "Plant samples were flash frozen and ground to a fine powder in liquid nitrogen .", "One milliliter extraction buffer ( EB: MeOH: H2O: formic acid ( FA ) ; 50: 50: 0 . 5% ) was added to 100 mg sample .", "Larval samples were weighed ( five larvae pooled per biological replicate ) and directly ground in the extraction buffer ( 1 mL EB per 100 mg tissue ) with a pellet pestle motor ( nD . balteata = 3 , nD . undecimpunctata = 8 , nD . virgifera = 5 ) .", "Larval exudates were extracted by rinsing third instar larvae with 25 µL distilled water and adding 25 µL MeOH: FA ( 99: 1% ) to the solution ( n = 6 ) .", "Larval frass were collected by placing 20 starved larvae on maize roots for 2 hr before transferring them in 1 . 5 mL pre-weighed tubes for 1 hr ( n = 7 ) .", "BXs processed by EPNs ( n = 6–9 ) and bacteria ( ncontrol = 3 , nbacteria = 6 ) were extracted by adding MeOH: FA ( 99: 1% ) to the EPN or bacteria solution ( v/v ) and ground using a pellet pestle motor for 30 s .", "All extracts were vortexed for 1 min and centrifuged at 14 , 000 rpm for 20 min , at 4°C .", "Supernatants were collected for HPLC-MS analyses .", "BX profiling of larvae of the three insect species and of D . virgifera larvae following disruption was conducted using an Agilent 1200 infinity system ( Agilent Technologies , Santa Clara , CA ) coupled to an API 3200 tandem spectrometer ( Applied Biosystems , Darmstadt , DE ) equipped with a Turbospray ion source following the method described elsewhere ( Handrick et al . , 2016 ) .", "BXs in plants , infected larvae , larval tissue and exudates were quantified using an Acquity UHPLC system coupled to a G2-XS QTOF mass spectrometer equipped with an electrospray source ( Waters ) .", "Gradient elution was performed on an Acquity BEH C18 column ( 2 . 1 × 50 mm i . d . , 1 . 7 μm particle size ) at 99–72 . 5% A over 3 . 5 min , 100% B over 2 min , holding at 99% A for 1 min , where A = 0 . 1% formic acid/water and B = 0 . 1% formic acid/acetonitrile .", "The flow rate was 0 . 4 mL/min .", "The temperature of the column was maintained at 40°C , and the injection volume was 1 μL .", "The QTOF MS was operated in negative mode .", "The data were acquired over an m/z range of 50–1200 with scans of 0 . 15 s at collision energy of 4 V and 0 . 2 s with a collision energy ramp from 10 to 40 V . The capillary and cone voltages were set to 2 kV and 20 V , respectively .", "The source temperature was maintained at 140°C , the desolvation was 400°C at 1000 L h-1 and cone gas flows was 50 L/hr .", "Accurate mass measurements ( <2 ppm ) were obtained by infusing a solution of leucin encephalin at 200 ng/mL at a flow rate of 10 μL/min through the Lock Spray probe ( Waters ) .", "BXs processed by EPNs and their endobiontic bacteria were analyzed with an Acquity UHPLC-MS system equipped with an electrospray source ( Waters i-Class UHPLC-QDA , USA ) .", "Compounds were separated on an Acquity BEH C18 column ( 2 . 1 × 100 mm i . d . , 1 . 7 μm particle size ) .", "Water ( 0 . 1% FA ) and acetonitrile ( 0 . 1% FA ) were employed as mobile phases A and B . The elution profile was: 0–9 . 65 min , 97–83 . 6% A in B; 9 . 65–13 min , 100% B; 13 . 1–15 min 97% A in B . The mobile phase flow rate was 0 . 4 mL/min .", "The column temperature was maintained at 40°C , and the injection volume was 5 μL .", "The MS was operated in negative mode , and data were acquired in scan range ( m/z 150–650 ) using a cone voltage of 10V .", "HDMBOA-Glc and DIMBOA-Glc were quantified in positive mode using single ion monitoring ( SIM ) at m/z 194 with cone voltage of 20V .", "All other MS parameters were left at their default values as suggested by the manufacturer .", "Absolute BX concentrations were determined using standard curves obtained from purified DIMBOA , MBOA , DIMBOA-Glc , HDMBOA-Glc and synthetized MBOA-Glc .", "Statistical analyses were performed using SigmaPlot 13 .", "Data were first tested for the heteroscedasticity of error variance and normality using Brown-Forsythe and Shapiro-Wilk tests .", "Data that did not fulfill the above assumptions were transformed or rank-transformed .", "Student t-tests and analyses of variance ( ANOVA ) were performed to assess differences between treatments .", "Repeated measures over time were analyzed using repeated measures ANOVAs ( RM-ANOVA ) .", "Preference data were analyzed by comparing the average difference of the proportions of EPNs choosing control and treatment sides to the null Hypothesis H0 = 0 using a one tailed t-test .", "Details on the data transformation , statistical tests and their outcome are available in the Summary Statistics ." ] ]
[ "Highly adapted herbivores can phenocopy two-component systems by stabilizing , sequestering and reactivating plant toxins .", "However , whether these traits protect herbivores against their enemies is poorly understood .", "We demonstrate that the western corn rootworm Diabrotica virgifera virgifera , the most damaging maize pest on the planet , specifically accumulates the root-derived benzoxazinoid glucosides HDMBOA-Glc and MBOA-Glc .", "MBOA-Glc is produced by D . virgifera through stabilization of the benzoxazinoid breakdown product MBOA by N-glycosylation .", "The larvae can hydrolyze HDMBOA-Glc , but not MBOA-Glc , to produce toxic MBOA upon predator attack .", "Accumulation of benzoxazinoids renders D . virgifera highly resistant to nematodes which inject and feed on entomopathogenic symbiotic bacteria .", "While HDMBOA-Glc and MBOA reduce the growth and infectivity of both the nematodes and the bacteria , MBOA-Glc repels infective juvenile nematodes .", "Our results illustrate how herbivores combine stabilized and reactivated plant toxins to defend themselves against a deadly symbiosis between the third and the fourth trophic level enemies ." ]
[ "The western corn rootworm is the most damaging pest of maize plants .", "Out of sight , the larvae of this beetle feed on maize roots , and cause billions of dollars worth of losses each year .", "One of the reasons why this pest remains such a problem is it can adapt and resist many crop protection strategies .", "Biological control refers to combating a pest using its own natural enemies – for example , its predators .", "Biological control of the western corn rootworm has been attempted using nematode worms .", "Normally , the nematodes locate and enter an insect larvae , release bacteria that kill it , and then feed and multiply within the dead larvae .", "Yet , the western corn rootworm seems at least partly able to resist these nematodes , and the success of biological control in the field has been variable .", "Several insect herbivores are known to accumulate , or sequester , plant toxins in their own body for self-defense .", "Previously , in 2012 , researchers reported that the western corn rootworm is resistant and attracted to the major toxins in maize roots , the benzoxazinoids .", "The blood-like fluid of the western corn rootworm also repels many predators .", "Could the western corn rootworm be sequestering maize benzoxazinoids to resist the biological control of nematodes and their bacterial partners ?", "Plants store benzoxazinoids in a non-toxic form .", "If herbivores damage the plant , these molecules quickly break down into compounds that are toxic to most insects .", "Now Robert et al . – who include two of the researchers involved in the 2012 study – show that the western corn rootworm uses a similar defense system to protect itself against biological control nematodes and their bacterial partners .", "First , the larvae convert a benzoxazinoid breakdown product by adding a glucose molecule .", "They then release large amounts of this modified molecule to repel young nematodes .", "Second , via an unknown mechanism , the larvae stabilize a second plant-derived benzoxazinoid , sequester its non-toxic form in their bodies , and activate it upon nematode attack .", "The resulting toxins can kill both nematodes and their bacterial partners .", "By combining different chemical strategies to stabilize and activate plant toxins , the western corn rootworm is able to resist the nematodes used for biological control .", "These findings can help to explain why biological control has had limited success against the western corn rootworm .", "In the long run , they may lead to more effective biological control programs , for instance by stopping the western corn rootworm from sequestering benzoxazinoids or by using natural enemies that are resistant to the insect’s toxins ." ]
2017
[ "Introduction", "Results", "Materials and methods" ]
[ "developmental biology", "cell biology" ]
Direct visualization of a native Wnt in vivo reveals that a long-range Wnt gradient forms by extracellular dispersal
elife-38325-v3
[ [ "Wnts are a family of secreted signaling proteins with critical roles in development , homeostasis , and disease ( Nusse and Clevers , 2017 ) , and anteroposterior gradients of Wnt activity are a common and ancient feature of animal development ( Darras et al . , 2018; Petersen and Reddien , 2009; Harterink et al . , 2011a; Scimone et al . , 2016; Nordström et al . , 2002; Kiecker and Niehrs , 2001 ) .", "Wnt proteins have often been thought to spread extracellularly to form concentration gradients and act directly at long ranges from their source cells ( Zecca et al . , 1996; Neumann and Cohen , 1997; Strigini and Cohen , 2000; Kiecker and Niehrs , 2001;Rhinn et al . , 2005 ) , but recent work has called this paradigm into question ( Alexandre et al . , 2014; Farin et al . , 2016; Stanganello et al . , 2015; Huang and Kornberg , 2015; Stanganello and Scholpp , 2016; Mattes et al . , 2018 ) .", "An increasingly prominent hypothesis is that Wnts do not freely spread over long distances in vivo and instead are primarily short-range signaling molecules ( Loh et al . , 2016 ) that are transferred at contacts between signaling and receiving cells that are either in close proximity or linked by dynamic plasma membrane extensions called cytonemes or signaling filopodia ( Stanganello et al . , 2015; Huang and Kornberg , 2015; Stanganello and Scholpp , 2016; Farin et al . , 2016; Mattes et al . , 2018 ) .", "Additionally , migrating cells can deliver membrane-bound Wnts ( Pfeiffer et al . , 2000; Serralbo and Marcelle , 2014 ) , and a Wnt gradient across a cell lineage can form by short-range ligand transfer followed by dilution of receptor-bound Wnt through cell divisions ( Farin et al . , 2016 ) .", "In contrast , free , extracellular spreading has never been directly shown for an endogenous Wnt .", "These findings are consistent with a model where free , extracellular Wnt spreading is rare , and long-range Wnt gradients are most likely generated by cytoneme/filopodia- or cell lineage-based mechanisms .", "Furthermore , experiments demonstrating that an endogenously membrane-tethered Wnt is sufficient for many aspects of patterning and growth control in flies ( Alexandre et al . , 2014 ) indicated that Wnt spreading between cells is not necessarily required for proper development in some contexts where Wnt protein gradients normally occur .", "However , basic aspects of Wnt dispersal and the existence and functions of free , extracellular spreading have been debated without the most direct types of evidence – visualization of endogenous Wnts in vivo and methods for limiting Wnt transfer between cells without altering intrinsic spreading ability .", "Two major challenges for investigating how Wnts disperse and how Wnt gradients form are the historical inability to visualize endogenous Wnts in living animals , and difficulties in observing potential cytonemes/signaling filopodia , which are often not preserved by standard tissue fixations or marked by cytoplasmic fluorescent proteins ( Sanders et al . , 2013; Kornberg , 2017 ) .", "The ideal system to test if Wnts spread extracellularly over long distances in vivo would allow for live imaging of endogenously tagged Wnt in concert with labeled signaling and receiving cell membranes .", "This type of experiment would make it possible to directly visualize how far Wnt proteins spread from source cells in vivo and the extent to which Wnts located far from their sources might be associated with cryptic cell membrane structures that could mediate contact-dependent signaling .", "However , for technical reasons live imaging experiments on Wnt gradient formation have relied on exogenously delivered fusion proteins , with their associated caveats , and Wnts have been challenging to fluorescently tag ( Willert and Nusse , 2012 ) .", "As a consequence , there are extremely limited data on Wnt localization in vivo , and the existence and potential roles of free , extracellular Wnt spreading are unresolved .", "Here , using biologically functional , fluorescently-tagged knock-in alleles for an endogenous Wnt , we report the first in vivo visualization of a native long-range Wnt gradient , provide evidence for free , extracellular Wnt dispersal , and demonstrate that free , extracellular spreading is required for aspects of long-range Wnt signaling in vivo ." ], [ "Based on our recently-demonstrated ability to generate a functional , mNeonGreen ( mNG ) knock-in tag for a C . elegans Wnt homolog ( mom-2 ) ( Heppert et al . , 2018 ) that is required for embryonic viability and endoderm specification at the 4 cell stage , we reasoned that C . elegans might be a tractable system to pursue the question of how native Wnts disperse in an animal amenable to in vivo imaging .", "We focused our attention on the Wnt homolog egl-20 , which is expressed in a cluster of posterior cells ( Harterink et al . , 2011a; Whangbo and Kenyon , 1999 ) , forms an anteroposterior gradient in transgenic experiments ( Whangbo and Kenyon , 1999; Coudreuse et al . , 2006 ) , and regulates cell migrations , fates , polarities , and axon guidance along the anteroposterior axis during embryonic and larval development ( Harris et al . , 1996; Maloof et al . , 1999; Whangbo and Kenyon , 1999; Coudreuse et al . , 2006; Prasad and Clark , 2006; Pan et al . , 2006; Green et al . , 2008; Yamamoto et al . , 2011; Harterink et al . , 2011a; Mentink et al . , 2014 ) .", "Previous work has shown that Wnt/EGL-20 processing and secretion relies on many of the same factors as Wnts in flies and vertebrates ( Coudreuse et al . , 2006; Bänziger et al . , 2006; Harterink et al . , 2011b ) , making it a useful paradigm to investigate mechanisms of long-range Wnt movement that are likely to be relevant to other animals .", "Here , we have engineered fluorescently-tagged , biologically functional knock-in alleles for Wnt/EGL-20 using Cas9-triggered homologous recombination ( Dickinson et al . , 2015 ) .", "We inserted the amphioxus-derived fluorescent protein mNG ( Shaner et al . , 2013 ) or the GFP/YFP derivative mYPET ( hereafter YPET ) at the C-terminus of Wnt/egl-20 similar to how we tagged Wnt/mom-2 .", "EGL-20::mNG and EGL-20::YPET knock-in worms had normal external morphologies and did not exhibit the characteristic defects in Q neuroblast migration seen in egl-20 mutants ( Harris et al . , 1996; Whangbo and Kenyon , 1999 ) ( Figure 1a , b ) .", "Subsequently , mNG and YPET tagged strains were used interchangeably depending on the relative importance of fluorescent protein photostability ( mNG ) versus brightness/signal:noise ratio ( YPET ) in different experiments ( Heppert et al . , 2016 ) .", "We first observed tagged Wnt/EGL-20 fluorescence in posterior cells in comma-stage embryos along with isolated punctae in more anterior regions ( Figure 1c , d ) and found that tagged Wnt spread from the posterior to the mid-body region of the embryo within 60 min ( Figure 1d ) .", "In early larvae , tagged Wnt/EGL-20 formed an anteroposterior gradient along approximately the posterior half of the worm ( Figure 1e–h ) with low levels of protein detectable along the entire body axis ( Figure 1f , h ) .", "Tagged Wnt localized most conspicuously to posterior cells near its source along with longitudinal body wall muscles , epithelial seam cells , neuroblasts , and ventral midline cells ( Figure 1e , g; Figure 1—figure supplements 1 and 2 ) .", "Because the mid-body seam cell precursors and most body wall muscles are already present and positioned ( Sulston et al . , 1983 ) by the time tagged Wnt/EGL-20 was first visible in comma-stage embryos , we can rule out the prospect that cell lineage-dependent mechanisms generate the observed anteroposterior gradient , which suggests that Wnt can move across multiple cells in this context .", "To determine the locations , shapes , and behaviors of Wnt/egl-20-expressing cells in vivo , we used the egl-20 upstream intergenic region ( hereafter Pegl-20> ) to drive a single copy plasma membrane reporter consisting of 2 copies of mKate2 fused to a PH domain from PLC1Δ1 , a plasma membrane marker previously used to visualize intricate cellular architectures in C . elegans ( Linden et al . , 2017 ) .", "In early larvae , this reporter was expressed in a cluster of posterior cells including rectal epithelial cells , the overlying dorsal and ventral body wall muscles , the stomatointestinal muscles , the anal depressor muscle , and P11/12 , along with weak expression in several head neurons ( Figure 1g , Figure 1—figure supplement 2 ) , which is largely consistent with smFISH data on egl-20 transcript localization ( Harterink et al . , 2011a ) and previous transgenes ( Whangbo and Kenyon , 1999 ) .", "This reporter also labeled several posterior neurons and their projections along the ventral nerve cord that terminated in the nerve ring ( Figure 1g; Figure 1—figure supplement 2 ) .", "Tagged Wnt protein clearly localized near reporter-labeled axons in the head ( Figure 1—figure supplement 2 ) , suggesting they could act as local sources of Wnt for ventral and head cells separately from the overall anteroposterior gradient .", "Despite extensive attempts , we did not observe extensions from Pegl-20-expressing cells resembling cytononemes/signaling filopodia .", "Although the stomatointestinal muscles have an elaborate shape including muscle arms that extend dorsally ( Figure 1—figure supplement 2 ) , this morphology is similar throughout development , and we did not observe dynamic behaviors of these cells .", "Comparing membrane reporter and tagged Wnt fluorescence intensity along the anteroposterior axis indicated that the protein formed a gradient emanating from the posterior source cells consistent with extracellular spreading ( Figure 1h ) .", "To ensure that we detected all cells that expressed egl-20 at any time of development - and therefore to assess the possibility of autocrine signaling - we marked the lineage of cells that expressed egl-20 by inserting flp::F2A upstream of endogenously tagged egl-20::mNG to engineer a bicistronic gene expressing both a FLP recombinase and tagged EGL-20 from its native locus ( Figure 1—figure supplement 3a ) .", "We combined this tool with a ubiquitously-expressed transgene that irreversibly converts a membrane marker from red to cyan after excision by FLP ( Figure 1—figure supplement 3a ) .", "This experiment confirmed our interpretation of the reporter transgene pattern including the previously undescribed neuronal expression and lack of expression in anterior body wall muscles ( Figure 1—figure supplement 3b , c ) that sometimes showed weak expression of Pegl-20-driven transgenes .", "These results demonstrate that broad , unseen expression at earlier stages does not underlie the post-embryonic EGL-20 distribution , and the results argue against the possibility of an autocrine/cellular memory mechanism for EGL-20 signaling as was described for Wnt signaling in the Drosophila wing disc ( Alexandre et al . , 2014 ) .", "Next , to test if cells known to respond to Wnt/EGL-20 possess cellular extensions that could directly mediate signaling , we used plasma membrane markers to investigate membrane architectures of Q neuroblasts , which migrate under the control of Wnt/EGL-20 signaling during early larval development ( Harris et al . , 1996; Whangbo and Kenyon , 1999; Maloof et al . , 1999 ) .", "In vivo imaging of these cell membranes in concert with Wnt/EGL-20 source cell membranes and tagged ligand showed that neither Wnt producing cells nor responding neuroblasts produced structures that could directly support contact-dependent signaling between them during Q neuroblast polarization through the early stages of migration ( Figure 2a–c ) .", "We did observe that tagged Wnt/EGL-20 localized to posteriorly directed , lamellipodia-like protrusions and short filopodia on polarizing QL neuroblasts ( Figure 2a , Figure 3a , b ) that shortly afterwards undergo a stereotypical posterior migration regulated by EGL-20 ( Harris et al . , 1996; Whangbo and Kenyon , 1999; Maloof et al . , 1999 ) .", "As a natural test of whether these protrusions might be required to capture Wnt/EGL-20 , we also examined tagged Wnt localization in QR neuroblast descendants .", "The QR and QL neuroblasts initially have identical axial positions , and the descendants of both cells respond to Wnt/EGL-20 ( Whangbo and Kenyon , 1999; Mentink et al . , 2014 ) .", "However , QR neuroblasts migrate towards the anterior and do not make posteriorly directed protrusions ( Middelkoop and Korswagen , 2014 ) .", "We found that tagged Wnt/EGL-20 also localized to QR descendants ( Figure 2—figure supplement 1a , b ) suggesting that posterior protrusions are not required for Q neuroblasts to capture and accumulate Wnt .", "We also found that tagged Wnt/EGL-20 localized to the posterior side of seam cells prior to their first division ( Figure 2a , b , Figure 2—figure supplement 1 ) , which is polarized in part by EGL-20 ( Yamamoto et al . , 2011 ) , and to their posterior daughters at later stages ( Figure 1—figure supplement 1 ) .", "Because receptor localization might provide additional insights into where Wnt signals are received in cells , we endogenously tagged two Frizzled homologs , MIG-1 and LIN-17 , that are involved in QL neuroblast migration ( Harris et al . , 1996 ) using a design previously used to tag the Frizzled homolog MOM-5 ( Heppert et al . , 2018 ) , and imaged them in concert with tagged Wnt/EGL-20 and labeled receiving cells in live animals .", "We did not observe defects in cell migrations or egg-laying in animals with tagged MIG-1 nor vulval or tail abnormalities in animals with tagged LIN-17 , suggesting these fusions are biologically functional .", "During QL neuroblast polarization , we observed tagged Frizzled punctae that overlapped with tagged Wnt/EGL-20 and sometimes localized to short , dorsally oriented filopodia and broad protrusions from the QL neuroblast prior to its posterior migration ( Figure 3a , b ) .", "We found that a tagged Dishevelled homolog also localized to QL neuroblast protrusions ( Figure 3—figure supplement 1 ) , consistent with them being a subcellular site of Wnt activation .", "While these findings imply that some neuroblast protrusions contribute to receiving the Wnt signal , the observed protrusions did not contact egl-20-expressing cells or resemble the thin and dynamic filopodia/cytonemes proposed to mediate Wnt signaling elsewhere ( Stanganello et al . , 2015; Huang and Kornberg , 2015 ) .", "Based on the overlap between Wnt/EGL-20 punctae and Frizzled receptors in responding Q neuroblasts , we wondered whether the Wnt punctae we observed more generally represented receptor-associated molecules , therefore most likely associated with individual receiving cells , versus some type of Wnt-containing particle that could be freely transferred between cells .", "Live imaging of Wnt/EGL-20::mNG simultaneously with tagged Frizzled/MIG-1 and Frizzled/LIN-17 revealed a striking overlap between tagged Wnt punctae and receptors within their respective expression domains , and most , if not all , tagged Wnt punctae visible by spinning disc confocal microscopy overlapped with one or both of the tagged receptors ( Figure 3—figure supplement 2 , Figure 3—figure supplement 3 ) .", "These results suggest that the vast majority of bright Wnt punctae visible in our experiments most likely represent receptor-bound molecules , suggesting to us that the fraction of Wnt capable of moving between cells is present in a more diffuse form that was not easily discernible by eye .", "We next sought to test if patterns of local cell-cell contacts or tissue continuity control long-range Wnt/EGL-20 spreading by investigating where Wnt secreted from specific cells was localized and it if was spatially restricted or spread broadly .", "To map dispersed Wnt molecules to their source cells , we tagged endogenous Wnt/EGL-20 with FRTmKate2::stopFRTmNG to generate a strain that converts irreversibly from a red to yellow fluorescent protein tag after excision by FLP recombinase ( Figure 4a ) .", "Then , to visualize both total Wnt/EGL-20 and specific Wnt molecules derived from only a small population of cells , we used an enhancer from the posterior Hox gene egl-5 to drive FLP expression in a small number of cells and imaged EGL-20::mKate2 and EGL-20::mNG in larval animals .", "These experiments showed that mNG-tagged Wnt spread from excised cells over long distances ( Figure 4b–d ) .", "We noted little difference in the distribution of mKate2- and mNG-tagged ligands ( Figure 4b–d ) , suggesting that there is a common pool of extracellular Wnt in much of the body derived from multiple cell types .", "Because the egl-5 enhancer we used does not drive FLP expression in stomatointestinal muscles or neurons , we were also able to rule out the possibility that ventral cord axons or stomatointestinal muscle arms ( see Figure 1—figure supplement", "2 ) are the main source of Wnt/EGL-20 in more anterior regions .", "To test the extent to which a Wnt can spread extracellularly from ectopic source cells with different shapes and cellular neighbors , we then mis-expressed Wnt/EGL-20::mNG in farther posterior tail epidermal cells using a fragment of another Wnt promoter ( lin-44 ) to ensure that the ectopically expressing cells were competent to secrete functional Wnts .", "Similar to the endogenous protein , Wnt/EGL-20::mNG expressed from a farther posterior source was detectable in QL neuroblast descendants , seam cells , and body wall muscle more than 75 μm and multiple cells away from source cells ( Figure 4—figure supplement 1 ) , suggesting that native cell contact patterns are not essential for ligand spreading to its normal target cells .", "To directly observe Wnt spreading in vivo , we used fluorescence recovery after photobleaching ( FRAP ) , to visualize tagged Wnt/EGL-20 spreading .", "After photobleaching a region of interest that included a responding QL neuroblast , we found that EGL-20::YPET fluorescence began to recover in biologically relevant cells within 30 s of photobleaching ( Figure 5a–c , Video 1 ) .", "Performing this experiment at a stage before the QL neuroblast began its protrusive behaviors confirmed that protrusions themselves are not essential for these cells to capture Wnt ( Figure 5a , b , Video 1 ) .", "Interestingly , some bright Wnt punctae recovered in approximately the same locations as before photobleaching ( Figure 5d ) , which suggested that mobile Wnt molecules not individually visible by spinning disc confocal microscopy in vivo were recruited to spatially stable clusters containing Wnt at the cell membrane .", "Recovery began rapidly but was incomplete ( Figure 5c ) , even over time scales of up to 81 min ( Figure 5—figure supplement 1 , Video 2 ) .", "This result is consistent with there being a relatively small fraction of highly mobile Wnt molecules and a larger proportion of more hindered or receptor-associated Wnts similar to what has been observed for nanoparticle-labeled FGF2 in cell culture ( Duchesne et al . , 2012 ) and transgenically-expressed dpp ( a BMP homolog ) in the Drosophila wing disc ( Zhou et al . , 2012 ) .", "Given that tagged Wnt punctae visible by spinning disc confocal microscopy also consistently overlapped with tagged receptors in our experiments , ( Figure 3—figure supplement 2 , Figure 3—figure supplement", "3 ) our data are consistent with proposed models for morphogen gradient formation based on free , extracellular movement with ligand dispersal hindered and shaped by binding to extracellular molecules including receptors ( Müller et al . , 2013 ) .", "Our findings also suggest that live imaging studies focusing on bright Wnt punctae associated with cytonemes/filopodia ( Stanganello et al . , 2015; Huang and Kornberg , 2015; Holzer et al . , 2012 ) may not have detected a significant fraction of extracellularly mobile Wnt , which could be a general caveat for studies using live imaging of potentially diffusible signaling proteins .", "As a whole , our findings led us to the working hypothesis that Wnt/EGL-20 spreads between cells in the extracellular environment , and that this spreading is a key factor in gradient formation .", "To conclusively determine whether extracellular Wnt/EGL-20 spreading shapes the endogenous anteroposterior gradient , and whether free ligand dispersal is important for normal development , we used a nanobody-based Morphotrap ( Harmansa et al . , 2015 ) approach to limit tagged Wnt movement between cells without otherwise altering the ligand itself .", "To capture and sequester extracellular Wnt/EGL-20::YPET , we used an extracellular 2x anti-GFP nanobody tethered to a CD8a transmembrane domain and intracellular mTurq2 ( Figure 6 ) , hereafter referred to as Morphotrap .", "Because ubiquitous Morphotrap expression caused intracellular accumulation of tagged Wnt in producing cells that suggested impaired secretion ( Figure 6—figure supplement 1 ) , we chose to express Morphotrap in body wall muscles using a myo-3 promoter ( Figure 6b–d ) to allow normal Wnt release from most source cells and to prevent potentially interfering with Wnt-receptor interactions in responding neuroblasts and seam cells .", "Because we did not alter the intrinsic ability of Wnt/EGL-20 to spread or manipulate Wnt capture or signal transduction in known responding cells , this approach allowed us to directly test the extent to which extracellular Wnt spreading across multiple cells is required for signaling .", "We first found that Morphotrap expression in body wall muscles altered tagged Wnt distribution in the body , resulting in a clearly altered anteroposterior gradient shape ( Figure 6a–f ) .", "We observed elevated levels of Wnt near source cells ( Figure 6c–e ) along with a relatively sharper decrease in levels along the anteroposterior body axis ( Figure 6f ) compared to YPET-tagged Wnt alone .", "In a region anterior to Wnt/EGL-20 producing cells , Morphotrap also significantly reduced tagged Wnt levels outside of body wall muscle ( Figure 6g , p<0 . 0001 , Mann-Whitney test ) .", "To assess the biological relevance of this manipulation , we imaged Q neuroblast migration and observed that Morphotrap reversed QL descendant migration ( Figure 6h , i ) in a significant number of worms ( n = 20/27 versus 0/23 , p<0 . 0001 , Fisher’s exact test ) .", "As Morphotrap phenocopied the QL migration defects characteristic of egl-20 loss of function mutants , we concluded that free , extracellular Wnt/EGL-20 spreading is required for signaling to QL descendants .", "We noticed that adult Pmyo-3>Morphotrap animals did not display the egg-laying defects seen in egl-20 mutants , suggesting that either signaling in body wall muscles is adequate for normal development in this context or that sufficient un-trapped ligand is able to reach the essential cells , possibly as a result of Morphotrap saturation .", "Efforts to express Morphotrap at higher levels using multi-copy extrachromosomal arrays were unsuccessful as this frequently led to aberrant shapes and behaviors of Morphotrap-expressing cells ( Figure 6—figure supplement 1 ) .", "Together , our Morphotrap results demonstrate that: ( 1 ) a long-range Wnt gradient is shaped by extracellular spreading; ( 2 ) Wnt molecules that localize to body wall muscle and other tissues are derived from a common extracellular pool , and; ( 3 ) free , extracellular Wnt spreading between cells is required for normal neuroblast migration .", "Taken together , our in vivo findings on endogenous Wnt localization and dispersal , along with visualization of membrane architectures of signaling and responding cells , provide the first direct evidence that a native , long-range Wnt gradient forms through free , extracellular dispersal .", "Our findings also challenge the idea that Wnts primarily act as short-range signals and establish that an endogenous Wnt can freely spread across multiple cells in vivo , which highlights the variety of mechanisms that animals can deploy to generate Wnt gradients .", "Furthermore , our Morphotrap results demonstrate that extracellular Wnt movement between cells is required for a developmental signaling event .", "While this result at first seems to conflict with the finding that a membrane-tethered Wnt is sufficient for essential aspects of signaling in Drosophila , there are key differences in Wnt expression that may explain at least some of the differential requirements for Wnt spreading in these cases .", "In particular , Drosophila wingless is broadly expressed at early third instar stages in prospective wing blade cells that are later patterned by Wnt signaling , before becoming restricted to a narrow stripe of expression at the dorsoventral boundary .", "The sufficiency of membrane-tethered Wingless for normal wing patterning is thought to be a consequence of this earlier autocrine signaling ( Alexandre et al . , 2014 ) , which is not necessarily a broadly applicable mechanism .", "In contrast , our lineage tracing experiments using flp::F2A inserted at the egl-20 locus showed that Wnt/egl-20 is never expressed in the majority of receiving cells or their neighbors , which makes the same mechanism unlikely in C . elegans .", "Given the evolutionarily conserved nature of the Wnt signaling pathway , our results support the idea that free , extracellular dispersal of Wnts could be a viable mechanism for long-range Wnt movement and Wnt gradient formation in many organismal contexts .", "While our findings argue against a cytoneme/filopodia-based mechanism for anteroposterior Wnt gradient formation here or ligand transfer by direct contact from source cells to responding Q neuroblasts and seam cells , they do not rule out roles for cell shapes or direct ligand transfer in different situations , and we predict that the architectures of many cells have important roles in cell-cell signaling .", "Indeed , our finding that a tagged Wnt and its receptors colocalized on neuroblast protrusions , which also contained tagged Dishevelled punctae , is consistent with a role for these structures in receiving the Wnt signal , possibly through interactions with the extracellular matrix or nearby cells .", "While we have demonstrated that Wnt/EGL-20 forms a long-range gradient in vivo , our findings do not necessitate that the gradient shape is itself critical for any biological outcome .", "However , our results taken together with earlier experiments showing that EGL-20 overexpression can also alter neuroblast migration ( Whangbo and Kenyon , 1999; Mentink et al . , 2014 ) establish that both extracellular spreading and proper Wnt levels are required for at least some aspects of normal development .", "Diffusion-based models for morphogen gradient formation in general have several perceived weaknesses ( Stanganello and Scholpp , 2016; Kornberg , 2017; Müller et al . , 2013; Wolpert , 2016 ) .", "First , diffusion alone is predicted to be inefficient over long distances in three dimensions , and there are questions about whether it could generate a robust gradient over the time scales and distances required for developmental patterning .", "Second , cell-cell signaling must be tightly controlled in space and time while contending with growth and morphogenesis , and it is not clear that a passive dispersal mechanism could produce the necessary precision or stability in vivo .", "In light of these issues , one possibility for how animals could produce robust and spatiotemporally controlled signaling protein gradients would be for source cells to produce non-limiting amounts of ligand and for the observed tissue- and cellular-level distributions to be shaped by factors acting in receiving cells and/or the intervening extracellular environment .", "In this case , factors such as secreted antagonists , binding to receptors , extracellular matrix interactions , and destruction , in combination with feedback control of gene expression , might transform a relatively imprecise dispersal process such as diffusion into a robust protein gradient ( Müller et al . , 2013 ) .", "One prediction of this hypothesis is that ligand abundance and spreading rates should not be the limiting factors in gradient formation , which will be important to test in the future .", "Signaling molecule dispersal also may not need to be actively directed to specific cells in order for accurate signaling to individual cells or tissues , and the cell-type specific and sometimes polarized Wnt/EGL-20 localization we observed in vivo suggests that responding cells themselves determine key features of signaling protein distribution and signaling activity within an organismal-scale gradient .", "Consistent with this possibility , cell autonomous factors such as timed expression of different Wnt receptors in migrating cells ( Mentink et al . , 2014 ) , autonomously-acting Syndecan-1 ( Saied-Santiago et al . , 2017 ) , and an Rnf43/Znrf3 homolog ( Moffat et al . , 2014 ) are known to modulate Wnt signaling activity in cells along the C . elegans anteroposterior axis .", "The Ror/CAM-1 receptor is also thought to have dual roles in both regulating Wnt activity cell-autonomously ( Song et al . , 2010 ) and non-autonomously restricting ligand availability to other cells ( Green et al . , 2007 ) , which could be a general function for other receptors as well .", "Due to their hydrophobic nature ( Willert et al . , 2003 ) , fully processed Wnts are not thought to be capable of freely moving over long distances in the extracellular space on their own ( Langton et al . , 2016 ) , and it remains to be determined where in the extracellular environment Wnt/EGL-20 spreads and if spreading involves association with cell surface or extracellular matrix proteins ( Han et al . , 2005; Fuerer et al . , 2010; Mii et al . , 2017 ) , structures such as lipoprotein particles ( Neumann et al . , 2009; Panáková et al . , 2005 ) or exosomes/microvesicles ( Korkut et al . , 2009; Gross et al . , 2012; Greco et al . , 2001 ) , and/or other interacting molecules to modulate Wnt solubility ( Mii and Taira , 2009; Mulligan et al . , 2012; Chang and Sun , 2014 ) .", "While we have shown that extracellular dispersal is required for shaping a long-range Wnt gradient , the endogenous factors that promote and/or hinder ligand spreading await further characterization .", "It is tempting to speculate that mixing of extracellular fluids could also contribute to ligand spreading in vivo , although there is not currently evidence for this .", "Activities of cells such as protrusions , migrations , divisions , morphogenetic movements , and muscular contractions could drive extracellular mixing , potentially helping to circumvent the theoretical limitations that diffusion alone places on speed and distance of ligand dispersal .", "Our direct visualization of a Wnt gradient here establishes that long-range Wnt spreading can occur through free , extracellular dispersal and lays a groundwork to investigate how endogenous Wnts are transferred in vivo between cells , and how other molecules , cellular processes , and biophysical factors might orchestrate Wnt movement to control the range and strength of signaling ." ], [ "Caenorhabditis elegans animals were cultured on Normal Growth Media ( NGM ) plates , fed E . coli ( OP50 strain ) , and grown at 20°C for experiments .", "Worms were grown at 25°C for incubation during strain construction or to accelerate development during crosses .", "Single copy Pwrt-2>2x mTurq2::PH ( cpIs89 ) or Pwrt-2>2x mKate2::PH ( cpIs130 ) transgenes were used to visualize seam cells and Q neuroblasts before and during migration .", "Single copy Pmec-7>2x mTurq2::PH ( cpIs92 ) and Pgcy-32>2x mKate2::PH ( cpIs129 ) transgenes were used to visualize positions of Q neuroblast progeny after their migrations .", "Knock-in strains were generated using Cas9-triggered homologous recombination with standard methods ( Dickinson et al . , 2015 ) .", "Fluorescent protein knock-ins were made at the C-terminus for egl-20 , mig-1 , and lin-17 , and flp::F2A was inserted directly upstream of the egl-20 start codon .", "Repair templates were constructed by inserting homology arm PCR products amplified from genomic DNA into plasmids containing a fluorescent protein and self-excising selection cassette using Gibson assembly ( New England Biolabs ) or SapTrap assembly as described in detail elsewhere ( Dickinson et al . , 2015; Schwartz and Jorgensen , 2016 ) .", "All C-terminal homologous repair templates included a nine amino acid flexible linker ( GASGASGAS ) between the endogenous coding sequence and fluorescent protein .", "To construct the FRTmKate2::stopFRTmNG cassette we used the let-858 3’UTR as a strong transcriptional terminator and placed FRT5T2 sites within synthetic introns between egl-20 and mKate2 and between the let-858 terminator and mNG .", "Cas9 guide RNA targeting sequences for each gene were selected using the CRISPR Design Tool ( Hsu et al . , 2013 ) ( http://crispr . mit . edu ) .", "Guide RNA target sites used were ( 5’−3’ ) : egl-20 N-terminus , GAGAATATTGCCCATAAACG AGG; egl-20 C terminus , GCAGTACATACATGCAAATA AGG; lin-17 C-terminus , TCTCGCTCAGACGACCTTAC TGG; mig-1 C-terminus , AGTTCGAAACGTCGACGCGT AGG; chromosome I near ttTi4348 site , GAAATCGCCGACTTGCGAGG AGG; chromosome II near ttTi5605 site , GATATCAGTCTGTTTCGTAA CGG .", "Guide RNA sequences were cloned into the Peft-3 >Cas9+sgRNA expression vector pDD162 using Q5 site-directed mutagenesis ( New England Biolabs ) and co-injected into adult germlines with repair templates and extrachromosomal array markers as described previously ( Dickinson et al . , 2015; Dickinson et al . , 2013 ) .", "Candidate knock-ins were selected by hygromycin B treatment , phenotypic identification ( roller ) , and the absence of fluorescent extrachromosomal arrays .", "Candidates were singled to new plates to establish homozygous lines , and PCR genotyping was used to confirm fluorescent protein knock-ins .", "To excise the selectable marker cassettes , early L1 larvae were heat-shocked at 32°C for four hours to induce Cre expression , and non-roller offspring were picked in the next generation .", "Backbones for single copy transgene insertions using Cas9-triggered homologous recombination were made by replacing the unc-119 ( + ) selectable marker in the MOSCI targeting vectors pCFJ150 ( ttTi5605 locus ) and pCFJ352 ( ttTi4348 locus ) with a self-excising selection cassette ( Dickinson et al . , 2015 ) flanked by loxN sites and deleting the guide RNA target sequences from the homology arms .", "Coding sequences for transgenes were codon optimized using the C . elegans codon adapter tool ( Redemann et al . , 2011 ) ( https://worm . mpi-cbg . de/codons/cgi-bin/optimize . py ) and synthesized as gBlock DNA fragments ( Integrated DNA Technologies ) .", "Transgene promoters were amplified from genomic DNA or existing plasmids and inserted into linearized plasmid backbones using Gibson assembly .", "The egl-5 K enhancer corresponding to syEx616 ( Teng et al . , 2004 ) was cloned upstream of a pes-10Δ minimal promoter .", "Primers are provided in Supplementary file 1 .", "For imaging post-embryonic stages , larval animals were anesthetized with 0 . 1 mmol/L levamisole in M9 buffer ( Chai et al . , 2012 ) or immobilized using 0 . 1 μm polystyrene nanoparticles ( Kim et al . , 2013 ) ( Polysciences ) and mounted on 5–10% ( wt/vol ) agarose pads and maintained at room temperature ( ~20°C ) .", "Larval animals were imaged within one hour of mounting , and images shown in figures were representative of at least ten animals imaged on at least two occasions .", "Numerous animals were mounted on each slide , and animals were selected for imaging based on developmental stage , orientation on the slide , and overall health .", "For embryo imaging , embryos were dissected from gravid adults in egg buffer and mounted on poly-L-lysine coated coverslips with 2 . 5% agarose pads .", "Our imaging systems consisted of: ( 1 ) Nikon TiE stand with CSU-X1 spinning disk head ( Yokogawa ) , 447 nm , 514 nm , and 561 nm solid state lasers , ImagEM EMCCD camera ( Hammamatsu ) , iLas2 ( Roper Scientific ) , and 100 × 1 . 49 NA objective , or; ( 2 ) a Nikon TiE stand with CSU10 spinning disk head ( Yokogawa ) , 514 nm and 561 nm solid state lasers , ORCA Flash sCMOS camera ( Hammamatsu ) , and 40 × 1 . 3 NA or 60 × 1 . 4 NA objectives .", "Images were acquired using MetaMorph software ( Molecular Devices ) with a 0 . 3 μm step size for z-stacks and varying exposure times and laser intensities depending on the strain and developmental stage .", "FRAP experiments were performed using an iLas2 system and software ( Roper Scientific ) .", "Images were acquired immediately before and after photobleaching followed by time-lapse imaging at defined intervals .", "To prepare figures , image stitching was performed using FIJI where necessary .", "Stitched images were not used for quantitative comparisons .", "Images were cropped and rotated , and brightness and contrast were adjusted using FIJI ( Schindelin et al . , 2012 ) .", "No statistical tests were used to predetermine sample size .", "Animals were selected for measurements based on developmental stage , orientation on the slides , and health .", "No animals were excluded from analyses post-hoc .", "Measurements from at least two imaging sessions of each worm strain were used for analyses .", "Results are presented as raw data points with mean and 95% confidence intervals for all graphs .", "P-values were considered significant at p<0 . 05 .", "Positions of Q neuroblast progeny after migration were quantified by using the non-motile URX neuron in the head and PLM neurons in the tail as fiducial markers .", "Relative positions of Q neuroblast progeny AQR , AVM , PVM , and PQR were calculated as a percentage of the distance between URX and PLM .", "Investigator was blinded to genotype before measurements , but egl-20 mutants were clearly distinguishable from the other genotypes .", "Statistical tests for differences between control ( Bristol N2 ) and tagged EGL-20 or EGL-20 mutant strains were performed using a one-way ANOVA with Sidak’s multiple comparisons test .", "Fluorescence intensity profile values for tagged EGL-20 and Pegl-20>2x mKate2::PH were obtained in FIJI ( Schindelin et al . , 2012 ) by drawing a line the width of the worm from head to tail and using the ‘plot profile’ function .", "Fluorescence intensity values for graphs were calculated by subtracting off-worm background in a nearby region from the raw pixel intensities .", "Where necessary for comparing shapes of fluorescence intensity profiles , intensity data were normalized in order to plot profiles of differing absolute intensities on the same graph .", "For kymographs of FRAP recovery , we drew a line the width of the worm along the anteroposterior axis including the photobleached and nearby unbleached regions and used the KymoResliceWide plugin in FIJI ( Schindelin et al . , 2012 ) to plot average intensity along the line as a function of time .", "Wnt/EGL-20::YPET levels outside of body wall muscle were calculated by measuring the mean pixel intensity of a region of interest anterior to the egl-20-expressing cells that did not include body wall muscles marked by a Pmyo-3-driven transgene .", "Control and Morphotrap worms were imaged with identical settings on the same slides , and off-worm background in a nearby region was subtracted from the pixel intensities before analyses .", "A Mann-Whitney test was used to assess statistical significance .", "The direction of Q neuroblast migration in Morphotrap and control animals was scored by visualizing migrating cells using a Pwrt-2 >2x mKate2::PH marker in L1 stage animals , and a Fisher’s exact test was used to assess statistical significance of migration reversals .", "All statistical tests were performed using Prism seven software ( GraphPad Software ) .", "Strains generated for this work will be made available through the Caenorhabditis Genetics Center ( CGC ) .", "Correspondence and requests for other materials should be addressed to A . M . P ." ] ]
[ "Wnts are evolutionarily conserved signaling proteins with essential roles in development and disease that have often been thought to spread between cells and signal at a distance .", "However , recent studies have challenged this model , and whether long-distance extracellular Wnt dispersal occurs and is biologically relevant is debated .", "Understanding fundamental aspects of Wnt dispersal has been limited by challenges with observing endogenous ligands in vivo , which has prevented directly testing hypotheses .", "Here , we have generated functional , fluorescently tagged alleles for a C . elegans Wnt homolog and for the first time visualized a native , long-range Wnt gradient in a living animal .", "Live imaging of Wnt along with source and responding cell membranes provided support for free , extracellular dispersal .", "By limiting Wnt transfer between cells , we confirmed that extracellular spreading shapes a long-range gradient and is critical for neuroblast migration .", "These results provide direct evidence that Wnts spread extracellularly to regulate aspects of long-range signaling ." ]
[ "Cells exchange signaling proteins that help them to communicate with each other .", "These signals control which genes are active , and how cells grow and specialize to do different tasks .", "Signals can also help cells to position themselves inside a body to form new tissues and organs .", "For example , the so-called Wnt signaling system is important for many processes in the body , from early development to the growth and maintenance of tissues .", "Signaling proteins are often thought to travel long distances between cells that produce them and the cells that respond to them .", "How these molecules move between cells has been challenging to study in a natural context .", "Signals may travel by diffusion – the random movement of molecules over time .", "But this had not been directly shown , and some studies suggest that thin , finger-like extensions from cells help carry the signals .", "Pani and Goldstein investigated how Wnt signals travel between cells in the round worm , Caenorhabditis elegans .", "The Wnts were labelled with fluorescent tags inserted into the genome , which made them glow under certain lights .", "The results showed that Wnts can travel quickly between remote cells by using diffusion .", "Diffusion can create gradients of Wnt over long distances , with higher levels near the cells that produce Wnt and lower in others .", "When the Wnts were prevented from spreading freely across cells , they could not travel as far or act on their regular target cells .", "Both Wnt molecules and Wnt receptor proteins clustered on thin cell extensions in some cells , but the extensions were not necessary for helping the molecules spread .", "This study helps us to understand one way that Wnt can traverse cells .", "A next step will be to examine if this aspect of Wnt signaling is similar between worms and humans .", "In humans , faulty Wnt signaling is implicated in many cancers .", "A better understanding of how this pathway normally works may help researchers develop ways to manipulate Wnt signaling in diseases ." ]
2018
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "cancer biology" ]
Regulation of localization and function of the transcriptional co-activator YAP by angiomotin
elife-23966-v1
[ [ "Angiomotin ( Amot ) was originally identified as an angiostatin-binding protein involved in the regulation of endothelial cell polarization , migration , proliferation , and angiogenesis ( Kikuno et al . , 1999; Levchenko et al . , 2003; Troyanovsky et al . , 2001 ) .", "Amot is expressed as two isoforms generated by alternative splicing , full-length Amot-p130 ( referred to as Amot in this manuscript ) and truncated Amot-p80 , which lacks the 409-amino acid N-terminus ( Ernkvist et al . , 2006 ) .", "Amot , Angiomotin-like 1 ( AmotL1 ) and Angiomotin-like 2 ( AmotL2 ) compose the Motin family of proteins , which share an N-terminus with conserved glutamine-rich domains and PPxY motifs , and a C-terminus containing conserved coiled-coil ( CC ) and PDZ-binding domains ( Bratt et al . , 2002; Nishimura et al . , 2002; Zheng et al . , 2009 ) .", "Although structurally similar , the functions of Motin family members appear to be distinctive , as divergent spatiotemporal and expression levels have been described across human and mouse tissues and cell lines for each of the family members ( Moleirinho et al . , 2014 ) .", "Functionally , Amot is required for endothelial cell migration , by binding to the Syx:Patj/Mupp1 polarity complex to localize RhoA activity to the leading edge of migratory cells ( Ernkvist et al . , 2009 ) .", "It has also been implicated in epithelial cell polarity , as an inhibitor of the GTPase-Activating Protein ( GAP ) Rich1 and shown to compromise the integrity of tight junctions by promoting Rich1-mediated hydrolysis of Rho GTPases Rac1 and Cdc42 ( Wells et al . , 2006; Yi et al . , 2011 ) .", "Amot regulates collective migration of epithelial cells as part of a signaling axis composed of Merlin-Amot-Rich1 and the Rac1 small GTPase ( Das et al . , 2015 ) .", "At tight junctions ( TJ ) , TJ-associated Merlin inhibits Rac1 activation through the Amot-Rich1 axis , relocalizing Merlin to the cytoplasm during cell migration and releasing Rac1 from a suppressed state ( Das et al . , 2015; Yi et al . , 2011 ) .", "Amot also directly binds to YAP , a central effector of the Hippo signaling pathway , via 106LPTY109/239PPxY242 motifs present in the N-terminal domain of Amot-p130 and WW domain present in YAP ( Yi et al . , 2013 , 2011; Zhao et al . , 2011 ) .", "The Hippo-YAP signaling pathway , originally characterized in Drosophila , is highly conserved in mammals and regulates organ size , cell contact inhibition , proliferation , apoptosis and polarity ( Ramos and Camargo , 2012; Yi and Kissil , 2010; Yu et al . , 2015 ) .", "At the core of the mammalian pathway , MST1/2 promote phosphorylation of WW45 ( also known as Sav1 ) , Lats1/2 , and MOB1 ( MOBKL1A/B ) .", "Once activated , Lats1/2 phosphorylates the primary downstream effector YAP , promoting ubiquitination and proteosomal degradation by a SCFbeta-TRCP E3 ligase and sequestering YAP from the nucleus , where it functions as a transcriptional co-activator ( Hao et al . , 2008; Zhao et al . , 2010b , 2007 ) .", "Among the different transcription factors activated by YAP , the DNA-binding TEA domain ( TEAD ) transcription factors are thought to mediate a YAP-driven pro-proliferative gene expression program ( Galli et al . , 2015; Zhao et al . , 2010b , 2007 ) .", "YAP’s involvement in cancer has been demonstrated in several tissues , including liver , intestine , heart , pancreas , and brain ( Yu et al . , 2015; Guerrant et al . , 2016 ) .", "Importantly , recent studies revealed YAP plays a key role in developing resistance to RAF- and MEK-targeted therapies in lung and colon cancer cells ( Lin et al . , 2015 ) and cancer relapse in KRAS-driven colon and pancreatic cancers ( Kapoor et al . , 2014; Shao et al . , 2014 ) .", "Upstream of the core kinase cassette is the tumor suppressor Merlin ( moesin-ezrin-radixin-like protein ) , which is inactivated in Neurofibromatosis type 2 ( NF2 ) ( McClatchey et al . , 1998 ) .", "Functions for Merlin have been described in the nucleus ( Li et al . , 2014 , 2010 ) as well as at the plasma membrane ( Yin et al . , 2013; Zhang et al . , 2010 ) .", "In the mouse-liver , homozygous deletion of Nf2 results in tumor formation .", "However , heterozygous deletion of Yap significantly suppresses the loss-of-Nf2 phenotype , thus implicating YAP as a major downstream effector of NF2 ( Zhang et al . , 2010 ) .", "Analysis of liver-specific Nf2 knockout mice and Nf2:Amot double knockout ( DKO ) mice showed Amot is required for hepatic ductal cell proliferation and tumor formation in the context of either Nf2 loss or DDC ( 3 , 5-diethoxycarbonyl-1 , 4-dihydrocollidine ) -induced injury .", "Additionally , substantially increased expression of Amot was observed in NF2-null human schwannomas samples , which primarily displayed localization of Amot in the nucleus ( Yi et al . , 2013 ) .", "Further evidence demonstrated that in renal cell carcinoma ( RCC ) , Amot promotes the proliferation of renal epithelial and RCC cells , and is crucial for YAP transcriptional activity by promoting its nuclear localization ( Lv et al . , 2016 ) .", "However , it has also been reported that Amot functions as a negative regulator of YAP activity , as direct association of Amot and YAP results in translocation to cytoplasm/cell junctions ( Leung and Zernicka-Goetz , 2013; Zhao et al . , 2011 ) .", "Moreover , a number of studies suggest that Amot acts as a scaffold protein to promote localization of YAP , as well as other Hippo/YAP core kinases , to the cytoplasm/cell junctions/actin cytoskeleton and promote Lats1/2-mediated phosphorylation of YAP ( Chan et al . , 2011; Dai et al . , 2013; Paramasivam et al . , 2011; Wang et al . , 2011 ) .", "One possible explanation for these opposing regulatory functions of Amot could be attributed to post-translational modifications .", "Phosphorylation of Ser175 on human Amot ( corresponding to Ser176 in mouse ) at the conserved HVRSLS motif by Lats1/2 has been reported as a key post-translational modification controlling Amot function and association with YAP ( Hirate et al . , 2013; Moleirinho et al . , 2014 ) .", "Here we show that Amot-p130 functions as a scaffold protein mediating YAP and Merlin association and that phosphorylation of Amot at Ser176 induces translocation of the complex from the cytoplasm and nucleus to the plasma membrane .", "This relocalization of the Amot/YAP/Merlin complex significantly impacts the activity of YAP and regulates YAP’s ability to promote cell proliferation and tumorigenesis .", "These results uncover an unrecognized layer of regulation in the Hippo-YAP pathway and resolve questions regarding the seemingly opposing functions of Amot ." ], [ "Previous work by multiple groups identified the p130 splice isoform of Angiomotin ( Amot ) as a binding partner of YAP and Merlin ( Chan et al . , 2011; Yi et al . , 2013 , 2011; Zhao et al . , 2011 ) .", "We first thought to assess whether Amot-p130 , YAP and Merlin form a complex and subsequently whether this is dependent on Angiomotin phosphorylation .", "We used HEK293 , as they express relatively high levels of endogenous Angiomotin .", "First , to determine the interaction between Merlin and YAP , HEK293 cells were transfected with expression vectors for HA-tagged Merlin and V5-tagged YAP .", "Reciprocal co-immunoprecipitation ( co-IP ) analyses using anti-HA or anti-V5 antibodies revealed association between YAP and Merlin ( Figure 1A ) .", "The Merlin-YAP interaction was also confirmed in another independent cell type – the hSC2λ immortalized human Schwann cells ( Figure 1B ) .", "Importantly , further validation of the association between YAP and Merlin was confirmed at the endogenous protein levels , using antibodies directed against Merlin or YAP ( Figure 1C ) . 10 . 7554/eLife . 23966 . 003Figure 1 . YAP associates with Merlin in the nucleus and cytoplasm . Co-immunoprecipitation of YAP and Merlin .", "( A ) HEK293 or ( B ) hSC2λ cells were co-transfected with expression plasmids for HA-Merlin or Flag-Merlin and V5-YAP .", "Total cell lysates ( Input ) and HA , Flag , YAP or V5 immunoprecipitates ( IP ) were subjected to immunoblotting analysis with anti-V5 , anti-HA , anti-Flag , Merlin or YAP antibodies as indicated .", "( C ) Association of endogenous YAP and Merlin .", "Total lysates from HEK293 cells ( input ) or IPs with anti-Merlin or anti-YAP antibodies were subjected to immunoblotting analysis with indicated antibodies .", "( D–F )", "YAP associates with Merlin in the nucleus and in the cytoplasm .", "( D ) HEK293 or ( E ) hSC2λ cells expressing Flag-Merlin and V5-YAP were fractionated into cytoplasmic , nuclear , and plasma membrane fractions .", "Cell lysates ( input ) and V5-IP or Flag-IP of each subcellular fraction were subjected to immunoblot analysis with indicated antibodies .", "GAPDH , Lamin A/C , and EGFR/ Na+/K+ATPase were used as fractionation controls for the cytoplasmic , nuclear , and plasma membrane fractions , respectively .", "IgG was used as a non-specific antibody control for IPs throughout .", "The blots shown are representative of three independent biological replicates ( n = 3 ) .", "( F ) HEK293 ( left ) or hSC2λ ( right ) cells were co-transfected with YAP and Merlin expression plasmids and subjected to immunofluorescence staining with anti-YAP and anti-Merlin antibodies .", "Hoechst was used for nuclei fluorescence staining .", "Pictures show fields at 63x magnification and representative of three independent biological replicates , in each of which 20 independent fields were examined .", "Scale bar = 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 003 As previous studies reported multiple cellular localizations for Merlin and YAP , we sought to determine the cellular compartments where YAP and Merlin co-localize .", "Flag-tagged Merlin and V5-tagged YAP were co-transfected in both HEK293 and hSC2λ cells and the localization and association of these proteins were examined by subcellular fractionation coupled to IP with an anti-Flag antibody .", "These subcellular fractionation studies showed an association between Merlin and YAP primarily in the nucleus and cytoplasm , and to a much lesser extent at the plasma membrane ( Figure 1D–E ) .", "Further confirmation of Merlin and YAP co-localization was provided by immunofluorescence ( IF ) staining , in HEK293 and hSC2λ cells , where we observed co-localization of IF signal predominantly in the cytoplasm and nucleus ( Figure 1F ) .", "Thus , we conclude that YAP associates with Merlin , co-localizing mostly in the cytoplasm and nucleus .", "Since Amot directly binds YAP and Merlin through two distinct domains ( Yi et al . , 2013 , 2011 ) , we hypothesized that Amot could act as a scaffold and recruit YAP and Merlin into the same complex .", "To confirm this hypothesis we generated HEK293 cells stably expressing a lentiviral short hairpin RNA specific to Amot ( HEK293-shAmot cells ) .", "Co-IP experiments with HA-tagged Merlin and V5-tagged YAP confirmed that in the absence of Amot , the complex is not formed as YAP and Merlin could no longer be co-immunoprecipitated ( Figure 2A ) .", "Similarly , knockdown of Amot using an siRNA smartpool impaired the association of Merlin and YAP ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 23966 . 004Figure 2 . YAP and Merlin association is dependent on Amot-p130 . ( A ) HEK293 cells stably infected with lentiviral vectors encoding a control shRNA ( shCtr ) or shRNA targeting Amot ( shAmot ) were co-transfected with expression plasmids for HA-Merlin and V5-YAP .", "Total lysates ( input ) and HA or V5 IPs were subjected to immunoblot analysis with anti-YAP and anti-Merlin antibodies as indicated .", "( B ) Graphical representation of Amot p130 and p80 isoforms .", "Amot-p130 N-terminus PPxY and LPxY motifs are shown , and the generated PY motif mutants are highlighted in red .", "See Figure 2—figure supplement", "1 . CC/BAR – Coiled-Coil/ ( Bin/Amphiphysin/Rvs ) domain .", "PDZ – Post synaptic density protein ( PSD95 , Drosophila disc large ( Dlg1 ) and Zonula occludens-1 ( ZO-1 ) domain .", "( C ) HEK293-shAmot cells were co-transfected with HA-Merlin , V5-YAP , and Flag-Amot-p80 .", "Total lysates ( Input ) and HA or V5 IPs were subjected to immunoblot analysis with anti-V5 and anti-HA antibodies as indicated .", "The blots shown are representative of three independent biological replicates ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 00410 . 7554/eLife . 23966 . 005Figure 2—figure supplement 1 . YAP and Merlin association is dependent on Amot-p130 . HEK293 cells were co-transfected with siRNA targeting Amot ( siAMOT ) or non-targeting control ( siCtrl ) and expression plasmids for HA-Merlin and V5-YAP .", "( A ) Merlin was IP’ed with anti-HA antibody and subjected to immunoblot analysis with anti-V5 antibodies .", "Total levels of Merlin or YAP ( A–input ) and Amot ( B ) were assessed using the indicated antibodies .", "Tubulin was used as a loading control .", "The blots shown are representative of three independent biological replicates ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 00510 . 7554/eLife . 23966 . 006Figure 2—figure supplement 2 . YAP/Merlin complex require Amot-p130 PPxY and LPxY motifs . HEK293-shAmot cells were co-transfected with YAP , Flag-Merlin and ( A–C ) single PY motif mutant Amot-p130 ( PY1* , PY2* , or PY3* ) , ( D–E ) double PY motif mutant Amot-p130 ( PY1 +3* or PY2 +3* ) , or ( F ) triple PY motif mutant Amot-p130 ( PY1 +2 + 3* ) .", "Total lysates ( input ) and YAP or Flag IPs were subjected to immunoblot analysis with anti-YAP and anti-Merlin antibodies as indicated .", "Immunoblot analysis was used to verify the transfection efficiency of the indicated Amot-p130 constructs in total lysates of HEK293-shAmot cells .", "The blots shown are representative of three independent experiments ( n = 3 ) .", "* indicates the number of PY mutations . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 006 To further validate the scaffolding role of Amot we evaluated whether Amot-80 can support the interaction between Merlin and YAP .", "Amot-p80 lacks the 409 amino acid N-terminus present in Amot , which harbors the PPxY motifs ( 239PPEY242 and 284PPEY287 ) , and an unconventional LPTY motif ( 106LPTY109 ) that mediate the interaction with YAP ( Ernkvist et al . , 2006; Wang et al . , 2012; Yi et al . , 2013 ) ( Figure 2B ) .", "As expected , attempting to IP HA-Merlin or V5-YAP demonstrated that Amot-p80 was unable to support the association between these proteins ( Figure 2C ) .", "Moreover , mutation of the individual PPEY/LPTY motifs or combinations thereof , significantly impaired the association between Merlin and YAP ( Figure 2—figure supplement 2 ) .", "Taken together , these results show that Amot functions as a scaffold that supports the YAP-Merlin association .", "Several reports have shown the importance of Amot post-translational modifications in regulating its function , specifically phosphorylation of Serine 176 ( Serine 175 in humans ) ( Adler et al . , 2013a; Chan et al . , 2013; Dai et al . , 2013; Hirate et al . , 2013; Paramasivam et al . , 2011 ) .", "We therefore explored whether Amot phosphorylation regulates formation of the Amot/YAP/Merlin complex .", "HEK293-shAmot cells were transfected with expression vectors for Flag-Merlin , V5-YAP and either HA-Amot-p130 , HA-Amot-p130S176A ( non-phosphorylated mutant ) or HA-Amot-p130S176E ( phosphomimetic mutant ) and similar levels of the different Amot alleles were confirmed ( Figure 3—figure supplement 1 ) .", "IPs were carried out using anti-Flag or anti-V5 antibodies and the presence of Merlin and YAP was determined by western blotting .", "These analyses demonstrated that all three forms of Amot support the formation of the complex , as evidenced by the co-IP of Merlin and YAP ( Figure 3A–C ) .", "Further validation of these results were demonstrated by co-IP experiments in which Amot was IPed using an anti-HA antibody ( Figure 3—figure supplement 2 ) .", "In addition , we examined whether phosphorylated Amot is still able to bind both YAP and Merlin , by using an antibody that specifically binds phosphorylated S176 ( p-S176 ) .", "The antibody was used to IP phosphorylated Amot and co-IP of Merlin and YAP was assessed by western blotting .", "These experiments showed that Amot phosphorylated at S176 binds to both YAP and Merlin .", "Similarly , IP with antibodies against YAP or Merlin also showed co-IP with phosphorylated Amot ( Figure 3D ) .", "Collectively , our findings demonstrate that Amot S176 phosphorylation state does not affect formation of the Amot/YAP/Merlin complex . 10 . 7554/eLife . 23966 . 007Figure 3 . Phosphorylation status of Amot-p130S176 does not impact formation of the Amot/YAP/Merlin complex . HEK293-shAmot cells were co-transfected with expression plasmids for Flag-Merlin , V5-YAP , and ( A ) HA-Amot-WT ( B ) HA-Amot-p130S176A or ( C ) HA-Amot-p130S176E .", "Total lysates ( input ) and Flag or V5 IPs were subjected to immunoblot analysis with anti-YAP and anti-Merlin antibodies as indicated .", "( D ) HEK293-shAmot cells were co-transfected with an expression plasmid for Amot-p130 .", "Total lysates ( input ) and IPs for phospho-Amot ( Ser176 ) , YAP , and Merlin were subjected to immunoblot analysis with indicated antibodies .", "The blots shown are representative of three independent biological replicates ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 00710 . 7554/eLife . 23966 . 008Figure 3—figure supplement 1 . Analysis of exogenous Amot expression levels and distribution .", "( A ) HEK293-shAmot cells were transfected with expression plasmids for Amot-WT , Amot-p130S176A or Amot-p130S176E .", "Relative levels of Amot expression were assessed by western blotting with anti-Amot antibody and compared to expression of the endogenous protein ( 293T ) .", "Tubulin was used as a loading control .", "( B ) HEK293T or HEK293-shAmot cells that were transfected with expression plasmids for FLAG-Amot-WT or HA-Amot-WT were fractionated in cytoplasmic ( C ) , nuclear ( N ) or plasma membrane ( PM ) fractions .", "Amot was detected by anti-Amot antibody and fractionation was validated by antibodies against EGFR , Lamin A/C and tubulin .", "The blots shown are representative of three independent experiments ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 00810 . 7554/eLife . 23966 . 009Figure 3—figure supplement 2 . Phosphorylation of Amot does not impact formation of YAP/Amot-p130 or Merlin/Amot-p130 complexes .", "( A–C )", "HEK293-shAmot cells were co-transfected with V5-YAP and ( A ) HA-Amot-WT or ( B ) HA-Amot-p130S176A or ( C ) HA-Amot-p130S176E .", "Total lysates ( input ) and V5 or HA IPs were subjected to immunoblot analysis with anti-YAP and anti-Amot antibodies as indicated .", "( D–F )", "HEK293-shAmot cells were co-transfected with Flag-Merlin and ( D ) HA-Amot-WT or ( E ) HA-Amot-p130S176A or ( F ) HA-Amot-p130S176E .", "Total lysates ( input ) and Flag or HA IPs were subjected to immunoblot analysis with anti-Merlin and anti-Amot antibodies as indicated .", "The blots shown are representative of three independent experiments ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 00910 . 7554/eLife . 23966 . 010Figure 3—figure supplement 3 . Phosphorylation of YapS127 does not impact the formation of the Amot/YAP/Merlin complex .", "( A ) HEK293 cells were co-transfected with expression plasmids for HA-Merlin and His-tagged constitutively active mutant of YAP ( His-YapS127A ) .", "Total lysates ( input ) and His or HA IPs were subjected to immunoblot analysis with anti-His and anti-HA antibodies as indicated .", "( B ) HEK293 cells were co-transfected with expression plasmids for Flag-Merlin and YAP .", "Total lysates ( input ) and Merlin or phospho-YAP IPs were subjected to immunoblot analysis with anti-phospho-YAP , Merlin and Flag antibodies as indicated .", "( C ) HEK293 cells were co-transfected with expression plasmids for HA-Merlin and Flag-tagged constitutively active mutant of YAP ( His-Yap5SA ) .", "Total lysates ( input ) and HA or Flag IPs were subjected to immunoblot analysis with anti-Flag and anti-HA antibodies as indicated .", "The blots shown are representative of three independent experiments ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 010 A major regulatory mechanism of YAP localization is through Lats1/2 phosphorylation of Ser127 promoting YAP cytoplasmic sequestration through 14-3-3 binding and concomitant sequestration from the nucleus ( Zhao et al . , 2010b , 2007 ) .", "We therefore evaluated whether YAP phosphorylation at Serine 127 plays a role in the establishment of the Amot/YAP/Merlin complex .", "Using similar IP approaches described above , we found that both phosphorylated YAP and constitutively active YAPS127A mutant retain their ability to associate with Amot/Merlin ( Figure 3—figure supplement 2 ) .", "Surprisingly , a YAP mutant where five major phosphorylation sites have been abolished ( YAP-5A [Zhao et al . , 2010b] ) was no longer associated with the complex ( Figure 3—figure supplement 2C ) .", "To determine whether phosphorylation induces a change in the sub-cellular localization of Amot/YAP/Merlin complex , 293-shAmot cells were transfected with expression vectors for Amot-p130 , Amot-p130S176A or Amot-p130S176E .", "The transfected cells were fractionated and the presence of Amot determined by Western blot analysis .", "In the cells expressing Amot-p130 , the majority of this protein localized to cytoplasmic and nuclear fractions , and to a lesser extent in the plasma membrane fraction .", "Interestingly , Amot-p130S176A showed a marked increase in nuclear localization and almost no presence at the plasma membrane .", "In contrast , Amot-p130S176E showed a clear shift in localization towards the cytoplasm and plasma membrane ( Figure 4A ) .", "Immunoblotting of total cell extracts confirmed similar expression levels of Amot-p130 , Amot-p130S176E and Amot-p130S176A ( Figure 4B ) .", "To determine the broader relevance of our findings in HEK293 cells to additional biological systems , we carried out a similar analysis in human hSC2λ Schwann cells and HepG2 hepatocellular carcinoma cells .", "Fractionation studies from these additional cell types followed a similar distribution pattern to the transfected 293-shAmot cells ( Figure 4—figure supplements 1A and 2A ) . 10 . 7554/eLife . 23966 . 011Figure 4 . Amot-p130S176 shifts localization of the YAP/Merlin complex .", "( A ) Phosphorylation of Amot-p130 shifts its localization at the plasma membrane .", "HEK293-shAmot cells were transfected with Amot-WT , Amot-p130S176A or Amot-p130S176E expression plasmids and fractionated into cytoplasm ( C ) , nuclear ( N ) and plasma membrane ( PM ) fractions .", "Immunoblot analysis was conducted using an anti-Amot antibody .", "GAPDH , Lamin A/C , and Na+/K+ATPase were using as controls for the cytoplasmic , nuclear , and plasma membrane fractions , respectively .", "( B ) IB analysis was used to verify the transfection efficiency of the indicated constructs in total lysates of HEK293-shAmot cells .", "Tubulin was used as loading control .", "Blots shown are representative of three independent biological experiments ( n = 3 ) .", "( C ) HEK293-shAmot cells were transfected with Amot-WT , Amot-p130S176A or Amot-p130S176E and subjected to immunofluorescence staining using an antibody against Amot .", "DAPI was used for nuclei fluorescence staining .", "Pictures show fields at 63x magnification and are representative of three independent biological replicates , in each of which 20 independent fields were examined .", "Scale bar = 10 μm .", "( D–F )", "Phosphorylation state of Amot-p130 mediates YAP localization .", "HEK293-shAmot cells were co-transfected with YAP and ( D ) Amot-WT , ( E ) Amot-p130S176A or ( F ) Amot-p130S176E and subjected to subcellular fractionation as in ( A ) .", "Cell lysates ( input ) and Amot IPs of each one of the subcellular fractions were subjected to immunoblot analysis with anti-YAP antibodies as indicated .", "Loading controls were as in ( A ) .", "All western blots shown are representative of three independent biological replicates ( n = 3 ) .", "( G ) Double-immunofluorescence staining with anti-YAP and anti-Merlin antibodies on HEK293-shAmot cells transfected with expression vectors for Amot-WT , Amot-p130S176A or Amot-p130S176E .", "Hoechst was used for staining of nuclei .", "Pictures show fields at 63x magnification and are representative of three independent biological replicates , in each of which 20 independent fields were examined .", "Scale bar = 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01110 . 7554/eLife . 23966 . 012Figure 4—figure supplement 1 . Amot phosphorylation regulates the localization of the Amot/YAP complex in human Schwann cells .", "( A ) Phosphorylation at Serine 176 shifts Amot-p130 localization .", "hSC2λ cells were transfected with Amot-WT , Amot-p130S176A or Amot-p130S176E expression plasmids and fractionated into cytoplasm ( C ) , nuclear ( N ) and plasma membrane ( PM ) fractions .", "Immunoblot analysis was conducted using an anti-Amot antibody .", "GAPDH , Lamin A/C , and Na+/K+ATPase were using as controls for the cytoplasmic , nuclear , and plasma membrane fractions , respectively .", "( B–D )", "Phosphorylation state of Amot-p130 mediates Amot/YAP complex localization .", "hSC2λ cells were co-transfected with YAP and ( B ) Amot-WT , ( C ) Amot-p130S176A or ( D ) Amot-p130S176E and subjected to subcellular fractionation as in ( A ) .", "Cell lysates ( input ) and Amot IPs of each one of the subcellular fractions were subjected to immunoblot analysis with anti-YAP antibodies as indicated .", "Loading controls were as in ( A ) .", "All western blots shown are representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01210 . 7554/eLife . 23966 . 013Figure 4—figure supplement 2 . Amot phosphorylation regulates the localization of the Amot/YAP complex in human hepatocellular carcinoma cells .", "( A ) Phosphorylation at Serine 176 shifts Amot-p130 localization .", "HepG2 cells were transfected with Amot-WT , Amot-p130S176A or Amot-p130S176E expression plasmids and fractionated into cytoplasm ( C ) , nuclear ( N ) and plasma membrane ( PM ) fractions .", "Immunoblot analysis was conducted using an anti-Amot antibody .", "GAPDH , Lamin A/C , and Na+/K+ATPase were using as controls for the cytoplasmic , nuclear , and plasma membrane fractions , respectively .", "( B–D )", "Phosphorylation state of Amot-p130 mediates Amot/YAP complex localization .", "HepG2 cells were co-transfected with YAP and ( B ) Amot-WT , ( C ) Amot-p130S176A or ( D ) Amot-p130S176E and subjected to subcellular fractionation as in ( A ) .", "Cell lysates ( input ) and Amot IPs of each one of the subcellular fractions were subjected to immunoblot analysis with anti-YAP antibodies as indicated .", "Loading controls were as in ( A ) .", "All western blots shown are representative of three independent biological replicates ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 013 To complement the biochemical fractionation studies , we assessed localization of the different Amot proteins by IF staining in the 293-shAmot cells .", "These analyses confirmed the fractionation findings , demonstrating a stronger intensity of Amot in the cytoplasm and nucleus of cells expressing Amot-p130 , a shift to nuclear staining in Amot-p130S176A expressing cells and cytoplasmic/plasma membrane localization in Amot-p130S176E expressing cells ( Figure 4C ) .", "We next examined if the status of S176 can regulate localization of the Amot/YAP/Merlin complex .", "First , we examined Amot/YAP localization using co-IP studies in fractions prepared from 293-shAmot cells transfected with expression vectors for Amot-p130 , Amot-p130S176A or Amot-p130S176E .", "In agreement with our previous results , in 293-shAmot cells transfected with Amot-p130 and YAP , Amot co-IPed with YAP mainly in the cytoplasm but also in the nucleus with little to no interaction detected at the plasma membrane ( Figure 4D ) .", "Importantly , in cells expressing the Amot-p130S176A allele , the Amot-YAP complex mainly localized to the nucleus with little to no interaction detected in the cytoplasm and plasma membrane ( Figure 4E ) .", "Significantly , in cells expressing the Amot-p130S176E allele an increase in the Amot-YAP association was detected at the plasma membrane , in addition to the cytoplasmic fraction .", "Little to no interaction was detected in the nucleus ( Figure 4F ) .", "Thus , the distribution of Amot/YAP complex mimics the distribution observed for Amot ( Figure 4A ) .", "In addition to the analysis in the 293-shAmot cells , a similar analysis carried out in the hSC2λ and HepG2 cells confirmed a similar distribution pattern ( Figure 4—figure supplements 1B–D and 2B–D ) .", "These findings were extended and confirmed by IF analysis of the YAP-Merlin association in 293-shAmot cells , where the localization of YAP and Merlin to the plasma membrane is significantly enhanced in cells expressing the Amot-p130S176E allele ( Figure 4G ) .", "Overall our findings demonstrate that while AmotS176 phosphorylation does not impact the formation of the Amot/YAP/Merlin complex , it mediates the localization of the complex through phosphorylation of Serine 176 that leads to recruitment of the complex to the plasma membrane or a shift to a nuclear localization in the hypophosphorylated state .", "Previous studies showed binding of Amot to the tight junction-associated proteins Pals1 and Patj at the apical membrane ( Ernkvist et al . , 2009; Sugihara-Mizuno et al . , 2007; Wells et al . , 2006; Yi et al . , 2011 ) .", "To further characterize the mechanisms by which phosphorylated Amot shifts from the cytoplasm and nucleus to the plasma membrane , we examined the interactions between Amot-p130 , Amot-p130S176A and Amot-p130S176E with Patj , Pals1 and E-cadherin .", "Using co-IP , we found that Flag-tagged Patj strongly associates with Amot-p130S176E , compared to weaker or null interaction with Amot-p130 and Amot-p130S176A , respectively .", "In a reciprocal approach , IP of the different Amot forms demonstrated a stronger association between Patj and Amot-p130S176E ( Figure 5A–C ) .", "Similar results were obtained when examining the association of Pals1 or E-cadherin with Amot .", "While we were consistently able to co-IP Pals1 or E-cadherin with Amot-p130S176E , this association was greatly diminished with Amot-p130S176A or wild type Amot-p130 ( Figure 5D–E ) .", "These findings suggest that localization of phosphorylated Amot-p130 to the cell membrane is mediated through interactions with components of junctional structures , which remain to be identified . 10 . 7554/eLife . 23966 . 014Figure 5 . AmotS176 status impacts binding to the junctional proteins PATJ , Pals1 and E-cadherin . HEK293-shAmot cells were co-transfected with Flag-PATJ and ( A ) HA-Amot-WT or ( B ) HA-Amot-p130S176A or ( C ) HA-Amot-p130S176E .", "Total lysates ( Input ) and Flag or HA IPs were subjected to immunoblot analysis with anti-HA and anti-Flag antibodies , as indicated .", "( D–E )", "HEK293-shAmot cells were co-transfected with ( D ) Flag-Pals1 or ( E ) E-cadherin and relevant Amot alleles as in panels ( A–C ) .", "Total lysates ( input ) and Flag IP were subjected to IB analysis with anti-Amot and anti-Flag antibodies as indicated .", "( F ) HEK293-shAmot cells were transfected with expression vectors for HA-Amot-WT , HA-Amot-p130S176A or HA-Amot-p130S176E .", "Cells were extracted in F-actin stabilizing buffer and F-actin was pulled down using Biotinylated-phalloidin and streptavidin-coupled magnetic beads .", "Pull downs were then alayzed by western blotting using anti-Amot or Actin antibodies .", "The blots shown are representative of three biological replicates ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 014 Previous reports concluded that phosphorylation of Amot at Serine 176 reduces the association between Amot and F-actin ( Chan et al . , 2013; Mana-Capelli et al . , 2014; Dai et al . , 2013 ) .", "To assess this directly in cells , we employed a co-precipitation approach relying on the high selectivity and binding affinity of phalloidin to F-actin , previously employed to identify F-actin binding proteins in cells ( Clarke and Mearow , 2013; Fulga et al . , 2007 ) .", "Briefly , biotinylated phalloidin was used to specifically pull-down F-actin from 293-shAmot cells transfected with expression vectors for Amot-p130 , Amot-p130S176A or Amot-p130S176E and levels of precipitated proteins were examined by western blotting .", "As shown in Figure 5F , similar amounts of all Amot proteins were present in the pull downs ( Upper panel ) .", "Importantly , similar levels of Actin were present in all pull downs indicating the expression of the different Amot alleles did not affect cellular levels of F-actin ( Lower panel ) .", "Given the impact of Amot-p130S176 phosphorylation on localization of the Amot/YAP/Merlin complex , we conducted loss- and gain-of-function studies to gain insight into the functional significance of this phosphorylation .", "First , we determined whether AmotS176 phosphorylation modulates cell proliferation .", "The 293-shAmot cells transfected with expression plasmids for Amot-p130 and Amot-p130S176A proliferated at increased rates compared to control transfected cells , as determined by cell counting and BrdU incorporation over a 96 hr period .", "In contrast , the Amot-p130S176E expressing cells exhibited a growth rate comparable to that of the control cells ( Figure 6A–B ) .", "Similar results were obtained in hSC2λ and HepG2 cells transfected with the different Amot alleles ( Figure 6—figure supplement 1A–B ) .", "These findings indicate that expression of Amot can promote the proliferation of these three different cell types and that this activity is diminished when Amot is phosphorylated at Serine 176 . 10 . 7554/eLife . 23966 . 015Figure 6 . AmotS176A promotes the proliferative and transcriptional activities of YAP .", "( A ) AmotS176 regulates cellular proliferation .", "HEK293-shAmot cells were transiently transfected with indicated expression plasmids and total cell numbers were counted over 4 days .", "Means of each data point were calculated from three independent biological replicates conducted in triplicate .", "Error bars represent ±S . D . Immunoblot analysis was used to verify the transfection efficiency of the indicated Amot-p130 constructs .", "Tubulin was used as a loading control .", "The blots shown are representative of three biological replicates .", "( B ) Amot expression drives proliferative phenotype that is YAP-dependent .", "HEK293-shAmot cells were co-transfected with the indicated expression plasmid and either a SMARTpool of siRNAs targeting YAP or a non-targeting control ( siCtr ) .", "Levels of BrdU incorporation compared to HEK293-shAmot+pcDNA+siCtr ( set to 1 ) were determined 48 hr , 72 hr , and 96 hr post co-transfection for all the conditions .", "Means were calculated from three biological replicates conducted in triplicate .", "Error bars represent ±S . D . Individual pairwise comparisons were assessed by Student's t-test , *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "Exact p-values are indicated in the figure .", "Immunoblot analysis to confirm efficient knockdown of YAP using two independent siRNAs ( siYAP-A and siYAP-B ) ( see Figure S6 ) and efficient overexpression of the indicated Amot-p130 constructs in HEK293-shAmot cells .", "Tubulin was used as a loading control .", "The blots shown are representative of three biological replicates ( n = 3 ) .", "( C ) Amot-p130 serine 176 does not affect Rac1 activation .", "IB analysis of cell lysates from HEK293-shAmot cells expressing Amot-WT , Amot-p130S176A or Amot-p130S176E with anti-Rac1-GTP , anti-Rac1 and anti-Amot antibodies as indicated .", "Tubulin was used as a loading control .", "Cells were serum starved overnight and stimulated with 10 ng/mL EGF for 5’ .", "The blots shown are representative of three biological replicates ( n = 3 ) .", "( D ) AmotS176 status regulates YAP transcriptional activity .", "HEK293 and HEK293-shAmot cells were transfected with indicated constructs and HIP-flash or HOP-flash reporters .", "Reporter’s firefly luciferase activity was normalized to the levels of Renilla luciferase used as an internal control .", "The means of luciferase activity were calculated from three biological replicates conducted in quadruplicate .", "Error bars represent ±S . D . Individual pairwise comparisons were assessed by Student's t-test , **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "Exact p-values are indicated in the figure .", "( E ) Immunoblot analysis showing efficient transfection of Amot-p130 , Amot-p130 mutants , and YAP in cell lysates used in ( D ) .", "Tubulin was used as a loading control .", "The blots shown are representative of three biological replicates .", "( F ) AmotS176 status regulates expression of endogenous YAP targets .", "Expression of the YAP target genes Areg and ApoE was probed in HEK293-shAmot cells expressing Amot-WT , Amot-p130S176A or Amot-p130S176E by quantitative real-time PCR .", "mRNA levels were compared with the empty vector control ( set to 1 ) .", "Means were calculated from Ct values in three independent biological replicates conducted in triplicate .", "GAPDH was used to normalize for variances in input cDNA .", "See Table", "1 . Error bars represent ±S . D . Individual pairwise comparisons were assessed by Student's t-test , **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "Exact p-values are indicated in the figure . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01510 . 7554/eLife . 23966 . 016Figure 6—source data 1 . Cell counts for HEK293 cells , treated as described Figure 6A . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01610 . 7554/eLife . 23966 . 017Figure 6—figure supplement 1 . AmotS176A promotes proliferation of human Schwann and hepatocellular carcinoma cells .", "( A ) hSC2λ or ( B ) HepG2 cells were cells were transiently transfected with indicated expression plasmids and total cell numbers were counted over 4 days ( top ) .", "Means of each data point were calculated from three independent biological replicates conducted in triplicate .", "Error bars represent ±S . D . Immunoblot analysis was used to verify the transfection efficiency of the indicated Amot-p130 constructs ( bottom ) .", "Tubulin was used as a loading control .", "The blots shown are representative of three biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01710 . 7554/eLife . 23966 . 018Figure 6—figure supplement 1—source data 1 . Cell counts for hSCλ cells , treated as described in Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01810 . 7554/eLife . 23966 . 019Figure 6—figure supplement 1—source data 2 . Cell counts for hSCλ cells , treated as described Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 01910 . 7554/eLife . 23966 . 020Figure 6—figure supplement 2 . Amot-p130S176 pro-proliferative phenotype is YAP dependent and regulates YAP transcriptional activity .", "( A ) HEK293-shAmot cells were co-transfected with the indicated plasmid DNAs and siRNAs .", "Fold variation of BrdU incorporation compared to HEK293-shAmot+pcDNA+siCtr ( set to 1 ) was determined 48 hr , 72 hr , and 96 hr post co-transfection for all the conditions .", "Means were calculated from three independent biological replicates conducted in triplicate ( n = 9 ) .", "Error bars represent ±S . D . ( n = 9 ) .", "Individual pairwise comparisons were assessed using Student's t-test , *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "( B ) HEK293 and HEK293-shAmot cells were transfected with indicated constructs and GTIIC-luc reporter .", "Reporter luciferase activity was normalized to levels of Renilla luciferase used as an internal reporter control .", "Means of luciferase activity were calculated from three independent biological replicates conducted in quadruplicate ( n = 12 ) .", "Error bars represent ±S . D . Individual pairwise comparisons were assessed using Student's t-test , *p<0 . 05; **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "Exact p-values are indicated in the figure . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02010 . 7554/eLife . 23966 . 021Figure 6—figure supplement 2—source data 1 . Counts for BrdU incoporation . Cells treated as described in Figure 6—figure supplement 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02110 . 7554/eLife . 23966 . 022Figure 6—figure supplement 2—source data 2 . Counts for luciferase activity . Cells treated as described in Figure 6—figure supplement 2B . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02210 . 7554/eLife . 23966 . 023Figure 6—figure supplement 3 . Amot-p130S176 regulates YAP transcriptional activity . Expression of the YAP target genes Areg and ApoE was assessed by quantitative real-time PCR in ( A ) hSC2λ or ( B ) HepG2 cells co-transfected with the indicated plasmid DNAs and siRNAs .", "Areg and ApoE mRNA levels were compared with the empty vector control ( pCDNA , set to 1 ) .", "Means were calculated from Ct values in three independent biological replicates conducted in triplicate .", "GAPDH was used to normalize for variances in input cDNA .", "See Table", "1 . Error bars represent ±S . D . Individual pairwise comparisons were assessed by Student's t-test , **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "Exact p-values are indicated in the figure . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02310 . 7554/eLife . 23966 . 024Figure 6—figure supplement 3—source data 1 . Source data for qPCR analysis of AREG expression in hSC-lambda cells . Analysis as described in Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02410 . 7554/eLife . 23966 . 025Figure 6—figure supplement 3—source data 2 . Source data for qPCR analysis of APOE expression in hSC-lambda cells . Analysis as described in Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02510 . 7554/eLife . 23966 . 026Figure 6—figure supplement 3—source data 3 . Source data for qPCR analysis of AREG expression in HepG2 cells . Analysis as described in Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02610 . 7554/eLife . 23966 . 027Figure 6—figure supplement 3—source data 4 . Source data for qPCR analysis of APOE expression in HepG2 cells . Analysis as described in Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02710 . 7554/eLife . 23966 . 028Table 1 . Primer sequences used in qPCR . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 028GenePrimersForwardReverseAmot5’-CAGCTTGCAGAGAAGGAATATGAG-3’5’-CTGGCTTTCTTTATTTTTTGCAAAG-3’ApoE5’-AGGAACTGAGGGCGCTGA-3’5’-AGTTCCGATTTGTAGGCCTTCA-3’Areg5’-TGATCCTCACAGCTGTTGCT-3’5’-TCCATTCTCTTGTCGAAGTTTCT-3’GAPDH5’ – GATCATCAGCAATGCCTCCT-3’5’ – TGTGGTCATGAGTCCTTCCA-3’ As work from our group and others has shown that Angiomotin functions as a regulator of small G-proteins from the Rac1/cdc42 family ( Wells et al . , 2006; Yi et al . , 2011 ) , we assessed whether the increased proliferation observed in the Amot-p130 and Amot-p130S176A expressing cells is mediated by activation of Rac1 .", "Towards this goal we analyzed the status of active Rac1 ( Rac1-GTP ) in these cells compared to control 293-shAmot cells .", "As expected , transfection with the different Amot alleles resulted in increased Rac1-GTP levels .", "However , we found no significant differences in Rac1-GTP levels between the cells , suggesting the status of Amot serine 176 phosphorylation does not affect Rac1 activation ( Figure 6C ) .", "We next determined whether the observed increased rates of cell proliferations induced by Amot-p130 and Amot-p130S176A expression are YAP-dependent , by introducing two independent siRNAs against YAP into these cells .", "Indeed , the increased proliferation afforded by Amot-p130 and Amot-p130S176A was completely inhibited by YAP knockdown ( Figure 6B and Figure 6—figure supplement 2A ) .", "The expression of Amot-p130 and Amot-p130S176A and reduction of YAP levels was confirmed by immunoblotting ( Figure 6B ) .", "Next , we determined if Amot Serine 176 phosphorylation regulates YAP-mediated transcriptional activation .", "We used two independent luciferase reporter assays , the HIP/HOP-flash and 8xGTIIC luciferase reporters ( Leask and Abraham , 2006; Zhao et al . , 2008 ) .", "The HIP ( Hippo-YAP signaling incompetent promoter ) /HOP ( Hippo-YAP signaling optimal promoter ) -flash reporters contain , respectively , seven mutated TEAD-binding sites and multimerized ( x8 ) TEAD-binding sites from the promoter of YAP’s direct target CTGF ( Leask and Abraham , 2006; Zhao et al . , 2008 ) .", "The former is a negative control for HOP-flash activity ( Kim and Gumbiner , 2015 ) .", "In agreement with our previously reported findings ( Yi et al . , 2013 ) , the activity of YAP in the HOP-reporter assay was suppressed in 293-shAmot cells , while the reintroduction of Amot fully rescued YAP activity ( Figure 6D–E ) .", "Moreover , the expression of Amot-p130S176A resulted in a substantial increase in the activation of the HOP-flash reporter when compared to Amot .", "Significantly , Amot-p130S176E was unable to rescue the function of YAP in the 293-shAmot cells ( Figure 6D–E ) .", "As expected , no significant changes in activation of the HIP reporter were observed under similar experimental conditions ( Figure 6D ) .", "These observations were further confirmed using the GTIIC luciferase system , which has eight copies of TEAD-binding sequence driving the expression of the luciferase gene ( Davidson et al . , 1988; Ota and Sasaki , 2008; Yi et al . , 2013 ) ( Figure 6—figure supplement 2B ) .", "To further evaluate the regulation of YAP’s activity by Amot , we investigated the impact of Amot-p130 , Amot-p130S176A and Amot-p130S176E expression in the 293-shAmot cells on known gene targets of YAP by quantitative real-time PCR .", "We focused on Areg ( amphiregulin ) and ApoE ( Apolipoprotein E ) as previous findings suggest Amot plays a role in their regulation ( Yi et al . , 2013 ) .", "While expression of both Amot-p130 and Amot-p130S176E had only minor effects on levels of Areg and ApoE , the expression of Amot-p130S176A significantly induced up-regulation of Areg and ApoE by 2 . 5 , and 3-fold respectively , relative to control cells ( Figure 6F ) .", "Similar results were obtained in hSC2λ and HepG2 cells transfected with the different Amot alleles ( Figure 6—figure supplement 3 ) .", "Previously it was shown that Amot is part of transcriptionally active YAP-Tead-containing complex in the nuclei of adult liver and HEK293 cells ( Yi et al . , 2013 ) .", "We hypothesized that Amot exerts its function in this complex in the dephosphorylated state .", "To test this , we performed reciprocal IP assays with antibodies against YAP or pan-Tead .", "We observed efficient co-IP of YAP and pan-Tead in the presence of Amot-p130 and Amot-p130S176A but not in the presence of Amot-p130S176E ( Figure 7A ) .", "Moreover , we conducted Chromatin immunoprecipitation ( ChIP ) with Amot antibodies in 293-shAmot cells transfected with Amot-p130 , Amot-p130S176A or an empty control vector , followed by RT-PCR analysis .", "These experiments showed a striking enrichment and binding of Amot-p130S176A to the promoter of ApoE and Areg , compared to Amot ( Figure 7B ) .", "These findings suggest that phosphorylation of Amot serine 176 prevents YAP-mediated transcriptional activation by inhibiting Amot nuclear function as a co-factor in a YAP-Tead transcriptional complex ( Figure 7C ) . 10 . 7554/eLife . 23966 . 029Figure 7 . AmotS176A but not AmotS176E is required for formation of the nuclear Yap-Tead complex . HEK293-shAmot cells were co-transfected with Amot-WT or Amot-p130S176A or Amot-p130S176E .", "Total lysates ( Input ) and Pan-Tead or YAP IPs were subjected to immunoblot analysis with anti-Pan-Tead or anti-YAP antibodies , as indicated .", "The blots shown are representative of three independent biological replicates .", "( B ) ChIP analysis of HEK293T-shAmot cells transfected with Amot-WT , Amot-p130S176A or an empty vector control .", "Real-time quantitative PCR was performed in eluted DNA using primers targeting the promoter regions of ApoE and Areg .", "See Table", "2 . The data show the means ±s . e . m . from three independent biological replicates ( n = 3 ) .", "Individual pairwise comparisons were assessed by Student's t-test , **p<0 . 01; ***p<0 . 001; n . s . – non-significant .", "Exact p-values are indicated in the figure .", "( C ) Proposed model for YAP-Merlin complex regulation by AmotS176 .", "Hypophosphorylation of AmotS176 promotes translocation of the Amot-p130/YAP/Merlin complex from the cytoplasm to the nucleus where it binds to TEADs and activates YAP-dependent transcriptional programs .", "Conversely , phosphorylation of AmotS176 induces cytoplasmic sequestration and plasma membrane localization of the tertiary complex .", "At the membrane , the complex associates with the junctional proteins Patj and Pals1 and YAP’s nuclear functions are inhibited .", "S176 in blue indicates phosphorylation . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 02910 . 7554/eLife . 23966 . 030Figure 7—source data 1 . Source data for qPCR analysis of ApoE expression in HEK293 cells . Analysis as described in Figure 7B . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 03010 . 7554/eLife . 23966 . 031Figure 7—source data 2 . Source data for qPCR analysis of AREG expression in HEK293 cells . Analysis as described in Figure 7B . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 03110 . 7554/eLife . 23966 . 032Table 2 . Primer sequences used in CHIP . DOI: http://dx . doi . org/10 . 7554/eLife . 23966 . 032GenePrimersForwardReverseApoEGCGTTCACTGTGGCCTGTCCAGCATGGAGGACAGCCCTGGCAregTGTTCTTCCCAGAAACCCTCTTTACCTACACCATCTCACAGC" ], [ "The role of Angiomotin regulating the Hippo/YAP pathway has so far been elusive , mainly due to conflicting reports suggesting that YAP regulation by Angiomotin is either positive or negative .", "To gain a deeper understanding of Amot function , we employed multiple cell types originating from tissues in which dysregulation of the Hippo-YAP pathway is observed under pathological conditions .", "Our study reveals that Angiomotin phosphorylation at serine 176 mediates YAP localization .", "This post-translational modification is the key event determining a promoting or repressing regulation of YAP by Angiomotin .", "We found that phosphorylation at serine 176 does not mediate Angiomotin’s scaffolding functions but does impact the localization of a tertiary complex composed by Amot , YAP and Merlin , which is present in both the cytoplasm and nucleus for the wild type protein .", "The serine 176 phosphomimetic mutant ( AmotS176E ) is recruited to the plasma membrane where it is found co-localized and associated with Patj , Pals1 and E-cadherin at junctional structures .", "Our previous studies indicate that Amot is required for YAP function in the nucleus ( Yi et al . , 2013 ) .", "Thus , the relocation of Amot out of the nucleus and sequestration of Amot/YAP complex to the plasma membrane , function to prevent YAP from operating as a growth-promoting transcriptional activator .", "The non-phosphorylated mutant ( AmotS176A ) is preferentially localized to the nucleus , where it facilitates YAP interactions with TEADs and concomitant activation of target gene transcription .", "Angiomotin’s involvement in regulation of Hippo-YAP signaling arises from studies showing that Amot functions as a scaffold protein for several components of this signaling cascade including YAP , Merlin , Kibra , Lats , and F-actin ( Moleirinho et al . , 2014 ) .", "Our findings extend Amot scaffolding functions to the formation of the YAP-Merlin complex independent of Amot Ser176 phosphorylation , although it is possible that additional PTMs might regulate the complex specifically at different sub-cellular localizations .", "Multiple reports describe the role of AmotS176 phosphorylation on Amot interactions with binding partners and function ( Adler et al . , 2013b; Chan et al . , 2013; Hirate et al . , 2013; Mana-Capelli et al . , 2014 ) .", "While these reports converge towards the idea that Amot serine 176 phosphorylation is mediated by Lats1/2 and that this impacts Amot’s binding to F-actin , they differ significantly when examining the influence on Amot localization and function .", "Whilst the discussion of these differences merits a separate review , we highlight a few points that agree or conflict with our current findings .", "In regards to the effect of Amot phosphorylation on binding to F-actin or YAP , previous reports conclude that serine 176 phosphorylation impairs the interaction between Amot and F-actin and suggest that this can favor binding to YAP ( Chan et al . , 2013; Mana-Capelli et al . , 2014; Dai et al . , 2013 ) .", "Our findings suggest that serine 176 phosphorylation does not impact association with F-actin .", "However , in agreement with one of these studies ( Dai et al . , 2013 ) our findings suggest that phosphorylation on Amot has little impact on the Amot-YAP association .", "Possible explanations for the differences between our current findings and previous reports could be attributed to the use of HEK293 cells expressing high levels of endogenous Amot , which might mask some of the interactions with the mutated alleles .", "To circumvent this possibility , our studies incorporated HEK293 cells in which endogenous Amot expression was knocked-down and the different mutant alleles reintroduced .", "Additionally , previous studies used different cell types , which not only could impact the stability of the Amot/YAP interaction but also explain the observed differences in Amot localization .", "In MCF10A , MCF7 and MDA-MB-468 cells , hypophosphorylated Amot showed junctional localization and co-localization with F-actin ( Adler et al . , 2013b; Chan et al . , 2013 ) .", "Although we cannot exclude differences in the actin cytoskeleton between the different cell lines used in those studies , we observe enrichment of AmotS176E to the plasma membrane , in agreement with Hirate et al . ( 2013 ) , and enrichment of AmotS176A to the nucleus .", "Furthermore , our studies confirm previous findings and show that Amot is required for YAP activity and that the AmotS176A mutant displays prominent nuclear function by means of increased levels of YAP target gene expression , increased cell proliferation rates and higher colony formation capacity ( Adler et al . , 2013b; Chan et al . , 2013; Hirate et al . , 2013 ) .", "Importantly , in vivo studies are needed to clarify the role of Amot in different cell and tissue types .", "We further extended the analysis of Amot phosphorylation on the Amot-YAP-Merlin complex , by determining whether its formation depends on YAPS127 phosphorylation site .", "In agreement with previous reports showing that the Amot-YAP interaction is independent of phosphorylation at YAPS127 ( Wang et al . , 2011; Zhao et al . , 2011 ) , we observed that the YAP-Merlin-Angiomotin complex also occurs independently of YAPS127 ( Figure 3—figure supplement 3A and B ) .", "Interestingly , mutation of serine in each of YAP’s five HxRxxS consensus sites ( S5A , Zhao et al . , 2010b ) precluded formation of the complex ( Figure 3—figure supplement 3C ) .", "The molecular mechanisms underlying the reduced association between the S5A and the Amot-merlin complex remain to be elucidated .", "However , this is not likely as a result of increased nuclear localization of YAP-S5A , since wild-type Amot can also be found in the nucleus in complex with wild type YAP .", "Therefore , it is likely that at least one additional post-translational modification of YAP could regulate the formation of the Amot-YAP-Merlin complex and future studies are needed to identify this modification .", "We found that AmotS176 phosphorylation induces association of the YAP-Merlin complex with Patj , Pals1 and E-cadherin , and that plasma membrane sequestration inhibits YAP activity .", "Several reports have described the localization of Amot at junctions in epithelial and endothelial cells ( Bratt et al . , 2005; Ernkvist et al . , 2008; Patrie , 2005; Wells et al . , 2006 ) and its colocalization with Patj/Pals1 via its C-terminal PDZ-binding motifs ( Wells et al . , 2006 ) .", "However , binding to Patj is not required for Amot’s localization to the plasma membrane , as a mutant lacking the PDZ binding motifs still localize to the cell cortex ( Sugihara-Mizuno et al . , 2007 ) .", "This suggests that an additional mechanism can regulate Amot membrane localization , which is in agreement with our findings where phosphorylation of AmotS176 induces localization of Amot-YAP-Merlin complex to the cell cortex .", "In HeLa , HEK293 and MDCK cells , Amot mediates YAP localization at the plasma membrane resulting in suppression of cell proliferation ( Chan et al . , 2011; Paramasivam et al . , 2011; Zhao et al . , 2011 ) .", "Thus , we propose that phosphorylation of Amot triggers a shift of the YAP-Merlin complex from the nucleus to the plasma membrane , thus suppressing the nuclear activity of YAP and exerting cell growth and tumor suppressive functions ( Figure 7C ) .", "Amot associates with Merlin and Rich1 at junctional structures and inhibits Rac1 and downstream signaling into the MAPK pathway ( Yi et al . , 2011 ) .", "Our results suggest that AmotS176 phosphorylation does not impact the ability to modulate Rac1 activity at the cell membrane .", "Yet , it is possible that AmotS176 phosphorylation modulates Merlin’s nuclear function .", "Previous studies showed that nuclear accumulation of Merlin results in inhibition of the CRL4DCAF1 E3 ubiquitin ligase .", "In NF2-mutant tumors , CRL4DCAF1 ubiquitinates Lats , suppressing phosphorylation and activating YAP ( Li et al . , 2014 , 2010 ) .", "In light of our findings , we can speculate that in the nucleus , Lats is inactivated by CRL4DCAF1-driven ubiquitylation and becomes unable to phosphorylate Amot , resulting in an increase of YAP transcriptional activity .", "However , if Lats remains active and Amot phosphorylation occurs , Amot-YAP-Merlin tertiary complex translocates from the nucleus to the cytoplasm and plasma membrane , resulting in suppression of YAP nuclear functions .", "Future studies will be required to shed light on this possibility .", "Another open question pertains to what mechanism/s regulate Angiomotin/YAP localization .", "Lats1/2 are obvious candidates , as they were shown to phosphorylate both YAP and Amot ( Adler et al . , 2013b; Chan et al . , 2013; Hirate et al . , 2013; Mana-Capelli et al . , 2014 ) .", "However , the role of Lats1/2 in regulation of these proteins and the relationship between Lats , YAP and Angiomotin is complex .", "For example , a number of reports suggest that Lats phosphorylates Amot , leading to reduced binding to F-actin and increased YAP binding and inhibition ( Chan et al . , 2013; Mana-Capelli et al . , 2014 ) .", "Moreover , Amot has been proposed to activate Lats2 and increase phosphorylation of YAP ( Paramasivam et al . , 2011; Chan et al . , 2013 ) .", "In contrast , we have previously shown that in multiple systems , Amot antagonizes the association of Lats and YAP and inhibits phosphorylation of YAP by Lats ( Yi et al . , 2013 ) .", "Given the complexity of the interactions between Amot and YAP , addressing the role of Lats1/2 in regulating the translocation and activity of the Amot-YAP complex will require future studies .", "Another potential regulatory mechanism stems from the lack of interaction between Amot and YAP-S5A mentioned above .", "This finding suggests that additional phosphorylation events could regulate the complex .", "As a number of other kinases such as AMPK and CK1delta/epsilon have been shown to phosphorylate YAP ( Wang et al . , 2015; Zhao et al . , 2010a ) .", "Further work will be required to determine the relevant phosphorylation sites and responsible kinases In conclusion , our studies suggest a mechanism to explain the previous conflicting observations regarding the role of Amot by demonstrating that AmotS176 phosphorylation state is a key event that dictates a positive or negative regulation of YAP by Amot by targeting the Amot-YAP-Merlin complex to the plasma membrane , sequestration in the cytoplasm or translocation to the nucleus ." ], [ "The expression plasmids for HA-Amot-p130 , HA-Amot-p130S176A and HA-Amot-p130S176E were a gift from Dr . Hiroshi Sasaki ( Kumamoto University , Japan ) .", "The following plasmids were previously described: psCMV-Pals1-Flag; PATJ-Flag ( Wells et al . , 2006; Yi et al . , 2011 ) ; Flag or HA-tagged Merlin ( Kissil et al . , 2002 , 2003 ) ; pCMV-Flag-Amot-p80 , pCMV-V5-YAP and pCMV-Amot-130 LPxY/PPxY mutants ( Yi et al . , 2011 , Yi et al . , 2013 ) .", "The pcDNA4/His-MaxB-YAP-S127A ( Plasmid #18988 ) and pCMV-Flag-YAP-5SA ( Plasmid #27371 ) were from Addgene .", "Human Amot-p130 shRNA vectors have been previously described ( Yi et al . , 2011 ) .", "siRNA duplexes targeting human YAP ( ID #s20366 ) as well as non-targeting control siRNAs ( ID #4390843 ) were from Thermo Fischer Scientific ( Carlsbad , CA ) .", "The second siRNA targeting human YAP1 ( 5 FlexiTube #SI02662954 ) was purchased from Qiagen .", "siRNA targeting human angiomotin ( ON-TARGETplus Human AMOT siRNA- smartpool L-015417-01-0005 ) or control pool of siRNA ( ON-TARGETplus Non-targeting pool-set of 4 LU-017595-01-0002 ) .", "HEK293 and HepG2 cells were purchased from the ATCC .", "hSC2λ cells were obtained from the laboratory of Dr . Margret Wallace ( Li et al . , 2016 ) .", "All cell lines were authenticated by short tandem repeat ( STR ) DNA profiling ( DDC Medical ) .", "Cells were tested every 3 months for mycoplasma contamination and confirmed free of contamination .", "Cells were maintained in low glucose Dulbecco’s Modified Eagle’s Medium ( DMEM ) ( Gibco ) supplemented with 10% fetal bovine serum ( Atlas Biologicals ) and antibiotics ( 100 units/ml penicillin and 100 μg/ml Streptomycin ) ( Gibco ) , at 37°C in a humidified atmosphere of 5% CO2 ( v/v ) .", "All experiments were carried out on cells grown to 70–80% confluency .", "Transfections were performed using Lipofectamine 2000 ( Invitrogen , Carlsbad , CA ) unless stated otherwise .", "Lentiviral infection of HEK293 cells was performed according to standard protocols .", "Briefly , HEK293T cells were co-transfected with packaging plasmids VSVG , Δ8 . 2 , and either with pLKO . 1-GFP or pLKO . 1-shAmot constructs .", "Supernatants were collected 48 hr and 72 hr after transfection , and cells were infected with 4 mL of viral supernatant containing 4 mL of polybrene ( 8 μg/mL ) .", "After 48 hr , transduced cells were selected with puromycin ( 2 μg/mL ) and this selection maintained for 72 hr .", "Rabbit polyclonal anti-Angiomotin was previously described ( Yi et al . , 2011 ) ( IB: 1:1500 ) .", "The following antibodies are available commercially: anti-phospho-Angiomotin ( ABS1045 from EMD Millipore; 1:2000 ) .", "Anti-HA tag ( sc805 , 1:1000 ) ; monoclonal anti-Merlin ( E-2 , 1:500 ) ; polyclonal anti-Merlin ( C-18 , 1:500 ) ; polyclonal anti-YAP ( H-125 , 1:1000 ) ; polyclonal anti-Lamin A/C ( sc-6215 ( N-18 ) , 1:500 ) ; polyclonal anti-EGFR ( sc03 , 1:1000 ) were from Santa Cruz Biotechnologies .", "Anti-Flag tag ( F1804 , 1:1000 ) ; anti-GAPDH ( 68795 , 1:10000 ) ; anti-tubulin ( T5168 , 1:1000 ) ; anti-actin ( A4700; 1:10000 ) were from Sigma .", "Anti-V5 tag ( ab27671 , 1:2000 ) from Abcam .", "Anti-6x His tag ( MA1-21315 , 1:2000 ) from Thermo Scientific .", "Anti-phospho-YAP ( 4911 , 1:1000 ) ; anti-pan-Tead ( 13295; 1:1000 ) from Cell Signaling Technologies .", "Anti-Na+/K+ATPase ( a-5 , 1:2500 ) from Developmental Studies Hybridoma Bank ( University of Iowa ) .", "For immunofluorescence the following antibodies were used at the stated concentrations: rabbit monoclonal anti-YAP D8H1X ( 14074 , 1:50 ) , Cell Signaling Technologies; mouse monoclonal anti-Merlin ( E-2; 1:50 ) , Santa Cruz Biotechnologies; Rabbit polyclonal anti-Angiomotin ( 1: 200 ) .", "150 cm diameter dishes were transiently transfected with 10 μg of plasmid DNAs using Lipofectamine 2000 ( Invitrogen ) .", "48 hr later cell lysates were collected , washed twice in ice-cold PBS , and lysed with radioimmuno precipitation assay buffer ( RIPA: 50 mM Tris-HCl; 150 mM NaCl; 1% NP40; 0 . 1% sodium dodecyl sulfate; 0 . 5% sodium deoxycholate; 1:25 protease inhibitor cocktail and phosphatase inhibitors ( Roche Applied Science ) ) .", "1 mg of protein was immunoprecipitated with Protein A/G resins ( #20333; #20398; Thermo Scientific ) and indicated antibody with gentle rotation at 4°C , overnight .", "Immunoprecipitates were washed four times in RIPA buffer , and bound proteins were dissociated in 25 μL of 1x loading dye ( 25 mM Tris-HCl pH 6 . 8 , 4% SDS , 5% glycerol , bromophenol blue ) .", "Eluted proteins were separated on SDS/10% polyacrylamide gel and transferred onto Immobilon-P membranes ( Millipore ) .", "To prevent nonspecific binding , membranes were incubated in blocking buffer ( 5% skimmed dried milk , 33 . 3 mM Tris-HCl , 16 . 68 mM Tris base , 138 mM NaCl , 2 . 7 mM KCl , 0 . 1% Tween-20 ) with agitation for 1 hr at room temperature , followed by immediate incubation with specific antibodies diluted in either 5% BSA or blocking buffer , overnight .", "Membranes were then washed three times in washing buffer ( 33 . 3 mM Tris-HCl , 16 . 68 mM Tris base; 138 mM NaCl; 2 . 7 mM KCl; 0 . 1% Tween-20 ) , incubated for 1 hr at room temperature with goat anti-mouse HRP-conjugated antibody ( sc-2005; 1:10000 ) or Protein A-HRP linked ( NA9120V; 1: 2000; GE Healthcare ) and protein expression was detected by chemiluminescence using ECL ( #RPN2106 or #RPN2236 , GE Healthcare ) .", "Cells were collected and lysed with actin stabilization buffer ( 1% Triton-X 100 , 0 . 1% SDS , 10 mM EDTA , 1% sodium deoxycholate , 200 pM sodium vanadate , 200 pM NaF , 1 complete protease inhibitor cocktail tablet , 0 . 5 mM ATP and Tris-Buffered Saline , pH 7 . 4 ) .", "Biotinylated-phalloidin ( 5 µg , Thermo-Fisher Scientific ) was added to samples and incubated for 60’ at 4°C with constant rotation .", "Subsequently , 20 µL of streptavidin-coupled magnetic Dynabeads were added , incubated for 60’ at 4°C with constant rotation .", "The magnetic beads were isolated with a magnet , washed 3X with PBS and analyzed as described .", "Immunofluorescence staining was carried out as previously described ( Li et al . , 2010 ) .", "Briefly , cells were grown on coverslips and transfected the next day using Lipofectamine .", "After 48 hr , coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and incubated in permeabilization buffer ( 0 . 3% sodium deoxycholate and 0 . 3% Triton-X in PBS ) for 30 min on ice .", "Coverslips were then washed twice in PBS and blocked in 5% goat serum/0 . 3% Triton-X in PBS for 1 hr at room temperature followed by overnight incubation with indicated primary antibodies .", "After three PBS washes , coverslips were incubated with goat anti-mouse Alexa Fluor 488 ( #A11029 , 1:400; Life Technologies ) , goat anti-rabbit Alexa Fluor 568 ( #A11011 , 1:400; Life Technologies ) or both secondary antibodies for 1 hr at room temperature .", "Cells were stained 5 min with 1 μg/mL of DAPI or Hoechst and mounted in Vectashield .", "Slides were examined using confocal microscopy ( LSM 780; Carl Zeiss; Plan Neofluar 63x/1 . 3 NA Korr differential interference contrast M27 objective in water ) at room temperature .", "Digitalized images were assembled using ZEN 2011 ( 64 bit ) software ( Carl Zeiss ) .", "Cells were incubated on ice for 20 min in ice-cold cytoplasmic buffer ( 20 mM Tris-HCl pH 7 . 4 , 150 mM KCl , 1 . 5 mM MgCl2 , 1 mM PMSF , 1 mM DTT , 0 . 5%Nonidet P-40 , protease inhibitor mixture ) , centrifuged at 4000 g for 5’ at 4°C .", "Supernatant was kept for the cytosolic and plasma membrane fractions .", "The pellet was washed five times with nuclear washing buffer ( 10 mM HEPES pH7 . 9 , 10 mM KCl , 1 . 5 mM MgCl2 , 0 . 34 M Sucrose , and complete protease inhibitor mixture ) , lysed on ice for 20 min in 400 μl of RIPA buffer and centrifuged at 13 , 000 g for 10 min at 4°C .", "Supernatant was kept as the nuclear fraction .", "For the cytosolic and plasma membrane fractions the supernatant was spun at 200 , 000 g for 30 min at 4°C and the resultant supernatant centrifuged again at 13 , 000g for 5 min at 4°C and kept as the cytosolic fraction .", "The pellet was washed in 500 μl lysis buffer ( 50 mM Tris pH 7 . 4 , 1 mM EDTA , 2 . 5 mm MgCl2 , 150 mM NaCl , and complete protease inhibitor mixture ) and re-sedimented at 200 , 000g for 30 min at 4°C .", "The pellet was then resuspended in ice-cold IP buffer ( 1 M Tris-HCl pH 7 . 4 , 4 M NaCl , 10% ( w/v ) Triton X-100 , and complete protease inhibitor mixture ) , incubated on a rocker for 30 min at 4°C , and cleared by centrifugation at 13 , 000g for 15 min at 4°C .", "The supernatant was kept as the plasma membrane fraction .", "HEK293-shAmot cells were transfected with 8 μg of plasmid DNA ( empty vector control/ wild type Amot-p130/AmotS176A/AmotS176E ) and 48 hr later lysed in RIPA buffer and precipitated with indicated antibody overnight .", "Rac1-GTP was pulled down according to the manufacturer’s instructions ( Millipore , #17–441 ) .", "HEK293-shAmot cells were seeded at a cell density of 2 × 105 cells per well in 6-well plates .", "On the following day cells were co-transfected with 40 nM of siRNAs ( targeting either YAP or a non-targeting siRNA duplex ( control ) ) , and 2 μg of plasmid DNA ( empty vector control/ wild type Amot-p130/AmotS176A/AmotS176E ) using TransIT-LT1 and TransIT-TKO ( Mirus , Fisher Scientific , Illinois ) according to manufacturer’s instructions .", "After 24 hr , cells were trypsinized and 2 × 104 cells/well were reseeded in sterile 96-well tissue culture plates .", "48 hr , 72 hr , and 96 hr after co-transfection , plates were processed and analysed using BrdU cell proliferation assay kit following manufacturer’s instructions ( Millipore , #2750 ) .", "Plates were read using a spectrophotometer microplate reader set to 450 nm ( SpectraMax M5; Molecular Devices ) .", "The HIP/HOP-flash luciferase reporter system was a kind gift from Dr . Barry Gumbiner ( Gumbiner and Kim , 2014 ) and the GTIIC-luciferase reporter was previously described ( Yi et al . , 2013 ) .", "HEK293 and HEK293-shAmot cells were seeded at a concentration of 40 × 103 cells/50 μl in 96-well plates .", "On the following day the indicated luciferase reporters and plasmids were transfected to a total of 160 ng and incubated overnight at 37°C .", "Luciferase activity was then measured with Dual Luciferase Reporter Assay System ( Promega; #E1910 ) according to manufacturer’s instructions .", "The reporter’s firefly luciferase activity was normalized to the levels of Renilla luciferase used as an internal control reporter .", "The relative luciferase activity displayed on the Y-axis indicates the ratio between Firefly/Renilla luciferase activities .", "Extraction of RNA from cell lysates was performed using Qiagen RNeasy kit ( Qiagen ) followed by cDNA synthesis of 1 μg DNase-digested RNA , using SuperScript III First-Strand Synthesis System for quantitative RT–PCR ( Invitrogen ) according to manufacturer’s instructions .", "Quantitative PCR of the synthesized cDNA was conducted using SYBR Green 2x Master Mix ( Applied Biosystems ) according to the manufacturer’s protocol .", "10 ng of each sample were used in each analysis .", "Real-time quantitative RT-PCR reactions were performed on StepOnePlus Real-Time PCR System ( Applied Biosystems ) and analysed using StepOne Software v2 . 2 . 2 .", "All measurements were conducted three times in triplicate and standardized to the levels of GADPH .", "Relative changes in gene expression were calculated according to the 2−ΔΔCT algorithm ( Livak et al . , 2001 ) .", "Sequence of the qPCR primers is provided in Table 1 .", "20 million HEK293T-shAmot cells were fixed with 1% formaldehyde for 10 min at RT .", "Fixation was halted with 125 mM glycine for 5 min at RT .", "Fixed cells were washed twice with cold PBS .", "Cell pellets were then resuspended in ChIP lysis buffer and chromatin was sheared with sonicator to obtain 0 . 3–0 . 5 kb DNA fragments .", "Angiomotin antibody ( 5 μg ) and Dynabeads Protein A were added to the cell lysate and incubated overnight at 4°C .", "Beads were washed with buffer 1 ( 150 mM NaCL , 20 mM TrisCl pH 8 . 0 , 5 mM EDTA , 65% w/v sucrose , 10% Triton-X-100 , 20% SDS ) and then washed with TE buffer .", "DNA was eluted by resuspending the beads in TE/1%SDS .", "ChIP DNA and Input were treated with RNase A ( 5 μg ) for 1 hr at 37°C .", "Proteinase K ( 0 . 5 mg/mL ) was added and incubated overnight at 65°C to reverse crosslinking .", "DNA was then purified phenol:chloroform and resuspended in a 30 μL of elution buffer .", "DNA was used for real time-PCR using SYBR Green PCR kit .", "A standard dilution curve was obtained for each Input and 1 μL of ChIP DNA was used in each PCR reaction .", "Melt curves were analyzed to confirm specificity of the amplified target ." ] ]
[ "The Hippo-YAP pathway is a central regulator of cell contact inhibition , proliferation and death .", "There are conflicting reports regarding the role of Angiomotin ( Amot ) in regulating this pathway .", "While some studies suggest a YAP-inhibitory function other studies indicate Amot is required for YAP activity .", "Here , we describe an Amot-dependent complex comprised of Amot , YAP and Merlin .", "The phosphorylation of Amot at Serine 176 shifts localization of this complex to the plasma membrane , where it associates with the tight-junction proteins Pals1/PATJ and E-cadherin .", "Conversely , hypophosphorylated Amot shifts localization of the complex to the nucleus , where it facilitates the association of YAP and TEAD , induces transcriptional activation of YAP target genes and promotes YAP-dependent cell proliferation .", "We propose that phosphorylation of AmotS176 is a critical post-translational modification that suppresses YAP’s ability to promote cell proliferation and tumorigenesis by altering the subcellular localization of an essential YAP co-factor ." ]
[ "Cells in animals and other multi-cellular organisms need to know when and where they should grow and divide .", "Individual cells communicate with their surrounding environment and each other via signaling pathways such as the Hippo-YAP pathway , which stimulates cells to grow and therefore influences the size of organs .", "When the Hippo part of the pathway is active it causes a protein known as YAP to move out of a compartment in the cell called the nucleus .", "Inside the nucleus , YAP helps to activate genes that promote cell growth .", "If the Hippo pathway can no longer respond to cues from the environment , YAP becomes over-active and can contribute to the development of various cancers .", "Therefore researchers are trying to better understand how it is regulated .", "Many signals both from inside and outside the cell influence YAP activity .", "For example , some signals block YAP from entering the nucleus , whereas others cause YAP to be broken down entirely .", "Several studies have recently identified a signal protein called angiomotin as a regulator of YAP .", "However , the studies provide conflicting reports as to whether angiomotin promotes or inhibits cell growth .", "Like many other proteins , angiomotin can be tagged with a small molecule called a phosphate group that can alter its activity .", "Moleirinho , Hoxha et al . studied human cells containing versions of angiomotin that mimic different forms of the protein with or without the phosphate .", "The experiments indicate that when a phosphate is attached at a particular position ( known as serine 176 ) , angiomotin predominantly interacts with YAP and another protein called Merlin at the cell surface .", "On the other hand , when angiomotin does not have a phosphate attached to it , all three proteins can move into the nucleus , where YAP is able to activate genes and promote cell growth .", "Overall , these findings indicate that adding a phosphate group to angiomotin can act as a switch to regulate where in the cell it and YAP are found and thus , whether YAP is active .", "Future experiments will investigate which enzymes add the phosphate group to serine 176 , and when they are able to do so ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "plant biology" ]
A non canonical subtilase attenuates the transcriptional activation of defence responses in Arabidopsis thaliana
elife-19755-v2
[ [ "Regulation of protein turnover plays a central role in the proper functioning of eukaryotic cells .", "Indeed , proteases of different families have been shown to be involved in the control of metabolism , physiology , growth and adaptive responses to biotic and abiotic stimuli ( van der Hoorn , 2008 ) .", "Subtilisin-like proteases ( subtilases ) are serine proteases featuring a catalytic triad characterized by the three amino acids aspartate , histidine and serine ( Dodson and Wlodawer , 1998 ) .", "According to the MEROPS ( http://merops . sanger . ac . uk ) classification , subtilases belong to the S8 family within the SB clan of serine proteases and are grouped into two subfamilies , subtilisins ( S8A ) and kexins ( S8B ) .", "Nine subtilases have been characterized in mammals , seven of which belong to the kexin group and two others to the S8A subfamily ( pyrolysins ) .", "With no representatives of the kexin type , plant subtilases exclusively belong to the pyrolysin group within the S8A subfamily .", "Pyrolysin-related subtilase families are largely expanded throughout the plant kingdom with a degree of complexity that exceeds that of their mammalian counterparts ( Schaller et al . , 2012 ) .", "In Arabidopsis the subtilase family comprises 56 members distributed in six distinct subgroups ( SBT1-6 ) ( Rautengarten et al . , 2005 ) .", "Despite their prevalence , our knowledge of the function of plant subtilases is rather poor .", "Subtilases are predicted to be secreted and have been involved in general protein turnover as well as in the highly specific regulation of plant development or responses to environmental changes and , more recently , in suppression of basal immunity and immune priming ( Schaller et al . , 2012; Figueiredo et al . , 2014 ) .", "As sessile organisms , plants must face the diversity of pathogens that they encounter in their habitat .", "Plants , unlike mammals , lack mobile defender cells and a somatic adaptive immune system .", "Instead , they rely on the innate immunity of each cell and on systemic signals originating from infection sites .", "Plant resistance to disease is a costly response , closely connected to plant physiological and developmental processes , and often associated with the so-called hypersensitive response ( HR ) , a form of programmed cell death that develops at attempted infection sites , allegedly to prevent pathogen propagation through the plant ( Coll et al . , 2011 ) .", "The sharp limit of the HR suggests the existence of tight regulatory mechanisms to restrict cell death development to the inoculated zone although the molecular actors involved in this process remain unknown for the most part .", "In line with the high cellular cost of triggering defence and cell death-associated responses , negative regulatory mechanisms are used by the plant to attenuate the activation of immune-related functions and allow a balanced allocation of resources upon pathogen challenge .", "Transcriptional reprogramming of the plant cell is a crucial step that allows mounting of efficient defence responses after pathogen attack .", "Transcription factors ( TFs ) and co-regulatory proteins play essential roles in launching and regulating the transcriptional changes that direct the plant defence response ( Buscaill and Rivas , 2014; Tsuda and Somssich , 2015 ) .", "MYB TFs of the R2R3 type ( 126 members in Arabidopsis ) mostly regulate plant-specific functions ( Dubos et al . , 2010 ) .", "Among the MYB TFs regulating defence-related transcription , Arabidopsis MYB30 is one of the best characterized .", "MYB30 promotes defence and cell death-associated responses through the transcriptional activation of genes related to the lipid biosynthesis pathway that leads to the production of very-long-chain fatty acids ( VLCFAs ) ( Raffaele et al . , 2008 ) .", "MYB30 is targeted by the effector protein XopD , from the bacterial pathogen Xanthomonas campestris pv .", "campestris ( Xcc ) , leading to suppression of MYB30-mediated transcriptional activation of plant resistance and thus underlining the important role played by MYB30 in plant defence regulation ( Canonne et al . , 2011 ) .", "A previously performed yeast two-hybrid ( Y2H ) screen using MYB30 as bait identified a secretory phospholipase ( AtsPLA2-α ) and a RING-type E3 ubiquitin ligase ( MIEL1 ) that exert negative spatial and temporal control on MYB30 transcriptional activity through distinct molecular mechanisms ( Froidure et al . , 2010; Marino et al . , 2013 ) .", "Here , we describe SBT5 . 2 , a serine protease of the subtilisin group , as a new MYB30-interacting partner .", "We demonstrate that the SBT5 . 2 transcript is alternatively spliced and that one of the two SBT5 . 2 splice variants , SBT5 . 2 ( b ) , whose expression pattern follows that of MYB30 after bacterial treatment , encodes an atypical subtilase that specifically mediates retention of MYB30 at endosomal vesicles .", "This phenomenon is independent of the integrity of the SBT5 . 2 ( b ) catalytic triad , requires N-terminal myristoylation of SBT5 . 2 ( b ) and results in attenuation of MYB30-mediated HR .", "Our work uncovers a novel regulatory mode for a subtilase protein and underlines the intricacy of the transcriptional regulation of plant responses to pathogen attack ." ], [ "In order to search for components involved in MYB30-mediated signalling , a Y2H screen was previously conducted using a MYB30 version deleted from its transcriptional activation domain ( MYB30ΔAD ) ( Froidure et al . , 2010 ) as bait .", "A cDNA clone encoding the last 103 amino acids of the Arabidopsis serine protease of the subtilisin group SBT5 . 2 ( At1g20160 ) was identified in this screen ( Figure 1 ) .", "SBT5 . 2 belongs to subgroup V ( 6 members ) within the classification of the Arabidopsis subtilase family ( Schaller et al . , 2012; Rautengarten et al . , 2005 ) . 10 . 7554/eLife . 19755 . 003Figure 1 . Specific interaction between MYB30 and SBT5 . 2 in yeast . Yeasts are shown after growth for five days on low stringency ( left; SD/-TL ) or high stringency ( right; SD/-TLHA ) media .", "Co-expression of MYB30 deleted from its C-terminal transcription activation domain ( MYB30△AD ) and the isolated cDNA clone encoding the last 103 amino acids of SBT5 . 2 ( SBT5 . 2628-730 ) resulted in yeast growth on selective medium .", "In a control experiment , yeast cells expressing MYB30△AD or SBT5 . 2628-730 with controls provided by Clontech ( T-antigen or P53 , respectively ) were not able to grow on selective medium .", "BD , GAL4 DNA-binding domain; AD , GAL4 activation domain . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 003 Two gene models are annotated in the TAIR database ( http://www . arabidopsis . org ) for At1g20160 , suggesting that the corresponding transcript is alternatively spliced ( Figure 2a ) .", "Comparison of the two SBT5 . 2 cDNA clones [designated SBT5 . 2", "( a ) and SBT5 . 2", "( b ) ] with the genomic sequence revealed that the first intron is specifically spliced in the SBT5 . 2", "( b ) cDNA ( Figure 2—figure supplement 1a ) .", "Reverse transcription PCR ( RT-PCR ) using specific cDNA primers confirmed the existence of both splice variants in planta ( Figure 2b ) .", "The sequence of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) 5’ ends was determined by 5’ RACE and cDNA sequencing ( Figure 2—figure supplement 1b ) .", "To gain knowledge on the relative abundance of both isoforms after bacterial inoculation , we monitored the expression of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) in Col-0 wild-type plants inoculated with Pseudomonas syringae pv .", "tomato DC3000 expressing the avirulence gene AvrRpm1 ( Pst AvrRpm1 ) .", "As shown in Figure 2c , expression of SBT5 . 2", "( a ) was downregulated after treatment with bacteria , whereas expression of SBT5 . 2", "( b ) was induced and displayed an expression profile highly similar to that of MYB30 ( Figure 2c ) .", "This data suggests that SBT5 . 2", "( b ) may have a MYB30-related function . 10 . 7554/eLife . 19755 . 004Figure 2 . SBT5 . 2 is alternatively spliced .", "( a ) Genomic structure of SBT5 . 2 alternatively spliced variants .", "Exons are shown as dark gray boxes ( E1-E9 ) , introns as black lines between exons and 5’ and 3’ UTRs are shown in light gray .", "( b ) RT-PCR analysis of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) transcripts in four-week old Col-0 Arabidopsis leaves .", "( c ) The relative expression of SBT5 . 2", "( a ) , SBT5 . 2", "( b ) and MYB30 at the indicated timepoints after inoculation of Col-0 plants with Pst AvrRpm1 ( 5 × 107 cfu/ml ) .", "Expression values were normalized using SAND family gene as internal standard and related to the value of each gene at time 0 , which is set at 1 . The SEM values were calculated from 4 independent experiments ( 4 replicates/experiment ) .", "The asterisks indicate statistically significant values for the three tested genes according to a Student’s t-test ( p<0 . 005 ) and with respect to gene expression values at time 0 .", "( d ) Schematic representation of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) protein sequences .", "The signal peptide ( SP ) and the pro-domain ( PD ) in the SBT5 . 2", "( a ) isoform are shown as black and grey boxes , respectively .", "Catalytically conserved Asp , His , Asn , and Ser residues are indicated .", "Putative N-glycosylation sites ( PGSs ) are indicated by black dots .", "( e ) Confocal images of epidermal N . benthamiana cells 36 hr after Agrobacterium-mediated transient expression of the indicated constructs .", "Accumulation of SBT5 . 2", "( a ) -RFP in apoplastic spaces is indicated by arrowheads .", "Bars = 10 µm .", "( f ) Western blot analysis of total protein extracts ( TE ) and intercellular fluids ( IF ) from N . benthamiana leaves expressing the intracellular protein MIEL1 alone ( left ) or co-expressed with HA-tagged SBT proteins ( right ) , as indicated .", "Molecular mass markers in kilodaltons are indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 00410 . 7554/eLife . 19755 . 005Figure 2—figure supplement 1 . Alternative splicing of SBT5 . 2 gives raise to two distinct variants .", "( a ) Alignment of the two SBT5 . 2 cDNA sequences with the genomic sequence .", "The consensus splice sequences flanking the intron that is retained in SBT5 . 2", "( a ) and shown in red in the genomic sequence are boxed .", "In the genomic sequence , ^ indicates 5’ and 3’ splice sites .", "In SBT5 . 2", "( a ) , the retained intron contains an ATG ( in bold ) encoding the first coding SBT5 . 2", "( a ) Met residue .", "Splicing of this intron gives rise to the SBT5 . 2", "( b ) isoform .", "Lower case in SBT5 . 2", "( a ) and SBT5 . 2", "( b ) cDNA sequences indicates untranslated 5’UTRs whereas upper case indicates coding sequences .", "Sequences used to specifically amplify SBT5 . 2", "( a ) or SBT5 . 2", "( b ) are underlined: for amplification of SBT5 . 2", "( a ) the primer was situated in the intron [that is absent in SBT5 . 2", "( b ) ] , whereas a primer in a sequence of the SBT5 . 2", "( b ) 5’UTR [that is absent in SBT5 . 2", "( a ) ] was used for SBT5 . 2", "( b ) amplification .", "Identical sequences are highlighted by a green box .", "From the ATG ( in bold ) in SBT5 . 2", "( b ) both nucleotide sequences are identical and , thus , not shown .", "( b ) Nucleotide sequences of 5’ends of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) obtained by 5’RACE amplification .", "The ATG encoding the first coding Met residue in each protein is shown in bold .", "Identical sequences are highlighted by a green box .", "From the ATG in SBT5 . 2", "( b ) both nucleotide sequences are identical and , thus , not shown .", "Sequences used to specifically amplify SBT5 . 2", "( a ) or SBT5 . 2", "( b ) are underlined .", "Every tenth residue is marked by | .", "( c ) Sequence alignment of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) proteins .", "Identical amino acids are highlighted in blue .", "The signal peptide and the prodomain in SBT5 . 2", "( a ) are boxed in red and yellow , respectively .", "Catalytical conserved residues are indicated by red dots .", "Putative N-glycosylation sites ( PGSs ) are indicated by blue dots .", "The 103 C-terminal amino acids encoded by the partial SBT5 . 2 cDNA clone identified in the yeast two-hybrid screen as interacting with MYB30ΔAD is boxed in blue .", "A putative myristoylation domain in SBT5 . 2", "( b ) is boxed in green with an asterisk indicating the putative myristoylated glycine residue .", "Every tenth residue is marked by | .", "DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 005 SBT5 . 2", "( a ) corresponds to a transcript of 2402 bp , which is predicted to encode a 769 amino acid preproenzyme containing a signal peptide ( SP ) followed a by prodomain ( PD ) , which acts as an intramolecular inhibitor , and a mature polypeptide ( Figure 2d; Figure 2—figure supplement 1c ) .", "In contrast , SBT5 . 2", "( b ) corresponds to a transcript of 2373 bp , predicted to encode a protein of 730 amino acids with no SP and lacking the first five amino acids of the PD ( Figure 2d; Figure 2—figure supplement 1c ) .", "Except for their N-terminal differences , the two corresponding encoded proteins are predicted to be identical and contain a catalytic triad with three amino acids ( D145 , H210 and S546 in SBT5 . 2", "( a ) , and D106 , H171 and S507 in SBT5 . 2", "( b ) ) conserved within serine proteases ( Figure 2d; Figure 2—figure supplement 1b ) .", "Alternative splicing ( AS ) of SBT5 . 2 may have important implications for the subcellular localization and function of the proteins encoded by the two transcripts .", "The presence of a SP and a PD in SBT5 . 2", "( a ) suggests that this protein may enter the secretory pathway and be secreted to the extracellular space .", "Indeed , secretion of SBT5 . 2", "( a ) was previously reported ( Engineer et al . , 2014; Kaschani et al . , 2012 ) .", "In contrast , the absence of the SP in SBT5 . 2", "( b ) may prevent secretion of the protein .", "In order to test this possibility , the subcellular localization of the two proteins was first investigated using Agrobacterium-mediated transient expression of RFP-tagged SBT5 . 2", "( a ) and SBT5 . 2", "( b ) under the control of a dexamethasone- ( Dex- ) inducible promoter in leaf epidermal cells of N . benthamiana .", "As expected , SBT5 . 2", "( a ) was found to be located in apoplastic spaces whereas SBT5 . 2", "( b ) was detected at vesicle-like structures inside cells ( Figure 2e ) .", "To obtain biochemical validation of the subcellular localization of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) , HA-tagged versions of both proteins were transiently expressed in N . benthamiana and intercellular fluid ( IF ) was isolated .", "In order to control the detection of intracellular proteins in the IF , the intracellular protein MIEL1 ( Marino et al . , 2013 ) was co-expressed with SBT5 . 2 proteins .", "As expected for an intracellular protein , MIEL1 was detected in the total extract fraction ( TE ) and not in the IF , confirming that the IF fraction did not contain intracellular proteins due to unintentional cellular lysis during IF isolation ( Figure 2f ) .", "SBT5 . 2", "( a ) was detected in the IF as two protein bands that may correspond to the processed and unprocessed forms of the protease , whereas SBT5 . 2", "( b ) was exclusively detected in the TE and never in the IF fraction ( Figure 2f ) .", "These results confirm the secretion of SBT5 . 2", "( a ) and the intracellular localization of SBT5 . 2", "( b ) .", "Most extracellular or secreted proteins are modified via N-glycosylation ( Moremen et al . , 2012 ) .", "Seven putative N-linked glycosylation sites ( PGSs; N in NxS/T motifs ) are present in SBT5 . 2 proteins [N225 , N363 , N467 , N525 , N636 , N650 and N678 in SBT5 . 2", "( a ) ] .", "In order to test whether SBT5 . 2 proteins are glycosylated in planta , protein extracts containing SBT5 . 2", "( a ) and SBT5 . 2", "( b ) were treated with PNGase F or EndoH and analysed for mobility shifts by Western Blot .", "At the end of their maturation in the secretory pathway , some plant N-linked glycans are modified , which renders them resistant to cleavage by glycosylases ( Lerouge et al . , 1998 ) .", "In agreement , an electrophoretic shift was only observed for the slow migrating , unprocessed form of SBT5 . 2", "( a ) , whereas migration of the fully processed form remained unaltered ( Figure 3a ) .", "In addition , SBT5 . 2", "( a ) bound to and was eluted from a concanavalin A resin ( Figure 3b ) , further suggesting that SBT5 . 2", "( a ) is a glycosylated protein .", "Finally , the increased electrophoretic mobility of SBT5 . 2", "( a ) in protein extracts from N . benthamiana leaves expressing HA-tagged SBT5 . 2", "( a ) and treated with tunicamycin , an inhibitor of N-linked glycosylation of newly synthesized glycoproteins in the ER ( Bassik and Kampmann , 2011 ) , further confirmed SBT5 . 2", "( a ) N-glycosylation in planta ( Figure 3c ) . 10 . 7554/eLife . 19755 . 006Figure 3 . SBT5 . 2", "( a ) , but not SBT5 . 2", "( b ) , is glycosylated in planta .", "( a ) SBT5 . 2", "( a ) , but not SBT5 . 2", "( b ) , is deglycosylated by PNGase F and Endo H . Protein extracts containing HA-tagged SBT5 . 2", "( a ) and SBT5 . 2", "( b ) transiently expressed in N . benthamiana were treated ( + ) or not ( − ) with PNGase F or Endo H as indicated .", "The arrowhead on the left indicates the fully processed form of SBT5 . 2", "( a ) whose mobility is not affected by the enzymatic treatment .", "( b ) HA-tagged SBT5 . 2", "( a ) , but not SBT5 . 2", "( b ) can be affinity purified using a concanavalin A resin .", "( c ) Glycosylation of HA-tagged SBT5 . 2", "( a ) , but not SBT5 . 2", "( b ) , is blocked by tunicamycin treatment ( + ) after transient expression in N . benthamiana .", "( d ) Electrophoretic mobility of individual HA-tagged PGS SBT5 . 2", "( a ) mutants .", "Mutated N to A residues are indicated .", "WT: wild-type SBT5 . 2", "( a ) proteins were interspersed to facilitate detection of the mobility shifts .", "In all cases , Western blot analyses were performed using anti-HA antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 006 We next analyzed individual PGS removal mutants ( in which the N residue was replaced by A ) on high resolution SDS-PAGE gels to determine their electrophoretic mobility as compared to wild-type SBT5 . 2", "( a ) .", "This analysis revealed a small but significant mobility shift for all SBT5 . 2", "( a ) PGS mutants ( Figure 3d ) , suggesting that all PGS in SBT5 . 2", "( a ) are used in planta .", "Despite the fact that SBT5 . 2", "( b ) contains the seven PGSs present in SBT5 . 2", "( a ) ,", "( i ) SBT5 . 2", "( b ) electrophoretic mobility was not modified after treatment with PNGase F or EndoH ( Figure 3a ) ;", "( ii ) neither SBT5 . 2", "( b ) binding to nor elution from a concanavalin A resin was observed ( Figure 3b ) ; and", "( iii ) no effect of tunicamycin leaf treatment on the SBT5 . 2", "( b ) electrophoresis profile was detected ( Figure 3c ) .", "These results , which are consistent with the absence of SP in SBT5 . 2", "( b ) and our previous observation that SBT5 . 2", "( b ) is not secreted , strongly suggest that SBT5 . 2", "( b ) does not enter the secretory pathway and is therefore not N-glycosylated .", "Subtilases , as other proteases , are typically able to catalyze their self-processing to render a mature active polypeptide .", "The presence of the three conserved amino acids in the catalytic triad of SBT5 . 2 proteins is consistent with these proteins displaying protease activity .", "When transiently expressing SBT5 . 2", "( a ) in leaf epidermal cells of N . benthamiana , two protein bands were detected that , as mentioned earlier , may correspond to the processed and unprocessed forms of the protease ( Figure 4a ) .", "In contrast , only a single band was detected for SBT5 . 2", "( b ) , suggesting that this protein is either not processed or fully processed in planta ( Figure 4a ) .", "In order to learn more about the proteolytic cleavage of SBT5 . 2 proteins , we engineered SBT5 . 2", "( a ) and SBT5 . 2", "( b ) mutant versions , in which the conserved histidine residue in the catalytic triad of both proteins was mutated to alanine , [SBT5 . 2", "( a ) H210A and SBT5 . 2", "( b ) H171A] .", "Following transient expression in N . benthamiana , mutation of the catalytic histidine residue did not affect migration of SBT5 . 2", "( b ) as compared to the wild-type protein , suggesting that this protein does not self-process in planta ( Figure 4a ) .", "In contrast , in the case of SBT5 . 2", "( a ) H210A , only the slow migrating band , that very likely corresponds to the unprocessed form of the protein , was detected ( Figure 4a ) .", "This observation suggests that SBT5 . 2", "( a ) is able to auto-process in planta and is thus active as a protease . 10 . 7554/eLife . 19755 . 007Figure 4 . SBT5 . 2", "( a ) , but not SBT5 . 2", "( b ) , shows serine protease activity .", "( a ) Western blot analysis shows expression of HA-tagged SBT5 . 2", "( a ) , SBT5 . 2", "( b ) and their catalytic mutant versions in N . benthamiana , as indicated .", "Ponceau S staining confirms equal loading .", "Molecular mass markers in kilodaltons are indicated on the right .", "( b ) Fluorimetric assay to detect protease activity following incubation of Arabidopsis protoplasts expressing the indicated proteins with fluorescein isothiocyanate ( FITC ) -conjugated casein ( top ) .", "RFU: relative fluorescence units .", "Error bars indicate SEM .", "Lowercase letters indicate significant differences as determined by Bonferroni-corrected p-values ( p<0 . 001 ) obtained after ANOVA and subsequent LSD post-hoc test .", "All proteins were detected by Western blot ( bottom ) .", "( c ) TAP-tagged MYB30 was expressed in N . benthamiana alone or with SBT5 . 2", "( a ) , SBT5 . 2", "( b ) and their catalytic mutant versions , as indicated .", "Western blot analysis shows the expression of TAP-tagged MYB30 and HA-tagged SBT proteins .", "Ponceau S staining confirms equal loading .", "Molecular mass markers in kilodaltons are indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 007 The catalytic activity of HA-tagged SBT5 . 2", "( a ) and SBT5 . 2", "( b ) was further investigated using a fluorimetric assay .", "Protein extracts from Arabidopsis protoplasts expressing HA-tagged SBT5 . 2", "( a ) , SBT5 . 2", "( a ) H210A , SBT5 . 2", "( b ) or SBT5 . 2", "( b ) H171A were incubated with the generic protease substrate casein conjugated to fluorescein isothiocyanate ( FITC ) .", "Increased fluorescence was observed for SBT5 . 2", "( a ) , reflecting proteolytic cleavage of the FITC-casein substrate ( Figure 4b ) .", "In contrast , fluorescence intensity in the case of SBT5 . 2", "( a ) H210A , SBT5 . 2", "( b ) and SBT5 . 2", "( b ) H171A was indistinguishable from that of protoplasts transformed with an empty vector ( Figure 4b ) , although their protein expression levels were comparable to those of SBT5 . 2", "( a ) ( Figure 4b ) .", "These results reinforce the idea that SBT5 . 2", "( a ) , but not SBT5 . 2", "( b ) , is correctly processed thus displaying protease activity .", "To determine whether SBT5 . 2", "( a ) or SBT5 . 2", "( b ) are involved in MYB30 proteolytic processing , the in planta accumulation of MYB30 when expressed alone or together with the different SBT5 . 2 proteins was analysed .", "As shown in Figure 4c , MYB30 accumulation was consistently unaltered in the presence of SBT5 . 2", "( a ) or SBT5 . 2", "( b ) as compared to the expression observed in the presence of the respective subtilase catalytic mutant versions .", "These results suggest that neither SBT5 . 2", "( a ) nor SBT5 . 2", "( b ) are able to proteolytically cleave MYB30 .", "MYB30 was previously localized to the nucleus of N . benthamiana and Arabidopsis cells ( Froidure et al . , 2010 ) .", "In order to investigate MYB30 potential colocalization with SBT5 . 2", "( a ) and/or SBT5 . 2", "( b ) , GFP-tagged MYB30 was co-expressed with RFP-tagged SBT5 . 2", "( a ) or SBT5 . 2", "( b ) .", "Confocal microscopy analysis of N . benthamiana leaves transiently co-expressing GFP-MYB30 and SBT5 . 2", "( a ) -RFP showed that these proteins do not co-localize in planta , as SBT5 . 2", "( a ) and MYB30 retain their respective extracellular and nuclear localization when expressed together ( Figure 5a ) .", "Surprisingly , when co-expressed with RFP-tagged SBT5 . 2", "( b ) , GFP-MYB30 was excluded from the nucleus and localized to the same vesicle-like structures where SBT5 . 2", "( b ) was localized , suggesting a possible in planta interaction between the two proteins outside the nucleus ( Figure 5a ) .", "Importantly , the unrelated MYB TF MYB123 retained its nuclear localization when co-expressed with SBT5 . 2", "( b ) , suggesting that SBT5 . 2", "( b ) -mediated MYB30 nuclear exclusion is specific ( Figure 5a ) .", "Moreover , SBT5 . 2", "( b ) -mediated specific nuclear exclusion of MYB30 was confirmed in Arabidopsis protoplasts ( Figure 5b ) . 10 . 7554/eLife . 19755 . 008Figure 5 . SBT5 . 2", "( b ) mediates retention of MYB30 in intracellular vesicles .", "( a ) Confocal images of epidermal N . benthamiana cells 36 hr after Agrobacterium-mediated transient expression of the indicated constructs .", "( b ) Confocal images of Arabidopsis protoplasts 16 hr after transformation of the indicated constructs .", "( c , d )", "GFP lifetime distribution of GFP-MYB30 in N . benthamiana cells", "( c ) or Arabidopsis protoplasts", "( d ) expressing SBT5 . 2", "( b ) .", "Histograms show the number of vesicles according to GFP-MYB30 lifetime classes in the presence of SBT5 . 2", "( b ) -HA ( green bars ) or SBT5 . 2", "( b ) -RFP ( magenta bars ) .", "The degree of overlap of GFP lifetime distribution is represented with magenta ( SBT5 . 2", "( b ) -RFP ) and green ( SBT5 . 2", "( b ) -HA ) arrows .", "Bars = 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 00810 . 7554/eLife . 19755 . 009Figure 5—figure supplement 1 . SBT5 . 2", "( b ) -mediated retention of MYB30 outside the nucleus is independent of C-terminal tagging of the subtilase and of SBT5 . 2", "( b ) catalytic triad .", "( a ) Confocal images of epidermal cells of N . benthamiana leaves 36 hr after Agrobacterium-mediated transient expression of GFP-tagged MYB30 and HA-tagged ( top ) or untagged ( down ) versions of SBT5 . 2", "( b ) Bars = 10 µm .", "( b ) Confocal images of Arabidopsis protoplasts 16 hr after transformation with the indicated constructs .", "Bars = 10 µm .", "( c ) GFP lifetime distribution of GFP-MYB30 in protoplasts expressing SBT5 . 2", "( b ) H171A .", "Histograms show the number of vesicles according to GFP-MYB30 lifetime classes in the presence of SBT5 . 2", "( b ) H171A-HA ( green bars ) or SBT5 . 2", "( b ) H171A-RFP ( magenta bars ) .", "The degree of overlap of GFP lifetime distribution is represented with magenta ( +SBT5 . 2", "( b ) H171A-RFP ) and green ( SBT5 . 2", "( b ) H171A-HA ) arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 00910 . 7554/eLife . 19755 . 010Figure 5—figure supplement 2 . MYB30 interacts with SBT5 . 2", "( b ) through the C-terminus of the subtilase .", "( a–c )", "Confocal images of epidermal cells of N . benthamiana leaves 36 hr after Agrobacterium-mediated transient expression of the indicated constructs .", "Bars = 10 µm .", "( d–f )", "GFP lifetime distribution of GFP-MYB30 , or GFP-MYB123 , in nuclei of N . benthamiana cells expressing the C-terminus of SBT5 . 2", "( b ) [SBT5 . 2", "( b ) 341-730] , or SBT5 . 1 [SBT5 . 1405-780] , as indicated .", "Histograms show the number of nuclei according to GFP-MYB30 , or GFP-MYB123 , lifetime classes when the MYB protein is expressed alone ( green bars ) or in the presence of RFP-tagged SBT5 . 2", "( b ) 341-730 , or SBT5 . 1405-780 , ( magenta bars ) , as indicated .", "The degree of overlap of GFP lifetime distribution is represented with magenta ( +RFP-tagged SBT constructs ) and green ( GFP-tagged MYB constructs ) arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 010 We next sought out to confirm the interaction between MYB30 and SBT5 . 2 in plant cells .", "We focused on the study of the interaction between MYB30 and SBT5 . 2", "( b ) because we were unable to detect a subcelullar co-localisation between MYB30 ( nuclear ) and SBT5 . 2", "( a ) ( secreted ) ( Figure 5a , b ) , which is a first requisite for the study of protein-protein interactions .", "The physical interaction between SBT5 . 2", "( b ) and MYB30 was investigated in FRET-FLIM assays using GFP- ( donor ) and RFP- ( acceptor ) tagged MYB30 and SBT5 . 2", "( b ) , respectively .", "In order to avoid potential changes in GFP lifetime due to differences in the molecular environments of two distinct subcellular compartments [MYB30 being nuclear when expressed alone or in vesicle-like structures when co-expressed with SBT5 . 2", "( b ) ] , the subcellular localization of GFP-tagged MYB30 when co-expressed with non-fluorescent HA-tagged , or an untagged version of SBT5 . 2", "( b ) , was therefore investigated .", "Importantly , both SBT5 . 2", "( b ) -HA and untagged SBT5 . 2", "( b ) , which are not able to act as acceptors for GFP fluorescence , also led to MYB30 retention in intracellular vesicle-like structures ( Figure 5—figure supplement 1a , b ) .", "A significant reduction of GFP lifetime was observed when GFP-MYB30 was co-expressed with RFP-tagged SBT5 . 2", "( b ) as compared to co-expression with SBT5 . 2", "( b ) -HA , both in N . benthamiana epidermal cells and in Arabidopsis protoplasts , thus confirming the physical interaction between the two proteins in intracellular vesicles ( Figure 5c , d; Table 1 ) .", "This interaction did not depend on the integrity of SBT5 . 2", "( b ) catalytic triad , as shown by the reduced GFP lifetime of GFP-MYB30 when co-expressed with RFP-tagged SBT5 . 2", "( b ) H171A ( Table 1; Figure 5—figure supplement 1c ) . 10 . 7554/eLife . 19755 . 011Table 1 . FRET-FLIM analysis shows that MYB30 physically interacts with SBT5 . 2", "( b ) at intracellular vesicles . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 011DonorAcceptorLifetime*SD†N‡E§p-value#N .", "benthamianaGFP-MYB30SBT5 . 2", "( b ) -HA2 . 5520 . 01357-GFP-MYB30SBT5 . 2", "( b ) -RFP2 . 2740 . 0195410 . 865 . 8 × 10−21GFP-MYB30-2 . 6690 . 00982GFP-MYB30SBT5 . 2", "( b ) 362-730-RFP2 . 2710 . 0165114 . 913 . 8 × 10−49GFP-MYB30SBT5 . 1", "( b ) 405-780-RFP2 . 5920 . 018442 . 864 . 2 × 10−05GFP-MYB123-2 . 5700 . 01359GFP-MYB123SBT5 . 2", "( b ) 362-730-RFP2 . 5440 . 013581 . 000 . 17A .", "thalianaGFP-MYB30SBT5 . 2", "( b ) -HA2 . 4910 . 00960-GFP-MYB30SBT5 . 2", "( b ) -RFP2 . 1510 . 0186013 . 651 . 2 × 10−33GFP-MYB30SBT5 . 2", "( b ) H171A-HA2 . 4730 . 01260-GFP-MYB30SBT5 . 2", "( b ) H171A-RFP2 . 1150 . 0236014 . 497 . 8 × 10−26* Mean lifetime in nanoseconds† Standard deviation‡ Total number of measured vesicles§ Percentage of FRET efficiency ( E = 1 - τDA/τD ) calculated by comparing the lifetime of the donor in the presence of the acceptor ( τDA ) with its lifetime in the absence of the acceptor ( τD ) .", "# p value of the difference between the donor lifetimes in the presence and in the absence of the acceptor ( t-test ) The identification of a partial SBT5 . 2 cDNA clone in yeast suggested that the MYB30-SBT5 . 2", "( b ) interaction is mediated by the C-terminus of SBT5 . 2", "( b ) .", "In order to confirm this idea , a truncated SBT5 . 2", "( b ) version containing the C-terminal end of the protein [SBT5 . 2", "( b ) 362-730] fused to the RFP was generated and transiently expressed in N . benthamiana .", "SBT5 . 2", "( b ) 362-730-RFP presents a nucleocytoplasmic localization and colocalises with MYB30 in the nucleus ( Figure 5—figure supplement 2a ) .", "A significant reduction of the average GFP lifetime was measured in nuclei coexpressing GFP-MYB30 and SBT5 . 2", "( b ) 362-730-RFP , as compared to nuclei expressing GFP-MYB30 alone ( Figure 5—figure supplement 2d; Table 1 ) , confirming that the C-terminus of SBT5 . 2", "( b ) is sufficient for the interaction with MYB30 .", "The specificity of this observation was highlighted by the lack of interaction between GFP-MYB30 and the equivalent C-terminal domain of the closest Arabidopsis SBT5 . 2 homolog , SBT5 . 1 , ( SBT5 . 1405-780-RFP ) ( Figure 5—figure supplement 2b , e; Table 1 ) .", "Moreover , no significant reduction of the average GFP lifetime was detected between nuclei expressing the unrelated TF MYB123 [whose nuclear localization was not affected in the presence of full length SBT5 . 2", "( b ) ( Figure 5a , b ) ] , when expressed alone or together with SBT5 . 2", "( b ) 362-730-RFP , despite the nuclear co-localization of the two proteins ( Figure 5—figure supplement 2c , f; Table 1 ) .", "Together , our data confirms that MYB30 specifically interacts with SBT5 . 2", "( b ) at vesicle-like structures .", "This interaction is mediated by SBT5 . 2", "( b ) C-terminus , does not require an intact SBT5 . 2", "( b ) catalytic triad and results in MYB30 nuclear exclusion .", "We next sought to determine the nature of the intracellular vesicle-like structures where SBT5 . 2", "( b ) resides .", "Given the mobile character and varied sizes of these vesicles , we conducted co-localization experiments with VHA-a1 and SYP61 , two markers for the trans-Golgi network/early endosomes ( TGN/EE ) ( Dettmer et al . , 2006; Tanaka et al . , 2009 ) and ARA6 and SYP21 , two markers for late endosomes/multivesicular bodies ( LE/MVB ) ( Ueda et al . , 2004; Uemura et al . , 2004 ) .", "Colocalization with of SBT5 . 2", "( b ) with these endosomal markers was clearly observed in N . benthamiana leaves ( Figure 6a , Figure 6—figure supplement 1 ) .", "In contrast , no colocalization of SBT5 . 2", "( b ) -RFP with the Golgi marker GmMan-GFP ( Nelson et al . , 2007 ) was detected ( Figure 6a ) . 10 . 7554/eLife . 19755 . 012Figure 6 . An N-terminal myristoylation site in SBT5 . 2", "( b ) is required for its localization to endosomes and MYB30 nuclear exclusion .", "( a , b )", "Confocal images of epidermal N . benthamiana cells 36 hr after Agrobacterium-mediated transient expression of the indicated constructs .", "Bars = 10 µm .", "( a ) SBT5 . 2", "( b ) localizes to endosomal vesicles .", "TGN/EE: trans-Golgi network/early endosomes; LE/MVB: late endosomes/multivesicular bodies .", "( b ) A free N-terminal myristoylation site in SBT5 . 2", "( b ) mediates its localization to endosomes and retention of MYB30 in endosomal vesicles .", "( c ) Relative fluorescence values of cytoplasmic MYB30 , expressed alone or with the indicated SBT5 . 2", "( b ) versions , represented as ratios between cytoplasmic and nuclear fluorescence values in individual cells .", "Mean and SEM values were calculated from two independent experiments in which fifteen fluorescence measurements were taken per experiment and construct combination .", "Lowercase letters indicate statistically significant differences as determined by Bonferroni-corrected p-values ( p<0 . 001 ) obtained after ANOVA and subsequent LSD post-hoc test . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 01210 . 7554/eLife . 19755 . 013Figure 6—figure supplement 1 . SBT5 . 2", "( b ) localizes to endosomal vesicles . Confocal images of epidermal cells of N . benthamiana leaves 36 hr after Agrobacterium-mediated transient expression of the indicated constructs .", "TGN/EE: trans-Golgi network/early endosomes; LE/MVB: late endosomes/multivesicular bodies .", "Bars = 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 013 The nucleocytoplasmic subcellular localization of SBT5 . 2", "( b ) 362-730 suggested that the N-terminal region of SBT5 . 2", "( b ) is required for its localization to endosomes ( Figure 5—figure supplement 2 ) .", "In order to further test this idea , an N-terminal deletion of SBT5 . 2", "( b ) was generated [SBT5 . 2", "( b ) 162-730] .", "RFP-tagged SBT5 . 2", "( b ) 162-730 indeed localized to the cytoplasm ( Figure 6b ) .", "Furthermore , an N-terminally tagged RFP fusion of SBT5 . 2", "( b ) also presented a cytoplasmic localization ( Figure 6b ) , suggesting that a free SBT5 . 2", "( b ) N-terminus is necessary for SBT5 . 2", "( b ) endosomal targeting .", "Close inspection of the SBT5 . 2", "( b ) N-terminal region uncovered the presence of a putative myristoylation site ( MGSASSA; Figure 2—figure supplement 1c ) .", "A SBT5 . 2", "( b ) version in which the putatively myristoylated Gly2 residue was mutated to Ala [SBT5 . 2", "( b ) G2A] also localized to the cytoplasm ( Figure 6b ) , confirming the importance of N-myristoylation for SBT5 . 2", "( b ) endosomal targeting .", "Finally , co-expression of SBT5 . 2", "( b ) 162-730-RFP , RFP-SBT5 . 2", "( b ) or SBT5 . 2", "( b ) G2A RFP with GFP-MYB30 did not affect MYB30 nuclear targeting ( Figure 6b ) .", "Interestingly , MYB30 accumulation in the cytoplasm was enhanced in the presence of these SBT5 . 2", "( b ) versions , consistent with the presence in the three proteins of an intact SBT5 . 2 C-terminal domain that mediates the interaction with MYB30 ( Figure 6c ) .", "An HA-tagged SBT5 . 2", "( b ) G2A version induced the same effect confirming that the enhanced detection of GFP-MYB30 in the cytoplasm is not an artefact due the presence of a second fluorophore .", "Together , our data strongly suggest that a free N-terminal myristoylated residue is responsible of SBT5 . 2", "( b ) targeting to the endosomes and of MYB30 retention in endosomal vesicles .", "In order to further characterize the simultaneous localization of SBT5 . 2", "( b ) to both early and late endosomes , HA-tagged versions of either SBT5 . 2", "( a ) , SBT5 . 2", "( b ) or SBT5 . 2", "( b ) G2A were co-expressed with both GFP-tagged VHA-a1 and RFP-tagged ARA6 .", "When expressed with SBT5 . 2", "( a ) , both subcellular markers conserved their distinct subcellular localization in TGN/EE and LE/MVB , respectively ( Figure 7 ) .", "In contrast , co-expression with SBT5 . 2", "( b ) led to co-localization of VHA-a1 and ARA6 in the same endosomal compartment , strongly suggesting that SBT5 . 2", "( b ) is able to interfere with endosomal trafficking .", "Myristoylation of SBT5 . 2", "( b ) appears to be essential to this effect , since expression of SBT5 . 2", "( b ) G2A did not affect the discrete endosomal populations tagged by VHA-a1 or ARA6 ( Figure 7 ) . 10 . 7554/eLife . 19755 . 014Figure 7 . SBT5 . 2", "( b ) leads to the formation of hybrid endosomal compartments in a myristoylation-dependent manner . Confocal images of epidermal N . benthamiana cells 36 hr after Agrobacterium-mediated transient expression of the indicated constructs .", "TGN/EE: trans-Golgi network/early endosomes; LE/MVB: late endosomes/multivesicular bodies .", "Bars = 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 014 To investigate the function of SBT5 . 2 in the plant response to bacterial inoculation , we used Arabidopsis sbt5 . 2 null mutants , sbt5 . 2–1 ( SALK_012113 ) and sbt5 . 2–2 ( SALK_132812C ) , both containing a T-DNA insertion in the last exon of SBT5 . 2 .", "Despite the severe reduction of SBT5 . 2 expression in the mutant lines ( Figure 8—figure supplement 1 ) , no obvious macroscopic phenotype was observed in these plants .", "The phenotype of these lines in response to bacterial inoculation was next analysed .", "Similar to MYB30-overexpressing ( MYB30OE ) , sbt5 . 2 mutant plants showed stronger HR cell death symptoms after inoculation with Pst AvrRpm1 as compared to Col-0 wild-type plants ( Figure 8a ) .", "This phenotype was quantified by ion leakage measurements in leaf disk assays .", "Conductivity values measured in sbt5 . 2 and MYB30OE plants were significantly higher than those displayed by Col-0 wild type plants after bacterial inoculation ( Figure 8b ) .", "In agreement with faster HR development , sbt5 . 2 plants showed increased resistance in response to inoculation with Pst AvrRpm1 , as compared to wild-type plants ( Figure 8c ) , confirming the role of SBT5 . 2 as a negative regulator of plant defence . 10 . 7554/eLife . 19755 . 015Figure 8 . SBT5 . 2", "( b ) is a negative regulator of resistance and HR responses in Arabidopsis in response to bacterial inoculation .", "( a ) Symptoms developed by the indicated Arabidopsis lines 60 hpi with Pst AvrRpm1 ( 2 × 106 cfu/ml ) .", "The pictures are representative of three independent experiments in which 4 plants of each line were infiltrated .", "( b ) Quantification of cell death by measuring electrolyte leakage of the indicated Arabidopsis lines in a time course of 24 hr .", "Plants were inoculated with Pst AvrRpm1 ( 5 × 106 cfu/ml ) .", "Mean and SEM values were calculated from four independent experiments in which three plants were inoculated ( four leaves/plant ) .", "( c ) Growth of Pst AvrRpm1 in the indicated Arabidopsis lines .", "Bacterial growth 0 ( white bars ) and three days ( blue bars ) was measured after inoculation ( 5 × 105 cfu/ml ) .", "Mean bacterial densities were calculated from 6 independent experiments with 6 individual plants ( 4 leaves/plant ) .", "Statistical differences using multiple factor analysis of variance ( ANOVA ) ( p<0 . 001 ) are indicated by letters . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 01510 . 7554/eLife . 19755 . 016Figure 8—figure supplement 1 . Characterization of sbt5 . 2 mutant lines before and after transformation with SBT5 . 2", "( a ) and SBT5 . 2", "( b ) overexpresing constructs .", "( a ) The expression of SBT5 . 2 is strongly repressed in sbt5 . 2 mutant lines .", "qRT-PCR analysis of the six members of the clade V of Arabidopsis subtilases in wild-type Col-0 ( black bars ) , sbt5 . 2–1 ( gray bars ) and sbt5 . 2–2 ( white bars ) Arabidopsis leaves .", "Expression values were normalized using SAND family gene as internal standard .", "Mean and SEM values were calculated from 4 independent replicates .", "Expression of SBT 5 . 4 and SBT5 . 5 was not detected in any line .", "( b ) The relative expression of SBT5 . 2 in Arabidopsis leaves in wild-type Col-0 , SBT5 . 2", "( a ) OE and SBT5 . 2", "( b ) OE lines was determined by qRT-PCR .", "The expression values were normalized by using SAND family gene as internal standard , and related to the value of Col-0 , which is set at 1 . Mean and SEM values were calculated from four individual plants per line .", "( c ) Western blot analysis showing SBT5 . 2", "( a ) -HA or SBT5 . 2", "( b ) -HA protein accumulation in Arabidopsis transgenic and Col-0 wild-type plants .", "Ponceau S staining confirms equal loading .", "Molecular mass markers in kiloDaltons are indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 016 Importantly , sbt5 . 2 mutant plants displayed higher expression of MYB30 VLCFA-related target genes FDH and CER2 ( Raffaele et al . , 2008 ) as compared to Col-0 wild-type plants 1 hr after inoculation ( Figure 9a ) .", "Moreover , this phenotype was abolished in the myb30 mutant background ( sbt5 . 2 myb30; Figure 9a ) and correlated with loss of increased HR in the sbt5 . 2 myb30 double mutant ( Figure 9b ) .", "Together , these data confirm that SBT5 . 2 negatively regulates Arabidopsis defence through repression of MYB30 transcriptional activity . 10 . 7554/eLife . 19755 . 017Figure 9 . SBT5 . 2", "( b ) attenuates MYB30-dependent transcriptional activation of VLCFA-related genes and hypersensitive cell death .", "( a ) Expression analysis of the MYB30 target genes FDH and CER2 in the indicated Arabidopsis lines 1 hr after inoculation with Pst AvrRpm1 ( 5 × 107 cfu/ml ) .", "Expression values of the individual genes were normalized using SAND family as internal standard .", "Mean and SEM values were calculated from 3 independent experiments ( 4 replicates/experiment ) .", "Statistical significance according to a Student’s t-test ( p<0 . 05 ) is indicated by letters .", "( b , c )", "Quantification of cell death by measuring electrolyte leakage of the indicated Arabidopsis lines before ( black bars ) and 24 hr after ( gray bars ) inoculation with Pst AvrRpm1 ( 5 × 106 cfu/ml ) .", "Mean and SEM values were calculated from four independent experiments ( three plants/experiment and four leaves/plant ) and related to the value displayed by wild-type Col-0 plants , which is set at 100% .", "Statistical differences using multiple factor analysis of variance ( ANOVA ) ( p<0 . 01 ) are indicated by letters .", "( d ) Representative pictures of accumulation of phenolic compounds ( blue coloration indicative of cell death ) in the indicated Arabidopsis lines detected by epifluorescence 24 hr after inoculation with Pst AvrRpm1 ( 2 × 105 cfu/ml ) .", "Bar = 100 µm .", "( e ) Blue pixels in the indicated lines were quantified using Image-Pro Plus and are shown as the percentage of the total number of blue pixels in each image .", "Boxplots are as follows: box limits , values between first and third quartiles; middle bar , median .", "Whiskers cover 1 . 5 times the interquartile distance and circles represent extreme values .", "Lowercase letters indicate significant differences with respect to the sbt5 . 5–2 line as determined by Bonferroni-corrected p-values ( p<0 . 01 ) obtained after ANOVA and subsequent LSD post-hoc test . DOI: http://dx . doi . org/10 . 7554/eLife . 19755 . 017 In sbt5 . 2 mutant plants , expression of both SBT5 . 2", "( a ) and SBT5 . 2", "( b ) is affected .", "To obtain additional proof of the negative role specifically played by SBT5 . 2", "( b ) on MYB30-mediated responses , sbt5 . 2 mutant plants were transformed with an HA-tagged version of either SBT5 . 2", "( a ) or SBT5 . 2", "( b ) under the control of the 35S promoter .", "Expression of SBT5 . 2 gene and protein was monitored by qRT-PCR and Western Blot analysis in two independent homozygous T4 lines for each construct ( Figure 8—figure supplement 1b , c ) .", "Importantly , the increased HR phenotype displayed by the sbt5 . 2 mutant was specifically suppressed in sbt5 . 2 plants overexpressing SBT5 . 2", "( b ) , but not SBT5 . 2", "( a ) ( Figure 9c ) .", "In order to obtain in vivo confirmation of the negative control of defence-related cell death exerted by SBT5 . 2", "( b ) , the above described lines were inoculated with a low dose of HR-inducing Pst AvrRpm1 .", "Enhanced accumulation of phenolic compounds , characteristic of HR cell death , was detected in MYB30OE ans sbt5 . 2 mutant lines as compared to wild-type Col-0 and this phenotype was suppressed both in sbt5 . 2 myb30 double mutants and by overexpressing SBT5 . 2", "( b ) , but not SBT5 . 2", "( a ) , in the sbt5 . 2 mutant background ( Figure 9d , e ) .", "Overall our data demonstrate that , in agreement with SBT5 . 2", "( b ) -mediated retention of MYB30 in endosomes , negative regulation of MYB30-mediated defence-related cell death is specifically controlled by SBT5 . 2", "( b ) ." ], [ "AS is a fundamental process that allows generating a large number of mRNA and protein isoforms from a genome of limited size ( Graveley , 2005 ) .", "Moreover , AS plays crucial functions in eukaryotic cells , as it determines the binding properties , intracellular localisation , enzymatic activity , protein stability and posttranslational modifications of a large number of proteins ( Stamm et al . , 2005 ) .", "In humans , the importance of AS is clearly highlighted by the fact that about 15% of genetic hereditary diseases are caused by mutations that affect splicing ( Kornblihtt et al . , 2013 ) .", "In plants , AS plays fundamental roles in regulating plant growth , development , and responses to environmental signals ( Staiger and Brown , 2013 ) .", "A genome-wide transcriptomic analysis in Arabidopsis plants inoculated with bacteria uncovered a surprisingly large number of AS events ( Howard et al . , 2013 ) .", "Although we are still far from understanding the functional implications of this transcriptome complexity , the function of numerous plant resistance genes , encoding immune receptors , appears to be regulated by AS ( Yang et al . , 2014; Gassmann , 2008 ) .", "To our knowledge , our work represents the first described example of AS affecting the function of a protein of the subtilase family .", "AS of SBT5 . 2 results in the production of the atypical subtilase SBT5 . 2", "( b ) , uncovering a novel mode of regulation of defence reactions and contributing to further our understanding of the varied roles of AS in the control of plant immunity .", "Following bacterial inoculation , the respective induction and repression of SBT5 . 2", "( b ) and SBT5 . 2", "( a ) expression suggests a functional role for SBT5 . 2", "( b ) during defence regulation .", "This idea is reinforced by the observed co-regulation of SBT5 . 2", "( b ) and MYB30 expression after bacterial treatment .", "Despite their pervasiveness , our current understanding of the functions of plants subtilases is still limited .", "Different studies suggest a role in both general protein turnover and regulation of plant development or responses to environmental cues ( Schaller et al . , 2012; Figueiredo et al . , 2014 ) .", "The first example of a plant subtilase potentially acting during plant-pathogen interactions was reported in tomato , where the expression of the subtilases P69B and P69C was induced by pathogen infection and treatment with salycilic acid ( Jordá et al . , 1999; Tornero et al . , 1997 ) .", "More recently , the Arabidopsis subtilase SBT3 . 3 was found to be involved in the regulation of immune signalling through chromatin remodelling of defence-related genes associated with the activation of immune priming ( Ramírez et al . , 2013 ) .", "Plant subtilases are usually synthesized in the form of preproprotein precursors , translocated via an N-terminal ER-targeting SP into the endomembrane system ( Schaller et al . , 2012 ) .", "In addition , using mass spectrometry ( Cedzich et al . , 2009 ) and structural analyses ( Murayama et al . , 2012 ) , plant subtilases were shown to be glycosylated in the secretory pathway and to accumulate extracellularly ( Schaller et al . , 2012 ) .", "In agreement with the preproprotein structure , the maturation of the active enzyme from its inactive precursor requires at least two processing steps .", "After cleavage of the SP , subtilases are ultimately activated by cleavage of the PD producing the mature active enzyme ( Taylor et al . , 1997 ) .", "Processing of the PD , an auto-inhibitor domain of plant subtilases ( Nakagawa et al . , 2010; Meyer et al . , 2016 ) , is an intramolecular autocatalytic reaction that occurs late in the ER or in the early Golgi ( Cedzich et al . , 2009 ) .", "Here , we show that AS of the SBT5 . 2 gene has important implications for the subcellular localization and activity of the resulting isoforms , despite the nearly total conservation between the two protein sequences .", "Consistent with harbouring all canonical features of a subtilase , we confirmed that SBT5 . 2", "( a ) enters the secretory pathway , where it is glycosylated , and is secreted to the extracellular space as shown before ( Engineer et al . , 2014; Kaschani et al . , 2012 ) .", "SBT5 . 2 was previously described as a negative regulator of stomatal density under high CO2 conditions through cleavage of the extracellular pro-peptide ligand EPF2 in the apoplast ( Engineer et al . , 2014 ) .", "In contrast , in agreement with its lacking a SP and the first five amino acids of the PD , SBT5 . 2", "( b ) does not enter the secretory pathway , its PD is thus not cleaved and may fold onto SBT5 . 2", "( b ) catalytic domain inhibiting its protease activity .", "Indeed , despite the presence of PGSs and catalytic residues , we were unable to detect SBT5 . 2", "( b ) glycosylation or protease activity .", "We also showed that MYB30 accumulation in planta is not affected by SBT5 . 2", "( a ) or SB5 . 2", "( b ) , indicating that these proteins do not proteolytically cleave the TF .", "In the case of catalytically active SBT5 . 2", "( a ) , this can be explained by the lack of co-localization of both proteins .", "Despite their subcellular co-localization and physical interaction , lack of modification of MYB30 accumulation in the presence of SBT5 . 2", "( b ) is consistent with SBT5 . 2", "( b ) being inactive as a protease , as underlined by the finding that both wild-type SBT5 . 2", "( b ) and catalytic mutant SBT5 . 2", "( b ) H171A are able to interact with and retain MYB30 at endosomal vesicles .", "These results suggest an alternative mode of action of SBT5 . 2", "( b ) on MYB30 activity , likely related to SBT5 . 2", "( b ) -mediated MYB30 nuclear exclusion .", "Examples of proteases playing proteolysis-independent functions have been described in mammals .", "The proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) plays a major regulatory role in cholesterol homeostasis but does not require its catalytic activity to induce degradation of low-density lipoproteins ( LDLs ) ( McNutt et al . , 2007 ) .", "In addition , γ-secretase , an aspartyl protease that performs the final proteolytic cleavage step in the processing cascade of the β-amyloid precursor protein ( βAPP ) ( Vassar et al . , 1999 ) , displays proteolysis-independent functions during the regulation of βAPP maturation and trafficking ( Wrigley et al . , 2005 ) .", "SBT5 . 2", "( b ) localises to both early and late endosomal compartments and this requires N-terminal myristoylation of the protein .", "Since co-expression of SBT5 . 2", "( b ) , but not SBT5 . 2", "( a ) , with both early and late endosome markers leads to colocalisation of both markers , it is tempting to speculate that SBT5 . 2", "( b ) is able to interfere with endosomal trafficking leading to the formation of hybrid endosomes .", "In mammals , expression of constitutively active Rab5 blocks the conversion of early to late endosomes , giving rise to hybrid endosomal compartments and deregulation of autophagy ( Rink et al . , 2005 ) .", "Similarly , overexpression of Arabidopsis sorting nexin SNX2b leads to the formation of large SNX2-containing endosome aggregations , which inhibits vesicle trafficking ( Phan et al . , 2008 ) .", "SBT5 . 2", "( b ) -mediated regulation of MYB30 activity supports the emerging idea that endosomal trafficking pathways are not only central regulators of plasma membrane protein homeostasis but also control multiple signalling pathways , including those involved in plant disease resistance ( Reyes et al . , 2011 ) .", "Enhanced susceptibility of plants impaired in regulators of endosome trafficking emphasizes the importance of endocytic processes in establishing defence ( Lu et al . , 2012 ) .", "In addition , plasma membrane receptors that sense apoplastic microbes are immune-related cargos of plant trafficking pathways ( Beck et al . , 2012; Ben Khaled et al . , 2015 ) , and a number of intracellular immune receptors have been shown to constitutively localize to endomembranes ( Takemoto et al . , 2012; Engelhardt et al . , 2012 ) , further underlining the prominent role of endomembrane systems in determining plant resistance .", "Although the role of endosomes in implementing defence signalling is not yet well understood , they have been proposed as central subcellular sites for orchestration of the HR .", "Indeed , relocalisation of the potato resistance protein R3a from the cytoplasm to endosomal compartments is required to trigger the HR ( Engelhardt et al . , 2012 ) .", "The importance of these compartments during immunity is further underlined by the fact that both plant and animal pathogens produce inhibitory effector proteins that specifically target endomembrane trafficking .", "For example , the Arabidopsis ADP ribosylation factor ( ARF ) -Guanine exchange factor ( GEF ) MIN7 is targeted and degraded by the bacterial effector HopM1 altering secretory trafficking ( Nomura et al . , 2006 , 2011 ) .", "In mammalian cells , the effector PipB2 from the bacterial pathogen Salmonella enterica causes a specific redistribution of late endosomes/lysosomes ( LE/Lys ) compartments to the cell periphery promoting formation of Salmonella-induced filaments ( Sifs ) ( Knodler and Steele-Mortimer , 2005 ) .", "Our finding that SBT5 . 2", "( b ) mediates MYB30 nuclear exclusion resulting in attenuation of MYB30 gene expression and HR , is in agreement with several reports describing controlled nuclear localisation as an efficient mechanism to regulate TF activity in eukaryotic cells .", "Subcellular compartmentalization of the E2F TF family , either in the nucleus or in the cytoplasm , is used to control cell cycle in differentiated skeletal cells ( Gill and Hamel , 2000 ) .", "p53 , a tightly regulated animal TF that acts as a hub for numerous signalling pathways including apoptosis , shifts from the nucleus to the cytoplasm in the presence of HDM2 , where it has important roles that are independent of its transcriptional activity ( Boyd et al . , 2000; Green and Kroemer , 2009 ) .", "Similarly , alternative functions for MYB30 in endosomal vesicles , other than HR regulation , cannot be excluded at this stage .", "In addition , nuclear import of 'dormant' TFs plays important roles in the regulation of gene expression .", "Upon exposure to environmental stresses , several membrane-bound TFs have been shown to be proteolytically activated by either ubiquitin-mediated proteasome activities or by specific membrane-bound proteases ( Hoppe et al . , 2001 ) , which may facilitate triggering quick transcriptional responses to ensure plant survival under stressful conditions ( Kim et al . , 2006; Seo et al . , 2010 ) .", "Notably , subtilases are known to be involved in the release of 'dormant' membrane-bound TFs .", "Arabidopsis SBT6 . 1 is able to cleave the ER-located TF bZIP17 that , once released , moves to the nucleus to activate transcription in response to salt stress ( Liu et al . , 2007 ) .", "Finally , nuclear exclusion by localization to small vesicle-like structures has been reported as a negative regulatory mechanism of TF activity .", "Indeed , the small interference protein MIF1 , promotes nuclear exclusion of the TF ZHD5 that regulates flower architecture and leaf development .", "As a result , ZHD5 is relocalised into cytoplasmic vesicle-like structures , which interferes with is transcriptional activity ( Hong et al . , 2011 ) .", "During the last few years , different regulatory mechanisms of MYB30-mediated HR have been uncovered ( Raffaele and Rivas , 2013 ) , including spatio-temporal control of MYB30 activity through the action of the secreted phospholipase AtsPLA2-α ( that specifically relocalises to the nucleus in the presence of MYB30 [Froidure et al . , 2010] ) and the RING-type E3 ligase MIEL1 ( that ubiquitinates MYB30 and leads to its proteasomal degradation [Marino et al . , 2013] ) .", "SBT5 . 2", "( b ) -mediated nuclear exclusion of MYB30 represents an additional regulatory mode of the activity of this TF , underlining the complexity of the regulatory modes of defence-related plant cell death responses .", "This intricate regulation of MYB30 is reminiscent of the tight control exerted on animal TFs such as p53 that is also multi-regulated through varied modes including protein-protein interactions , ubiquitination and , notably , nuclear exclusion .", "This sophisticated fine-tuning , which provides an efficient means to regulate fundamental cellular processes , appears as a general feature underlying the transcriptional control in eukaryotic cells ." ], [ "Plasmids used in this study were constructed by Gateway technology ( GW; Invitrogen , Waltham , MA , USA ) following the instructions of the manufacturer .", "PCR products flanked by the attB sites were recombined into the pDONR207 vector ( Invitrogen ) via a BP reaction to create the corresponding entry clones with attL sites .", "Inserts cloned into the entry clones were subsequently recombined into the destination vectors via an LR reaction to create the expression constructs .", "A fusion of fluorescent proteins to MYB30 , SBT5 . 2", "( b ) , SBT5 . 2", "( b ) G2A , VHA-a1 , SYP61 , ARA6 and SYP21 was accomplished using a multisite GATEWAY cloning strategy ( Invitrogen ) described previously ( Serrano et al . , 2014 ) .", "Briefly , the full-length open reading frames of the indicated proteins were amplified from a plasmid template and cloned into the donor vector pBSDONR P1-P4 or the pBSDONR P4r-P2 by BP reaction ( ampicillin-resistant vectors derived from pDONR221 from Invitrogen ) ( Gu and Innes , 2011 ) .", "eGFP ( Cormack et al . , 1996 ) and RFP ( Campbell et al . , 2002 ) were cloned into pBSDONR P1-P4 for N-terminal fusion and into pBSDONR P4r-P2 for C-terminal fusion .", "To fuse SBT5 . 2", "( a ) , SBT5 . 2", "( b ) , SBT5 . 2", "( b ) 162-730 , SBT5 . 2", "( b ) G2A , VHA-a1 , SYP61 , ARA6 and SYP21 with the epitope tags , the P1-P4 clones were mixed with corresponding P4r-P2 and the desired destination vectors and recombined using Gateway LR Clonase II ( Invitrogen ) .", "All the above pBSDONR constructs were recombined with the destination vector pEarleyGate100 ( Earley et al . , 2006 ) with the exception of SBT5 . 2", "( a ) and SBT5 . 2", "( b ) , for which the corresponding pBSDONR constructs were recombined with the steroid-inducible destination vector pBAV154 ( Vinatzer et al . , 2006 ) .", "For yeast assays , the GAL4-BD-MYB30ΔAD fusion was previously described ( Froidure et al . , 2010 ) .", "AD-SBT5 . 2", "( b ) 628-730 construct was generated from recombination of the corresponding entry constructs with the pGAD-AD-GW vector ( Froidure et al . , 2010 ) .", "Point mutations were generated using the QuickChange mutagenesis kit ( Stratagene , Santa Clara , CA , USA ) using the pENTR-SBT5 . 2 as a template and following the manufacturer’s instructions .", "Primers used for mutagenesis are shown in Supplementary file 1 .", "The yeast two-hybrid screen and methods used for identification of SBT5 . 2 were previously described ( Froidure et al . , 2010 ) .", "Briefly , an Arabidopsis thaliana Gal4 yeast two-hybrid cDNA prey library ( MatchMaker; Clontech ) was generated from mRNA isolated from leaves of four-week-old plants ( Ws-4 ecotype ) syringe-infiltrated with the Xanthomonas campestris pv .", "campestris 147 strain .", "An MYB30 version deleted from its C-terminal activation domain ( amino acids 1 to 234 ) was used as bait for screening 2 × 106 independent transformants exhibiting His auxotrophy on selective plates .", "5’ ends of SBT5 . 2 mRNA were determined using the GeneRacerTM RACE Ready kit ( Invitrogen , France ) according to manufacturers’ instructions , using RNA from Col-0 leaves and gene specific primers indicated in Supplementary file 1 .", "PCR products were cloned in pGEM-T Easy vector ( Promega Corporation , Fitchburg , WI , USA ) and sequenced .", "Arabidopsis lines used in this study were in the Columbia background and grown in Jiffy pots under controlled conditions in a growth chamber at 21°C , with a 9-hr light period and a light intensity of 190 µmol . m−2 . s−1 .", "The MYB30ko line ( SALK_122884 ) was reported before ( Marino et al . , 2013 ) .", "For transient expression of proteins in N . benthamiana , overnight bacterial cultures of Agrobacterium tumefaciens strain C58C1 or GV3101 carrying the vector of interest were harvested by centrifugation .", "Cells were resuspended in induction buffer ( 10 mM MgCl2 , 10 mM MES , pH 5 . 6 , and 150 µM acetosyringone ) to an OD600 of 0 . 5 .", "After 2 hr at 22°C , cells were infiltrated into leaves of four-week-old N . benthamiana plants .", "Two days after A . tumefaciens infiltration , leaf discs used for experiments were harvested and processed , or frozen immediately in liquid nitrogen and stored at –80°C .", "For tunicamycin treatment , 24 hr after Agrobacterium-mediated transient expression , leaf discs of N . benthamiana leaves were incubated in a solution of 10 µM tunicamycin for 20 hr at room temperature and later frozen in liquid nitrogen before processing .", "When testing the effect of SBT5 . 2 proteins on MYB30 accumulation , to minimize differences in protein expression , which are inherent to transient assays , MYB30 was co-expressed with SBT5 . 2", "( a ) , or SBT5 . 2", "( b ) , and the respective catalytic mutant side by side in the same N . benthamiana leaf .", "To avoid ex planta protein degradation , the leaves were pre-treated with the protease inhibitor PMSF ( 1 mM ) 30 min before harvesting the tissue for protein extraction .", "Arabidopsis four-week-old plants were kept at high humidity 12 hr before inoculation and injected with a bacterial suspension of Pst AvrRpm1 at the indicated bacterial densities using a blunt syringe on the abaxial side of the leaves .", "For determination of in planta bacterial growth , the leaf samples were harvested 0 and three days after inoculation and ground on sterile water .", "A predetermined dilution for each sample was plated on King’s B medium and incubated at 28°C for two days .", "The data were submitted to a statistical analysis using Statgraphics Centurion XV . II Professional Software ( Statpoint Technologies Inc . , Warrenton , VA , USA ) .", "Normality of residues was verified by the Kolmogorov-Smirmov test .", "The effect of the genotype was tested by Multiple Factor ANOVA .", "GFP and RFP fluorescence was analyzed with a confocal laser scanning microscope ( TCS SP2-AOBS; Leica ) using a x63 water immersion objective lens ( numerical aperture 1 . 20; PL APO ) .", "GFP fluorescence was excited with the 488 nm ray line of the argon laser and recorded in one of the confocal channels in the 505 to 530 nm emission range .", "RFP fluorescence was excited with the 561 nm line ray of the He-Ne laser and detected in the range between 595 and 620 nm .", "Images were acquired in the sequential mode using Leica LCS software ( version 2 . 61 ) .", "In order to quantify the fluorescence of MYB30 in the nucleus and cytoplasm , fluorescence intensity was measured using Leica LCS software ( version 2 . 61 ) .", "Region of interest ( ROIs ) were defined for each photograph and the mean value was taken as a fluorescence measure .", "Five fluorescence measures were obtained from 6 photographs taken from two independent experiments ( n = 30 ) .", "The fluorescence lifetime of the donor was experimentally measured in the presence and absence of the acceptor .", "The FRET efficiency ( E ) was calculated by comparing the lifetime of the donor in the presence ( tDA ) or absence ( tD ) of the acceptor: E = 1- ( tDA ) / ( tD ) .", "Statistical comparisons between control ( donor ) and assay ( donor + acceptor ) lifetime values were performed by Student t test .", "FRET-FLIM measurements were performed using a FLIM system coupled to a streak camera ( Krishnan et al . , 2003 ) .", "The light source ( l = 850 nm ) was a pulsed pulsed femtosecond IR laser ( Spectra-Physics , USA ) .", "All images were acquired with a 60x oil immersion lens ( Plan Apo 1 . 4 numerical aperture , IR ) mounted on an inverted microscope ( Eclipse TE2000E , Nikon , Japan ) coupled to the FLIM system .", "The fluorescence emission was directed back out into the detection unit through a band pass filter .", "The detector was composed of a streak camera ( Streakscope C4334 , Hamamatsu Photonics , Japan ) coupled with a fast and high sensitivity CCD camera ( model C8800-53C , Hamamatsu ) .", "For each region of interest ( vesicle and nucleus ) , average fluorescence decay profiles were plotted and lifetimes were estimated by fitting data with exponential function using a non-linear least-squares estimation procedure .", "Proteins were extracted in 50 mM Tris-HCl , pH 7 . 5 , 150 mM NaCl , 10% [v/v] glycerol , 1 mM PMSF , and 1% plant protease inhibitor cocktail ( Sigma-Aldrich , St Louis , MO , USA ) and centrifuged at 14 , 000g for 10 min at 4°C .", "Proteins in the supernatant were denatured and then incubated with PNGase F or Endo H ( New England Biolabs , Ipswich , MA , USA ) following the instructions of the manufacturer .", "Deglycosylation reactions were performed for 30 min and stopped by adding SDS-PAGE loading buffer and boiling .", "Proteins were detected by Western blot using anti-HA antibodies as described below .", "Proteins were extracted in 50 mM Tris-HCl , pH 7 . 5 , 150 mM NaCl , 10% [v/v] glycerol , 1 mM PMSF , and 1% plant protease inhibitor cocktail ( Sigma-Aldrich ) and centrifuged at 14 , 000 g for 10 min at 4°C .", "The supernatant was equilibrated in concanavalin A buffer ( 0 . 2 M Tris-HCl pH 7 . 5 , 1 M NaCl , 200 mM MgCl2 , 200 mM CaCl2 ) and applied to concanavalin A-agarose resin from Canavalia ensiformis ( Sigma-Aldrich ) pre-equilibrated in concanavalin A buffer .", "After three steps of washing with concanavalin A buffer , glycosylated proteins were eluted in concanavalin A buffer supplemented with 0 . 75 M α-D-methyl-glucoside and 0 . 75 M α-D-methylmannoside .", "The presence of HA-tagged SBT5 . 2 in the eluted proteins was confirmed by Western blot using anti HA antibodies as described below .", "N . benthamiana leaves transiently expressing the proteins of interest were harvested 48 hr after agroinfiltration and infiltrated with water .", "Intercellular fluids ( IF ) were isolated by centrifugation at 3000 g as previously described ( de Wit and Spikman , 1982 ) .", "Antibodies used for Western blotting were anti-HA-HRP ( 3F10 , Roche , Germany , 1:5000 ) , and PAP anti-rabbit-HRP ( Sigma , 1:10 , 000 ) .", "Proteins were visualized using the Immobilon kit ( Millipore , Billerica , MA , USA ) under standard conditions .", "The isolation and transient transfection of leaf mesophyll cell protoplasts from Arabidopsis plants ( four weeks-old ) was performed at room temperature following published procedures ( Yoo et al . , 2007 ) .", "A total of 10 μg plasmid DNA was used for each transfection experiment and plasmids were mixed in an equal ratio for cotransfections .", "FITC casein was used for the detection of proteolytic activity ( Twining , 1984 ) .", "sbt5 . 2 Arabidopsis protoplasts transfected with SBT5 . 2", "( a ) , SBT5 . 2", "( a ) H210A , SBT5 . 2", "( b ) , SBT5 . 2", "( b ) H171A or empty vector were lysed and used for the activity assay .", "A concentration of 400 µg/ml of FITC casein was used in a final volume of 25 µl of 150 mM NaCl , 20 mM phosphate buffer pH 7 . 6 .", "Samples were incubated for 1 hr at 25°C and fluorescence was measured at excitation 485 nm and emission 520 nm using a microtiter fluorimeter ( FL600 , Bio-Tek , Highland Park , VT , USA ) .", "For electrolyte leakage measurement , four leaf discs ( 6-mm diameter ) were harvested at the indicated timepoints after plant inoculation , washed , and incubated at room temperature in 5 ml of distilled water before measuring conductivity .", "The production of phenolic compounds was monitored under UV light using a Zeiss Axioplan microscope 24 hr after inoculation .", "Material for RNA analysis was grounded in liquid nitrogen and total RNA was isolated using the Nucleospin RNA plant kit ( Macherey-Nagel , Germany ) according to the manufacturer’s recommendations .", "Reverse transcription was performed using 1 . 5 µg of total RNA .", "Real-time quantitative PCR was performed on a Light Cycler 480 II machine ( Roche Diagnostics , France ) , using Roche reagents .", "Primers used for Q-RT-PCR are provided as Supporting Information .", "Relative expression was calculated as the ∆Cp between each gene and the internal controls SAND family ( At2g28390 ) .", "Average ∆Cp was related to the value of each gene in each line at time 0 ." ] ]
[ "Proteases play crucial physiological functions in all organisms by controlling the lifetime of proteins .", "Here , we identified an atypical protease of the subtilase family [SBT5 . 2", "( b ) ] that attenuates the transcriptional activation of plant defence independently of its protease activity .", "The SBT5 . 2 gene produces two distinct transcripts encoding a canonical secreted subtilase [SBT5 . 2", "( a ) ] and an intracellular protein [SBT5 . 2", "( b ) ] .", "Concomitant to SBT5 . 2", "( a ) downregulation , SBT5 . 2", "( b ) expression is induced after bacterial inoculation .", "SBT5 . 2", "( b ) localizes to endosomes where it interacts with and retains the defence-related transcription factor MYB30 .", "Nuclear exclusion of MYB30 results in its reduced transcriptional activation and , thus , suppressed resistance .", "sbt5 . 2 mutants , with abolished SBT5 . 2", "( a ) and SBT5 . 2", "( b ) expression , display enhanced defence that is suppressed in a myb30 mutant background .", "Moreover , overexpression of SBT5 . 2", "( b ) , but not SBT5 . 2", "( a ) , in sbt5 . 2 plants reverts the phenotypes displayed by sbt5 . 2 mutants .", "Overall , we uncover a regulatory mode of the transcriptional activation of defence responses previously undescribed in eukaryotes ." ]
[ "Like animals , plants have evolved numerous ways to protect themselves from disease .", "When a plant detects an invading microbe , it massively changes which genes it expresses to establish a defensive response .", "This is possible thanks to the action of a type of protein , named transcription factors , which are able to bind to DNA in the cell nucleus and regulate gene expression .", "However , triggering such a response comes at a cost , and so plants must keep their defensive response in check such that they can allocate resources in a balanced way .", "In the model plant Arabidopsis , a protein named MYB30 is one transcription factor that is able to promote disease resistance .", "Previous research identified some proteins that can reduce the activity of this transcription factor to avoid triggering a response when it is not needed , for example , when no infectious microbes are present .", "However , it was likely that other proteins were also involved in the process .", "Now , Serrano et al . report that an enzyme called SBT5 . 2 is an additional negative regulator of MYB30 activity .", "SBT5 . 2 belongs to a family of protein-degrading enzymes called subtilases , which are typically localized outside cells .", "As such , it was unclear how SBT5 . 2 could interact and regulate a transcription factor that is found inside the nucleus of plant cells .", "Nevertheless , Serrano et al . found that the gene that encodes SBT5 . 2 actually gives rise to two distinct proteins .", "The first is a classical subtilase that is indeed located outside of the cell , and so cannot interact with MYB30 and does not affect its activity .", "The second protein is an atypical subtilase that localises to bubble-like compartments called vesicles within the cell and is able to highjack MYB30 on its way to the nucleus .", "When the atypical subtilase interacts with MYB30 at vesicles , it stops MYB30 from entering the nucleus .", "As a result , MYB30 cannot bind to the DNA nor activate its target genes .", "This means that the defensive response that normally depends on MYB30 is weakened .", "The work of Serrano et al . uncovers a new way to regulate the expression of defence-related genes .", "Further unravelling the molecular mechanisms involved in the fine-tuning of gene expression represents a challenging task for future research ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics" ]
Phosphatidic acid modulation of Kv channel voltage sensor function
elife-04366-v2
[ [ "Voltage-gated potassium ( Kv ) channels shape and terminate action potentials .", "While membrane voltage is the fundamental stimulus for Kv channel gating , other stimuli such as protein phosphorylation ( Vacher and Trimmer , 2011 ) , intracellular Ca2+ ( Gamper et al . , 2005 ) and accessory proteins also regulate various Kv channels .", "In retrospect , given the lipid complexity of cell membranes , it is not surprising to learn that specific lipid molecules are among the regulators of membrane proteins generally and ion channels specifically ( Hilgemann et al . , 2001; Dart , 2010 ) .", "Even the structurally simple bacterial K+ channel KcsA requires anionic phospholipids in order to open ( Heginbotham et al . , 1998 ) , while the more complex eukaryotic inward rectifier K+ channels are so dependent on the lipid PIP2 ( see abbreviations section for lipid and detergent definitions ) that they might have been called the PIP2-regulated K+ channels ( Huang et al . , 1998; Suh and Hille , 2005; Hansen et al . , 2011; Whorton and MacKinnon , 2011 ) .", "These are just two examples from a growing list of ion channels whose function depends on the presence of specific lipid molecules .", "In the latter example , since PIP2 levels vary as a function of the physiological state of a cell , PIP2 is known as a ‘signaling lipid’ because it triggers or signals the action of molecules to which it binds , such as inward rectifier K+ channels .", "The function of certain Kv channels is also strongly influenced by the membrane's lipid composition .", "For example , the archeal KvAP channel requires phospholipids in order to open; they remain completely inactive in non-phospholipid membranes ( Schmidt et al . , 2006 ) .", "Eukaryotic Kv7 channels , also known as M current channels , depend on the presence of PIP2 in the membrane ( Falkenburger et al . , 2010; Kruse et al . , 2012; Rodriguez-Menchaca et al . , 2012; Telezhkin et al . , 2012 ) .", "Stimulation of Gq-coupled G protein coupled receptors activates phospholipase C , which depletes PIP2 from the membrane's inner leaflet and closes Kv7 channels ( Dickson et al . , 2013 ) .", "Thus , PIP2 functions as a signaling lipid in regulating Kv7 channel activity , just as for inward rectifier K+ channels .", "In this study we report our findings from a systematic analysis of Kv1 channel dependence on membrane lipid composition .", "Channels were reconstituted into planar bilayer membranes , which allow complete control of lipid composition ( Miller , 1986 ) .", "We identified one lipid , phosphatidic acid ( PA ) , which uniquely affects channel gating .", "We next studied the mole fraction dependence , membrane sidedness , and chemical characteristics of PA necessary to mediate its effect .", "We further show that PA similarly alters gating in the distantly related Kv channel KvAP .", "Experiments with mutant KvAP channels point to a mechanism whereby the primary phosphate group on PA stabilizes voltage sensor arginine residues in the closed conformation .", "Because PA is a naturally occurring ‘signaling lipid’ in the inner leaflet of cell membranes , we think we have likely uncovered a new and biologically relevant mode of Kv channel regulation ." ], [ "Figure 1 introduces the fundamental observation that this study seeks to understand: phosphatidic acid is an outlier among tested lipids in its ability to influence voltage-dependent gating of a K+ channel . 10 . 7554/eLife . 04366 . 003Figure 1 . POPA modifies Kv channel gating .", "( A ) Representative family of currents recorded from Kv channels in DPhPC bilayers .", "Voltage is stepped from a holding voltage of −110 mV to increasingly more positive depolarization voltages ( −110 mV to +80 mV; ΔV = 10 mV ) and then returned to the holding voltage of −110 mV .", "( B ) Normalized tail currents ( mean ± SEM ) from current families recorded from Kv channels in DPhPC bilayers are fit with a Boltzmann function with half activation voltage Vmid = −71 ± 1 mV , Z = 4 . 2 , N = 8 .", "( C ) Representative family of currents recorded from Kv channels in DPhPC:POPA ( 3:1 ) bilayers from a holding voltage of −80 mV to increasingly more positive depolarization voltages ( −80 mV to +40 mV; ΔV = 10 mV ) and then returned to the holding voltage of −80 mV .", "( D ) Normalized tail currents ( mean ± SEM ) from current families recorded from Kv channels in different lipid mixtures are fit with Boltzmann functions ( DPhPC:POPA ( 3:1 ) Vmid = −40 ± 2 mV , Z = 2 . 6 , N = 7; DPhPC:POPC ( 3:1 ) Vmid = −66 ± 1 mV , Z = 4 . 0 , N = 6; DPhPC:POPE ( 3:1 ) Vmid = −64 ± 1 mV , Z = 3 . 0 , N = 7; DPhPC:POPG ( 3:1 ) Vmid = −62 ± 1 mV , Z = 2 . 9 , N = 9; DPhPC:POPS ( 3:1 ) Vmid = −59 ± 1 mV , Z = 3 . 8 , N = 6; DPhPC:PI ( 3:1 ) Vmid = −63 ± 1 mV , Z = 2 . 6 , N = 7; DPhPC:Sphingomyelin ( 3:1 ) Vmid = −66 ± 1 mV , Z = 3 . 9 , N = 6; DPhPC:Cardiolipin ( 3:1 ) Vmid = −60 ± 1 mV , Z = 3 . 4 , N = 6; DPhPC Vmid = −71 ± 1 mV , Z = 4 . 2 , N = 8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 00310 . 7554/eLife . 04366 . 004Figure 1—figure supplement 1 . Representative families of currents recorded from Kv channels . Representative families of currents recorded from Kv channels in ( A ) DPhPC:POPC ( 3:1 ) bilayers , ( B ) DPhPC:POPE ( 3:1 ) bilayers , ( C ) DPhPC:POPG ( 3:1 ) bilayers , ( D ) DPhPC:POPS ( 3:1 ) bilayers , ( E ) DPhPC:PI ( 3:1 ) bilayers , ( F ) DPhPC:Sphingomyelin ( 3:1 ) bilayers , and ( G ) DPhPC:Cardiolipin ( 3:1 ) bilayers .", "Voltage is stepped from a holding voltage of −110 mV to increasingly more positive voltages ( −110 mV to +80 mV; ΔV = 10 mV ) and then returned to the holding voltage of −110 mV for A–D , F or −90 mV for E and G . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 004 The K+ channel under study is a mutant of the rat Kv1 . 2 channel in which the helix-turn-helix segment termed the voltage sensor paddle was replaced by the corresponding segment from Kv2 . 1 , a closely related K+ channel ( Long et al . , 2007 ) .", "This ‘paddle chimera’ mutant is more stable biochemically but otherwise is functionally very similar to wild type ( Tao and MacKinnon , 2008 ) .", "The α ( conduction pore and voltage sensor-forming ) subunit was expressed and purified with its β subunit , an aldo-keto reductase-like domain , attached to the cytoplasmic surface ( Gulbis et al . , 1999 ) .", "We refer to the α-β complex of the mutant channel simply as the Kv channel .", "When reconstituted into planar lipid bilayers the Kv channel opens upon membrane depolarization from a negative ( inside relative to outside ) holding voltage ( Figure 1A ) .", "Figure 1B graphs tail currents ( normalized to maximal current ) , which are measured shortly after stepping negative from the depolarization voltage , as a function of the depolarization voltage .", "This voltage-dependent ‘activation curve’ shows that channels begin to open around −100 mV and reach near maximal activation by −40 mV , with a half activation voltage ( Vmid ) of approximately −70 mV .", "These currents were recorded in DPhPC bilayers , which were chosen as the ‘baseline’ lipid in this study because they form exceptionally stable bilayers .", "Figure 1D shows the influence of mixing different lipids at a mole fraction of 0 . 25 into the DPhPC lipid ( Figure 1—figure supplement 1 ) .", "In all but one case the activation curves are similar , with Vmid around −70 mV .", "POPA is unique: it produces an activation curve that is less steep and has a Vmid around −40 mV .", "At a mole fraction of 0 . 25 , POPA induced a rightward shift of the activation curve ( Figure 1D ) that is associated with slowed activation kinetics ( Figure 1A , C ) .", "Figure 2 shows to what extent channel gating is altered as the mole fraction of POPA is varied ( Figure 2A , B , Figure 2—figure supplement 1 ) .", "At a mole fraction of 0 . 05 the effect of POPA on Vmid is already substantial , and by 0 . 1 it is nearly complete .", "Thus , POPA influences gating according to an approximately saturating function with a steep dependence in the low ( less than 0 . 1 ) mole fraction range .", "The functional relationship is similar whether POPA is added to DPhPC or POPE membranes , although , due to the intrinsic instability of pure POPE bilayers , gating was not assessable at the origin in POPE ( Figure 2B ) . 10 . 7554/eLife . 04366 . 005Figure 2 . Concentration dependence of Kv channel activation by POPA .", "( A ) Normalized tail currents ( mean ± SEM ) from current families recorded from Kv channels in DPhPC:POPA mixtures are fit with Boltzmann functions ( DPhPC Vmid = −71 ± 1 mV , Z = 4 . 2 , N = 8; DPhPC:POPA ( 19:1 ) Vmid = −60 ± 1 mV , Z = 3 . 7 , N = 8; DPhPC:POPA ( 9:1 ) Vmid = −43 ± 2 mV , Z = 2 . 7 , N = 5; DPhPC:POPA ( 3:1 ) Vmid = −40 ± 2 mV , Z = 2 . 6 , N = 7; DPhPC:POPA ( 1:1 ) Vmid = −33 ± 1 mV , Z = 3 . 5 , N = 6; POPA Vmid = −31 ± 2 mV , Z = 2 . 7 , N = 6 ) .", "( B ) Plot of Vmid determined from fit of tail currents to the Boltzmann equation vs mole fraction of POPA for Kv channels in bilayers containing DPhPC:POPA ( red ) or POPE:POPA ( green ) mixtures . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 00510 . 7554/eLife . 04366 . 006Figure 2—figure supplement 1 . Representative families of currents recorded from Kv channels . Representative families of currents recorded from Kv channels in ( A ) DPhPC bilayers , ( B ) DPhPC:POPA ( 19:1 ) bilayers , ( C ) DPhPC:POPA ( 9:1 ) bilayers , ( D ) DPhPC:POPA ( 3:1 ) bilayers , ( E ) DPhPC:POPA ( 1:1 ) bilayers and ( F ) POPA bilayers .", "For A–C , voltage is stepped from a holding voltage of −110 mV to increasingly more positive voltages ( −110 mV to +80 mV; ΔV = 10 mV ) and then returned to a holding voltage of −110 mV .", "For D–F , voltage is stepped from a holding voltage of −90 mV to increasingly more positive voltages ( −90 mV to +70 mV; ΔV = 10 mV ) and then returned to the holding voltage of −90 mV . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 006 The data graphed in Figure 3 show how chemical variation within the lipid head-group or acyl chain affects Vmid ( Figure 3—figure supplement 1 ) .", "Here , as in Figure 2 , the lipid under study was added to DPhPC and Vmid was graphed as a function of the added lipid mole fraction .", "In Figure 3A five lipids with identical or similar acyl chains but different head-groups are compared ( Figure 3A , E , Figure 3—figure supplement 1 ) .", "Only POPA has a large effect .", "POPG , POPS and PI , similar to POPA , have a net −1 charged head-group .", "Thus , the large gating effect of POPA on Vmid is not attributable to the −1 charge of the head-group . 10 . 7554/eLife . 04366 . 007Figure 3 . Concentration dependence of Kv channel activation by phospholipids .", "( A ) Plot of Vmid determined from a fit of tail currents to the Boltzmann equation vs phospholipid mole fraction for Kv channels in bilayers containing DPhPC:POPA ( red ) , DPhPC:POPC ( black ) , DPhPC:POPG ( teal ) , DPhPC:POPS ( purple ) and DPhPC:PI ( blue ) mixtures .", "( B ) Plot of Vmid determined from a fit of tail currents to the Boltzmann equation vs phospholipid mole fraction for Kv channels in bilayers containing DPhPC:POPA ( red ) , DPhPC:DMPA ( green ) , DPhPC:DOPA ( burgundy ) and DPhPC:BrPOPA ( orange ) mixtures .", "( C ) Plot of Vmid determined from a fit of tail currents to the Boltzmann equation vs phospholipid mole fraction for Kv channels in bilayers containing DPhPC:POPA ( red ) , DPhPC:DOPMe ( pink ) and DPhPC:DOPEth ( blue ) mixtures .", "( D ) Plot of Vmid determined from a fit of tail currents to the Boltzmann equation vs phospholipid mole fraction for Kv channels in bilayers containing DPhPC:DOPP ( orange ) , DPhPC:DOPA ( burgundy ) , DPhPC:PIP ( violet ) and DPhPC:PI ( blue ) mixtures .", "( E ) Molecular structures of phospholipids analyzed in A–D with primary phosphates highlighted in red . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 00710 . 7554/eLife . 04366 . 008Figure 3—figure supplement 1 . Representative families of currents recorded from Kv channels . Representative families of currents recorded from Kv channels in ( A ) DPhPC:DMPA ( 3:1 ) bilayers , ( B ) DPhPC:DOPA ( 3:1 ) bilayers , ( C ) DPhPC:BrPOPA ( 3:1 ) bilayers , ( D ) DPhPC:DOPMe ( 3:1 ) bilayers , ( E ) DPhPC:DOPEth ( 3:1 ) bilayers ( F ) DPhPC:DOPP bilayers and ( G ) DPhPC:PIP bilayers .", "For A , B and F , voltage is stepped from a holding voltage of −90 mV to increasingly more positive voltages ( −90 mV to +80 mV; ΔV = 10 mV ) and then returned to the holding voltage of −90 mV .", "For C , voltage is stepped from a holding voltage of −90 mV to increasingly more positive voltages ( −90 mV to +80 mV; ΔV = 10 mV ) and then returned to −70 mV .", "For D , E and G , voltage is stepped from a holding voltage of −110 mV to increasingly more positive voltages ( −110 mV to +70 mV; ΔV = 10 mV ) and then returned to the holding voltage of −110 mV . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 008 In Figure 3B four lipids with different acyl chains but the same primary phosphate head-group are compared ( Figure 3—figure supplement 1 ) .", "These lipids are indistinguishable with respect to their effect on Vmid .", "Addition of either a methyl ( DOPMe ) or ethyl ( DOPEth ) group to the phosphate abolished the effect on gating ( Figure 3C , Figure 3—figure supplement 1 ) .", "On the other hand , addition of a second phosphate did not abolish the effect: the phosphodiester lipid DOPP appears to have a somewhat more potent effect on gating than DOPA ( Figure 3D , Figure 3—figure supplement 1 ) .", "When a free phosphate was present far away from the acyl chain , as in PIP , a Vmid shift occurred but to a much lesser extent than in DOPA or DOPP .", "Thus , it would appear that Vmid is mainly responsive to the presence of a primary phosphate group located relatively near the glycerol backbone .", "In our experience Kv channels incorporate randomly into planar bilayers with approximately half the channels oriented outside-out ( defined as extracellular surface of the channel facing the ground electrode ) and half inside-out ( defined as intracellular surface of the channel facing the ground electrode ) .", "If the membrane is held at −110 mV relative to ground ( 0 mV ) and then stepped toward more positive voltages , only the outside-out channels open because the inside-out channels are inactivated .", "If the same membrane is held at +110 mV relative to ground and stepped toward more negative voltages , only the inside-out channels open because the outside-out channels are inactivated .", "Figure 4A shows the activation curves for outside-out and inside-out channels in the same membrane .", "The curves are indistinguishable .", "This is expected because in these experiments the lipid bilayer and ionic solutions on both sides of the membrane were symmetrical with respect to the inner and outer membrane leaflets . 10 . 7554/eLife . 04366 . 009Figure 4 . Kv activation in Phospholipase D1-treated DPhPC bilayers .", "( A ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer ( black–outside-out facing channels , Vmid = −72 mV; red–inside-out facing channels , Vmid = −69 mV ) are fit to the Boltzmann equation .", "( B ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer following addition of 50 units/ml S . chromofuscus phospholipase D1 to the extracellular membrane are fit to the Boltzmann equation ( black 0 min , Vmid = −65 mV; blue 10 min , Vmid = −66 mV; purple 20 min , Vmid = −68 mV; burgundy 30 min Vmid = −77 mV; red 40 min , Vmid = −87 mV ) .", "( C ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer following addition of 50 units/ml S . chromofuscus phospholipase D1 to the intracellular side of the membrane are fit to the Boltzmann equation ( black 0 min , Vmid = −60 mV; blue 10 min , Vmid = −43 mV; purple 20 min , Vmid = −38 mV; burgundy 30 min , Vmid = −23 mV; red 40 min , Vmid = −6 mV ) .", "( D ) Average change in Vmid following addition of 50 units/ml S . chromofuscus phospholipase D1 to the intracellular or extracellular side of the membrane ( ΔVmid = Vmid ( t ) –Vmid ( t = 0 ) ; 10 min intracellular ΔVmid = 11 mV , extracellular ΔVmid = −4 mV , N = 5; 20 min intracellular ΔVmid = 15 mV , extracellular ΔVmid = −7 mV , N = 5; 30 min intracellular ΔVmid = 24 mV , extracellular ΔVmid = −15 mV , N = 4; 40 min intracellular ΔVmid = 39 mV , extracellular ΔVmid = −17 mV , N = 2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 00910 . 7554/eLife . 04366 . 010Figure 4—figure supplement 1 . Representative families of currents recorded from Kv channels in Phospholipase D1-treated DPhPC bilayers . Representative families of currents recorded from Kv channels in DPhPC bilayers ( A ) 0 and ( B ) 40 min after addition of 50 units/ml S . chromofuscus phospholipase D1 to the extracellular side of the membrane .", "Representative families of currents recorded from Kv channels in DPhPC bilayers ( C ) 0 and ( D ) 40 min after addition of 50 units/ml S . chromofuscus phospholipase D1 to the intracellular side of the membrane .", "For A , voltage is stepped from a holding voltage of −110 mV to increasingly more positive voltages ( −110 mV–0 mV; ΔV = 10 mV ) then returned to the holding voltage of −110 mV .", "For B , voltage is stepped from a holding voltage of −130 mV to increasingly more positive voltages ( −130 mV–0 mV; ΔV = 10 mV ) then returned to the holding voltage of −130 mV .", "For C , voltage is stepped from a holding voltage of +110 mV to increasingly more negative voltages ( +110 mV–0 mV; ΔV = −10 mV ) then returned to the holding voltage of +110 mV .", "For D , voltage is stepped from a holding voltage of +80 mV to increasingly more negative voltages ( +80 mV to −50 mV; ΔV = −10 mV ) then returned to the holding voltage of +80 mV . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 010 When phospholipase D1 from Streptomyces chromofuscus was added to one side of a DPhPC membrane an asymmetry was generated because this enzyme cleaves the choline head-group and generates PA on the side to which it is added ( Ramu et al . , 2006 ) .", "Figure 4B shows activation curves for outside-out channels recorded over time after addition of phospholipase D1 to the ground electrode side of the membrane ( Figure 4—figure supplement 1 ) .", "Because the ground electrode side of outside-out channels corresponds to the physiological extracellular surface of the channel , it is evident that the activation curve shifted to more negative voltages ( Vmid −65 to −87 mV ) as PA was generated in the physiological extracellular leaflet ( Figure 4B , Figure 4—figure supplement 1 ) .", "Figure 4C shows activation curves for inside-out channels after phospholipase D1 was added to the ground electrode side of the membrane ( Figure 4—figure supplement 1 ) .", "Here we observe that as PA was generated on the intracellular side of the channels the activation curve shifted toward more positive voltages .", "The Vmid shift toward more positive voltages ( ∼+40 mV ) was greater than the shift toward more negative voltages ( ∼−15 mV ) ( Figure 4D ) .", "PA thus has an opposite and greater effect on channel gating when acting on the intracellular membrane leaflet .", "The PA composition of individual inner and outer membrane leaflets were altered another way .", "After formation of a DPhPC bilayer POPA was added in the form of vesicles to one side of the membrane .", "Figure 5A shows an activation curve for channels before and after addition of POPA to the ground electrode side .", "The effect of the POPA on outside-out and inside-out channels is shown .", "Here , as in the experiment in which PA was generated by enzymatic cleavage , shifts in the midpoint voltage of the activation curve were observed .", "POPA on the extracellular side ( outside-out channels ) produced a modest negative Vmid shift ( ∼−20 mV ) while POPA on the intracellular side ( inside-out channels ) produced a large positive Vmid shift ( ∼+60 mV ) ( Figure 5E ) .", "These data are explicable if fused vesicles add their lipids predominantly to one leaflet .", "Accordingly , addition of POPA vesicles to the extracellular and intracellular leaflets had the same effect as adding phospholipase D1 to the extracellular and intracellular sides , respectively .", "Notably , both methods of PA addition had a greater effect on Vmid when PA was altered on the intracellular leaflet . 10 . 7554/eLife . 04366 . 011Figure 5 . Kv activation in DPhPC bilayers fused with phospholipid vesicles .", "( A ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer before ( black , Vmid = −65 mV ) and after fusion of POPA vesicles to the extracellular ( red , Vmid = −80 mV ) or intracellular ( blue , Vmid = −17 mV ) surface of the bilayer are fit to the Boltzmann equation .", "( B ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer before ( black , Vmid = −61 mV ) and following fusion of POPC vesicles to the extracellular ( red , Vmid = −63 mV ) or intracellular ( blue , Vmid = −69 mV ) surface of the bilayer are fit to the Boltzmann equation .", "( C ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer before ( black , Vmid = −65 mV ) and following fusion of POPG vesicles to the extracellular ( red , Vmid = −76 mV ) or intracellular ( blue , Vmid = −48 mV ) surface of the bilayer are fit to the Boltzmann equation .", "( D ) Normalized tail currents from representative current families recorded from Kv channels in a DPhPC bilayer before ( black , Vmid = −63 mV ) and following fusion of POPS vesicles to the extracellular ( red , Vmid = −83 mV ) or intracellular ( blue , Vmid = −43 mV ) surface of the bilayer are fit to the Boltzmann equation .", "( E ) Average change in Vmid following addition of phospholipid vesicles to the intracellular or extracellular side of the membrane ( ΔVmid = Vmid ( vesicle fusion ) –Vmid ( no fusion ) ; POPA extracellular ΔVmid = −18 mV , intracellular ΔVmid = 58 mV , N = 4; POPG extracellular ΔVmid = −17 mV , intracellular ΔVmid = −18 mV , N = 4; POPS extracellular ΔVmid = −16 mV , intracellular ΔVmid = 19 mV , N = 4; POPC extracellular ΔVmid = −1 mV , intracellular ΔVmid = −1 mV , N = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 011 Addition of electrically net neutral phospholipid POPC vesicles to either side of the membrane had little effect on Vmid ( Figure 5B ) .", "In contrast , addition of the −1 charged phospholipid POPG produced an approximately −20 mV shift when added to the extracellular side of the membrane and a +20 mV shift when added to the intracellular side ( Figure 5C ) .", "A similar effect was observed when the −1 charged phospholipid POPS was used in place of POPG ( Figure 5D ) .", "The near absence of an effect of neutral POPC and approximately equal positive and negative shifts in response to asymmetrically applied −1 charged POPG and POPS to the intracellular and extracellular surfaces , respectively , is consistent with a surface charge voltage offset .", "A pictorial description of a surface charge voltage offset is shown in Figure 6A–C .", "Voltage sensors of Kv channels respond to the electric field inside the membrane , which is a function of both the applied voltage across the membrane and the surface potentials at the membrane–water interface .", "( The electric field is also a function of dipole potentials at the membrane water interface , but these , being essentially identical but oppositely oriented on the two sides of the membrane , cancel . )", "To interpret Figure 6A–C , consider that the channel responds to Vmem , the value of the voltage difference across the membrane where the voltage sensors reside .", "We the experimenters do not know the value of Vmem , but set the command voltage , Vi − Vo , with our amplifier .", "The pictures illustrate how changing surface charge on the membrane gives rise to different values of Vmem under a constant command voltage according to the expression Vmem = ( Vi − Vo ) + ( Φi − Φo ) , where Φi is the surface potential on the inside and Φo is the surface potential on the outside .", "Now consider a channel whose half open probability occurs at a particular value , Vmemʹ .", "In the absence of surface charge the experimenter observes the half open probability at a command voltage ( Vi − Vo ) mid = Vmemʹ .", "Upon addition of surface charge to the inner or outer leaflets the experimenter observes the half open probability at command voltage ( Vi − Vo ) mid = Vmemʹ + ( Φo − Φi ) .", "Thus , negative surface charge on the outside shifts Vmid towards more negative values and negative surface charge on the inside shifts Vmid towards more positive values . 10 . 7554/eLife . 04366 . 012Figure 6 . Surface charge voltage offset in phospholipid membranes .", "( A ) In symmetric membranes lacking charged phospholipids , Vmem , the voltage to which channels respond , is equal to the command voltage , Vi − Vo , set on the amplifier .", "( B ) In asymmetric membranes containing anionic lipids exclusively in the outer leaflet of the membrane , Vmem is equal to the command voltage , Vi − Vo , minus the surface potential of the outer membrane , Φo .", "( C ) In asymmetric membranes containing anionic lipids exclusively in the inner leaflet of the membrane , Vmem is equal to the command voltage , Vi − Vo , plus the surface potential of the inner membrane , Φi . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 012 The relationship between membrane surface potential Φ ( mV ) and charge density σ ( electron charges per Å2 ) in a monovalent electrolyte ( e . g . KCl ) at concentration c ( M ) is ( 1 ) Φ=2KBTe0 sinh−1 ( 136σc ) , where KB is Boltzmann's constant , T is absolute temperature and eo the charge of an electron ( McLaughlin et al . , 1970 ) .", "Using this expression and the mean surface area of a DPhPC molecule ( ∼80 Å2 ) ( Tristram-Nagle et al . , 2010 ) the 20 mV shifts in Figure 5C , D point to a surface charge density of about 1 eo/900 Å2 ( neglecting contributions due to decane in the bilayer ) , which corresponds to a ratio of 1 POPG ( or POPS ) molecule per 11 DPhPC molecules , or a mole fraction of about 0 . 09 .", "The equal magnitude but opposite direction shift produced by extracellular and intracellular POPG ( or POPS ) is consistent with a pure surface charge voltage offset ( Figure 5C , D ) .", "The shift produced by extracellular POPA—similar in direction and magnitude to the shifts produced by extracellular POPG and POPS—is also consistent with a surface charge voltage offset .", "In contrast , the larger shift produced by intracellular POPA implies that an additional ‘PA-specific’ voltage offset is occurring ( Figure 5E ) .", "In the experiments shown in Figures 2 and 3 both membrane leaflets contained the same lipid composition and therefore surface charge effects on the voltage sensor should have largely canceled .", "Therefore the Vmid shift in these experiments must have resulted from a non-surface charge , PA-specific effect .", "In the experiments behind Figures 4 and 5 the lipid composition of the two leaflets was different and therefore different surface charge densities should have contributed to the Vmid shift .", "As noted earlier , POPA in the inner leaflet caused a greater shift than POPA in the outer leaflet or than POPG/POPS in either leaflet ( Figures 4B–D and 5A–E ) .", "This can be understood in terms of a specific offset added to a surface charge offset for the case of POPA in the inner leaflet .", "In fact the shift produced by inner leaflet POPA ( ∼+50 mV , Figures 4C and 5A ) was approximately equal to the surface charge shift ( ∼+20 mV , Figure 5C , D ) plus the PA-specific shift ( ∼+30 mV , Figure 3A ) .", "Therefore POPA , like other anionic lipids , imposes a bias on the voltage sensor because it creates a layer of negative charge on the membrane surface .", "But unlike other lipids POPA also exerts an additional bias from the inner membrane leaflet .", "The surface charge effect is a simple electrostatic consequence of a fixed charge layer on the membrane surface .", "What is the origin of the PA-specific effect ?", "A strong interaction between guanidinium and phosphate groups has been described ( Woods and Ferre , 2005 ) .", "We therefore wondered whether PA in the inner membrane leaflet might stabilize a closed conformation of the voltage sensor through interactions with one or more of the arginine residues on the charge-bearing S4 helix of the voltage sensor .", "To test this possibility we turned to mutagenesis .", "Mutations in the paddle chimera Kv channel are not well tolerated so we tested whether the PA-specific effect is present in another distantly related Kv channel that can be more easily mutated .", "Figure 7A shows a PA-specific ( both membrane leaflets contain PA so the surface charge effect should be absent ) positive Vmid shift in the activation curve of the archeal channel KvAP ( Figure 7—figure supplement 1 ) .", "The shift is smaller ( +17 mV ) than that observed in the eukaryotic Kv channel ( +30 mV ) but clearly present ( Figures 2A and 7A ) .", "Figure 7B graphs the Vmid shift brought about by DPhPA in wild type KvAP and in several mutants within S4 ( Figure 7—figure supplement 1 ) .", "Replacement of the arginine at position 133 by either lysine or alanine abolished the DPhPA-induced shift .", "Introduction of an arginine at position 136 restored the shift in the absence of an arginine at 133 . 10 . 7554/eLife . 04366 . 013Figure 7 . Phosphatidic acid modifies KvAP activation .", "( A ) Normalized tail currents ( mean ± SEM ) from current families recorded from KvAP in DPhPC bilayers ( black , Vmid = −25 ± 1 mV , Z = 2 . 6 , N = 8 ) , DPhPC:DPhPA ( 1:1 ) bilayers ( red , Vmid = −6 ± 1 mV , Z = 2 . 2 , N = 6 ) and DPhPC:POPA ( 3:1 ) bilayers ( blue , Vmid = −7 ± 2 mV , Z = 2 . 2 , N = 5 ) are fit to the Boltzmann equation .", "( B ) Average difference in Vmid between DPhPC and DPhPC:DPhPA ( 1:1 ) membranes for KvAP and KvAP mutant channels ( ΔVmid = Vmid ( DPhPC:DPhPA 1:1 ) –Vmid ( DPhPC ) ) .", "The bar heights correspond to KvAP 18 mV , N = 5; R123K 24 mV , N = 5; R126K 12 mV , N = 5; R133K −1 mV , N = 6; R133A 0 mV , N = 5; K136A 12 mV , N = 5; K136R 8 mV , N = 5; R133A K136A −1 mV , N = 6; R133K K136A −1 mV , N = 5; R133K K136R 15 mV , N = 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 01310 . 7554/eLife . 04366 . 014Figure 7—figure supplement 1 . Representative families of currents recorded from KvAP channels . Representative families of currents recorded from Kv channels in ( A ) DPhPC bilayers , ( B ) DPhPC:DPhPA ( 1:1 ) bilayers , ( C ) DPhPC:POPA ( 3:1 ) bilayers .", "Voltage is stepped from a holding voltage of −100 mV to increasingly more positive voltages ( −100 mV to +80 mV; ΔV = 10 mV ) returned to the holding voltage of −100 mV for A and B and −80 for C . ( D ) Vmid determined from fits to the Boltzmann equation for KvAP and KvAP mutant channels ( KvAP: DPhPC Vmid = −25 ± 1 mV , Z = 2 . 6 , N = 8; DPhPC:DPhPA ( 1:1 ) Vmid = −6 ± 1 mV , Z = 2 . 2 , N = 5; R123K: DPhPC Vmid = −42 ± 3 mV , Z = 2 . 6 , N = 6; DPhPC:DPhPA ( 1:1 ) Vmid = −17 ± 3 mV , Z = 2 . 1 , N = 5; R126K: DPhPC Vmid = −6 ± 2 mV , Z = 3 . 1 , N = 5; DPhPC:DPhPA ( 1:1 ) Vmid = 6 mV , Z = 2 . 5 , N = 6; R133K: DPhPC Vmid = −29 ± 2 mV , Z = 3 . 1 , N = 6; DPhPC:DPhPA ( 1:1 ) Vmid = −30 ± 2 mV , Z = 3 . 6 , N = 7; R133A: DPhPC Vmid = 13 ± 1 mV , Z = 2 . 8 , N = 5; DPhPC:DPhPA ( 1:1 ) Vmid = 13 ± 3 mV , Z = 1 . 9 , N = 6; K136A: DPhPC Vmid = −3 ± 2 mV , Z = 3 . 4 , N = 5; DPhPC:DPhPA ( 1:1 ) Vmid = 9 ± 1 mV , Z = 2 . 6 , N = 6; K136R: DPhPC Vmid = −32 ± 1 mV , Z = 2 . 8 , N = 5; DPhPC:DPhPA ( 1:1 ) Vmid = −24 ± 2 mV , Z = 2 . 3 , N = 6; R133A K136A: DPhPC Vmid = 57 ± 1 mV , Z = 3 . 6 , N = 6; DPhPC:DPhPA ( 1:1 ) Vmid = 57 ± 2 mV , Z = 2 . 9 , N = 6; R133K K136A: DPhPC Vmid = −5 ± 1 mV , Z = 3 . 7 , N = 5; DPhPC:DPhPA ( 1:1 ) Vmid = −5 ± 2 mV , Z = 2 . 7 , N = 6; R133K K136R: DPhPC Vmid = −34 ± 1 mV , Z = 2 . 8 , N = 8; DPhPC:DPhPA ( 1:1 ) Vmid = −19 ± 2 mV , Z = 2 . 3 , N = 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 014 A plausible interpretation is that the S4 arginine at position 133—and at position 136 in the context of no arginine at position 133—can interact with a primary phosphate group in the membrane's inner leaflet .", "Figure 8A shows x-ray crystal structures of the paddle chimera and KvAP voltage sensors ( Jiang et al . , 2003; Long et al . , 2007 ) .", "Arginine 133 in KvAP and the corresponding arginine in paddle chimera are located close to the center of the membrane bilayer ( Figure 8A ) .", "But these structures correspond to ‘open’ conformations of voltage sensors .", "In closed conformations the centrally located arginine residues likely approach the membrane's inner leaflet where they could interact with PA to help stabilize that conformation . 10 . 7554/eLife . 04366 . 015Figure 8 . Structure and sequence alignment of Kv voltage sensor domains .", "( A ) View from the membrane plane of the voltage sensor domains of rat Kv1 . 2–2 . 1 paddle chimera and KvAP .", "The S4 transmembrane helices and their positively charged residues are highlighted in yellow .", "The dashed grey line marks the approximate positions of the phospholipid head groups .", "( B ) Sequence alignment of the S4 transmembrane helices from rat Kv1 . 2–2 . 1 paddle chimera , KvAP and D . melanogaster Shaker K+ channel .", "Numbering is according to KvAP sequence . DOI: http://dx . doi . org/10 . 7554/eLife . 04366 . 015" ], [ "Phosphatidic acid is present in many cellular membranes including the inner leaflet of the plasma membrane where it plays essential roles in cellular pathways such as mTOR complex stability and signaling ( reviewed in Foster , 2013 ) , growth factor receptor signaling ( reviewed in Gomez-Cambronero , 2010 ) and hormone signaling ( Garrido et al . , 2009 ) .", "In its signaling role , PA is most commonly generated from either phosphatidyl choline via phospholipase D cleavage or from diacylglycerol via diacylglycerol kinase ( Foster , 2013 ) .", "Once generated , PA can then be rapidly depleted from the plasma membrane by phosphatidic acid phosphatase or a variety of phospholipases , thus allowing tight cellular control over its abundance ( Foster , 2013 ) .", "The precise control of plasma membrane PA concentrations in response to intracellular and extracellular stimuli combined with its influence on Kv channel function situate PA at a potential interface between cellular and global metabolic signaling pathways and membrane excitation .", "Given the importance of PA to cellular signaling we suspect that the unique ability of PA to alter Kv channel gating is of biological significance .", "PA's effect is exerted by two mechanisms .", "The surface charge component is expected of any charged lipid disposed asymmetrically over the two membrane leaflets ( Ramu et al . , 2006; Xu et al . , 2008 ) .", "The PA-specific component requires a primary phosphate group on the lipid molecule and acts only from the inner leaflet .", "The mutational data support a hypothesis that PA in the inner leaflet interacts with specific arginine residues in the voltage sensor .", "It seems plausible that hydrogen bonding between a primary phosphate group and arginine guanidinium group could hold the voltage sensor closed , requiring stronger depolarization to open ( i . e . a Vmid shift to more positive voltages ) .", "We have determined crystal structures of the paddle chimera mutant in the presence of brominated PA .", "We typically observe electron density for lipid molecules in the crystal structure , but none with a specific signal for bromine .", "Therefore we do not know whether PA is specifically bound to a groove on the surface of the channel .", "Given the somewhat surprising observation that PA influences gating similarly in both the eukaryotic Kv channel and KvAP—channels that are only distantly related—we imagine that PA's specific effect might be mediated through the guanidinium–phosphate interaction alone ( i . e . without other interactions between the lipid tail and the channel ) .", "In other words , we imagine in a closed conformation the arginine could be released from its hydrogen bond pairing with counter charges on the channel and become anchored in the membrane's inner leaflet through hydrogen bonding with a primary phosphate group .", "The magnitude of the voltage shift is definitely large enough to influence the electrical properties of an excitable cell .", "A shift towards more positive voltages brought about by increasing PA in the inner leaflet will ‘silence’ K+ channels over an otherwise active voltage range .", "This silencing will lead to increased membrane excitability .", "The sensitivity of Kv channels to PA seems a likely link between metabolic pathways coupled to lipid metabolism and membrane excitability ." ], [ "A mutant of the rat Kv1 . 2 channel in which the helix-turn-helix segment termed the voltage sensor paddle was replaced by the corresponding segment from Kv2 . 1 , known as the paddle chimera Kv channel , was expressed and purified as described previously ( Long et al . , 2007; Tao and MacKinnon , 2008 ) , with minor modifications .", "In brief , the paddle chimera Kv channel was co-expressed with the rat β2-core gene in Pichia pastoris .", "The channel complex was extracted from membranes with DDM ( Anatrace , Maumee , OH ) and purified with a cobalt affinity column followed by size exclusion chromatography on a Superdex-200 gel filtration column ( GE Biosciences , Pittsburgh , PA ) .", "The size exclusion buffer was composed of 20 mM Tris–HCl , pH 7 . 5 , 150 mM KCl , 6 mM DM ( Anatrace ) , 2 mM Tris ( 2-carboxyethyl ) phosphine , 2 mM dithiothreitol , and 0 . 1 mg/ml POPC:POPE:POPG 3:1:1 ( mass ratio ) ( Avanti Polar Lipids , Alabaster , AL ) .", "Purified channel complexes were reconstituted into OM ( Anatrace ) -solubilized 3:1 ( wt:wt ) POPE:POPG lipid vesicles as described ( Long et al . , 2007 ) .", "Detergent was removed by dialysis for 5 days against detergent-free buffer containing 10 mM HEPES-KOH , pH 7 . 5 , and 450 mM KCl , and 2 mM dithiothreitol at 4°C , with daily buffer exchanges .", "After 5 days , all residual detergent was removed by incubating the reconstituted channels with Bio-Beads ( Bio-Rad , Hercules , CA ) for 2 hr at room temperature .", "The reconstituted channels were aliquoted and flash frozen into liquid nitrogen prior to storage at −80°C .", "KvAP was expressed and purified as described previously ( Ruta et al . , 2003 ) .", "Briefly , the channel was extracted from Escherichia coli membranes with DM ( Anatrace ) and purified with a cobalt affinity column followed by size exclusion chromatography on a Superdex-200 gel filtration column ( GE Biosciences ) with 20 mM Tris–HCl , pH 8 . 0 , 100 mM KCl , 4 mM DM ( Anatrace ) .", "Purified KvAP channels were reconstituted into DM-solubilized 3:1 ( wt:wt ) POPE:POPG lipid vesicles at a 1:10 ( wt:wt ) protein:lipid ratio .", "Detergent was removed by dialysis for 3 days against detergent-free buffer containing 10 mM HEPES , 4 mM N-methylglucamine , pH 7 . 4 , and 450 mM KCl , with twice daily buffer exchanges .", "The reconstituted channels were aliquoted and flash frozen into liquid nitrogen prior to storage at −80°C .", "Planar lipid bilayer experiments were performed as described previously ( Miller , 1986; Ruta et al . , 2003 ) .", "Lipids of desired compositions were prepared by dissolving argon-dried lipids in decane to a final concentration of 20 mg/ml .", "Lipid solutions were painted over a 300 μm hole in a polystyrene partition that separated the two chambers to form the planar lipid bilayer .", "The chamber ( cis ) contained 4 ml of 150 mM KCl and 10 mM HEPES-KOH , pH 7 . 5 , while the cup ( trans ) contained 3 ml of 15 mM KCl and 10 mM HEPES-KOH , pH 7 . 5 .", "Reconstituted channels were pipetted onto the chamber side of the bilayer after thinning of a planar lipid bilayer had been detected via monitoring of electrical capacitance .", "Once channels were successfully fused with the bilayer , 135 mM KCl was added to the cup side to equilibrate the K+ concentrations across the bilayer .", "Membranes were held at a negative holding voltage , stepped to more depolarized voltages in 10-mV increments and then back to the negative holding voltage to close the channels .", "Shortly after the return to the negative holding voltage , inward current called ‘tail current’ is measured .", "The fraction of maximal activation at each depolarization voltage can be determined by graphing the inward tail current , normalized by the maximum value , as a function of the preceding depolarization voltage and fit to a two-state Boltzmann equation: ( 2 ) IImax=11+e−ZFRT ( V−Vmid ) , where I/Imax is the fraction of maximal current , V is the command depolarization voltage to open the channels , Vmid is the command voltage at which the channels have reached 50% of their maximal current , F is the Faraday constant , R is the gas constant , T is the absolute temperature and Z is the apparent valence of the voltage dependence .", "All recordings were performed using the voltage-clamp method in whole-cell mode .", "Analogue signals were filtered at 1 kHz using a low-pass Bessel filter on an Axopatch 200B amplifier ( Molecular Devices ) in whole-cell mode and digitized at 10 kHz using a Digidata 1400A analogue-to-digital converter ( Molecular Devices ) .", "The pClamp software suite ( Molecular Devices ) was used to control membrane voltage and record current .", "Following successful incorporation of paddle chimera channels into a DPhPC bilayer , 4 μl of 50 , 000 units/ml phospholipase D1 purified from S . chromofuscus ( Sigma , St . Louis , MO ) were pipetted into the ground side of the bilayer and mixed thoroughly with a pipette , resulting in a final concentration of 50 units/ml .", "Electrophysiological recordings were conducted using both the forward and reverse protocols every ten minutes until the membrane became unstable .", "10 mg of desired lipid in chloroform were dried down under constant Argon stream and then resuspended into 0 . 5 ml of 450 mM KCl , 10 mM HEPES pH 7 . 5 by sonication , resulting in a final concentration of 20 mg/ml .", "Following incorporation of paddle chimera channels into a DPhPC bilayer , 1 . 5 μl of the vesicle solution was pipetted onto the chamber side of the bilayer .", "After 10 min , electrophysiological recordings were performed using both the forward and reverse protocols from the same bilayer .", "DPhPC–1 , 2-diphytanoyl-sn-glycero-3-phosphocholine , POPA–1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate , POPC–1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine , POPG 1–palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol , PI–Bovine liver L-α-phosphatidylinositol , POPS–1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine , DMPA–1 , 2-dimyristoyl-sn-glycero-3-phosphate , DOPA–1 , 2-dioleoyl-sn-glycero-3-phosphate , BrPOPA–1-palmitoyl-2- ( 9 , 10-dibromo ) stearoyl-sn-glycero-3-phosphate , DOPMe–1 , 2-dioleoyl-sn-glycero-3-phosphomethanol , DOPEth–1 , 2-dioleoyl-sn-glycero-3-phosphoethanol , DOPP–1 , 2-dioleoylglycerol pyrophosphate , PIP–Porcine brain L-α-phosphatidylinositol-4-phosphate , PIP2-phosphatidylinositol-4 , 5-bisphosphate , Cardiolipin–Bovine heart Cardiolipin , Sphingomyelin–Porcine brain Sphingomyelin , DPhPA–1 , 2-diphytanoyl-sn-glycero-3-phosphate , OM_octyl-β-D-maltopyranoside , DDM–n-dodecyl-β-D-maltopyranoside , DM–n-decyl-β-D-maltopyranoside , HEPES–N- ( hydroxyethyl ) piperazine-Nʹ-2-ethanesulfonic acid ." ] ]
[ "Membrane phospholipids can function as potent regulators of ion channel function .", "This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating .", "Using the method of reconstitution into planar lipid bilayers , in which protein and lipid components are defined and controlled , we characterize two effects of phosphatidic acid .", "The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces .", "The second is specific to the presence of a primary phosphate group , acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor .", "Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation .", "These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid ." ]
[ "The electrical signals that carry information through the nervous system rely on positively charged potassium ions moving in and out of neurons .", "These ions move through proteins called voltage-gated potassium channels that are embedded in the plasma membrane that surrounds the neurons .", "The potassium channels contain pores that can be opened and closed to control the movement of the potassium ions .", "The main factor that controls the opening and closing of these channels—a process known as ‘gating’—is the voltage across the membrane .", "However , the channels can also be controlled by proteins , or by other molecules .", "The plasma membrane is made of several different types of molecules called phospholipids .", "Some of these phospholipids are known to be involved in gating potassium channels , but the roles of other phospholipids remain unclear .", "To investigate the role of a phospholipid called phosphatidic acid , Hite et al . placed potassium ion channels in artificial plasma membranes .", "These experiments revealed that phosphatidic acid alters the gating of potassium ion channels in two ways .", "The first way is generic: the negative charge in phosphatidic acid shifts the membrane voltage .", "The second way is specific to phosphatidic acid: the end of the molecule with the negative charge interacts with the part of the potassium channel that senses changes in voltage to keep the pore closed .", "The next challenge is to understand how neurons shift their phosphatidic acid levels to regulate their electrical activity ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "ecology", "evolutionary biology" ]
Living with relatives offsets the harm caused by pathogens in natural populations
elife-66649-v1
[ [ "High relatedness between individuals can favour the evolution of cooperative interactions that increase reproductive success and survival ( Hamilton , 1964a; Hamilton , 1964b ) .", "For example , it has been repeatedly shown that individuals can pass on their genes indirectly by providing vital resources to relatives and assisting them with tasks that are difficult to do alone , such as caring for offspring ( Alexander , 1974; Rubenstein and Abbot , 2017; West et al . , 2007 ) .", "However , living with relatives can also increase susceptibility to pathogens that spread more easily among genetically similar individuals , with similar immune defences ( Anderson et al . , 1986; Baer and Schmid-Hempel , 1999; Hamilton , 1987; Liersch and Schmid-Hempel , 1998; Schmid-Hempel , 1998; Sherman et al . , 1998 ) .", "This phenomenon has been referred to as the ‘monoculture effect’ ( Elton , 1958 ) in agricultural settings after it was observed that clonal crops were highly susceptible to disease outbreaks ( Garrett and Mundt , 1999; Tooker and Frank , 2012; Wolfe , 1985; Zhu et al . , 2000 ) .", "More recently it has also been established that such effects occur in natural populations , with higher genetic similarity between individuals increasing rates of parasitism ( Ekroth et al . , 2019 ) .", "What remains unclear is whether this translates into higher rates of mortality , or whether the benefits of living with relatives are large enough to offset the costs of increased disease risk ( Hughes et al . , 2002 ) .", "Previous research into the effects of relatedness on disease spread have been conducted on an expansive range of species including bacteria , plants , and animals .", "These studies have revealed remarkable variation in how changes in relatedness influence parasitism and mortality .", "For example , in honeybees , Apis melifera , high relatedness among individuals increases the risk of disease and colony death ( Tarpy et al . , 2013 ) , whereas in Pharoah ants , Monomorium pharaonis , high relatedness reduces the abundance of pathogens ( Schmidt et al . , 2011 ) .", "Such differences between species may in part be due to how data are collected .", "In some studies , relatedness and pathogens have been experimentally manipulated , whereas in other studies relatedness and the abundances of pathogens are only observed ( ‘observational studies’ ) .", "In observational studies , results can be variable and difficult to interpret because the causality behind relationships is uncertain ( Lively et al . , 2014 ) .", "For instance , a negative relationship between relatedness and the abundance of pathogens can occur either because groups of relatives are less susceptible to pathogens , or because groups of relatives die from pathogens and so are rarely observed ( Ben-Ami and Heller , 2005; King et al . , 2011; Teacher et al . , 2009 ) .", "Additionally , species may vary in their susceptibility to pathogens because of differences in past selection to control disease spread among individuals ( Loehle , 1995; Romano et al . , 2020 ) .", "In species where relatives frequently interact , selection is predicted to favour the evolution of strategies that mitigate the impacts of pathogens ( Loehle , 1995; Romano et al . , 2020 ) .", "Limiting social interaction through group-level organisation , such as task partitioning and other mechanisms of the so-called ‘social immunity’ , can prevent disease spread among relatives ( Camargo et al . , 2007; Cremer and Sixt , 2009; Liu et al . , 2019; Ugelvig et al . , 2010; Waddington and Hughes , 2010 ) .", "However , whether species that typically live with kin are better able to cope with pathogens when relatives interact , compared to species that live with non-kin , is unclear .", "The spread of disease through populations also depends on how variable pathogen abundances are across groups .", "Pathogen abundances are expected to be more variable among groups of relatives because they either contain resistant or susceptible genotypes ( Boomsma and Ratnieks , 1996; van Baalen and Beekman , 2006 ) .", "Groups of unrelated individuals , on the other hand , will contain a mix of susceptible and resistant genotypes , leading to more predictable pathogen abundances and rates of mortality across groups .", "Such differences in variation across groups of related and unrelated individuals are nevertheless predicted to depend on pathogen diversity .", "When there are many different pathogens , groups of relatives are more likely to be susceptible to at least one pathogen , which can reduce variation in total pathogen abundance to a level that is similar to groups of unrelated individuals ( Ganz and Ebert , 2010; van Baalen and Beekman , 2006 ) .", "While both increases and decreases in variation in rates of parasitism and mortality have been found in specific study species ( Ganz and Ebert , 2010; Johnson et al . , 2006; Seeley and Tarpy , 2007; Thonhauser et al . , 2016 ) , whether variation among groups of relatives is generally higher across species remains to be tested .", "Here , we use phylogenetic meta-analysis to first examine whether the benefits of living with relatives counteract the costs of increased susceptibility to pathogens .", "Second , we tested if the ability to detect such effects is dependent upon the experimental manipulation of pathogens and within-group relatedness .", "Third , we examined if species that typically live with kin have evolved mechanisms to reduce pathogen spread among relatives compared to species that typically live with non-kin .", "Finally , we investigated whether variation in the abundance of pathogens and rates of mortality is higher across groups of relatives .", "The influence of relatedness on mortality and pathogen abundances were quantified by extracting effect sizes ( Pearson’s correlation coefficients r ) from 75 published studies across 56 species ( Supplementary file 1—Tables S1-S3 ) .", "Variation in pathogen abundances and rates of mortality were measured using a standardised effect size of variance that accounts for differences in means , the coefficient of variation ratio ( CVR ) , which was possible to estimate for 25 species ( Supplementary file 1—Table S4 ) ." ], [ "Across species , within-group relatedness had highly variable effects on rates of mortality and the abundance of pathogens ( Figure", "1 . Bayesian Phylogenetic Multi-level Meta-regression ( BPMM ) : Mean effect size [posterior mode ( PM ) ] of Zr = 0 . 06 , credible interval [CI] = −0 . 12 to 0 . 26 , pMCMC = 0 . 40 .", "Supplementary file 1—Table S5 ) .", "Such variation was ubiquitous across all taxonomic groups and was largely independent of phylogenetic history ( % of variation in Zr explained by phylogeny PM ( CI ) = 8 . 20 ( 0 . 11 , 31 . 59 ) .", "Figure 1; Supplementary file 1—Table S5 ) .", "Mortality was , however , consistently higher in groups of relatives in the presence of pathogens compared to when they were absent ( Figure", "2 . Zr pathogens absent versus present PM ( CI ) = −0 . 29 ( −0 . 44 , –0 . 12 ) , pMCMC = 0 . 002 .", "Supplementary file 1—Table S6 ) .", "Similar effects of within-group relatedness on pathogen abundances were found ( Zr pathogen abundance versus mortality PM ( CI ) = 0 . 01 ( –0 . 10 , 0 . 19 ) , pMCMC = 0 . 51 .", "Supplementary file 1—Table S6 ) , but these were much weaker ( Zr pathogen abundance PM ( CI ) = 0 . 10 ( –0 . 10 , 0 . 33 ) , pMCMC = 0 . 31 .", "Supplementary file 1—Table S6 ) .", "There was evidence that pathogens causally increased mortality in groups of relatives ( Figure 2; Supplementary file 1—Table S7 ) .", "In studies where pathogens were experimentally manipulated , groups of relatives had significantly higher mortality when pathogens were present compared to when they were absent ( Zr pathogens absent versus present PM ( CI ) = −0 . 40 ( −0 . 57 , –0 . 21 ) , pMCMC = 0 . 001 .", "Figure 2; Supplementary file 1—Table S7 ) .", "The contrasting effects of relatedness in the presence and absence of pathogens meant that overall the effect of relatedness on mortality did not significantly differ from zero ( Zr pathogens present PM ( CI ) = 0 . 17 ( −0 . 09 , 0 . 38 ) , pMCMC = 0 . 15 .", "Zr pathogens absent PM ( CI ) = −0 . 23 ( −0 . 49 , 0 . 03 ) , pMCMC = 0 . 11 .", "Figure 2; Supplementary file 1—Table S7 ) .", "Therefore the greater susceptibility of groups of relatives to pathogens appears to be masked by kin selected benefits of living with relatives when pathogens are rare .", "This may also explain why in observational studies the effect of relatedness on mortality , both in the presence and absence of pathogens , was close to zero ( Zr pathogens present PM ( CI ) = 0 . 06 ( −0 . 10 , 0 . 27 ) , pMCMC = 0 . 36 .", "Zr pathogens absent PM ( CI ) = 0 . 10 ( −0 . 11 , 0 . 50 ) , pMCMC = 0 . 26 .", "Figure 2; Supplementary file 1—Table S7 ) .", "Experimental manipulations of relatedness were less important for detecting the effects of relatedness on mortality than manipulations of pathogens ( Supplementary file 1—Table S8 ) .", "Studies that experimentally manipulated within-group relatedness found similar reductions in survival in groups of relatives when pathogens were present to observational studies ( Experimental studies: Zr pathogens absent versus present PM ( CI ) = −0 . 19 ( −0 . 42 , –0 . 08 ) , pMCMC = 0 . 01 .", "Observational studies: Zr pathogens absent versus present PM ( CI ) = −0 . 30 ( −0 . 77 , –0 . 10 ) , pMCMC = 0 . 02 .", "Supplementary file 1—Table S8 ) .", "Next , we tested whether species that typically live with relatives have evolved mechanisms to limit the negative effects of pathogens when within-group relatedness is high .", "To do this , species that typically live with relatives under natural conditions ( r > 0 . 25 referred to as ‘kin’ ) were compared to those that associate with unrelated individuals ( r < 0 . 25 referred to as ‘non-kin’ . See Materials and methods for details of data used to classify species ) .", "When pathogens were present , the effect of relatedness on rates of mortality did not differ between species that live with kin and non-kin ( Zr kin versus non-kin pathogen present PM ( CI ) = 0 . 09 ( −0 . 31 , 0 . 37 ) , pMCMC = 0 . 79 .", "Supplementary file 1—Table S9 ) .", "However , when pathogens were absent , high relatedness reduced mortality in species that live with kin , but increased mortality in species that live with non-kin ( Zr kin versus non-kin pathogen absent PM ( CI ) = −0 . 57 ( −1 . 11 , 0 . 02 ) , pMCMC = 0 . 03 .", "Figure 3 , Supplementary file 1—Table S9 ) .", "For example , in the red flour beetle , Tribolium castaneum , and the tube worm , Galeolaria caespitosa , that typically interact with non-kin , mortality was two to four times higher when individuals were placed in groups of relatives compared to when individuals were unrelated ( Agashe , 2009; McLeod and Marshall , 2009 ) .", "These results show that species that typically associate with non-kin suffer reductions in fitness when placed in groups of relatives , but only when pathogens are rare .", "Conversely , species that live with kin have higher fitness in groups of relatives when pathogens are absent , but such benefits disappear when pathogens are present ( Zr pathogens absent versus present PM ( CI ) = −0 . 33 ( −0 . 53 , –0 . 16 ) , pMCMC = 0 . 001 .", "Supplementary file 1—Table S9 ) .", "Variation in rates of mortality and the abundance of pathogens were influenced by relatedness in opposing ways ( Figure 4; Supplementary file 1—Table S10-S13 ) .", "Experimental manipulations of pathogen presence were important for detecting these effects ( Supplementary file 1—Table S12 ) .", "In observational studies , relatedness within groups had no effect on variance in mortality , either in the presence or absence of pathogens , and did not influence variance in pathogen abundances ( Mortality pathogens absent: LnCVR PM ( CI ) = 0 . 44 ( −0 . 32 , 0 . 94 ) , pMCMC = 0 . 32 .", "Mortality pathogens present: LnCVR PM ( CI ) = −0 . 03 ( −0 . 74 , 0 . 65 ) , pMCMC = 0 . 93 .", "Pathogen abundance: LnCVR PM ( CI ) = −0 . 15 ( −0 . 71 , 0 . 50 ) , pMCMC = 0 . 83 .", "Figure 4 , Supplementary file 1—Table S12 ) .", "In contrast , in experimental studies mortality was more variable across groups of relatives when pathogens were present ( LnCVR PM ( CI ) = 0 . 88 ( 0 . 21 , 1 . 41 ) , pMCMC = 0 . 02 .", "Figure 4 , Supplementary file 1—Table S12 ) .", "The opposite pattern was true for pathogen abundances , with groups of relatives being less variable .", "This meant that overall , mortality was significantly more variable than the abundance of pathogens among groups of related versus unrelated individuals ( LnCVR PM ( CI ) = 1 . 18 ( 0 . 56 , 1 . 88 ) , pMCMC = 0 . 001 .", "Figure 4 , Supplementary file 1—Table S12 ) .", "These results suggest that pathogens spread more uniformly across groups of relatives , but effects on mortality are more variable than across groups of unrelated individuals ." ], [ "Our analyses show that pathogens can increase rates of mortality in groups of relatives .", "The detrimental effects of pathogens were , however , counteracted by high relatedness reducing mortality when pathogens were rare , particularly in species that live in kin groups .", "Such contrasting effects of relatedness meant that experimental manipulations were crucial for detecting the costs and benefits of living with relatives when the presence of pathogens varied .", "Additionally , high relatedness resulted in more even abundances of pathogens across groups , but more variable rates of mortality , highlighting the importance of population genetic structure in explaining the epidemiology of diseases .", "We discuss these findings in relation to the environments favouring the evolution of different social systems , the mechanisms that have evolved to prevent disease spread in social groups , and the types of study system where more experimental data are required .", "The interaction between kin selected benefits and mortality caused by pathogens has important implications for our understanding of the ecological distributions of species and the evolutionary origins of different social systems .", "In some lineages , such as birds , cooperative species that live in families have been found to inhabit areas that are hot and dry ( Cornwallis et al . , 2017; Jetz and Rubenstein , 2011; Lukas and Clutton-Brock , 2017 ) .", "This has been attributed to the benefits of cooperative offspring care being higher in environments that are challenging for independent reproduction ( Emlen , 1982 ) .", "An additional , potentially important explanation is that the costs imposed by pathogens when living with relatives may be lower in such environments ( Campbell-Lendrum et al . , 2015 ) .", "Parallel arguments have been made for social insects .", "Species with sterile worker castes , that only evolved in groups with high levels of relatedness , are thought to have arisen in environments protected from pathogens ( Hamilton , 1987 ) .", "For example , sterile soldier castes have evolved at least six independent times in clonal groups of aphids , and the majority of these cases form galls that provide protection against pathogens ( Hamilton , 1987; Stern and Foster , 1996 ) .", "Escape from pathogens may therefore be a general feature governing the evolutionary origin , as well as the current ecological niches , of species living in highly related groups .", "The benefits of living with relatives are predicted to generate selection for increased resistance or tolerance to disease spread ( Loehle , 1995; Romano et al . , 2020 ) .", "Adaptations to limit pathogen transmission in kin groups have been documented in some species .", "For example , in leaf cutter ants , Acromyrmex spp .", ", workers outside the colony , where pathogens are more prevalent , do not enter the inner colony ( Camargo et al . , 2007 ) .", "Contamination of food by pathogens is also limited by workers outside the colony performing dedicated tasks , such as foraging versus waste management ( Waddington and Hughes , 2010 ) .", "Changing the organisational structure of groups or living in smaller groups can therefore increase social distancing and reduce pathogen transmission ( Loehle , 1995; Romano et al . , 2020; Liu et al . , 2019 ) .", "While examples of social immunity exist , there was little evidence that species that live with kin have generally evolved mechanisms to limit the harm caused by pathogens .", "Species that live in kin groups suffered similar reductions in survival from pathogens to species that live with non-kin ( Figure 3 ) .", "One explanation is that individuals respond to greater pathogen pressure by forming more genetically diverse groups ( Schmid-Hempel , 1998; Sherman and Morton , 1988 ) .", "For example , increases in mating promiscuity under higher disease risk can lower relatedness among offspring recruited to groups ( Busch et al . , 2004; Singh et al . , 2015; Soper et al . , 2014 ) .", "Such responses can reduce disease spread , but also weakens selection for adaptations that limit pathogen spread among related individuals .", "The relative costs of decreasing the effects of pathogens by reducing relatedness versus other mechanisms remains unclear , but may provide insight into why different responses to pathogens have evolved across species .", "High relatedness was associated with higher and more variable rates of mortality in the presence of pathogens , but had little effect on variation in pathogen load .", "Such differences may arise because pathogen abundances are often weakly related to the virulence of pathogens ( Leggett et al . , 2012 ) .", "Genotypes can also be equally susceptible to pathogens , but vary in their ability to clear infections , which may explain why within-group relatedness influenced mortality rates without strongly affecting variation in pathogen abundances ( Best et al . , 2008; Howick and Lazzaro , 2014; Koskela et al . , 2002 ) .", "The effects of relatedness on mortality rates were only evident in experiments .", "There are a number of possible , non-mutually exclusive , explanations for this .", "It is possible that observational studies fail to capture the true effect of pathogens because of sampling biases: groups of relatives infected with pathogens can quickly die resulting in their effects being underestimated ( Ben-Ami and Heller , 2005; King et al . , 2011; Teacher et al . , 2009 ) .", "The diversity and abundance of pathogens may also differ between experimental and observational studies .", "Although experiments often reported that pathogens were manipulated in biologically realistic ways , it is possible that pathogen abundances are generally higher in experiments leading to larger effect sizes .", "Additionally , experiments generally only manipulated single pathogens whereas observational studies on natural populations often involve communities of pathogens .", "Low pathogen diversity is predicted to increase variation across groups of relatives ( Boomsma and Ratnieks , 1996; van Baalen and Beekman , 2006 ) .", "The lack of an effect of relatedness on variance in mortality in observational studies may therefore be due to the diversity of pathogens being higher .", "In our dataset , there was only one experimental study that manipulated multiple pathogens .", "In Daphnia magna it was found that variance in parasitism was higher in groups of relatives ( ‘clonal’ versus ‘polyclonal’ populations ) , but this diminished as the number of pathogens increased ( Ganz and Ebert , 2010 ) .", "This suggests that where pathogen diversity is high , groups of relatives become increasingly susceptible to pathogens , reducing variance across groups ( Boomsma and Ratnieks , 1996; Parsche and Lattorff , 2018; van Baalen and Beekman , 2006 ) .", "How relatedness among individuals influences pathogen spread has been investigated in a diverse range of species making our analyses possible .", "Nevertheless , experiments manipulating pathogen presence , abundance , and diversity across species with different ecological niches and social systems , especially those that typically associate with non-kin , remain limited .", "In-depth analyses comparing species with ancestrally versus derived levels of high and low relatedness will also help shed light on the importance of current versus past evolutionary responses to pathogens .", "We hope that our results stimulate further research in these areas which appears crucial to understanding the impact of pathogens on natural populations ." ], [ "A systematic literature review was performed to identify studies that have examined the relationship between within-group relatedness and rates of mortality or the abundance of pathogens .", "One challenge with locating relevant literature was that some studies use the term relatedness while others use the term genetic diversity .", "Genetic diversity encompasses studies that have examined within-individual genetic diversity ( e . g . heterozygosity ) , as well as genetic diversity of groups .", "The aims of our study only relate to variation in genetic diversity of groups ( relatedness ) .", "All studies where estimates of within-group genetic diversity were potentially influenced by within-individual genetic diversity were excluded ( see below ) .", "The literature search was performed using the Web of Science ( WoS ) including articles published up to the 27 July 2020 .", "Searches were restricted to articles in English and the WoS categories were restricted to Behavorial Sciences , Ecology , Biology , Evolutionary Biology , Ecology , Multidisciplinary Sciences , Genetics & Heredity , Biodiversity & Conservation , Entomology , Zoology as a preliminary study ( Bensch MSc thesis ) showed these categories to be the ones of interest .", "WoS searches included the following combinations of terms in the topic field: ( ( ( ‘genetic diversity’ OR ‘genetic variability’ OR ‘genetic diversities’ ) AND parasite∗ ) OR ( ( ‘genetic diversity’ OR ‘genetic variability’ OR ‘genetic diversities’ ) AND disease∗ ) OR ( ( ‘genetic diversity’ OR ‘genetic variability’ OR ‘genetic diversities’ ) AND pathogen∗ ) OR ( ( ‘genetic diversity’ OR ‘genetic variability’ OR ‘genetic diversities’ ) AND survival ) OR ( ( ‘genetic diversity’ OR ‘genetic variability’ OR ‘genetic diversities’ ) AND mortality ) OR ( relatedness AND pathogen∗ ) OR ( relatedness AND disease∗ ) OR ( relatedness AND parasite∗ ) OR ( relatedness AND mortality ) OR ( relatedness AND survival∗ ) OR ‘monoculture effect’ OR ‘Monoculture effect’ ) AND ( population∗ OR group∗ OR colony ) .", "Initial exploration of search terms included other words ( ‘clone’ , ‘clonal’ , ‘social’ ) .", "However , these terms inflated the number of search hits and papers with relevant data were retrieved using other terms included in our search criteria ( ‘group’ , ‘colony’ , or ‘relatedness’ ) .", "The search yielded a total of 4616 returns , 4615 after removing a duplicate .", "To aid finding relevant papers , abstracts were downloaded and imported into R for text analysis using the quanteda package ( Benoit et al . , 2018 ) .", "The frequency of words in each abstract was calculated and used to create a relevance score according to the number of words with positive and negative interest for this study .", "The following words had positive associations ( listed in order of priority ) : ‘genetic’ , ‘diversity’ , ‘diversities’ , ‘variation’ , ‘relatedness’ , ‘related’ , ‘unrelatedness’ , ‘unrelated’ , ‘diverse’ , ‘parasite’ , ‘ectoparasite’ , ‘ectoparasites’ , ‘parasites’ , ‘pathogen’ , ‘pathogenic’ , ‘pathogens’ , ‘disease’ , ‘diseases’ , ‘diseased’ , ‘mortality’ , ‘survival’ , ‘resistance’ , ‘infection’ , ‘infections’ , ‘prevalence’ , ‘tolerance’ , ‘transmission’ , ‘population’ , ‘group’ , ‘colony’ , ‘groups’ , ‘colonies’ , ‘populations’ .", "The following words had negative associations: ‘human’ , ‘humans’ , ‘hospital’ , ‘cancer’ , ‘hiv’ , ‘patients’ .", "Papers were sorted according to their relevance scores and then manually screened to examine whether they contained data that could be used to calculate an effect size of relatedness and mortality and/or pathogen abundance .", "We did not include studies examining the relationship between within-group relatedness and other fitness-related measures , such as fecundity or competitive ability , because such measures are influenced by many factors other than pathogens .", "We stopped screening after 2102 papers as number of new papers selected for in-depth screening decreased to less than 1% per 100 references ( Figure 1—figure supplement 1 ) .", "In addition to WoS searches , reference lists of key studies and the papers from which we extracted effect sizes were screened for relevant primary literature .", "PDF files of articles selected based on abstract screening were downloaded for in-depth examination of full texts .", "A preferred reporting items for meta-analyses diagram ( Moher et al . , 2009 ) of the literature screening process is shown in Figure 1—figure supplement 2 .", "In total our dataset consisted of 210 effect sizes from 75 studies and 56 species ( Abdi et al . , 2020; Agashe , 2009; Aguirre and Marshall , 2012a; Aguirre and Marshall , 2012b; Altermatt and Ebert , 2008; Anton et al . , 2007; Baer and Schmid-Hempel , 2001; Baer and Schmid-Hempel , 1999; Ben-Ami and Heller , 2005; Bensch and Cornwallis , 2017; Bichet et al . , 2015; Byrne and Robert , 2000; Byrne and Whiting , 2011; Cook‐Patton et al . , 2017; Cook-Patton et al . , 2011; Crutsinger et al . , 2006; Crutsinger et al . , 2008; Dagan et al . , 2017; Dagan et al . , 2013; de Morais , 2020; Desai and Currie , 2015; de Vere et al . , 2009; Dobelmann et al . , 2017; Ellison et al . , 2011; Field et al . , 2007; Franklin et al . , 2012; Fraser et al . , 2010; Gamfeldt and Källström , 2007; Ganz and Ebert , 2010; Gardner et al . , 2007; He and Lamont , 2010; Hoggard et al . , 2013; Hughes and Stachowicz , 2004; Hughes and Boomsma , 2006; Hughes and Boomsma , 2004; Johansson et al . , 2007; Johnson et al . , 2006; Kapranas et al . , 2016; Keeney et al . , 2009; King et al . , 2011; Kotowska et al . , 2010; Lambin and Krebs , 1993; Liersch and Schmid-Hempel , 1998; Mattila et al . , 2012; McLeod and Marshall , 2009; Mott et al . , 2019; Neumann and Moritz , 2000; Page et al . , 1995; Parker et al . , 2010; Parsche and Lattorff , 2018; Pearman and Garner , 2005; Reber et al . , 2008; Robinson et al . , 2013; Schmidt et al . , 2011; Seeley and Tarpy , 2007; Sera and Gaines , 1994; Shykoff and Schmid-Hempel , 1991; Siemens and Roy , 2005; Solazzo et al . , 2014; Strauss et al . , 2017; Tarpy , 2003; Tarpy and Seeley , 2006; Tarpy et al . , 2013; Teacher et al . , 2009; Thonhauser et al . , 2016; Trouvae et al . , 2003; Ugelvig et al . , 2010; van Houte et al . , 2016; Vanpé et al . , 2009; Walls and Blaustein , 1994; Wauters et al . , 1994b; Weyrauch and Grubb , 2006; Winternitz et al . , 2014; Woyciechowski and Król , 2001 ) .", "Studies were included if they presented data on the abundance/presence of pathogens and relatedness for four or more groups .", "Relatedness was estimated from breeding designs , pedigrees , and using molecular markers .", "A group was defined as three or more individuals as it has been shown to be sufficient for group-level defences ( Hughes and Stachowicz , 2004 ) .", "That said , only three studies used groups with three individuals ( 4% ) with over 93% of studies using groups with five or more individuals .", "Some studies manipulated levels of relatedness by experimentally creating groups ( referred to as ‘experimental relatedness’ ) , whereas other studies measured relatedness on already established groups ( referred to as ‘observational relatedness’ ) .", "The presence and abundance of pathogens was also experimentally manipulated in some studies ( referred to as ‘experimental pathogens’ ) whereas in others pathogens were measured without any manipulations ( referred to as ‘observational pathogens’ ) .", "Studies on plants were included that examined the effect of pathogens and herbivores , as it has previously been argued that herbivory is equivalent to parasitism ( see Price , 1980; Siemens and Roy , 2005 for discussion of herbivores as pathogens ) .", "One study was included from unpublished data collected by the authors on ostriches , Struthio camelus ( Supplementary file 1—Tables S19 ) .", "Studies were excluded if they were on domestic species or where there was the potential for within-individual genetic diversity , including inbreeding , to influence estimates of within-group relatedness .", "In some studies , inbreeding was not explicit but potentially possible ( Supplementary file 1—Table S1 ) .", "We tested the sensitivity of our results to any potential inbreeding effects by removing these effect sizes and repeating our analyses ( see verification analyses; Supplementary file 1—Tables S14 and S15 ) .", "If data of interest were missing in the text or figures , authors were contacted for supplementary data or clarification .", "If authors did not respond within 3 months , the effect sizes were excluded .", "If studies provided multiple measures of pathogen load and/or mortality , separate effect sizes were extracted .", "Where studies presented abundances of specific pathogens as well as total abundance of pathogens , the total was used .", "The relationship between within-group relatedness and mortality and/or pathogen abundance was analysed by comparing groups with high and low relatedness ( relatedness as a categorical variable ) , or by analysing variation in average within-group relatedness as a continuous variable .", "Information from both types of study was used to calculate a standardised effect size of the correlation between within-group relatedness and mortality/pathogen abundance: Pearson’s correlation coefficient , r .", "The statistical tests presented in studies were converted to r using the online meta-analysis calculator ( Morris , 2019 ) and the R package ‘esc’ ( Lüdecke , 2019 ) .", "Measures of r were transformed to Zr using ‘escalc’ function in the R package metafor ( Viechtbauer , 2010 ) .", "In some studies , it was not possible to obtain effect sizes directly from the statistics reported in studies , but r could be calculated from data presented in the text and/or figures in two ways .", "First , in studies where groups with high and low relatedness were compared , means ± SD of mortality or pathogen abundances were used to calculate r .", "Second , in studies where descriptive statistics ( e . g . means ± SD ) were reported for multiple groups that varied in relatedness , we conducted our own Pearson’s correlations in R ( see R script ‘EffectSizeCalculations’ and Supplementary file 1—Table S2 column ‘Effect size Rscript reference’ ) .", "In such cases , variation in measures of relatedness , mortality , and pathogen abundances were included by creating distributions from descriptive statistics that were sampled to create 1000 datasets .", "For each of these 1000 datasets , r was calculated and an average taken across the 1000 datasets .", "The effect of relatedness on variance in mortality and pathogen abundances was calculated using the natural logarithm of the ratio between the coefficient of variation from groups with high and low relatedness ( LnCVR: Nakagawa et al . , 2015 ) .", "LnCVR provides a standardised measure of differences in the variability of two groups accounting for differences in the means between groups .", "LnCVR was used because estimates of variation increased with the mean ( Figure 4—figure supplement 1 ) .", "LnCVR was calculated from studies that presented means and SDs ( converted to SD if studies presented SEs or CIs ) across groups when relatedness was low and high .", "This provides a standardised measure of the effect of relatedness on variability across groups , not within groups ( SDs were from across groups , not individuals ) .", "For each effect size extracted , we collected information on: ( 1 ) whether pathogens were present or absent; ( 2 ) whether pathogens were experimentally manipulated; ( 3 ) whether relatedness was experimentally manipulated; ( 4 ) the method used for measuring relatedness ( pedigree or molecular markers ) ; and ( 5 ) whether pathogen abundance or mortality were measured ( where survival estimates were presented , the sign of the effect size was reversed ) .", "If there was no mention of pathogens in the paper , then pathogens were assumed to be present when studies were conducted in the field and absent if conducted in the laboratory .", "For all species in our dataset we searched for whether they typically associate with kin ( ‘kin’ ) or not ( ‘non-kin’ ) during the life stage that effect sizes were measured .", "Species were categorised as kin if they lived in groups where r was estimated to be equivalent to 0 . 25 or higher and ‘non-kin’ if they live in groups where relatedness was estimated to be lower than 0 . 25 ( Supplementary file 1—Table S4 ) .", "Three sources of information were used to estimate levels of relatedness among individuals: ( 1 ) estimates of relatedness acquired either directly from molecular genetic analyses or records of groups of individuals with known relatedness; ( 2 ) information on the mating system; and ( 3 ) typical dispersal patterns , as low dispersal from groups increases relatedness .", "The relevant information was collected using Google Scholar including each species name combined with ‘genetic diversity’ , ‘relatedness’ , and ‘group’ as search terms to collect measures of within-group relatedness; ‘mating system’ and ‘paternity’ for information on mating system; and ‘dispersal’ and ‘philopatry’ for information on dispersal .", "The categorisation of each species as kin or non-kin along with evidence and the list of literature to support these classifications can be found in Supplementary file 1—Table S4 ( Abdi et al . , 2020; Aguirre and Marshall , 2012a; Aguirre and Marshall , 2012b; Amiri et al . , 2017; Anton et al . , 2007; Arnaud , 1999; Avise and Tatarenkov , 2015; Barrett et al . , 2005; Bee , 2007; Beermann et al . , 2015; Ben-Ami and Heller , 2005; Bryja et al . , 2008; Byrne and Robert , 2000; Byrne and Whiting , 2011; Chapuisat et al . , 2004; Croshaw et al . , 2009; M . Crutsinger et al . , 2008; Dagan et al . , 2013; Dean et al . , 2006; de Morais , 2020; de Vere , 2007; de Vere et al . , 2009; Dobelmann et al . , 2017; Edenbrow and Croft , 2012; Farentinos , 1972; Ficetola et al . , 2010; Field et al . , 2007; Franklin et al . , 2012; Fredensborg et al . , 2005; Gamfeldt and Källström , 2007; Gardner et al . , 2007; Getz et al . , 1993; Goldberg et al . , 2013; Goulson et al . , 2002; Goymann , 2009; Graham , 1941; Griffin , 2012; Haag et al . , 2002; He et al . , 2004; Head and Yu , 2004; Heppleston , 1972; Heske and Ostfeld , 1990; Hoffmann et al . , 2003; Hoggard et al . , 2009; Hughes and Stachowicz , 2004; Johnson , 2007; Johnson et al . , 2006; Kapranas et al . , 2016; Kawamura et al . , 1991; Keeney et al . , 2009; Kelly et al . , 1999; Keough , 1989; Keough and Chernoff , 1987; Kimwele and Graves , 2003; King et al . , 2011; Kozakiewicz et al . , 2009; König , 1993; Lambin and Krebs , 1991; Laurila and Seppa , 1998; Lepais et al . , 2010; Liker et al . , 2009; Liu et al . , 2013; Mackiewicz et al . , 2006; McLeod and Marshall , 2009; Meling-lópez and Ibarra-Obando , 1999; Myers et al . , 2011; Oettler and Schrempf , 2016; Osváth-Ferencz et al . , 2017; Pai and Bernasconi , 2007; Pietrzak et al . , 2010; Platt et al . , 2010; Reusch et al . , 1999; Rice et al . , 2009; Rock et al . , 2007; T . Russell et al . , 2004; Schmid-Hempel and Crozier , 1999; Schmid-Hempel and Schmid-Hempel , 2000; Schmidt et al . , 2011; Schmidt et al . , 2016; Schradin et al . , 2010; Schrempf et al . , 2006; Seppä and Walin , 1996; Seppä et al . , 2009; Shapiro and Dewsbury , 1986; Siemens and Roy , 2005; Simeonovska-Nikolova , 2007; Solomon et al . , 2004; Stürup et al . , 2014; Sutcliffe , 2010; Svane and Havenhand , 1993; Tarpy , 2003; Tatarenkov et al . , 2007; Thonhauser et al . , 2016; Trouvae et al . , 2003; Vanpé et al . , 2009; Verrell and Krenz , 1998; Walck et al . , 2001; Waldman , 1982; Walls and Blaustein , 1994; Wauters et al . , 1990; Wauters and Dhondt , 1992; Wauters et al . , 1994a; Zenner et al . , 2014 ) .", "We also collected data on whether species always lived in social groups ( ‘obligately social’ ) or whether species were only social during specific life stages ( ‘periodically social’ ) .", "However , it was not possible to analyse these data as experimental manipulations of pathogens , a key factor influencing the relationship between relatedness and mortality and pathogen abundances , were only performed for one periodically social species ( Rana latastei ) .", "Our dataset highlighted that there are several key variables where data are limited and where further empirical work would be extremely useful .", "In particular , information on the following is currently limited: ( 1 ) species that typically live with non-kin ( r: kin = 41 , non-kin = 15 . LnCVR: kin = 18 , non-kin = 7 ) ; ( 2 ) studies that quantify the effect of relatedness on rates of mortality in the absence of pathogens , particularly under natural conditions .", "Out of 75 studies , pathogens were excluded in 16 laboratory studies and no studies tried to explicitly exclude pathogens under field conditions .", "For LnCVR , pathogens were only excluded in seven laboratory studies out of a total of 32 studies; and ( 3 ) variation across groups in rates of mortality and pathogen abundance ( out of 210 mean effect sizes , variance could only be examined in 106 ) ." ] ]
[ "Living with relatives can be highly beneficial , enhancing reproduction and survival .", "High relatedness can , however , increase susceptibility to pathogens .", "Here , we examine whether the benefits of living with relatives offset the harm caused by pathogens , and if this depends on whether species typically live with kin .", "Using comparative meta-analysis of plants , animals , and a bacterium ( nspecies = 56 ) , we show that high within-group relatedness increases mortality when pathogens are present .", "In contrast , mortality decreased with relatedness when pathogens were rare , particularly in species that live with kin .", "Furthermore , across groups variation in mortality was lower when relatedness was high , but abundances of pathogens were more variable .", "The effects of within-group relatedness were only evident when pathogens were experimentally manipulated , suggesting that the harm caused by pathogens is masked by the benefits of living with relatives in nature .", "These results highlight the importance of kin selection for understanding disease spread in natural populations ." ]
[ "Living in a group with relatives has many advantages , such as helping with child rearing and gathering food .", "This has led many species to evolve a range of group behaviours; for example , in honey bee populations , worker bees sacrifice themselves to save the colony from incoming enemies .", "But there are also downsides to living with family .", "For example , bacteria , viruses and other disease-causing pathogens will find it easier to spread between relatives .", "This is because individuals with the same genes have similar immune defences .", "So , is it better to live with relatives who can help with life’s struggles or live with unrelated individuals where there is a lower chance of getting sick ?", "To help answer this question , Bensch et al . analysed data from 75 studies which encompassed 56 different species of plants , animals , and one type of bacteria .", "This showed that creatures living in family groups experienced more disease and had a higher risk of death .", "However , if groups had a low chance of encountering pathogens , individuals living with relatives were more likely to survive .", "This cancels out the disadvantages family groups face when pathogens are more common .", "The analysis by Bensch et al . provides new insights into how pathogens spread in species with different social systems .", "This information can be used to predict how diseases occur in nature which will benefit ecologists , epidemiologists , and conservation biologists ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology" ]
Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy
elife-50634-v2
[ [ "Nitric oxide ( NO ) is a critical primary signaling molecule in eukaryotic organisms ( Palmer et al . , 1987; Moncada et al . , 1991 ) .", "Cells detect this diatomic gas through the NO receptor soluble guanylate cyclase ( sGC ) , which is activated by NO to catalyze the cyclization of 5′-guanosine triphosphate ( GTP ) to 3′ , 5′-cyclic guanosine monophosphate ( cGMP ) ( Arnold et al . , 1977; Stone and Marletta , 1994; Russwurm and Koesling , 2004; Derbyshire and Marletta , 2012; Montfort et al . , 2017; Horst and Marletta , 2018 ) .", "The NO signaling pathway , with sGC as a central component , plays crucial roles in the cardiovascular and neurological systems in mammals ( Lucas et al . , 2000; Prast and Philippu , 2001; Benarroch , 2011 ) .", "Furthermore , aberrations in these signaling pathways can lead to pathologies that include various forms of hypertension , cardiovascular disease , and neurodegeneration ( Bredt , 1999; Friebe and Koesling , 2009; Lundberg et al . , 2015; Hervé et al . , 2014; Wallace et al . , 2016 ) .", "sGC is a heterodimer composed of two subunits , denoted α and β , that are each composed of four domains: an N-terminal heme nitric oxide/oxygen ( H-NOX ) domain , a Per/Arnt/Sim ( PAS ) -like domain , a coiled-coil ( CC ) domain , and a catalytic ( CAT ) domain ( Derbyshire and Marletta , 2012; Montfort et al . , 2017 ) .", "Although each subunit contains an H-NOX domain , only the β H-NOX domain binds a heme cofactor , with direct ligation occurring through a conserved histidine residue ( Zhao et al . , 1998 ) .", "The CC domains , together with the PAS domains , are thought to form a structured assembly upon dimerization of sGC ( Campbell et al . , 2014 ) .", "The CAT domains form a wreath-like structure with the active site at the dimer interface ( Derbyshire and Marletta , 2012; Hurley , 1998 ) .", "Biochemical aspects of sGC activation by NO have been studied in great detail .", "Without a ligand bound to the β H-NOX domain , sGC has a low basal activity .", "A stoichiometric equivalent of NO relative to the sGC heterodimer results in the cleavage of the proximal histidine-iron bond and the formation of a distal five-coordinate ferrous nitrosyl enzyme with 15% of maximal activity ( Russwurm and Koesling , 2004; Cary et al . , 2005; Fernhoff et al . , 2009; Herzik et al . , 2014 ) .", "This low-activity state of sGC will be referred to here as the 1-NO state; importantly , the KD of the ferrous nitrosyl heme is 1 . 2 × 10–12 M thus in this state NO remains bound to the heme ( Zhao et al . , 1999 ) .", "The activity of the 1-NO state can be increased to a maximally active state either by the addition of excess NO ( xsNO ) , or by addition of small-molecule stimulators ( Stasch et al . , 2011 ) .", "The benzylindazole compound YC-1 was the first reported small-molecule sGC stimulator ( Ko et al . , 1994 ) .", "It was identified in a screen of compounds that inhibit platelet aggregation , one of the physiological responses of sGC activation .", "The molecular mechanisms by which NO and small molecule stimulator binding leads to enzyme activation remain unclear , despite the fact that the sGC-targeted drug Adempas discovered through a small molecule screen based on the YC-1 scaffold was approved by the FDA in 2013 .", "Visualization of the molecular steps in sGC activation has been a longstanding challenge .", "Crystal structures of individual truncated domains from various homologues of sGC have been reported ( Pellicena et al . , 2004; Purohit et al . , 2013; Ma et al . , 2010; Winger et al . , 2008 ) .", "Additionally , structures of related heterodimeric and multidomain proteins have provided insight into the higher order connectivity of sGC .", "The heterodimeric catalytic domain from Homo sapiens was solved in an inactive conformation ( Allerston et al . , 2013; Seeger et al . , 2014 ) .", "Structures of membrane-bound adenylate cyclases have been solved that contain catalytic domains as well as portions of or the entire CC domains , helping to orient the C-terminal domains of sGC ( Vercellino et al . , 2017; Qi et al . , 2019 ) .", "However , the precise quaternary structural arrangement of domains in the N-terminal portion of sGC is not known .", "Consequently , the mechanism by which NO and small-molecule stimulators couple binding through the protein for activation are poorly understood .", "In the absence of a high-resolution full-length structure , previous work has used alternative methods in attempts to understand interdomain interactions and how small molecules activate sGC .", "Crosslinking experiments and negative stain electron microscopy support the hypothesis that sGC is a flexible dumbbell-shaped particle , in which the CC domains serve to connect the H-NOX and PAS domains on one end of the dumbbell to the CAT domain on the other ( Campbell et al . , 2014; Fritz et al . , 2013 ) .", "Hydrogen deuterium exchange mass spectrometry ( HDX-MS ) has implicated the linker region between the PAS domains and the CC domains as critical in the activation mechanism , as both regions change in H/D exchange upon NO binding to sGC ( Underbakke et al . , 2014 ) .", "Truncations of sGC have suggested that the β H-NOX domain directly inhibits the CAT domains ( Winger and Marletta , 2005 ) .", "Point mutagenesis was used to identify several residues thought to transmit the ligand occupancy of the gas-binding H-NOX domain to the catalytic domains , including β D102 and β D106 ( Baskaran et al . , 2011a; Underbakke et al . , 2013 ) .", "None of these methods , however , afford sufficient resolution to discern domain organization and interdomain communication for the full-length protein .", "Here , we report the first full-length structures of sGC with and without activating ligands using cryoelectron microscopy ( cryo-EM ) to 5 . 1 and 5 . 8 Å resolution , respectively .", "We find that sGC adopts two dramatically different conformations , revealing a large-scale global conformational change in response to NO and YC-1 .", "Density consistent with YC-1 is observed in the active state map directly between the β H-NOX and both CC domains .", "We also show the functional relationship of NO binding to sGC on overall organization of sGC in solution by small angle X-ray scattering ( SAXS ) .", "By visualizing the quaternary structural changes that activate this NO receptor , we can now answer the long-standing question as to how the ligand binding in the regulatory domains of sGC is communicated to the catalytic domains ." ], [ "We selected Manduca sexta ( Ms ) sGC as a biochemically tractable protein which has 36% and 59% sequence identity when compared to the human homolog for the α and β monomers , respectively .", "A schematic of the domain organization is shown in Figure 1a .", "Additionally , the regulatory properties in response to NO and YC-1 mirror those of its human counterpart and YC-1 had been shown to increase the Ms sGC 1-NO state to maximal activity ( Hu et al . , 2008 ) .", "While full-length Ms sGC has only been previously reported to be expressed in E . coli , we used a baculoviral expression protocol to produce reliable quantities of stable , full-length protein ( Figure 1—figure supplements 1 and", "2 ) ( Hu et al . , 2008 ) .", "UV–visible absorption spectra of Ms sGC without ligands ( unliganded , U ) , with xsNO , and with carbon monoxide ( CO ) show the expected ligand-dependent shifts of the Soret and Q bands , indicating the β H-NOX domain is competent to bind diatomic gases ( Figure 1b ) .", "We confirmed that Ms sGC displays catalytic activity similar to other well-characterized mammalian homologues , including Homo sapiens sGC .", "Ms sGC exhibits a low , basal activity in the unliganded state ( 71 ± 36 nmol/min/mg ) and partial activation in the 1-NO state ( 309 ± 77 nmol/min/mg ) ( Figure 1c , Figure 1—figure supplement 3 ) , consistent with previous reports of mammalian sGCs ( Fernhoff et al . , 2009 ) .", "Maximal activity could be achieved by adding excess NO to the 1-NO sample ( 1988 ± 131 nmol/min/mg ) , or by the addition the sGC stimulator YC-1 to the 1-NO state ( 1522 ± 38 nmol/min/mg ) ( Figure 1c , Figure 1—figure supplement 4 ) .", "Taken together , Ms sGC displays biochemical properties comparable to well-characterized mammalian sGCs .", "Cryo-EM was used to solve the structure of full-length inactive Ms sGC .", "Two-dimensional classification after removing poor particles showed intact density with two lobes with connective density between them similar to previously reported EM envelopes obtained by negative stain ( Figure 2—figure supplements 1–3 ) ( Campbell et al . , 2014 ) .", "Three-dimensional classification and refinement yielded a single reconstruction with a 5 . 1 Å nominal resolution ( Figure 2—figure supplement", "4 ) that exhibited a clear dimer interface and distinct CCs ( Figure 2a , Supplementary file 1 - Table 1 ) .", "While particles display a slight orientation preference , local resolution is uniform throughout the density map ( Figure 2—figure supplements 5 and 6 ) .", "Additionally , the overall map exhibits well-defined helices and continuous density .", "The two lobes of the structure are termed the ‘regulatory’ lobe and the ‘catalytic’ lobe , with the CC domains acting as a bridge between them ( Figure 2a ) .", "The long axis of the regulatory lobe is positioned perpendicular to the catalytic lobe and is predicted to contain the H-NOX/PAS bundle , while the CAT domains are predicted to form the catalytic lobe ( Campbell et al . , 2014 ) .", "Several features enabled conclusive assignment of the α and β subunit domains in the density map , despite the intermediate resolution and pseudosymmetry between the two subunits ( Figure 2b , Video 1 ) .", "A rigid body global search of potential H-NOX positions revealed two possible positions for H-NOX domains within the regulatory lobe ( Pettersen et al . , 2004 ) .", "The αF helix of the β H-NOX domain ( Figure 1—figure supplement", "5 ) binds a heme cofactor while the α H-NOX does not .", "Only the position closest to the catalytic lobe contains density for the heme cofactor ( Figure 2c ) , hence this was assigned to the β subunit .", "The two CAT domains could be differentiated because the α CAT domain contains a C-terminal extension ( residues 661–699 ) which is absent in the β CAT domain .", "Only one of the domains in the catalytic lobe contains extra C-terminal density and thus the CAT domains could be assigned unambiguously ( Figure 2—figure supplement 7 ) .", "Connective density from the CAT domains enabled clear differentiation of the two CC and PAS domains ( Figure 2d , green ) .", "As a result , the α PAS is assigned as the top domain , while β is the bottom domain ( Figure 2b ) .", "Homology models for Ms sGC domains were combined representing residues α 51–250 , 279–699 and β1–183 , 205–597 to create a full-length model ( described in Materials and methods ) .", "The CC domains are in a parallel orientation , and both domains have a clear bend , distinct from a previously determined CC X-ray structure ( Ma et al . , 2010 ) .", "A linker extends from the C-terminus of the PAS domains and forms a rigid loop connecting the bent helix to the H-NOX/PAS domains ( Figure 2d , light and dark green ) .", "The CC density bends sharply at residues α A422 and β L343 .", "This bent region forms a buried helix , and along with the C-terminal PAS linker , creates an interaction nexus between the H-NOX , PAS , and CC domains ( Figure 2d , highlighted in light and dark purple ) .", "The bent N-terminal portion of the CC domains ( α 403–415 and β 333–345 ) , is highly conserved across sGC homologues ( Figure 2—figure supplement 8 ) .", "The CAT dimer displays a wreath-like fold with the monomers related by a twofold axis , typical for class III nucleotide cyclase domains ( Hurley , 1998; Zhang et al . , 1997 ) .", "The CAT domains align well with a previous structure of an inactive guanylate cyclase ( Cα RMSD of 1 . 3 Å to PDB ID: 4NI2 ) , displaying an inaccessible nucleotide-binding pocket that would require a significant rearrangement for activation ( Figure 2—figure supplement 9 ) .", "In the absence of a substrate-bound structure of a guanylate cyclase , we compared our model to a substrate-bound adenylate cyclase .", "The alignment of the α chain of an active adenylate cyclase structure ( PDB ID: 1CJK , gray ) with the α CAT domain of our inactive Ms sGC structure reveals that the Cα of β N538 from the β CAT domain is within 2 Å of the bound nucleotide analog in the adenylate cyclase structure ( Figure 2e ) .", "This state is thus in a closed conformation that is sterically incompatible with nucleotide binding , explaining the lack of guanylate cyclase activity .", "To elucidate conformational changes associated with sGC activation , a structure of sGC bound to NO and the small molecule stimulator YC-1 was determined .", "We elected to supplement the xsNO Ms sGC with YC-1 to generate the most stable activated conformation .", "Particles of active Ms sGC were well-dispersed in vitreous ice ( Figure 3—figure supplements 1 and 2 ) .", "Cleaned two-dimensional class averages exhibit a two-lobed density that is distinct from the inactive structure ( Figure 3—figure supplement 3 ) .", "The final 3D reconstruction of the active state at 5 . 8 Å resolution displays clear density for the CCs , β H-NOX , and α helices throughout the structure ( Figure 3a , Video 2 , Figure 3—figure supplements 4–6 ) .", "Only diffuse density was present for the α H-NOX , likely due to increased flexibility of this domain and it was thus omitted in the model ( Figure 3b ) .", "The orientation of the domains in the regulatory lobe is maintained from the inactive state to the active state , and the β H-NOX position is readily identified via the bound heme cofactor density ( Figure 3c ) .", "A triangular-shaped lobe of unassigned density is present in the active state map but not in the inactive state map ( Figure 3c , arrow ) .", "This density is located directly between the β H-NOX domain and the α and β CC domains , adjacent to either the A or B pyrrole of the heme ( opposite of the propionate chains ) .", "The volume likely corresponds to YC-1 ( 304 . 3 Da ) and with the current resolution two orientations of this small molecule are possible ( Figure 3c ) .", "Residues within 5 Å of the density include α CC 422–427 ( AQDGLR ) , and β V39 , F77 , C78 , Y112 , and Q349 .", "Although the density for the β H-NOX and PAS domains is similar between the inactive and active states , the overall shape of the molecule is more linear ( Figure 3b ) .", "Continuous , linear helices from α L406–L457 and β A333–Y386 are seen in the active state ( Figure 3d , highlighted in purple ) .", "This conformation is more aligned with predicted CC length and is more similar to the previously published crystal structures in terms of the range of residues seen in an unbent coiled-coil ( Ma et al . , 2010; Qi et al . , 2019 ) .", "The CAT heterodimer in the active state still forms a wreath-like geometry , but with the two monomers moved apart from one another roughly perpendicular to the CC domain axis ( Figure 3e ) .", "An alignment of the α chain of an active adenylate cyclase structure ( PDB ID: 1CJK , gray ) with the α CAT domain of our active Ms sGC structure shows the Cα of β N538 is now greater than 4 Å from the bound nucleotide analog , providing sufficient space for the nucleotide to bind in the active site .", "The Ms sGC CAT domain thus adopts an open conformation with the apparent capacity to bind substrate .", "Binding of NO to β H-NOX initiates the signal transduction event , thus interfaces were examined between the inactive and active states .", "A significant rearrangement of the quaternary structure of Ms sGC occurs upon activation with the regulatory domain rotating 71° and the catalytic domain rotating 40° ( Figure 4a , Video 3 ) .", "In both inactive and active states , the β H-NOX domain is the closest domain of the regulatory lobe to the CAT domains ( Figure 4a ) ; however , in neither model does the β H-NOX domain come into direct contact with the CAT domains .", "In the inactive state , the Cα of β I47 is 12 Å away from the Cα of β H399 ( Figure 4a , gray ) , too far for direct contact .", "The closest Cα-Cα distance of residues between the β H-NOX domain and the CAT domains in the active state is between β M33 and α W468 at 21 Å ( Figure 4a , black ) , remaining too far for direct contact between the β H-NOX and the CAT dimer .", "Therefore , allosteric communication is transmitted through changes in CC positioning , not through direct H-NOX:CAT interactions .", "Interaction surfaces , defined by a ≤ 10 Å Cα to Cα distance ( Donald et al . , 2011 ) , occur between the β H-NOX and the PAS or CC region in both inactive and active states ( Figure 4b , c ) .", "In the inactive state , the αE , αF , and β2 secondary structural elements of the β H-NOX ( 82–126 , green ) form an extensive interface with the β PAS ( 270–275 , red ) and the α PAS-CC linker ( 400–419 , blue ) regions ( Figure 4b , Figure 1—figure supplement 5 ) .", "Overall , these interfaces are maintained in the active state ( Figure 4c ) , in spite of the straightening by the CC domains .", "Activation of sGC induces the formation of an interface between β CC ( 355-367 ) and β H-NOX ( 33-41 ) ( Figure 4c , purple ) .", "These residues on the β H-NOX comprise a loop located between helix αB and αC in the β H-NOX domain which was extended away from the CC in the inactive state .", "The large conformational change of the H-NOX/PAS bundle is necessary for the formation of the new interface in the active site , and thus this interaction could be critical for stabilizing the active sGC conformation .", "In the active structure , the CCs undergo a significant conformational change and rotation relative to the CAT dimer ( Figure 4d ) .", "The active state CC domains are completely extended ( 56 Å and 63 Å for αCC and βCC respectively in the inactive state , to 74 Å for both CC in the active state ) , as the bend present in the inactive state moves away from the CAT domains to be in line with the C-terminal portion of both CC domains .", "The CC conformational change upon activation twists the CC interface such that the β CC helix rotates relative to the α CC by 72 degrees ( Figure 4d ) .", "The dramatic rearrangements of the H-NOX/PAS bundle leads to unbending of the CC domains , a change in their orientations as they project from the regulatory lobe , and finally to opening of the nucleotide-binding pocket ( Figure 4e ) .", "Alignment to the α CAT domain shows a clear rotation of the β CAT domain away from α CAT domain by 40° ( Figure 4e , left ) .", "This rotation entails the pinching together of the base of the CC domains ( Figure 4e , middle ) .", "In combination , these motions open the GTP-binding pocket by moving the β CAT active site helix ( 535-545 ) away from the α CAT domain and into an active conformation .", "Small angle X-ray scattering ( SAXS ) was used to interrogate conformational changes with and without NO in solution .", "Experiemnts with sGC stimulators were not included as the limited solubility of YC-1 is precludes the collection of scattering data .", "Size exclusion chromatography ( SEC ) -SAXS and SEC-multi angle light scattering ( MALS ) chromatograms show a symmetrical peak for both samples with little variation in radius of gyration ( Rg ) and estimated MW across the peaks , indicating sample homogeneity ( Figure 5—figure supplement 1 ) .", "The UV-visible absorption of the Soret band of the heme and of the protein itself were used to evaluate the ligation state of Ms sGC in the SAXS experiment .", "The ratios of Soret band to protein peak in the full UV-visible absorption spectra are 0 . 85 , 0 . 35 , and 0 . 20 in the unliganded , 1-NO , and xsNO states , respectively ( Figure 1b , Figure 5—figure supplement 1 ) .", "The ratios of these two peaks in the SEC-SAXS experiments were 0 . 77 with NO and 0 . 32 without NO ( Figure 5—figure supplement 1 ) .", "The 432/280 ratio in the SAXS sample without NO is less than in the full UV-Visible spectra , indicating that the sample had a slightly lower heme incorporation .", "The ratio for the SAXS sample with NO was lower than that expected for 1-NO , but much larger than predicted for the xsNO ratio , indicating that the SAXS samples were in the unliganded and 1-NO state during elution , respectively .", "The Rg for the unliganded and 1-NO state were 43 . 1 ± 0 . 4 and 43 . 8 ± 0 . 2 Å , respectively , representing an elongation of the structure ( Figure 5—figure supplement 1 , Supplementary file 2 - Table S2 ) .", "Additionally , the Pair-distance function ( P ( r ) ) , which shows a composite of the inter-atomic distances , displays a distinct shift between the unliganded and 1-NO states in the region from ~70 to~100 Å .", "This corresponds to an extension of the regulatory lobe from catalytic lobe ( Figure 5a ) .", "Shifts in the secondary peaks of the Kratky plots ( Figure 5—figure supplement", "2 ) further suggest separation of the regulatory and catalytic lobes upon activation .", "Using the inactive model obtained from cryo-EM as an initial Cα model , a rigid-body modeling pipeline was developed to systematically explore conformational space ( see Online Methods ) ( Pelikan et al . , 2009 ) .", "A minimal ensemble search was performed over thousands of sGC conformations and corresponding scattering curves were calculated and compared to the experimental data .", "The result of this minimal ensemble search confirmed the presence of a single sGC conformation while in an inactive state ( Figure 5b , Figure 5—figure supplement 3 ) , which overlays with the inactive structure with an RMSD of 2 . 0 Å .", "The 1-NO state was best modeled as an ensemble of two states , with the majority ( 72% ) of the sample consistent with the inactive model ( Figure 5b ) .", "However , the remainder of the sample ( 28% ) is consistent with a more elongated conformation , one that is in between the inactive and active state obtained by cryo-EM .", "The Cα RMSD of the partially active SAXS model and the active state cryo-EM model is 13 . 5 Å .", "This analysis shows that sGC adopts a mixture of inactive and active conformations in the 1-NO state , suggesting that NO binding at the heme establishes an equilibrium between these two conformational extremes ( Figure 5—figure supplement 3 ) ." ], [ "The structures of the inactive and active states of full-length sGC provide detailed insight into the molecular mechanism of activation of Ms sGC .", "The most unexpected feature of the inactive state of the enzyme are the bends in the CC domains .", "Straightening of the bent CCs is the clearest structural link between the regulatory and catalytic lobes during activation .", "Previous HDX-MS data showed a differential exchange pattern for the CC domains , where the N-terminal portion of the α CC and the C-terminal portion of the β CC increase in H/D exchange upon NO binding , while the C-terminal portion of the α CC and the N-terminal portion of the β CC decrease in H/D exchange ( Figure 3—figure supplement 7 ) ( Underbakke et al . , 2014 ) .", "The changes seen in HDX in the CCs near the bend are thus consistent with the large-scale rearrangements of the CCs that we have now observed in the cryo-EM .", "Furthermore , the bent portion of the CC domain is highly conserved among sGC homologues , consistent with its centrality to allosteric communication .", "The C-terminal portions of the CC domains undergo a 72° twist and contain a motif known as the signaling helix ( or S-helix ) ( Anantharaman et al . , 2006 ) .", "Similar to sGC , proteins with this motif contain both receptor and output domains that are connected by dimeric coiled-coils .", "The inactive and active sGC structures are the first full-length enzyme with a S-helix motif to be characterized .", "This motif aligns with C-terminal part of the CC domain , part of the 72° twist described above ( Figure 2—figure supplement 10 ) .", "It is possible that the twisting motion of these domains is a more general mechanism of activation for proteins with S-helices .", "In transitioning between the inactive and active states , the H-NOX/PAS bundle rotates 71° in order for β H-NOX residues 33–41 to form an interface with residues β 355–367 of the β CC domain ( Figure 4c , purple ) .", "The αH helix of the β H-NOX domain contains the proximal histidine that binds to the heme cofactor ( Zhao et al . , 1998 ) .", "Crystal structures of bacterial H-NOX domains with and without NO show a rotation in the αF helix of the β H-NOX .", "However , in our full-length structures , the αF of the β H-NOX retains its interface with the β PAS and the α CC domain in both states ( Figure 4b and c ) .", "More strikingly , binding of YC-1 at a site bridging the β H-NOX and CC domain highlights how stabilization of this contact drives CC unbending ( Figure 3c ) .", "This interface corroborates specific point mutations known to decrease activity of the full-length protein; specifically , single point mutants of β I41E in the β H-NOX domain retain basal activity but are only minimally activate with excess NO , implying that these sGC variants are catalytically competent but not as sensitive to stimulation as the wildtype enzyme ( Underbakke et al . , 2013; Baskaran et al . , 2011b ) .", "The structural finding that residue Ile41 of the β H-NOX form contacts with the unbent β CC that are specific for the active conformation explains these phenotypes .", "Destabilizing these contacts by mutating Ile41 thus blocks this interface and prevents sGC from reaching the active conformation .", "While this paper was under review , a cryo-EM map of activated H . sapiens sGC was reported , where the activation was achieved by using xsNO ( Kang et al . , 2019 ) .", "This xsNO map and the xsNO + YC-1 map reported here overlay well , except for the proposed density for YC-1 .", "This lends support to our assignment of the YC-1 binding site at the β H-NOX- β CC interface .", "These insights suggest that the β H-NOX- β CC contact may be the most critical allosteric switch in the regulatory lobe of sGC .", "While conformational changes are readily observed by comparing the two cryo-EM structures , the SAXS implies that the 1-NO state can sample similar conformations ( Figure 5a ) .", "Although only a fraction of the population exhibits an extension , inspection of the Soret to protein UV-visible absorption indicates that the protein is in the 1-NO state .", "These data support the hypothesis that when the first NO binds to the heme of the β H-NOX , sGC adopts an equilibrium between the inactive and a partially extended conformation , with a Keq = [active]/[inactive] = [0 . 72]/[0 . 28]=0 . 39 .", "This is corroborated by the activity data from the 1-NO state , which exhibits 15% of the maximal activity .", "The conformational heterogeneity observed in SAXS analysis could represent the physiological state of sGC at basal cellular conditions .", "In the absence of increased NO concentrations , sGC stimulators can maximally activate cGMP production and are now used to treat forms of pulmonary hypertension ( Koglin et al . , 2002; Follmann et al . , 2013 ) .", "YC-1 was present in the sample used to generate the active state reconstruction of Ms sGC , and extra density is seen near the new β H-NOX: β CC interface ( Figure 3c ) .", "Distinct changes in both resonance Raman and electron paramagnetic resonance spectroscopy signatures for heme ligands have been detected with both YC-1 and BAY 41–2272 supporting this proposed binding site ( Derbyshire , 2008 ) .", "Previously , a stimulator binding site was proposed between helices αA and αD in the β H-NOX domain based on cross linking and NMR data ( Wales et al . , 2018 ) .", "However , there is no density for YC-1 present in the active state reconstruction between αA and αD helices .", "We note that our study used YC-1 compared to IWP-854 , IWP-051 and BAY 41–2272 previously used , which could explain the difference in binding sites .", "Visualization of YC-1 bound to sGC is an important development as a proof of concept that small molecule sGC stimulators can now be characterized structurally .", "Adenylyl cyclases ( AC ) catalyze a similar reaction mechanism as sGC .", "Mammalian membrane-associated ACs contain two catalytic domains which form the intrapolypeptide equivalent of a heterodimer .", "The catalytic domains in active mammalian AC heterodimer rotate by 7° with respect to an inactive homodimeric counterpart bound to two molecules of forskolin ( Zhang et al . , 1997; Tesmer et al . , 1997; Hurley , 1999 ) .", "We observe a rotation of the sGC CAT domains about the same axis , although the magnitude is larger for sGC , at 40° .", "A full-length membrane bound AC ( AC-9 ) was recently solved in the active nucleotide-bound state ( Qi et al . , 2019 ) .", "The CC domains between the full-length active sGC and AC9 structures overlay well , consistent with a common active geometry for GCs and ACs .", "The AC9 has a very different angle between the CC and CAT domains .", "The rotation of the sGC regulatory lobe is sterically incompatible with the presence of a membrane , thus by their different natures as soluble and membrane-associated enzymes , sGC and ACs must differ in the detailed modes of allosteric communication .", "To date , a detailed understanding of sGC activation has been hampered by the lack of full-length structures .", "The inactive and active structures in tandem with established studies lead to formation of new hypotheses for the activation of sGC .", "First , activity assays with sGC truncations suggested that direct interaction of the β H-NOX domain and the CAT dimer was responsible for sGC inhibition ( Winger and Marletta , 2005 ) .", "However , the structural data shows no direct interaction between the β H-NOX and the CAT domain in either conformation .", "Instead , the formation of the new β H-NOX/β CC interface in the active state stimulates the active CAT conformation .", "Activation leads to a global conformational rearrangement of the heterodimer , elongating the structure; however , only a single equivalent of NO is required to cleave the bond between the heme Fe center and the ligating histidine residue .", "Maximal activation of sGC involves either the binding of a second NO molecule to a non-heme site on sGC or a small molecule stimulator stabilizing the active state ( Guo et al . , 2017; Horst and Marletta , 2018 ) .", "Figure 6 depicts the proposed physiological activation sequence of sGC , where unliganded sGC adopts a more compact conformation with bent CC domains .", "Upon NO binding to the heme , an equilibrium of conformational states is established , with the partially elongated state affording about 15% of the maximal activity .", "Given the very tight association between NO and the heme , this represents the basal cellular activity .", "Finally , in the presence of either an increase in NO concentration or sGC stimulating molecules , the completely extended , fully active state is reached and sGC reaches maximal catalytic activity .", "Using these structures as a starting point , new avenues of exploration can now be undertaken , to elucidate the molecular mechanisms of excess NO activation .", "Critical residues for interdomain interactions along with the proposed stimulator binding site can be characterized in more detail .", "Having resolved the two structural states of an important therapeutic target , the structures of sGC will influence rational design of improved drugs for diseases associated with NO signaling impairment ." ], [ "All chemicals were purchased from commercial vendors and used without further purification , unless otherwise noted .", "Primers and gBlocks were obtained from Integrated DNA Technologies ( Coralville , IA ) .", "Gibson Master Mix was purchased from New England Biolabs ( Ipswich , MA ) .", "PrimeSTAR Max DNA polymerase and TALON superflow resin was purchased from Takara Bio USA ( Mountain View , CA ) .", "DNA purification kits were purchased from Qiagen ( Germantown , MD ) .", "ExCell-420 media , sodium dithionite ( Na2S2O4 ) , DNAse I from bovine pancreas , β-mercaptoethanol , sodium phosphate , dibasic ( Na2HPO4 ) , sodium carbonate ( Na2CO3 ) , potassium phosphate ( K2HPO4 ) , guanosine 5′-triphosphate sodium salt ( GTP ) ( >95% , HPLC ) , and zinc acetate ( Zn ( CH3CO2 ) 2 ) were purchased from Millipore Sigma ( Burlington , MA ) .", "4- ( 2-aminoethyl ) benzenesulfonyl fluoride hydrochloride ( AEBSF ) was purchased from Research Products International ( Mount Prospect , IL ) .", "Isopropyl β-D-1-thiogalactopyranoside ( IPTG ) , 4- ( 2-hydroxyethyl ) piperazine-1-ethanesulfonic acid ( HEPES ) , and the Pierce BCA Protein Assay Kit were purchased from Thermo Fisher Scientific ( Waltham , MA ) .", "Benzamidine , sodium chloride ( NaCl ) , magnesium chloride ( MgCl2 ) , and glycerol were purchased from VWR Life Science ( Visalia , CA ) .", "Dithiothreitol ( DTT ) was purchased from Bachem ( Bubendorf , Switzerland ) .", "Imidazole was purchased from Oakwood Chemical ( West Columbia , SC ) .", "Vivaspin spin concentrators were purchased from Sartorius ( Concord , CA ) .", "BioSpin six desalting columns were purchased from BioRad ( Hercules , CA ) .", "Diethylamine NONOate ( DEA NONOate ) , PROLI NONOate , diethylamintriamine NONOate ( DETA NONOate ) , and YC-1 were purchased from Cayman Chemical Company ( Ann Arbor , MI ) .", "Carbon monoxide ( CO , >99% ) gas was purchased from Praxair Inc ( Danbury , CT ) .", "Manduca sexta sGC α1 ( Uniprot: O77105 ) and β1 ( Uniprot: O77106 ) genes were purchased as gBlocks from IDT with a C-terminal 6x His tag on the α1 gene with Golden Gate cloning sites .", "The gBlocks were subcloned into a Golden Gate entry vector ( gift of the Tullman-Ercek lab , Northwestern University ) .", "PCR was used to add regions of homology to pFastBac ( Bac-to-Bac Baculovirus Expresion System , Thermo Fisher Scientific ) , and Gibson Assembly was performed to generate pFastBac_Ms_sGC_α1_His6 and pFastBac_Ms_sGC_β1 .", "The genes were transposed into a baculovirus bacmid using DH10Bac-GFP cells .", "The bacmid was isolated , validated , and then transfected into SF9 cells ( Berkeley Cell Culture Facility ) to generate recombinant baculovirus for both genes .", "SF9 cells were maintained in monolayer and in suspension in ExCell-420 media at 27°C ( cells were shaken at 135 rpm ) .", "The recombinant baculovirus was amplified until the titer was greater than 1 × 108 cfu/mL .", "Five liters of SF9 cells were coinfected with 50 mL of amplified virus per liter and allowed to express for 72 hr .", "Cells were spun down at 4300 g for 20 min , snap frozen in liquid nitrogen , and stored at –80°C .", "The following steps were performed at 4°C .", "Cell pellets were thawed in ice water and resuspended in lysis buffer: Buffer A ( 50 mM Na2HPO4 , pH 8 . 0 , 200 mM NaCl , 1 mM imidazole , 1 mM benzamidine , 5% ( v/v ) glycerol , 0 . 22 μm filtered ) supplemented with 10 mM benzamidine , 1 mM AEBSF , 0 . 5 mg/mL bovine DNAse I , and 5 mM β-mercaptoethanol .", "Cells were lysed in a bead beater ( BioSpec ) with 0 . 5 mm glass beads .", "The resulting cell lysate was clarified by spinning at 4300 g for 5 min , followed by spinning at 158 , 000 g for 2 hr ( Ti45 rotor , Beckman Coulter ) .", "The lysate was passed through a column containing 2 mL TALON superflow at 0 . 5 mL/min , and the flow-through was collected .", "The column was washed with 15 column volumes of Buffer A supplemented with 5 mM β-mercaptoethanol at 0 . 5 mL/min .", "The protein was eluted with 10 CV of Buffer B ( 50 mM Na2HPO4 , pH 8 . 0 , 200 mM NaCl , 150 mM imidazole , 1 mM benzamidine , 5% ( v/v ) glycerol , 0 . 22 μm filtered ) supplemented with 5 mM β- mercaptoethanol .", "Fractions with yellow color were concentrated to <2 mL using a 30 , 000 molecular weight cutoff spin concentrator , supplemented with 5 mM DTT and 5 mM EDTA , and stored overnight .", "Next , the sample was diluted to 9 mL with Buffer C ( 25 mM triethanolamine , 25 mM NaCl , 5 mM DTT , 5% ( v/v ) glycerol , 0 . 22 µm filtered ) , and applied to a POROS HQ2 anion exchange column ( Thermo Fisher Scientific ) .", "The column was washed with 3 CV of Buffer C , and then a gradient to 50% Buffer D ( 25 mM triethanolamine , 750 mM NaCl , 5 mM DTT , 5% ( v/v ) glycerol , 0 . 22 µm filtered ) was established over 17 CV at 0 . 5 mL/min .", "Fractions with purified sGC were concentrated to 5–50 µM and stored in liquid nitrogen .", "A typical yield for this expression and purification procedure is 100 µg sGC per liter of insect cells .", "Purified proteins were buffer-exchanged into 25 mM HEPES , pH 7 . 5 , 25 mM NaCl using three rounds of dilution and concentrations in a Vivaspin 500 ( 30 , 000 MWCO ) spin concentrator .", "Samples were filtered with a 0 . 22 μM spin filter ( Millipore ) .", "The final sample concentration was approximately 5 µM .", "Samples were separated with an Agilent 1200 series high-pressure liquid chromatography ( HPLC ) system over a ProSwift column ( ThermoFisher Scientific ) , and subsequently analyzed by an Agilent 6224 time-of-flight ( TOF ) mass spectrometer with a Turbospray ion source in positive ion mode .", "Samples were reduced in an anaerobic glove bag ( Coy ) with 5 mM Na2S2O4 for 15 min , and then desalted with a BioSpin6 column equilibrated with Buffer E ( 50 mM HEPES , pH 7 . 5 , 150 mM NaCl , 5% ( v/v ) glycerol , 0 . 22 µm filtered ) .", "CO-saturated buffer ( 950 μM CO ) was prepared by sparging 3 mL of anaerobic Buffer E for 15 min in a Reacti-Vial ( Thermo Fisher Scientific ) .", "CO was added to the sample to achieve a final concentration of 425 μM .", "Nitric oxide was added to a concentration of 500 μM by addition of DEA NONOate , based on 1 . 5 moles of NO released per mole of NONOate .", "Protein-ligand complexes were incubated for 15 min at room temperature to establish equilibrium , and no further spectral changes were observed after this time .", "Samples were placed in a septum-sealed 1 cm pathlength quartz cuvette inside the glove bag , and UV–Vis spectra were recorded on a Cary 300 spectrophotometer ( Agilent Technologies ) .", "Steady-state kinetics for Ms sGC were measured by quantifying the amount of cGMP produced in duplicate endpoint activity assays , performed in at least biological triplicate .", "Samples were reduced in an anaerobic glove bag with 5 mM Na2S2O4 for 15 min , and then desalted with a BioSpin6 column equilibrated with Buffer E . Ms sGC with 1-NO and xsNO were prepared by first adding PROLI NONOate to 50 µM , based on 2 moles of NO released per mole of PROLI NONOate .", "This sample was then buffer exchanged into Buffer E through a BioSpin6 column to generate the 1-NO state .", "To generate the xsNO state , PROLI NONOate was added back to a portion of the 1-NO sample and allowed to equilibrate for 5 min .", "YC-1 was added from a 100x stock solution in DMSO to a final concentration of 150 µM ( final DMSO concentration 1% ) .", "The protein concentration was determined using the reduced heme Soret peak at 433 nm ( 149 , 000 M–1 cm–1 ) ( Hu et al . , 2008 ) .", "Protein concentration was adjusted after desalting the excess NO by comparing the A280 peaks to the unliganded protein .", "Activity assays were conducted at 25°C and pH 7 . 5 in Buffer D , supplemented with 5 mM DTT and 5 mM MgCl2 .", "Reactions were initiated with 2 mM GTP and timepoints from were quenched with 125 mM Zn ( CH3CO2 ) 2 , followed by 125 mM Na2 ( CO3 ) to adjust the pH to 10 . 5 .", "Samples were frozen at –80°C until quantification .", "Quenched assays were thawed , and the zinc precipitate was spun down for 10 min at 23 , 150 g .", "The reactions were diluted by one to three orders of magnitude , and the cGMP was quantified in duplicate using an extracellular cGMP Enzyme Linked Immunosorbent Assay , following the manufacturer’s instructions ( Enzo Life Sciences ) .", "Concentrations of cGMP were determined from a standard curve , generated over 0 . 16–500 pmol/mL .", "Initial rates were calculated from the linear phase of the time course , where 5–10% of the GTP substrate was consumed .", "The experiment was repeated at least three times to ensure reproducibility .", "Samples were reduced in an anaerobic glove bag with 5 mM Na2S2O4 for 15 min , and then desalted with a BioSpin6 column equilibrated with Buffer F ( 25 mM triethanolamine , pH 7 . 5 , 25 mM NaCl , 5 mM DTT , 0 . 22 µM filtered ) .", "The inactive protein sample was diluted after thawing from a single frozen stock to ~2–4 µM in 1–3% trehalose .", "The sample ( 3 . 5 µL ) was applied to glow discharged UltrAUfoil 2/2 200 mesh gold grids ( Quantifoil ) and plunge-frozen using a vitrobot Mark IV ( Thermo Fischer ) .", "Blotting was performed under 100% humidity with zero blot force for 3–6 s .", "sGC was dispersed in vitreous ice which displayed clear contrast transfer function information ( Figure 2—figure supplement 1a .", "Active sample was prepared in a similar manner , but 500 µM NO ( from DEA NONOate ) and 150 µM YC-1 were added ( Figure 3—figure supplement 1a-b ) .", "Grids were screened and imaged on a Talos Arctica ( Thermal Fischer ) operated at 200 kV .", "Complete imaging conditions are described in Supplementary file 1 .", "Micrographs were collected at 36 , 000X nominal magnification on a K3 direct electron detector ( Gatan ) in super-resolution counted mode at 0 . 5685 å/pix .", "Serial EM was used for automated image shift data collection of 2841 and 9330 movies for the inactive and active sample , respectively .", "Movies were taken in 100 ms frames , totaling an electron dose of 60 electrons per movie .", "Inactive sGC movies were drift-corrected , gain-corrected , and binned to 1 . 137 Å/pix in 7 × 5 patches using MotionCor2 ( Zheng et al . , 2017 ) .", "Micrographs were CTF-corrected using CTFFIND4 and single particles were manually picked for initial 2D classification within Relion 3 . 0 ( Rohou and Grigorieff , 2015; Scheres , 2012; Nakane et al . , 2018 ) .", "Class averages representing the full-length complex ( Figure 2—figure supplement 1c ) were used for template picking with Relion autopicker , resulting in 675 , 956 particles .", "Particles were imported into Cryosparc2 and pruned using 2D classification and 3D ab initio classification ( Punjani et al . , 2017 ) .", "Initial 2D classification yielded a large number of falsely picked background particles and particles which represented broken sGC dimers .", "We suspect the background particles are due to the size of the complex , while the broken particles likely stem from damage at the air-water interface ( Figure 2—figure supplement 1c ) .", "These poor class averages were removed from further processing steps .", "In total , 59 , 165 final particles were refined using non-uniform refinement with default parameters ( Figure 2—figure supplement 1b ) .", "Active sGC movies were binned to 2 . 274 Å/pix before template-free Laplacian picking for initial particle selection ( Nakane et al . , 2018 ) .", "Micrographs were manually cleaned by visual inspection and FFT quality .", "Initial single particles were pruned using iterative 2D and 3D techniques ( Figure 3—figure supplement 1b , c ) , as described above for the inactive state .", "Similarly , many of the initial picked particles were background or broken in the Active dataset ( Figure 3—figure supplement 1c ) Blurred density at low thresholds was seen in a predicted region for the alpha-HNOX but did not resolve during processing and was masked away during the final reconstruction .", "Of note , both Inactive and Active datasets underwent exhaustive processing schemes , including Bayesian polishing , multibody refinement and focused classification , none of which improved the resolution or showed evidence of multiple conformations in a single dataset .", "Homology models for each domain were built using Phyre2 and correspond to the following PDB entries: α H-NOX ( 2O0C ) , α PAS ( 4GJ4 ) , α CC ( 3HLS ) , α CAT ( 3UVJ ) , β H-NOX ( 2O0C ) , β PAS ( 4GJ4 ) , β CC ( 3HLS ) , β CAT ( 2WZ1 ) ( Kelley et al . , 2015 ) .", "Domains ( with side chains removed ) were rigid-body docked into the inactive reconstruction using the fit_in_map function of Chimera ( Pettersen et al . , 2004 ) .", "Clear density for heme in the β H-NOX domain enabled clear distinction of the two H-NOX domains .", "Linkers between the H-NOX and PAS domains are missing in the density , likely due to flexibility , and were left out of the model ( α 238–278 and β184–206 ) .", "Initial placement of the CAT dimer was based on a significant extension in the sequence of the α CAT C-terminus , which is visible as unmodeled density .", "Continuous density from the CAT dimer enabled assignment of the CC and PAS domains .", "The linker region between the PAS domains and the CC were manually modeled in COOT based on secondary structure predictions from PSIPRED ( Emsley et al . , 2010; Vynne , 1997 ) .", "Refinement using iterative rounds of phenix . real_space_refine and inspection in COOT led to the final carbon back bone trace of the inactive state .", "Active state modeling was based on rigid-body fitting of domains based on the inactive state .", "Helical density was apparent throughout the structure ( with the exception of the α H-NOX , as mentioned above ) .", "Linker regions between domains were corrected with COOT and phenix . real_space_refine .", "Final model idealization was carried out using phenix . model_idealization and validated using Molprobity ( Chen et al . , 2010 ) .", "Samples were reduced in an anaerobic glove bag with 5 mM Na2S2O4 for 15 min , concentrated to 60 µL at 50 µM ( ~7 . 5 mg/mL ) , and then three rounds of dialysis were performed in Buffer G ( 50 mM KH2PO4 , pH 7 . 4 , 150 mM NaCl , 2% glycerol ) .", "The activated sample was prepared with 500 µM NO ( from DEA NONOate ) .", "The samples were sealed and run within 3 hr of being prepared; samples did not undergo freeze–thaw after the sample was prepared ( the protein was freeze–thawed once after purification for initial storage ) .", "In situ sample purification was accomplished through SEC to isolate well-folded proteins from aggregates and other impurities immediately before exposure to synchrotron X-ray radiation using a custom designed flow cell .", "SEC-SAXS was collected at the SIBYLS beamline ( bl12 . 3 . 1 ) at the Advanced Light Source at Lawrence Berkeley National Laboratory , Berkeley , California ( Classen et al . , 2010; Classen et al . , 2013 ) .", "X-ray wavelength was set at λ = 1 . 127 Å and the sample-to-detector distance was 2105 mm , as determined by silver behenate calibration , resulting in scattering vectors , q , ranging from 0 . 01 Å–1 to 0 . 4 Å–1 .", "The scattering vector is defined as q = 4πsinθ/λ , where 2θ is the scattering angle .", "Data was collected using a Dectris PILATUS3 × 2M detector at 20°C and processed as previously described ( Dyer et al . , 2014; Hura et al . , 2009 ) .", "Briefly , a custom-made SAXS flow cell was directly coupled with an Agilent 1260 Infinity HPLC system using a Shodex KW-803 column .", "The column was equilibrated with running Buffer E with a flow rate of 0 . 5 mL/min for inactive sGC and 0 . 55 mL/min for activated sGC .", "To achieve activation conditions , the buffer was continuously sparged with nitrogen gas and the column was equilibrated for at least 2 hr to maintain an anaerobic environment .", "Several NONOates with various half-lives were added to the running buffer to achieve activation of sGC .", "These NONOates include DEA NONOate and DETA NONOate , which spontaneously release NO with half-lives of 16 min and 56 hr , respectively .", "Each sample was run through the SEC-SAXS system and 3 s X-ray exposures were collected continuously over the 30 min elution .", "The SAXS frames recorded prior to the protein elution peak were used to correct all other frames .", "The corrected frames were investigated by radius of gyration Rg derived by the Guinier approximation I", "( q ) =I ( 0 ) exp ( –q2Rg2/3 ) with the limits q*Rg <1 . 3 ( Figure 5—figure supplement 2 ) .", "The elution peak was mapped by comparing the integral of ratios to background and Rg relative to the recorded frame using the program SCÅTTER ( Figure 5—figure supplement 1 ) .", "The frames in the regions of least Rg variation were averaged and merged in SCÅTTER to produce the highest signal-to-noise SAXS curves .", "These merged SAXS curves were used to generate the Guinier plots , volumes-of-correlation ( Vc ) , pair distribution functions , P", "( r ) , and normalized Kratky plots .", "The Guinier plot indicated an aggregation-free state of the protein ( Figure 5—figure supplement 2 ) .", "The P", "( r ) function was used to determine the maximal dimension of the macromolecule ( Dmax ) and estimate inter-domain distances ( Figure 4A ) ( Putnam et al . , 2007 ) .", "P", "( r ) functions were normalized based on the molecular weight ( MW ) of the assemblies , as determined by the calculated Vc ( Rambo and Tainer , 2013 ) .", "Eluent was subsequently split ( 4 to 1 ) between the SAXS line and a multiple wavelength detector ( UV-vis ) , set to 432 and 280 nm , multi-angle light scattering ( MALS ) , and refractometer .", "The ratios of the protein ( 280 nm ) and Soret band ( 432 nm ) of the heme from SEC were used to evaluate the ligation state of Ms sGC upon NO binding ( Figure 5—figure supplement 1 ) .", "MALS experiments were performed using an 18-angle DAWN HELEOS II light scattering detector connected in tandem to an Optilab refractive index concentration detector ( Wyatt Technology ) .", "System normalization and calibration was performed with bovine serum albumin using a 60 μL sample at 10 mg/mL in the same SEC running buffer and a dn/dc value of 0 . 185 and 0 . 15 mL/g for inactive and active sGC respectively .", "The MALS data was used to compliment the MWs calculated by the SAXS analysis and , being furthest downstream , the MALS peaks were used to align the SAXS and UV-vis peaks along the x-axis ( elution volume in mL/min ) to compensate for fluctuations in timing and band broadening ( Figure 5—figure supplement 1 ) .", "UV–vis data was integrated using Agilent Chemstation software and baseline corrected using Origin 9 . 1 ( Figure 5—figure supplement 1 ) .", "MALS and differential refractive index data was analyzed using Wyatt Astra seven software to monitor the homogeneity of the sample molecular weights across the elution peak , complementing the SEC-SAXS signal validation ( Figure 5—figure supplement 1 ) .", "The cryo-EM refined crystal structure for inactive sGC was used to build an initial atomistic model; all missing loops and terminal residues were added using MODELLER ( Fiser et al . , 2000 ) .", "A purpose-designed rigid body modeling pipeline was applied to the completed structure using BILBOMD to systematically explore conformational space for both the inactive and activated states of sGC ( Video 4 ) ( Pelikan et al . , 2009 ) .", "To obtain an inactive state model , the α H-NOX domain and both α terminal tails were moved , then all α and β domains were moved by allowing flexibility at the hinge region where the CCs meet the CAT domains , while holding the CAT domains fixed and still permitting flexibility of the terminal tails .", "This was then used to generate the 1-NO state model by moving the same regions as the inactive state in reverse order .", "Theoretical SAXS curves were produced by FOXS ( Schneidman-Duhovny et al . , 2010; Schneidman-Duhovny et al . , 2013 ) .", "Each model generated through BILBOMD as compared to the experimental SAXS profiles to assess the goodness of fit .", "Multistate model ensembles for activated sGC were determined using MultiFOXS ( Schneidman-Duhovny et al . , 2016 ) ." ] ]
[ "Soluble guanylate cyclase ( sGC ) is the primary receptor for nitric oxide ( NO ) in mammalian nitric oxide signaling .", "We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy .", "NO and the sGC-specific stimulator YC-1 induce a 71° rotation of the heme-binding β H-NOX and PAS domains .", "Repositioning of the β H-NOX domain leads to a straightening of the coiled-coil domains , which , in turn , use the motion to move the catalytic domains into an active conformation .", "YC-1 binds directly between the β H-NOX domain and the two CC domains .", "The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering ( SAXS ) .", "These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO ." ]
[ "In humans and other animals , as the heart pumps blood around the body , the blood exerts pressure on the walls of the blood vessels , much like water flowing through a hose .", "Our blood pressure naturally varies over the day , generally increasing when we are active and decreasing when we rest .", "However , if blood pressure remains high for extended periods of time it can lead to heart attacks , strokes and other serious health conditions .", "In 2013 , a new drug known as Adempas was approved to treat high blood pressure in the lungs .", "This drug helps a signaling molecule in the body called nitric oxide to activate an enzyme that widens blood vessels and in turn lower blood pressure .", "Previous studies have found that the enzyme – called soluble guanylate cyclase ( sGC ) – contains several distinct domains and that nitric oxide binds to a domain known as β H-NOX .", "However , it was not clear how β H-NOX and the other three domains fit together to make the three-dimensional structure of the enzyme , or how nitric oxide and Adempas activate it .", "To address this question , Horst , Yokom et al . used a technique called cryo-electron microscopy to determine the three-dimensional structures of the inactive and active forms of a soluble guanylate cyclase from a moth known as Manduca sexta .", "To produce the active form of the enzyme , soluble guanylate cyclase was incubated with both nitric oxide and a molecule called YC-1 that works in similar way to Adempas .", "The structures revealed that nitric oxide and YC-1 caused β H-NOX and another domain to rotate by 71 .", "This in turn caused the remaining two domains – known as the coiled-coil domains – to change shape , and all of these movements together led to the activated enzyme .", "The structures also revealed that YC-1 bound to a site on the enzyme between β H-NOX and the coiled-coil domains .", "Understanding how a drug for a particular condition works makes it much easier to develop new drugs that are more effective at treating the same condition or are tailored to treat other diseases .", "Therefore , these findings will allow pharmaceutical companies and other organizations to develop new drugs for high blood pressure and other cardiovascular diseases in a much more precise way ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "microbiology and infectious disease", "immunology and inflammation" ]
Chronic ethanol consumption compromises neutrophil function in acute pulmonary Aspergillus fumigatus infection
elife-58855-v2
[ [ "Ethanol abuse is a leading cause of mortality worldwide ( World Health Organisation , 2014 ) .", "Chronic alcohol consumption has been correlated , as a comorbidity , to a wide range of health conditions , including alcoholic liver diseases , cirrhosis and cancers ( Szabo and Saha , 2015; Pritchard and Nagy , 2005; Szabo , 1999; Bautista , 1999; Luján et al . , 2010 ) .", "Moreover , alcohol abusers are prone to develop severe lung inflammatory and infectious diseases , including acute respiratory distress syndrome ( ARDS ) ( Liang et al . , 2012 ) , pneumonia caused by Streptococcus pneumoniae ( Trevejo-Nunez et al . , 2015; Tsuchimoto et al . , 2015; Boé et al . , 2001 ) , Klebsiella pneumoniae ( Yeligar et al . , 2014; Ohama et al . , 2015 ) , Respiratory Syncytial Virus ( RSV ) infection ( Meyerholz et al . , 2008 ) and aspergillosis ( Blum et al . , 1978; Gustot et al . , 2014 ) .", "The mechanisms associated with this increased risk of disease and death are poorly understood , however studies have suggested that certain aspects of immune function may be altered by chronic ethanol intake ( D'Souza El-Guindy et al . , 2007; Lippai et al . , 2013; Yen et al . , 2017; Zhang et al . , 2015; Gurung et al . , 2009 ) .", "Aspergillus fumigatus is a ubiquitous and saprophytic fungus whose conidia are inhaled by humans on a daily basis ( Latgé , 1999 ) .", "Immunocompromised individuals are considered the risk group to develop the pulmonary invasive aspergillosis ( IA ) ( Latgé , 2001 ) and mortality rates reach up to 95% ( Brown et al . , 2012 ) .", "In normal conditions , inhaled conidia are cleared through mucociliary actions .", "However , if conidia pass through the initial barrier , alveolar macrophage ( AM ) phagocytosis takes place , resulting in a cascade of cytokine and chemokine release to recruit neutrophils to prevent fungal development ( Dagenais and Keller , 2009; Caffrey-Carr et al . , 2017 ) .", "In all these circumstances , an altered leukocyte function may be a major risk factor for IA .", "Despite all advance in diagnosis and treatment , aspergillosis’ morbidity and mortality remain very high .", "Mildly immunocompromised conditions such as diabetes mellitus , low-dose corticosteroid therapy and alcoholism has been considered as predisposing factors ( Blum et al . , 1978; Baddley , 2011; Kousha et al . , 2011 ) .", "Neutrophils have been shown to be essential to control fungal and bacterial burden in the site of infection and avoid the spread of these microbes and consequently , survival of the host ( Gazendam et al . , 2016; Romani , 2000; Robertson et al . , 2008; Ng et al . , 2019 ) .", "During a pathogen-triggered inflammatory response , neutrophils are the earliest immune effector cells recruited to a site of infection ( Kolaczkowska and Kubes , 2013; Sun et al . , 2014; Rios-Santos et al . , 2007 ) .", "Neutrophil migration starts with the tethering and rolling of these cells on endothelial cells , a process mediated by selectins and their carbohydrate ligands on neutrophils and endothelial venules ( Soethout et al . , 2002 ) .", "These interactions , together with chemokines signals such as CXCL1 and CXCL2 lead neutrophils to a crawling state through the endothelial vase ( Kolaczkowska and Kubes , 2013; Phillipson and Kubes , 2011 ) .", "G protein-coupled receptor on rolling neutrophils binds to the chemokines and changes β2 integrins conformation on the leukocyte surface , allowing a high affinity interaction with endothelial cells ( Zemans et al . , 2009 ) .", "Here , we describe that chronic ethanol consumption facilitates pulmonary infection by both A . fumigatus in mice .", "Mechanistically , chronic ethanol consumption impairs the normal neutrophil migration to the site of infection via release of high levels of circulating chemokine CXCL1 after infection , followed by downregulation of its receptor 2 ( CXCR2 ) .", "Additionally , ethanol consumption is responsible for an impaired neutrophil function characterized by less phagocytosis , killing , and oxidative burst leading to elevated lung pathology in mice and accentuated mortality rates after infection ." ], [ "To assess the relevance of ethanol chronic consumption , we characterized several parameters during chronic ethanol consumption on host before the infection ( Figure 1A ) .", "First , we checked weight change , food and liquid consumption during ethanol treatment .", "We found that there was no weight change in mice during the treatment with ethanol ( Figure 1B ) .", "Moreover , food and liquid consumption were diminished in ethanol-treated mice compared with control group ( Figure 1C and D ) .", "Second , blood ethanol levels in mice were about 200 mg of ethanol per deciliter of whole blood while control group ethanol level were not detectable , measured by gas chromatography after 12 weeks of ethanol treatment , ( Figure 1E ) .", "In order to verify if ethanol consumption affected hematopoiesis we analyzed bone marrow precursors and blood cell counts .", "Mononuclear and neutrophil counts in peripheral blood did not show differences between groups ( Figure 1F ) .", "Chronic ethanol consumption did not affect granulocyte progenitors in bone marrow ( Supplementary file 1 ) .", "In addition , no changes were observed in ALT levels in mice serum after the ethanol treatment compared to the control mice group ( data not shown ) .", "Finally , serum levels of inflammatory mediators , such as TNF-α , IL-1β and CXCL1 , have not been changed in ethanol-fed mice ( Figure 1G–I ) .", "After ethanol treatment , mice were intranasally infected with A . fumigatus conidia ( Figure 2A ) .", "Ethanol-treated mice showed an increased lethality compared to control mice group ( Figure 2B ) .", "In addition , weight loss was significantly higher in ethanol treated mice from days 3 to 7 after infection ( Figure 2C ) .", "These clinical signs were accompanied by higher pulmonary fungal burden in ethanol-fed mice , demonstrating an impaired fungal clearance .", "Moreover , we were also able to identify hyphae into the airways of infected ethanol-treated mice , showed by red arrows at 24 hr after infection compared to control group ( Figure 2D–F ) .", "Altogether , these results demonstrate that chronic ethanol consumption increased susceptibility to pulmonary A . fumigatus infection along with an impaired ability to clear the pathogen .", "Next , we assessed cytokine and chemokine levels by ELISA after A . fumigatus infection to determine whether levels of inflammatory mediators in BALF supernatants were altered after ethanol consumption .", "We found no significant differences in the levels of neutrophil chemotactic mediator CXCL1 after A . fumigatus infection in BALF of ethanol-treated mice compared to control group ( Figure 3A ) .", "Interestingly , another CXCR2 agonist and neutrophil chemotactic mediator , CXCL2 were increased at 24 hr of infection in BAL fluid of ethanol-treated mice compared to control mice ( Figure 3B ) .", "Alcohol consumption declined IL-17 levels after 24 hr of infection ( Figure 3C ) .", "We also observed no differences in TNF levels between control and ethanol-fed mice group ( Figure 3D ) .", "Moreover , ethanol consumption was able to down modulate IL-1β and IL-10 levels after fungal infection ( Figure 3E and F ) .", "These results suggest that chronic ethanol consumption dysregulate cytokine and chemokine release post A . fumigatus infection .", "To clarify whether the diminished fungal clearance is due to an impaired inflammatory response , we next observed cell influx into the site of A . fumigatus infection .", "Although both groups exhibited a large recruitment of total cells into the airways , ethanol-treated mice had a significant reduction of recruited cells , compared to control group 24 hr after infection .", "However , at 48 hr after infection , there was a similar cell migration into the site of infection in both groups ( Figure 4A ) .", "We demonstrate that almost all cells migrated to the airways represent neutrophils , one of the most important cells involved in killing and control of A . fumigatus infection ( Erwig and Gow , 2016 ) .", "Neutrophils and lymphocytes into airways were significantly decreased in ethanol-treated mice at 24 hr post infection ( Figure 4B and C ) .", "In contrast , there were no differences in macrophages and eosinophils recruited to the site of infection ( Figure 4D and E ) .", "Regarding to inflammatory cells recruited to lung tissue , we also observed a diminished neutrophils migration into the lung parenchyma in ethanol-treated mice , by MPO measurement , at 24 hr after infection ( Figure 4F ) .", "Similar to alveoli , eosinophils and macrophages in pulmonary tissue exhibited no differences after A . fumigatus infection ( Figure 4G and H ) .", "In addition , both ethanol-treated and control groups exhibited similar number of leukocytes in blood ( Figure 4I ) .", "Besides , we characterized the populations of lymphocyte migrating to the airways of infected mice .", "We assessed CD3+CD4+IL17+ cells and results demonstrate that chronic ethanol consumption strongly reduced lymphocytes and IL-17 production in the site of infection after 24 hr of infection ( Figure 4J–L ) .", "These results indicate that chronic ethanol consumption mostly affected specifically neutrophils recruitment to airways after A . fumigatus infection .", "We assessed histopathology to determine the effect of ethanol consumption in pulmonary tissue after infection .", "Tissue sections of infected mice revealed a massive leukocyte recruitment into the lungs at 24 hr after infection , in which the inflammatory infiltrate covers a large part of the pulmonary parenchyma structure , including alveoli and perivascular regions that decreases after 48 hr of infection in the control group .", "The cellular infiltrate tissue remains more prominent after 48 hr of infection only in ethanol-fed mice ( Figure 5A ) .", "Histopathology score results showed that both ethanol-treated and control groups had similar levels of inflammatory infiltrate , interstitial and alveolar edema and hemorrhage scores at 24 hr of infection .", "However , after 48 hr of infection , ethanol consumption showed a remaining cellular infiltrate , higher edema and hemorrhage , which increased the total pathology scores ( Figure 5B–D ) .", "In order to investigate the transmigration process of neutrophils upon chemotaxis in vivo , we first performed an intravital microscopy to visualize migratory cells ( Figure 6—source data 1 ) ( Figure 6-rich media videos ) .", "Intracrostal administration of LPS was not able to induce a strong increase in rolling and adherence of cells to post capillary venules in ethanol-fed group after 2 hr of stimulation , compared to stimulated control group ( Figure 6A–D ) .", "In addition , to confirm the impaired migratory ability of neutrophils was caused by ethanol consumption in mice , we performed ex vivo chemotaxis assay towards CXCL2 , with mouse bone marrow-derived neutrophils ( Figure 6E ) .", "As we expected because of previously results showed in Figure 4B , neutrophils from ethanol-treated mice had an impaired migration towards the chemokine stimuli compared to neutrophils from control non-treated mice ( Figure 6F ) .", "Taken together , these results demonstrate that chronic ethanol ingestion can affect neutrophil rolling , adhesion and recruitment in different tissues .", "Neutrophils chemotaxis and recruitment is a complex process that requires leukocyte-endothelial interactions as well as inflammatory mediators’ release ( Kolaczkowska and Kubes , 2013 ) .", "Moreover , the main regulator of neutrophil migration in acute inflammation is CXCR2 .", "To further investigate whether the impaired neutrophil migration is due a deficient neutrophil activation status during infection , we accessed the expression of the markers CD11b , CD62L and CXCR2 in circulating neutrophils after A . fumigatus infection using flow cytometry .", "While CD11b is mobilized from specific granules to the cell surface , enzymatic shedding in activated polymorphonuclear ( PMN ) rapidly down regulates CD62L .", "Both markers have their constitutive expression in resting PMNs .", "We found that neutrophils activation status was compromised , with CD62L up regulation and CD11b down regulation , in the peripheral blood neutrophils of infected ethanol-treated mice compared to infected non-treated mice ( Figure 7A–C ) .", "In sepsis , the reduction of neutrophils migration is related to the down regulation of CXCR2 protein expression on circulating neutrophils surface ( Alves-Filho et al . , 2009 ) .", "Indeed , CXCL1 serum levels were strongly augmented in ethanol-treated mice compared to the control group at 24 hr post infection ( Figure 7D ) .", "We next analyzed the role of chronic ethanol intake in regulating CXCR2 expression in circulating neutrophils .", "At 24 hr after A . fumigatus infection , ethanol-treated mice exhibited significantly fewer circulating neutrophils expressing CXCR2 in the surface compared to the non-treated group ( Figure 7E and F ) .", "For the proper killing of A . fumigatus , fungal phagocytosis and ROS production by neutrophils are key events ( Philippe et al . , 2003 ) .", "In this sense , we accessed phagocytosis and killing of A . fumigatus conidia by bone marrow-derived neutrophils from ethanol-treated and non-treated mice .", "We found that phagocytosis was significantly reduced in ethanol-treated mice either in vivo , evaluated by BALF recruited cells , or ex vivo , in bone marrow neutrophils , compared to control group ( Figure 8A and B ) .", "Fungal killing was also reduced in ethanol intake mouse neutrophils ( Figure 8C ) .", "Moreover , to evaluate the effect of chronic ethanol consumption in the promotion of respiratory burst of bone marrow-derived neutrophils we performed chemiluminescence experiments using luminol , which served as a probe for superoxide ( O2●− ) 49 .", "We observed that neutrophils from ethanol-fed mice produced lower levels of ROS triggered by A . fumigatus conidia compared to neutrophils from non-treated mice ( Figure 8D ) .", "These results suggest that phagocytosis , killing and ROS production functions in neutrophils were affected by chronic ethanol consumption ." ], [ "For several centuries , chronic ethanol consumption has been associated to increased susceptibility to infections as well as increased morbidity and mortality after injury ( Lieber , 2005; Molina et al . , 2010 ) .", "Numerous studies have shown the effects of acute or chronic exposure to ethanol in inflammatory infections such as in models of K . pneumoniae infection and gut bacteria-associated sepsis ( Ohama et al . , 2015 ) , intravenous Escherichia coli challenge ( Bagby et al . , 1998 ) , S . pneumoniae ( Trevejo-Nunez et al . , 2015; Boé et al . , 2001 ) and a few studies reporting this effect in Aspergillus infection ( Blum et al . , 1978 ) .", "In the present study , we demonstrate how chronic ethanol consumption alters immune and inflammatory pulmonary response after A . fumigatus infection .", "This involves several immunological phenomena , including defective leukocytes rolling and adhesion , impaired neutrophil migration by down regulation of CXCR2 , failed neutrophil activation , impaired neutrophil effector functions ( phagocytosis and killing ) , and reduction of polarized-Th17 innate response .", "Those features are responsible for an increased susceptibility of mice to A . fumigatus infection .", "Chronic ethanol consumption did not alter the basal levels of inflammatory cytokines and chemokines before infection .", "Ethanol ingestion had an important role in modulating immune response after infection , which is in accordance with previous findings ( D'Souza El-Guindy et al . , 2007; Bhatty et al . , 2011 ) .", "Ethanol consumption also alters IL-6 and TNF expression of LPS-stimulated Kupffer cells ( Maraslioglu et al . , 2014 ) .", "In the present work , we found reduction in IL-1β and IL-17 production after A . fumigatus infection in ethanol-fed mice , suggesting that the immunomodulatory effects caused by chronic ethanol consumption may contribute to the impaired inflammatory response against A . fumigatus .", "It has been demonstrated that chronic ethanol consumption causes cellular abnormality in mice lung resident cells .", "Alveolar macrophages from ethanol-fed mice had deregulation in NADPH oxidase system , impairing phagocytosis and killing against K . pneumoniae ( Yeligar et al . , 2012 ) .", "In fact , an impaired NADPH oxidase activity is a well-known risk factor to develop invasive aspergillosis and other life threatening diseases , as seen in Chronic Granulomatous Disease ( CGD ) patients ( Cohen et al . , 1981; King et al . , 2016; Segal and Romani , 2009 ) .", "Another study showed both lymphocytes and neutrophils response impaired by ethanol consumption in a model of cutaneous infection by Staphylococcus aureus , in which chronic ethanol-fed mice showed great skin lesions and bacteremia associated with reduced IL-17 and IL-1β production , suggesting that Th17-mediated neutrophilic response was impaired ( Parlet et al . , 2015 ) .", "Neutrophil recruitment to the site of infection is essential for the control of invading extracellular pathogens ( Kolaczkowska and Kubes , 2013; Sun et al . , 2014; Rios-Santos et al . , 2007 ) .", "Neutrophils are major cell type recruited for A . fumigatus conidia and hyphae killing and neutropenic patients are more susceptible to systemic fungal infections ( Dagenais and Keller , 2009; Gazendam et al . , 2016 ) .", "In our data , neutrophil recruitment to infection site , in ethanol-fed mice , was impaired even though we did not observe decrease in both CXCL1 and CXCL2 BALF levels , indicating that ethanol consumption is responsible for compromising neutrophils activation and migration .", "Moreover , our data showed reduced leukocyte rolling and adhesion after LPS stimuli in ethanol-treated mice outside the airways .", "CXCL1 and CXCL2 chemokines bind to CXCR2 displaying an essential role in neutrophils activation and ensuing adhesion to endothelium ( Kolaczkowska and Kubes , 2013; Phillipson and Kubes , 2011 ) .", "In fact , it was demonstrated in vitro that chronic ethanol exposure impacted tight junction structures in epithelial cells , leading them vulnerable to endotoxemia ( Wood et al . , 2013 ) .", "However , in our study vascular permeability was similar in both ethanol-fed mice and control mice groups ( data not shown ) .", "It has been well established that the decrease of CXCR2 expression impairs neutrophil migration ( Russo et al . , 2009 ) , especially in sepsis ( Rios-Santos et al . , 2007; Alves-Filho et al . , 2009 ) , in which a great amount of inflammatory cytokines and chemokines is released to the blood , causing a complex systemic inflammation ( Pierrakos and Vincent , 2010 ) .", "In a model of severe sepsis , it was found that the migration failure and consequent mortality of individual was associated with the diminished expression of neutrophils CXCR2 , which was due to great release of systemic CXCL1 ( 39 ) .", "As seen in sepsis , our findings showed decrease CXCR2 expression and great amount of systemic CXCL1 levels in serum of ethanol-fed mice after A . fumigatus infection .", "To our knowledge , this is the first report establishing a relation between chronic ethanol intake and downregulation of CXCR2 receptor in mouse neutrophils .", "It is also important to mention that the cooperation between the GPCR receptor CXCR2 and P-selectin ligand , the P-selectin glycoprotein ligand-1 ( PSGL-1 ) is essential to a successful neutrophil migration .", "The signaling events that ensure the adhesion cascade include the conversion of integrin αLβ2 from a low-affinity conformation to an extended high-affinity conformation that causes arrest and , consequently , perivascular crawling ( Yago et al . , 2018 ) .", "Further studies are needed to address the role of chronic ethanol consumption in expression of P-selectin and PSGL-1 .", "Our data indicate that chronic ethanol consumption drives host to a sepsis-like phenotype and this mechanism is responsible for impaired neutrophil migration .", "The mechanisms whereby CXCR2 is downregulated need further investigation .", "To conclude , the findings presented here indicate a new paradigm in how chronic ethanol consumption strongly impairs neutrophils host pulmonary defense against A . fumigatus infection .", "This infection in mice causes a great inflammatory response , with release of cytokines and chemokines that act in favor to recruit neutrophils into the alveoli and these neutrophils are able to clear the fungus .", "In contrast , in a condition of chronic ethanol consumption , despite the correct induction of inflammatory response , neutrophils exhibit failure in activation , through the down regulation of CD11b and up regulation of CD62L in blood neutrophils and by accentuated release of CXCR2 ligands in blood flow .", "This leads to CXCR2 down regulation , which culminated in impaired neutrophils recruitment , increased fungal load and exacerbated lung pathology in mice .", "We also associate the lower neutrophils levels into the airways with lower innate polarized-Th17 immune response and reduced phagocytosis and killing of A . fumigatus conidia .", "In fact , we observed growing conidia and substantial fungal burden in lung from ethanol-fed mice , contributing to the highest susceptibility of ethanol-treated mice to A . fumigatus infection ( Figure 9 ) ." ], [ "All animal experiments received prior approval from the Animal Ethics Committee ( CEUA ) of Universidade Federal de Minas Gerais ( UFMG ) , Brazil ( Protocol number: 4/2015 ) .", "Male C57BL/6J mice were randomly allocated into experimental groups .", "Mice were maintained in specific pathogen-free conditions .", "The chronic ethanol consumption model used was previously described ( Ceron et al . , 2018; Simplicio et al . , 2017 ) .", "Briefly , mice received ethanol 5% ( v/v ) in the first week , followed by 10% ( v/v ) in the second week ( to help mice acclimate with this intervention ) and were treated during 10 weeks with 20% ( v/v ) of ethanol in their drinking water .", "Control group received water .", "This protocol replicates blood ethanol levels after chronic ethanol consumption in human subjects , as described in studies with C57BL/6J or BALB/c mice ( Ceron et al . , 2018; Simplicio et al . , 2017; Urso et al . , 1981 ) .", "Weight change , food and liquid consumption were measured weekly during ethanol treatment .", "Cytokine and chemokine levels ( TNF-α , IL-1β , CXCL1 , CXCL2 , IL-17 , IL-10 ) were quantified in BAL , serum or plasma fluid using DuoSet ELISA kits ( R and D Systems ) , in accordance to the manufacturer’s instructions .", "The determination of blood ethanol levels was made as previously described ( Gonzaga et al . , 2015 ) .", "Briefly , mice were anesthetized and 100 μL of blood was collected and transferred into headspace vials .", "Blood ethanol levels were measured by gas chromatograph using a gas chromatographer as previously described ( Ceron et al . , 2018 ) .", "To determine the impact of chronic alcohol intake in fungal pulmonary infection , male C57BL/6J mice were treated for 12 weeks .", "After the last day of treatment , mice were infected intranasally with Aspergillus fumigatus A1163 strain ( Fedorova et al . , 2008 ) .", "The fungus was grown in complete media for 48 hr at 37°C ( Malacco et al . , 2019 ) .", "Fungal conidia were harvested by washing the media with sterile phosphate-buffered saline ( PBS ) .", "After filtering , conidia were centrifuged at 1400 x g resuspended and counted in Neubauer chamber .", "Mice were infected with 3 × 108 conidia/animal , prepared in PBS .", "After 24 or 48 hr of infection , mice were anesthetized with ketamine ( 100 mg/kg ) and xilazine ( 6 mg/Kg ) and blood smear and serum or plasma were collected .", "After that , mice were euthanized and bronchoalveolar lavage fluid ( BALF ) was harvested as previously described ( Malacco et al . , 2019 ) .", "BALF total cell counts were determined by counting leukocytes in Neubauer chamber .", "Differential cell count and in vivo phagocytosis count were obtained from cytospin preparations ( Shandon III ) .", "BALF supernatants were used for cytokines , chemokines and total protein measurements .", "Protein amounts were quantified in BALF samples using the Bradford assay ( Bradford , 1976 ) .", "At the indicated time points , lungs were collected .", "The right lobes were removed and frozen for subsequent analysis of myeloperoxidase ( MPO ) ( Huang et al . , 2016 ) , N-acetyglucosaminidase ( NAG ) ( Reiner et al . , 1981 ) , eosinophil peroxidase ( EPO ) ( Strath et al . , 1985 ) or measurement of fungal burden .", "The left lobes were fixed in formalin 4% ( v/v ) for histopathological analysis .", "Formalin-fixed tissue was dehydrated gradually in ethanol , embedded in paraffin , and 4 µm sections were stained with Hematoxylin and Eosin ( H and E ) or Grocott’s methenamine silver ( GMS ) .", "The total histopathology score considered inflammatory infiltrate , interstitial and alveolar edema and hemorrhage ( Hubbs et al . , 1997 ) .", "The percentage of germination of A . fumigatus conidia was counted in 200 to 300 fungal conidia in GMS-stained slides at x 100 magnification microscope .", "Leukocytes obtained from BAL or blood samples were subjected to hypotonic lysis to remove residual erythrocytes , as described previously ( Russo et al . , 2009 ) .", "Briefly , cells were treated with Fc block ( R and D Systems ) , labeled with relevant antibodies , namely: CD3 - fluorescein isothiocyanate ( FITC ) , CD4 - APC , IL-17 - phycoerythrin ( PE ) , Ly6G - brilliant violet 421 ( BV421 ) , CXCR2 - PE , CD62L – APC and CD11b – FITC or isotype control .", "At least 30 , 000 events were acquired in a FACScan cytometer , and data were analyzed using FlowJo ( Tree Star , Ashland , OR , USA ) software .", "The relevant populations were gated , using accepted criteria for granularity , and sized and evaluated for staining of relevant surface and intracellular markers .", "The mouse cremaster preparation was used to study the behavior of leukocytes in the microcirculation and adjacent connective tissue , as previously described ( Pinho et al . , 2007 ) .", "Briefly , 2 hr prior the surgery , mouse cremaster muscle were injected with LPS ( 250 ng/mL ) diluted in saline .", "Then an incision was made in the scrotal skin to expose the cremaster muscle , which was then carefully removed from the associated fascia .", "A lengthwise incision was made on the ventral surface of the cremaster muscle using a cautery .", "The testicle and the epididymis were separated from the underlying muscle and were moved into the abdominal cavity .", "The muscle was then spread out over an optically clear viewing pedestal and was secured along the edges with a 4–0 suture .", "The exposed tissue was superfused with warm PBS .", "An intravital microscope ( Olympus BX50F4 ) with x 20 magnification objective lens and x 10 times magnification eyepiece was used to examine the cremasteric microcirculation .", "A video camera ( 5100 HS; Panasonic ) was used to project the images onto a monitor , and the images were recorded for playback analysis .", "The numbers of rolling and adherent leukocytes were determined offline during the video playback analyses .", "Leukocytes were considered adherent to the venular endothelium if they remained stationary for at least 30 s .", "Rolling leukocytes were defined as white cells moving at a velocity slower than that of the erythrocytes within a given vessel .", "After isolation of mouse bone marrow ( BM ) from femur and tibia in RPMI 1640 medium , neutrophils were separated by density gradient centrifugation using Histopaque 1077 ( density , 1 . 077 g/ml ) in a 15 ml conical tube .", "Then , erythrocytes were lysed using ACK lysing buffer and the neutrophils were counted .", "Neutrophils purity was over than 80% ( Swamydas and Lionakis , 2013 ) .", "Phagocytosis and killing assay were performed by incubation of BM-derived neutrophils from mice treated and non-treated with ethanol with A . fumigatus conidia for 4 hr ( phagocytosis ) or 6 hr ( killing ) at 37° with 5% of CO2 in the ratio of 5:1 .", "Phagocytosis was evaluated in cytospin preparations .", "To determine A . fumigatus killing , cells were lysed with distilled water and the diluted samples were plated in fungal medium and colony-forming units ( CFU ) were determined after overnight incubation at 37°C , and the percentage of killing was calculated as a percentage of the viability after incubation without neutrophils .", "Luminometry assays were performed to evaluate the production of ROS by BM-derived neutrophils ( Goes et al . , 2016 ) .", "Neutrophils ( 1 × 106 cells/well ) were resuspended in complete RPMI medium without phenol red .", "Then the cells were plated in 96 well opaque plates ( NUNC , Rochester , NY , USA ) with 0 . 05 mM luminol ( 5-Amino-2 , 3-dihydro-1 , 4-phthalazinedione; Sigma-Aldrich ) and A . fumigatus conidia in the proportion of 10 conidia to one neutrophil .", "Measurements were taken for 60 min with 2 min interval .", "Production of ROS was assayed by the light intensity generated by the reaction between ROS and luminol and expressed as fluorescent relative units .", "A modified Boyden chamber assay to examine the neutrophil chemoattractant response to CXCL-2 ( kindly provided by Dr . José Carlos Alves-Filho , Universidade de São Paulo , Ribeirão Preto , Brazil ) was performed using a 48-well microchamber ( Neuro Probe ) as previously described ( Pinho et al . , 2007 ) .", "Murine bone marrow neutrophils were isolated as above described and resuspended in RPMI .", "Recombinant mouse CXCL-2 ( 20 ng/mL ) diluted in running buffer ( for wells containing neutrophils ) or appropriate buffer control was added to the lower chambers of the apparatus .", "A 5-µm-pore polycarbonate membrane ( Neuro Probe ) was placed between the upper and lower chambers , and 5 × 104 cells in a volume of 50 µL were added to the top chambers of the apparatus .", "Cells were allowed to migrate into the membrane for 1 hr per treatment at 37°C with 5% CO2 .", "Following incubation , the chamber was disassembled and the membrane was scraped and washed three times in PBS to remove nonadherent cells before being fixed in methanol and stained using the Diff-Quik system ( Dade Behring ) .", "Each well-associated membrane area was scored using light microscopy to count the intact cells present in five random fields .", "All experiments were made at least twice ( biological replication ) by independent experiments .", "The sample size estimation was done with G*Power 3 . 1 Software ( Jacob Cohen's A power primer , 1992 in Psychological Bulletin Journal ) .", "Statistical analysis was performed with Graph Pad Prism six software ( Graph Pad Prism Software , Inc , Sandiego , CA ) .", "Data are presented as the mean ± SD and were analyzed using One-way analysis of variance ( ANOVA ) followed by Tukey post-test to compare different groups .", "Student’s t test was used to compare two groups .", "Survival analysis was made by Log Rank test .", "Statistical significance was set as < 0 . 05 .", "For more information , please see the transparent reporting form ." ] ]
[ "Chronic ethanol consumption is a leading cause of mortality worldwide , with higher risks to develop pulmonary infections , including Aspergillus infections .", "Mechanisms underlying increased susceptibility to infections are poorly understood .", "Chronic ethanol consumption induced increased mortality rates , higher Aspergillus fumigatus burden and reduced neutrophil recruitment into the airways .", "Intravital microscopy showed decrease in leukocyte adhesion and rolling after ethanol consumption .", "Moreover , downregulated neutrophil activation and increased levels of serum CXCL1 in ethanol-fed mice induced internalization of CXCR2 receptor in circulating neutrophils .", "Bone marrow-derived neutrophils from ethanol-fed mice showed lower fungal clearance and defective reactive oxygen species production .", "Taken together , results showed that ethanol affects activation , recruitment , phagocytosis and killing functions of neutrophils , causing susceptibility to pulmonary A . fumigatus infection .", "This study establishes a new paradigm in innate immune response in chronic ethanol consumers ." ]
[ "Alcoholism is a chronic disease that has many damaging effects on the body .", "Over long periods , excessive alcohol intake weakens the immune system , putting consumers at increased risk of getting lung infections such as pneumonia .", "Some forms of pneumonia can be caused by the fungus Aspergillus fumigatus .", "This microbe does not tend to be a problem for healthy individuals , but it can be fatal for those with impaired immune systems .", "Here , Malacco et al . wanted to find out why excessive alcohol consumers are more prone to pneumonia .", "To test this , the researchers used two groups of mice that were either fed plain water or water containing ethanol .", "After 12 weeks , both groups were infected with Aspergillus fumigatus .", "The results showed that alcohol-fed mice were more susceptible to the infection caused by strong inflammation of the lungs .", "Normally , the immune system confronts a lung infection by activating a group of defense cells called neutrophils , which travel through the blood system to the infection site .", "Once in the right spot , neutrophils get to work by releasing toxins that kill the fungus .", "Malacco et al . discovered that after chronic alcohol consumption , neutrophils were less reactive to inflammatory signals and less likely to reach the lungs .", "They were also less effective in dealing with the infection .", "Neutrophil released fewer toxins and were thus less able to kill the microbial cells .", "These findings demonstrate for the first time how alcohol can affect immune cells during infection and pave the way for new possibilities to prevent fatal lung infections in excessive alcohol consumers .", "A next step would be to identify how alcohol acts on other processes in the body and to find a way to modulate or even revert the changes it causes ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "structural biology and molecular biophysics" ]
VEGFR-2 conformational switch in response to ligand binding
elife-13876-v1
[ [ "Angiogenesis , the development of new blood vessels from pre-existing ones , plays a critical role in embryogenesis , organ development , and wound healing ( Qutub et al . , 2009; Nessa et al . , 2009; Adams et al . , 2007 ) .", "In addition , angiogenesis is associated with diabetic and age-related macular degeneration and is required for the growth of solid tumors , which recruit blood vessels to ensure adequate supply with oxygen and nutrients ( Luo et al . , 2008; Tsuzuki et al . , 2001 ) .", "Angiogenesis is regulated by the ligands and receptors of the vascular endothelial growth factor ( VEGF ) signaling network ( Ferrara et al . , 2003; Matsumoto et al . , 2001; Koch et al . , 2011; Olsson et al . , 2006; Shibuya et al . , 2006 ) .", "Of the three VEGF receptors , VEGFR-2 is the primary regulator of endothelial cell proliferation and migration ( Takahashi et al . , 2005; Gerhardt et al . , 2003 ) .", "VEGFR-2 is expressed in all human vascular endothelial cells and is often overexpressed in highly malignant solid tumors ( Smith et al . , 2010; Yamagishi et al . , 2013 ) .", "Aggressive cancerous phenotypes correlate with enhanced VEGFR-2 signaling ( Chatterjee et al . , 2013 ) .", "VEGFR-2 is a receptor tyrosine kinase ( RTK ) which transduces biochemical signals via lateral dimerization in the plasma membrane .", "Like most RTKs , VEGFR-2 is composed of an extracellular ( EC ) domain , a transmembrane ( TM ) domain , and an intracellular ( IC ) domain consisting of a kinase domain and sequences required for downstream signaling .", "The EC domain consists of seven immunoglobulin homology ( Ig ) domains , termed D1 ( at the N-terminus ) to D7 ( closest to the membrane ) .", "VEGFR-2 binds to , and is activated by the ligands VEGF-A , VEGF-E , and a number of processed forms of VEGF-C and VEGF-D ( Olsson et al . , 2006; Matsumoto et al . , 2001 ) .", "Ligand binding to VEGFR-2 is mediated by Ig-domains 2 and 3 and the linker between D2 and D3 ( Brozzo et al . , 2012; Leppanen et al . , 2011; Kisko et al . , 2011; Hyde et al . , 2012; Ruch et al . , 2007 ) .", "All VEGF ligands form disulfide-linked anti-parallel homodimers ( Muller et al . , 1997 ) .", "VEGF-A exists as different isoforms of various lengths , all containing the binding site for VEGFR-2 , located within the N-terminal 121 amino acids .", "All isoforms exhibit similar binding affinity to VEGFR-2 ( Mac Gabhann et al . , 2010; Qutub et al . , 2009 ) .", "The longer VEGF-A isoforms possess additional sequences which enable binding to co-receptors such as neuropilins and heparan sulfate proteoglycans ( Qutub et al . , 2009; Vempati et al . , 2014 ) .", "At present , there is no universal consensus model of RTK-mediated transmembrane signaling .", "Instead , two models , which are not necessarily mutually exclusive , are discussed .", "The first is the 'diffusion-based' or 'canonical' model , in which RTKs exist as monomers in the absence of ligand because they do not have a sequence-specific propensity for lateral interactions .", "Ligand binding induces RTK dimerization , which brings the two catalytic domains into close proximity so they can cross-phosphorylate each other ( Fantl et al . , 1993 ) .", "The second model is the 'pre-formed dimer model' , in which the receptors can form dimers in the absence of ligand because they have a sequence-encoded propensity to interact laterally even in the absence of ligand , and thus they exist in a monomer-dimer equilibrium in the absence of ligand .", "Ligand binding promotes structural changes in the EC domain that trigger transmembrane signaling by reorienting the catalytic kinase domains ( Belov et al . , 2012; Bocharov et al . , 2008; 2013; Sarabipour and Hristova , 2016 ) .", "VEGFR-2 has been believed to follow the canonical model of ligand-induced dimerization and activation , with dimeric VEGF ligands binding to freely diffusing monomeric receptors and promoting receptor dimerization ( Kisko et al . , 2011; Hyde et al . , 2012; Ruch et al . , 2007; Shibuya et al . , 2006; Koch et al . , 2011 ) .", "In the active dimers , homotypic receptor-receptor contacts in domains D4-7 were observed that appear critical for efficient phosphorylation of the VEGFR-2 receptors in the presence of ligand ( Yang et al . , 2010; Hyde et al . , 2012; Ruch et al . , 2007 ) .", "Ligand-independent dimer formation has been observed for several RTKs , such as EGFR , FGFR , and Trk ( Low-Nam et al . , 2011; Maruyama et al . , 2012; Mischel et al . , 2002; Lin et al . , 2012; Sarabipour and Hristova , 2016 ) .", "The dimerization propensity of VEGFR-2 in the absence of ligand , however , has not been measured to date .", "Here , we examined VEGFR-2 dimerization , as well as the mechanism of VEGFR-2 ligand-mediated activation .", "We used a quantitative FRET-based method and biochemical assays to study the structure , stability , and activation of VEGFR-2 dimers ." ], [ "We investigated whether full-length VEGFR-2 is capable of forming dimers in the absence of ligand using an established quantitative FRET method ( Chen et al . , 2010 ) .", "The interactions between VEGFR-2 molecules , labeled with the fluorescent proteins YFP or mCherry ( a FRET donor-acceptor pair ) , were characterized in plasma membrane-derived vesicles generated from transfected CHO cells in response to osmotic stress ( Del Piccolo et al . , 2012; Sarabipour et al . , 2015 ) .", "While the plasma membrane topology of intact cells is complex ( Adler et al . , 2010; Parmryd et al . , 2013 ) , plasma-membrane-derived vesicles exhibit a simple membrane geometry , allowing us to determine the two-dimensional membrane receptor concentrations required for quantitative dimerization measurements ( Chen et al . , 2010 ) .", "To determine the thermodynamic parameters describing VEGFR-2 dimerization , we varied the receptor concentration , and we measured the concentrations of the receptors and the FRET efficiencies in individual plasma membrane-derived vesicles ( Figure 2 ) .", "This method yields the dimerization constant K and thus the dimerization free energy ( or dimer stability ) ( Chen et al . , 2010 ) .", "In addition , the method allows to extract structural information from the 'Intrinsic FRET' value , which depends on the separation and the orientation of the fluorescent proteins in the dimer but not on the dimerization propensity ( Chen et al . , 2010; Sarabipour and Hristova , 2015 ) .", "As shown previously , Intrinsic FRET values provide structural information by reporting changes in the spatial separation of the fluorescent proteins in receptor dimers ( Del Piccolo et al . , 2015; Sarabipour and Hristova , 2015 ) ( see Equation 7 ) . 10 . 7554/eLife . 13876 . 003Figure 1 . The plasmid constructs used in this study .", "( A ) The constructs used in the FRET experiments .", "The full-length receptors had fluorescent proteins attached to their C-termini via a flexible GGS linker .", "The truncated receptors had the intracellular domain substituted with a fluorescent protein , which was attached to the TM domain via a longer flexible ( GGS ) 5 linker .", "SP: signal peptide , EC: extracellular domain , TM: transmembrane domain , IC: Intracellular domain of VEGFR-2 .", "Fluorescent protein was either YFP or mCherry .", "Amino acid residue numbers are shown above the constructs .", "( B ) The constructs used in the Western blotting experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 00310 . 7554/eLife . 13876 . 004Figure 2 . FRET measurements of VEGFR-2 dimerization in CHO plasma membranes .", "( A ) FRET efficiencies as a function of acceptor concentration for full length VEGFR-2 ( solid red diamonds ) , EC+TM VEGFR-2 ( solid black diamonds ) , and TM VEGFR-2 ( open olive circles ) .", "Two hundred to 500 individual vesicles were imaged in at least three independent experiments .", "Each data point here corresponds to a single vesicle .", "The solid line is the so-called 'stochastic' or 'proximity' FRET which arises when donors and acceptors approach each other within distances of 100 Å or so , in the absence of specific interactions ( King et al . , 2014 ) .", "( B ) FRET efficiencies corrected for stochastic FRET ( King et al . , 2014 ) .", "( C ) Donor concentration versus acceptor concentration in individual vesicles , for the three VEGFR-2 constructs .", "( D ) .", "Dimeric fractions versus total receptor concentrations , for the full-length VEGFR-2 ( solid red diamonds ) , EC+TM VEGFR-2 ( solid black diamonds ) , and the TM domains only ( open olive circles ) .", "The measured dimeric fractions are binned and are shown with the symbols , along with the standard errors .", "The solid lines are the best fits of a monomer-dimer equilibrium model to the single-vesicle FRET data .", "Full-length VEGFR-2 has a significant propensity for dimerization in the absence of ligand .", "The contribution of the intracellular ( IC ) domain to dimerization is favorable , while the contribution of extracellular ( EC ) domain is inhibitory ( see Table 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 00410 . 7554/eLife . 13876 . 005Figure 2—figure supplement 1 . ( A ) CHO cells do not express VEGFR-2 endogenously .", "( B ) CHO cells do not express VEGF endogenously .", "Here , CHO cells , HEK293T cells , and MECs ( microvascular endothelial ) cells ) were stained for VEGF-A .", "Lysates were reduced before loading . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 00510 . 7554/eLife . 13876 . 006Figure 2—figure supplement 2 . A single vesicle imaged and analyzed in the FRET , acceptor , and donor channels . Images were acquired with a Nikon laser scanning confocal microscope .", "The intensity across the membrane ( open blue symbols ) is fit to a Gaussian ( solid line ) after background correction .", "Shown also is the residual from the fit ( Chen et al . , 2010 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 00610 . 7554/eLife . 13876 . 007Figure 2—figure supplement 3 . Cross-linking of CHO cells expressing full-length wild-type VEGFR-2 . Cells were starved for 24 hr to ensure that there was no ligand present .", "Staining with anti-VEGFR-2 antibodies shows the presence of a glycosylated monomer band at MW ~240 kDa and glycosylated dimer band at MW ~480 kDa .", "These results support the FRET results that VEGFR-2 forms ligand-independent dimers in the plasma membrane . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 007 The fluorescent proteins YFP and mCherry were attached to the C-terminus of VEGFR-2 via a flexible GGS linker ( see Figure 1 ) , to allow for their free rotation ( Evers et al . , 2006 ) .", "CHO cells , which do not endogenously express measurable amounts of VEGFR-2 ( Figure 2—figure supplement 1 ) were co-transfected with plasmids encoding VEGFR-2-YFP and VEGFR-2-mCherry .", "Following VEGFR-2 expression and trafficking to the plasma membrane , the cells were vesiculated by applying osmotic stress .", "Each vesicle was imaged in the donor , acceptor , and FRET channels ( see Figure 2—figure supplement 2 ) .", "The FRET efficiency , the donor concentration , and the acceptor concentration in each individual vesicle were determined as described in Chen et al . ( 2010 ) and in 'Materials and methods' .", "The measured FRET efficiency for VEGFR-2 is shown as a function of acceptor concentration ( VEGFR-2-mCherry ) in Figure 2A ( red solid symbols ) .", "The solid black line shows the so-called 'bystander' or 'stochastic' FRET , which occurs due to random approach of donors and acceptors in the absence of specific interactions ( Wolber et al . , 1979 ) .", "The magnitude of stochastic FRET is well understood and characterized , both theoretically and experimentally ( King et al . , 2014 ) .", "The measured FRET efficiencies significantly exceed this stochastic FRET contribution , demonstrating the existence of specific interactions between the full-length VEGFR-2 molecules in the cell membrane .", "VEGFR-2 dimerization in the absence of ligand is supported by VEGFR-2 cross-linking data ( see Figure 2—figure supplement 3 ) .", "Consistent with data in the literature , two monomeric bands were observed , corresponding to partially and fully glycosylated VEGFR-2 variants .", "We also observed weak bands at twice the VEGFR-2 molecular weight , corresponding to cross-linked dimers .", "We are aware that cross-linking efficiencies not only depend on dimerization , but also on the presence of suitable reactive groups in close proximity .", "Thus , the fact that we observed bands corresponding to dimers , albeit weak , provides support for our FRET results documenting VEGFR-2 dimerization in the absence of ligand .", "The measured FRET in Figure 2A was corrected for the stochastic contribution , and the corrected FRET is plotted as a function of total receptor concentration in each vesicle in Figure 2B with the red solid symbols .", "The donor ( VEGFR-2-YFP ) concentration versus the acceptor ( VEGFR-2-mCherry ) concentration in each vesicle is shown in Figure 2C with the red solid symbols .", "The FRET data were then used to calculate the dimeric fraction as a function of total concentration as discussed in 'Materials and methods' and in previous publications ( Chen et al . , 2010 ) .", "A model describing monomer-dimer equilibrium was fitted to the single vesicle data , yielding the optimal values for the dimerization constant K and the Intrinsic FRET .", "The dissociation constant Kdiss=1/K , the dimerization free energy △G ( calculated using Equation 10 ) and the Intrinsic FRET value are shown in Table 1 .", "The best fit-dimerization curve , plotted for the optimal parameters , is shown in Figure 2D in red , along with the experimentally measured binned dimeric fractions . 10 . 7554/eLife . 13876 . 008Table 1 . Dimerization free energies , ΔG , and Intrinsic FRET efficiencies E~ , obtained from least-square parameter fits to the single-vesicle FRET data for the different VEGFR-2 constructs studied here .", "d is the distance between the fluorescent proteins in the dimers , calculated from the the Intrinsic FRET values under the assumption of free fluorescent protein rotation .", "The uncertainties are 67% confidence intervals ( standard errors ) calculated from the fit in Matlab . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 008VEGFR-2 variantDissociation constant , Kdiss ( rec . μm-2 ) △G ( kcal . mol-1 ) I-FRETd ( Å ) full length35 ( 16 to 53 ) -6 . 1 ( -5 . 8 to -6 . 5 ) 0 . 82 ( 0 . 78 to 0 . 85 ) 41 . 3 ( 39 . 8 to 43 ) EC+TM3200 ( 2712 to 3750 ) -3 . 4 ( -3 . 3 to -3 . 5 ) 0 . 61 ( 0 . 55 to 0 . 68 ) 49 . 3 ( 46 . 8 to 51 . 4 ) TM310 ( 220 to 415 ) -4 . 8 ( -4 . 6 to -5 . 0 ) 0 . 61 ( 0 . 59 to 0 . 65 ) 49 . 3 ( 47 . 9 to 50 . 0 ) EC+TM +VEGF-A121100% dimer100% dimer0 . 45 ± 0 . 0255 ± 1EC+TM +VEGF-A165a100% dimer100% dimer0 . 43 ± 0 . 0256 ± 1EC+TM +VEGF-A165b100% dimer100% dimer0 . 42 ± 0 . 0256 ± 1EC+TM +VEGF-C100% dimer100% dimer0 . 45 ± 0 . 0255 ± 1EC+TM +VEGF-D100% dimer100% dimer0 . 43 ± 0 . 0256 ± 1EC+TM +VEGF-E100% dimer100% dimer0 . 44 ± 0 . 0255 ± 1EC+TM ( E764I-T771I-F778I ) 100% dimer100% dimer0 . 31 ± 0 . 0261 ± 1EC+TM ( E764I-T771I-F778I ) + VEGF-A121100% dimer100% dimer0 . 38 ± 0 . 0258 ± 1EC+TM ( N762I-V769I-G770I ) 3100 ( 2090 to 3600 ) -3 . 4 ( -3 . 3 to -3 . 7 ) 0 . 61 ( 0 . 58 to 0 . 65 ) 49 . 3 ( 47 . 9 to 50 . 3 ) EC+TM ( N762I-V769I-G770I ) + VEGF-A121100% dimer100% dimer0 . 24 ± 0 . 0264 ± 1EC ( D4→D5 ) +TM390 ( 271 to 480 ) -4 . 7 ( -4 . 5 to -4 . 9 ) 0 . 82 ( 0 . 78 to 0 . 89 ) 41 . 4 ( 37 . 5 to 43 . 0 ) EC ( D4→D5 ) + TM+VEGF-A121210 ( 130 to 350 ) -5 . 0 ( -4 . 7 to 5 . 3 ) 0 . 83 ( 0 . 77 to 0 . 89 ) 41 . 0 ( 37 . 5 to 43 . 4 ) EC ( C482R ) +TM100% dimer100% dimer0 . 45 ± 0 . 0255 ± 1EC ( C482R ) +TM+VEGF-A121100% dimer100% dimer0 . 47 ± 0 . 0254 ± 1EC ( C482R ) +TM ( N762I-V769I-G770I ) 100% dimer100% dimer0 . 31 ± 0 . 0261 ± 1 The VEGFR-2 dimerization free energy , i . e . dimer stability , in the absence of ligand was -6 . 1 ± 0 . 4 kcal . mole-1 ( Table 1 ) .", "To evaluate the significance of VEGFR-2 dimerization in the absence of ligand under physiological conditions , we note that", "( i ) VEGFR-2 expression levels in endothelial cells are 104 to 105 copies per cell , corresponding to 10 to 100 receptors per square micron ( Napione et al . , 2012; Lee et al . , 2007; Imoukhuede et al . , 2011; 2012 ) , and that", "( ii ) the measured dimerization curve in Figure 2D shows 30% to 60% VEGFR2 dimers in this expression range .", "Thus , VEGFR-2 has a strong dimerization propensity even in the absence of ligand .", "Next , the contributions of the three VEGFR-2 domains , the EC , the TM , and the IC domain , to the stability of dimers were evaluated in the absence of ligand .", "Experiments were performed with two truncated VEGFR-2 constructs , one lacking the IC domain ( EC+TM construct ) and one lacking both the EC and IC domains .", "The VEGFR-2 TM domain construct contained a ( GGS ) 5 flexible linker , and a fluorescent protein .", "This type of attachment of the fluorescent probe to the TM domains has previously been used successfully and has been demonstrated not to affect dimerization propensity ( Sarabipour and Hristova , 2015 ) .", "The EC+TM construct also included the VEGFR-2 EC domain .", "Both truncated constructs included the VEGFR-2 signal peptide which is cleaved during receptor processing .", "The FRET results for these two constructs are shown in Figure 2 with the olive and black symbols and are summarized in Table 1 .", "The data given in Figure 2 show that:", "( i ) the isolated TM domain forms dimers ( dimer stability △G = -4 . 8 ± 0 . 2 kcal . mole-1 ) .", "( ii ) the EC domain inhibits dimerization ( △△G = +1 . 4 ± 0 . 3 kcal . mole-1 ) , such that the stability of the EC+TM construct dimer is reduced to -3 . 4 ± 0 . 2 kcal . mole-1 .", "This result is consistent with previous findings showing that the phosphorylation of VEGFR-2 is increased upon removal of the EC domain ( Manni et al . , 2014a ) .", "( iii ) The IC domains stabilize the dimer by -2 . 7 ± 0 . 3 kcal . mole-1 .", "Thus , we show that the EC domain inhibits VEGFR-2 dimerization in the absence of ligand , while the TM and IC domains enhance its dimerization propensity .", "The FRET data further show that the TM and EC+TM constructs exhibit the same Intrinsic FRET value , 0 . 61 , suggesting that the inclusion of the EC domain in these constructs does not change the separation between the fluorescent proteins in the dimer .", "Since the fluorescent proteins are attached directly to the TM domains via flexible linkers , this suggests that the addition of the EC domain does not have a measurable effect on the TM domain dimer .", "Our results thus far showed that a fraction of VEGFR-2 forms phosphorylated dimers in the absence of ligand , in a concentration dependent manner .", "Next , we investigated if ligand binding alters the structure of the VEGFR-2 dimer .", "We hypothesized that ligand-induced structural changes in the EC domain propagate to the TM domain , and we reasoned that such a structural change could manifest itself as a change in Intrinsic FRET for the EC+TM VEGFR-2 constructs , where the reporter fluorescent proteins are attached to the TM domains via ( GGS ) 5 flexible linkers .", "The Intrinsic FRET for EC+TM of VEGFR-2 in the absence of ligand was 0 . 61 ( Table 1 ) .", "We performed FRET experiments in the presence of very high , saturating ligand concentrations , expected to yield 100% ligand-bound dimers .", "Under these conditions , the measured FRET signal would be independent of the receptor concentration and , instead , would depend solely on the donor-to-acceptor ratio and on the value of the Intrinsic FRET .", "Thus , the Intrinsic FRET can be measured directly in individual vesicles .", "FRET data , measured in the presence of 3 μg . ml-1 of VEGF , are shown in Figure 3 .", "In Figure 3A , B , and C , we show single vesicle FRET results for the ligands VEGF-A121 and VEGF-A165a .", "Results for VEGF-A165b , VEGF-C , VEGF-D , and VEGF-E were very similar .", "Consistent with our hypothesis , the corrected FRET efficiency did not vary with receptor concentration , indicative of constitutive dimerization .", "The Intrinsic FRET value was thus calculated for each individual vesicle , by dividing the corrected FRET efficiency by the acceptor fraction ( see Equation 6 ) .", "Figure 3D shows histograms of the Intrinsic FRET values for single vesicles in the presence of the six ligands .", "The histograms are well described by Gaussian distributions .", "The center of the Gaussians , ~0 . 45 , corresponding to a separation between the fluorescent proteins by ~56 Å , was the same for all six ligands .", "Since the fluorescent proteins were attached to the TM domains via flexible linkers , we conclude that the TM domain configuration is similar in the presence of the six ligands .", "On the other hand , the binding of any of the ligands significantly changed Intrinsic FRET for the EC+TM construct from 0 . 61 to 0 . 45 , thus increasing the separation of the fluorescent proteins by 7 ± 1 Å , from 49 ± 2 Å to 56 ± 1 Å .", "This demonstrates a ligand-induced structural change in the VEGFR-2 dimer , which promotes an increase in the spatial separation of the TM domain C-termini . 10 . 7554/eLife . 13876 . 009Figure 3 . A conformational change in the TM domain dimer upon ligand binding increases VEGFR-2 phosphorylation .", "( A ) FRET efficiency measured as a function of acceptor concentration for EC+TM VEGFR-2 , in the absence of ligand and in the presence of 3 μg . ml-1 VEGF-A121 and VEGF-A165a .", "Two hundred to 500 individual vesicles were imaged in at least three independent experiments .", "Each data point corresponds to a single vesicle .", "The stochastic FRET contribution ( King et al . , 2014 ) is shown as a solid line .", "Black stars: FRET data without ligand .", "( B ) FRET efficiencies for individual vesicles , corrected for the stochastic FRET contribution .", "There is no dependence on receptor concentration , indicative of constitutive dimerization .", "( C ) Donor concentrations versus acceptor concentrations .", "( D ) Intrinsic FRET values , measured for VEGFR-2 EC+TM in the presence of 3 μg . ml-1 of VEGF-A121 , VEGF-A165a , VEGF-A165b , VEGF-C , VEGF-E , and VEGF-D .", "( E ) Graphical representation of the ligand-induced changes in distance between fluorescent proteins , and the inferred changes in TM domain structures .", "( F ) The six VEGF ligands increase VEGFR-2 phosphorylation , to the same extent .", "A representative Western Blot comparing the phosphorylation of Tyr 1175 in the absence of ligand , and in the presence of six VEGF ligands , at concentrations of 3 μg . ml-1 .", "Only the top bands , corresponding to mature fully glycosylated receptors , are considered here .", "Phosphorylation is significantly increased upon ligand addition , with the increase being as high as 10 times .", "( G ) Quantification of Western blot results for the fully glycosylated receptors ( top bands ) , in the presence of the six ligands .", "VEGFR-2 phosphorylation in the presence of the six ligands is very similar . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 009 We next compared the phosphorylation of full-length VEGFR-2 in the absence of ligand , and in the presence of 3 μg . ml-1 VEGF-A121 , VEGF-A165a , VEGF-A165b , VEGF-C , VEGF-E , and VEGF-D .", "Figure 3F/G shows that receptor phosphorylation is the same for all six ligands , and increases as much as 10-fold compared to the phosphorylation of VEGFR-2 in the absence of ligand .", "The FRET and activation results thus clearly demonstrate a correlation between EC+TM VEGFR-2 dimer structure and receptor activation: the lower the Intrinsic FRET , that is , the larger the separation between the TM domain C-termini , the higher the receptor phosphorylation .", "Furthermore , the identical Intrinsic FRET values observed in the presence of the six ligands at saturating concentrations correlate with identical VEGFR-2 phosphorylation .", "Thus , the TM domain structure exerts control over VEGFR-2 phosphorylation and activation .", "The structure of the isolated wild-type VEGFR-2 TM domain dimer was recently solved in lipid bicelles ( Manni et al . , 2014b ) .", "To evaluate whether this structure agrees with the FRET data , we created two sets of EC+TM mutant constructs tagged with YFP and mCherry .", "First , amino acids E764 , T771 , and F778 were mutated to Ile to create the E764I-T771I-F778I mutant .", "These three amino acids stabilize the TM helix contacts in the NMR-derived TM dimer model ( Manni et al . , 2014b ) .", "Separately , a N762I-V769I-G770I mutant was generated from the EC+TM-YFP and EC+TM-mCherry constructs .", "These residues do not contribute to the TM domain dimer interface in the NMR-derived model as they face the lipid side instead .", "The FRET results for the two EC+TM mutants are summarized in Figure 4 ( raw FRET data are shown in Figure 4—figure supplement 1 ) .", "Dimerization in the absence of ligand was enhanced and the Intrinsic FRET was altered for the E764I-T771I-F778I mutant , as compared to wild-type .", "Thus , the E764I-T771I-F778I set of mutations affected the structure of the TM dimer in the absence of ligand .", "On the other hand , the N762I-V769I-G770I mutations had no effect on the FRET efficiencies measured for these constructs in the absence of ligand , suggesting that the published NMR structure of the isolated wild-type TM domain dimer represents the TM dimer configuration of the unliganded EC+TM constructs embedded in the plasma membrane . 10 . 7554/eLife . 13876 . 010Figure 4 . The published NMR structure of the isolated VEGFR-2 TM domain ( Manni et al . , 2014b ) is consistent with the unliganded TM dimer structure observed in the FRET experiments .", "( A ) Amino acid sequence of wild-type VEGFR-2 TM domain .", "The amino acids that mediate helix-helix contacts in the NMR dimer structure ( Manni et al . , 2014b ) are shown in red .", "( B ) The NMR structure , with two sets of amino acids highlighted .", "Left: a space-fill model of the wild-type VEGFR-2 TM dimer , based on NMR experiments .", "Center: E764 , T771 , and F778 help mediate helix-helix contacts in the NMR structure ( Manni et al . , 2014b ) ( also shown bold and underlined in ( A ) ) .", "Right: N762 , V769 , and G770 face away from the dimer interface , into the membrane .", "Two sets of mutations: a E764I-T771I-F778I set and a N762I-V769I-G770I set , were engineered .", "Table: Results of FRET experiments for the two sets of mutants ( see also Table 1 ) .", "The dimerization of the unliganded EC+TM construct was affected by the E764I-T771I-F778I but not by the N762I-V769I-G770I set of mutations , suggesting that the unliganded VEGFR-2 TM dimer is stabilized by contacts involving E764 , T771 , and/or F778 , as in the NMR structure .", "At least one of the amino acids N762 , V769 , and G770 forms direct intermolecular contacts in the TM domain in the ligand-bound state . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 01010 . 7554/eLife . 13876 . 011Figure 4—figure supplement 1 . FRET data , reporting on the effects of the E764I-T771I-F778I and N762I-V769I-G770I sets of mutations , engineered in the EC+TM VEGFR-2 plasmids . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 01110 . 7554/eLife . 13876 . 012Figure 4—figure supplement 2 . All mutations in the TM domain decrease VEGFR-2 phosphorylation . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 012 The N762I-V769I-G770I mutant significantly affected the Intrinsic FRET of the EC+TM dimer in the VEGF-A121 bound state .", "The ligand-induced phosphorylation of the full-length N762I-V769I-G770I mutant was lower as compared to wild-type VEGFR-2 ( Figure 4—figure supplement 2 ) .", "These findings suggest that", "( i ) at least one of the amino acids N762 , V769 , or G770 mediate helix-helix contacts in the ligand-bound state , and", "( ii ) the conformation of the ligand-bound EC+TM dimer differs from that observed in the wild-type VEGFR-2 TM domain NMR structure .", "Our mutagenesis data thus show that different contacts between the TM domains stabilize the unliganded and the ligand-bound VEGFR-2 dimers .", "The data also provide direct experimental support for the concept that the TM domain dimer switches between two different configurations when transitioning from the unliganded to the ligand-bound state , as suggested previously ( Manni et al . , 2014b ) .", "The current model of VEGFR-2 activation postulates that the ligand-bound EC domain dimer is stabilized by ligand binding to domains D2 and D3 .", "In addition , homotypic contacts between Ig domains D4-D7 have been observed in the isolated ligand-bound dimeric EC domains ( Ruch et al . , 2007 ) .", "These contacts were proposed to promote and stabilize a specific conformation of the two receptor monomers in ligand-bound VEGFR-2 dimers .", "Mutagenesis demonstrated that these contacts are indispensable for activation of the intracellular kinase domains ( Hyde et al . , 2012 ) .", "Here , we investigated how D4 and D7 affect dimerization of the EC +TM construct in the ligand-free and ligand-bound states .", "We examined two EC+TM VEGFR-2 mutants in which D4 or D7 were substituted with unrelated domains from VEGFR-1 , D4 ( VEGFR-2 ) →D5 ( VEGFR-1 ) and D7 ( VEGFR-2 ) →D6 ( VEGFR-1 ) .", "FRET data for these mutant constructs are shown in Figure 5 .", "The FRET efficiencies of the mutants were much higher than for the wild-type EC+TM construct indicating a change in dimer stability and/or conformation . 10 . 7554/eLife . 13876 . 013Figure 5 . The D4 and D7 mutants do not respond to ligand .", "( A ) FRET measurements for the D4 and D7 mutants , in the absence of ligand and in the presence of VEGF-A121 .", "The two mutants exhibit much higher FRET efficiencies , compared to the wild-type .", "The ligand , at 3 μg . ml-1 , has no effect on receptor dimerization .", "( B ) VEGF-A121 does not bind to the D4→D5 mutant , when it binds to the wild-type and the D7→D6 mutant .", "Here , a single vesicle containing YFP-tagged mutant receptors ( two top rows ) or wild-type receptors ( bottom row ) is shown after incubation with 3 μg . ml-1 AlexaFluor 594-labeled VEGF-A ( VEGF-A121-AF594 ) ( SibTech Inc . , CT ) .", "Note the differences in the middle column , showing the fluorescence of the bound ligand .", "( C ) Western blots comparing the phosphorylation of the full-length D7→D6 mutant in the absence and presence of VEGF-A121 .", "The top band corresponds to the mature fully glycosylated form of VEGFR-2 .", "The ligand does not increase the phosphorylation . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 01310 . 7554/eLife . 13876 . 014Figure 5—figure supplement 1 . Dimerization curves for the wild-type VEGFR-2 in the absence of ligand , the D4→D5 mutant in the absence of ligand , and the D4→D5 mutant in the presence of VEGF-A121 . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 014 A monomer-dimer equilibrium model was successfully fitted to the FRET data for the EC+TM VEGFR-2 D4 ( VEGFR-2 ) →D5 ( VEGFR-1 ) construct ( Figure 5—figure supplement 1 ) .", "Dimerization of the D4 mutant was stronger than for the wild type , and the mutant dimer exhibited much higher Intrinsic FRET .", "Thus , the substitution of the D4 domain caused a structural change in the unliganded VEGFR-2 EC domain favoring non-productive dimer formation in the absence of ligand .", "On the other hand , a monomer-dimer model could not be fitted to the D7 mutant data .", "This is indicative of the formation of higher order oligomers ( King et al . , 2016 ) .", "Thus , the substitution of both D4 and D7 significantly altered the association of the VEGFR-2 molecules in the absence of ligand .", "The FRET efficiencies measured for the two mutants remained unchanged upon treatment with saturating amounts ( 3 μg . mL-1 ) of VEGF-A121 ( Figure 5A ) , suggesting that neither of the mutants respond to ligand .", "To investigate whether these mutants were capable of binding ligand , we incubated the vesicles expressing these mutants with fluorescently labeled ligand ( VEGF-A121-AF594 ) .", "In the case of ligand binding , vesicle fluorescence would be observed both in the green channel , because of the presence of wild-type or mutant VEGFR-2-YFP , and in the red channel , because of VEGF-A121-AF594 binding to the receptor .", "No fluorescence would be observed in the red channel , however , if ligand does not bind .", "The results in Figure 5B showed that ligand binds to the D7 mutant and to the wild-type receptor , but not to the D4 mutant .", "We thus conclude that substitutions of D4 and D7 domains rendered the VEGFR-2 unresponsive to ligand , but in different ways .", "The D4 mutant dimers were structurally different from wild-type and were incapable of binding ligand .", "The D7 mutants formed oligomers in the absence of ligand , and ligand binding did not reverse oligomerization and did not promote transition to the correctly assembled ligand-bound dimeric state of the receptor .", "The latter conclusion is supported by results in Figure 5C , showing that ligand binding to the D7 mutant does NOT increase the phosphorylation of the D7 mutant .", "Taken together , results in the absence and presence of ligand demonstrate that domains D4 and D7 are essential for establishing the properly oriented unliganded , as well as ligand-bound , VEGFR-2 dimers .", "We therefore propose that the unliganded state is a critically important intermediate in VEGFR-2 activation .", "The C482R mutation in D5 of VEGFR-2 has been implicated in infantile hemangioma , characterized by disorganized angiogenesis in infants ( Boye et al . , 2009 ) .", "The FRET data for the mutant are shown in Figure 6 A–D .", "The fraction of dimeric C482R EC+TM receptor was similar in the presence and absence of ligand and was not dependent on receptor concentration .", "The Intrinsic FRET values measured in the presence and absence of ligand were the same , and similar to those measured for ligand-bound wild-type VEGFR-2 dimers .", "Thus , it appears that this mutation locked the VEGFR-2 dimer in the ligand-bound conformation in the absence of ligand .", "Consistent with these structural observations , the phosphorylation in the presence and absence of ligand was the same ( Figure 6E ) , despite the fact that this mutant was capable of binding fluorescent ligand ( Figure 6F ) .", "The data in Figure 6 therefore demonstrate the formation of constitutive C482R mutant dimers both in the absence and the presence of ligand .", "They also reveal a lack of structural transition upon ligand binding .", "Importantly , the phosphorylation of the mutant was higher than the phosphorylation of the wild-type in the absence of ligand ( Figure 6E ) , demonstrating that this mutation causes pathological ligand-independent constitutive VEGFR-2 activation . 10 . 7554/eLife . 13876 . 015Figure 6 . The C482R mutation mimics the effect of bound ligand by promoting a structural change in the EC+TM VEGFR-2 dimer .", "( A ) FRET efficiencies determined in individual plasma-membrane-derived vesicles .", "( B ) Corrected FRET as a function of receptor concentration .", "The corrected FRET for the mutant does not depend on receptor concentration , demonstrating that the mutant is a constitutive dimer in the presence and absence of ligand .", "( C ) Measured donor versus acceptor concentrations in each vesicle .", "( D ) Histograms of measured FRET efficiencies for the mutant , yielding Intrinsic FRET values of ~0 . 42 .", "( E ) Western blots showing an increase in phosphorylation due to the C482R mutation , in the absence of ligand , and no further increase upon ligand treatment .", "The top bands correspond to the mature fully glycosylated form of VEGFR-2 .", "( F ) Confocal images of VEGF-A121-AF594 binding to EC+TM VEGFR-2 C482R in CHO membrane vesicles , demonstrating that the C482R mutant is capable of ligand binding . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 015 To confirm that the unliganded C482R dimer structure is similar to the ligand-bound wild-type VEGFR-2 dimer structure , we engineered the N762I-V769I-G770I mutations in the C482 mutant construct .", "The FRET data for the mutant EC+TM , shown in Figure 7A and B , demonstrate that the TM domain mutations altered the Intrinsic FRET for the C482 mutant , suggesting that at least one of these amino acids mediate helix-helix contacts in the C482 mutant , reminiscent of the case of the ligand-bound wild-type dimer .", "Consistent with this result , the TM domain mutations decreased the phosphorylation of the full-length C482R mutant receptor , as observed for the ligand-bound wild-type VEGFR-2 dimer ( Figure 7 C ) . 10 . 7554/eLife . 13876 . 016Figure 7 . The N762I-V769I-G770I set of mutations in the TM domain of the C482R mutant receptor alters dimer structure and activity , as in the case of the ligand-bound wild-type ( see Figure 4 ) .", "( A ) Corrected FRET efficiencies for the EC+TM C482R-N762I-V769I-G770I mutant as a function of receptor concentration , indicative of constitutive dimers .", "( B ) Histograms of Intrinsic FRET measured for the EC+TM C482R mutant and the EC+TM C482R-N762I-V769I-G770I mutant .", "The TM domain mutations alter Intrinsic FRET and thus the structure of the C482R dimer .", "( C ) The N762I-V769I-G770I set of mutations obliterate the activity of the C482R mutant , as in the case of the ligand-bound wild type . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 01610 . 7554/eLife . 13876 . 017Figure 7—figure supplement 1 . Left: Western blots under reducing conditions , for full length C482R VEGFR-2 and for the C482R EC+TM-YFP VEGFR-2 construct . Right: Western blots under non-reducing conditions .", "No dimeric bands were observed under any conditions .", "The constitutive dimerization of the C482R mutant is not due to cysteine-induced intermolecular cross-linking mediated by unpaired cysteines . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 017 We next investigated whether intermolecular disulfide bonds between possible unpaired cysteine residues in the full-length and EC+TM C482R mutant receptors promote constitutive dimerization of the receptor .", "We analyzed the dimerization state of the receptors under reducing and oxidizing conditions using Western blotting .", "The results , shown in Figure 7—figure supplement 1 , demonstrate that receptors were not disulfide-linked both in the presence and absence of reducing agent .", "Thus , the C482 mutation did not lead to direct intermolecular disulfide bond formation , which suggests that the structural effects of the C482R mutation are more complex .", "Perhaps , this mutation alters the protein fold .", "In addition , this mutation may affect interactions with other proteins such as co-receptors ." ], [ "VEGFR-2 has been believed to exist in a monomeric form in the absence of ligand and has been hypothesized to follow the canonical model of ligand-induced dimerization and activation ( Yang et al . , 2010; Hyde et al . , 2012; Koch et al . , 2011; Matsumoto et al . , 2001; Olsson et al . , 2006 ) .", "Using a quantitative FRET-based methodology , specifically designed to probe receptor interactions in the plasma membrane , we demonstrated that full-length VEGFR-2 forms dimers in the absence of ligand .", "A substantial fraction of the receptors is dimeric for receptor expression levels reported for endothelial cells ( 10 to 100 receptors per square micron ) .", "Furthermore , these unliganded dimeric receptors are phosphorylated , albeit to a low degree .", "The basal phosphorylation occurs on Y1054/1059 and Y1175 ( Figure 6E ) , as well as on Y1214 ( Lamalice et al . , 2006 ) .", "Consistent with these observations , phosphorylation of downstream kinases has been reported in endothelial cells in the absence of ligand ( Oubaha et al . , 2009; Labrecque et al . , 2003 ) .", "Recent work has demonstrated that other RTKs , such as EGFR and the FGF receptors ( Chung et al . , 2010; Low-Nam et al . , 2011; Sarabipour and Hristova , 2016; Lin et al . , 2012; Comps-Agrar et al . , 2015 ) also form unliganded dimers .", "A new model of RTK activation , which includes unliganded dimers of various stabilities as intermediates , has been proposed ( Sarabipour and Hristova , 2016 ) .", "The behavior of VEGFR-2 observed here is consistent with this new model .", "Through mutagenesis , domain deletions and domain substitutions , our study provides insights into the interactions that stabilize VEGFR-2 dimers in the plasma membrane in the absence of ligand .", "First , there are sequence-specific contacts between the TM domains in the dimers .", "In particular , at least one of the amino acids E764 , T771 , or F778 participates in helix-helix contacts , as the substitution of these three amino acids with Ile changed the structure and the stability of the EC+TM VEGFR-2 dimers in the absence of ligand .", "Second , these dimers are further stabilized by contacts between the IC domains , since the removal of the IC domains decreased dimer stability by 2 . 7 kcal . mole-1 .", "Therefore , specific interactions between the IC domains regulate receptor dimerization and ensure control of receptor activity in the absence of ligand .", "Thus , our observations that VEGFR-2 phosphorylation can take place in the absence of ligand , along with the FRET results , point to a significant role of the IC domains in dimer stabilization .", "Third , we discovered a new role for domains D4 and D7 in unliganded VEGFR-2 dimerization , by showing that they stabilize VEGFR-2 dimers in the absence of ligand .", "Indeed , the substitution of Ig-domains D4 or D7 significantly altered both dimerization and Intrinsic FRET in the absence of ligand .", "Fourth , we showed that the wild-type VEGFR-2 EC domain , as a whole , inhibits receptor dimerization , as inferred from the fact that deletion of the entire EC domain increased the stability of the dimers by 1 . 4 kcal . mole-1 .", "Overall , this work documents a complex role for the EC domain in VEGFR-2 dimer stabilization and establishes the existence of specific dimer stabilizing contacts in the EC , the TM , and the IC domains of VEGFR-2 .", "To monitor ligand-induced conformational changes in the TM domain , we performed experiments with truncated VEGFR-2 constructs carrying fluorescent proteins attached via flexible ( GGS ) 5 linkers to the C-termini of the TM domains .", "The Intrinsic FRET decreased from 0 . 61 to 0 . 42 , corresponding to an increase in separation between the fluorescent proteins in these dimers from 49 to 56 Å , upon ligand binding .", "This reflects an increase in the spatial separation between the TM domain C-termini by 7 Å in response to ligand binding .", "Through mutagenesis analysis , we gained information about the structural changes in the TM domain of the VEGFGR-2 dimers that occur in response to ligand binding ( see Figure 8 ) .", "The configuration of the TM domain of the ligand-free EC+TM VEGFR-2 dimer was consistent with the published NMR spectroscopy-derived structural model of isolated wild-type VEGFR-2 TM domain dimers in lipid bicelles ( Manni et al . , 2014b ) .", "Ligand binding induces a rotation of the TM helices , leading to the engagement of at least one of the amino acids N762 , V769 , G770 in the helix-helix interface .", "These residues were chosen for mutagenesis because they face away from the TM helix interface in the NMR-derived model for the wild-type ( Manni et al . , 2014b ) .", "Substitution of these residues by Ile had no effect on dimerization in the absence of ligand , but had a significant effect in the presence of ligand . 10 . 7554/eLife . 13876 . 018Figure 8 . Proposed model for VEGFR-2 activation . VEGFR-2 pre-dimerizes in the absence of ligand .", "Under physiological conditions , corresponding to 10 to 100 VEGFR-2 molcules per square micron , 30 to 60% of the receptors are dimeric .", "The dimers are stabilized by homotypic contacts in D4-7 in the EC domain , TM interactions that involve amino acids E764 , T771 , and/or F778 , and contacts between the intracellular domains .", "The EC domain , as a whole , inhibits dimerization .", "The ligand-free dimers are phosphorylated , to a low degree .", "( B ) Ligand binding induces a rotation in the TM helices and the formation of a different TM dimer configuration , such that amino acids E764 , T771 , and F778 now face the lipid membrane , away from the TM dimer interface .", "These structural changes increase the separation between the C-termini of the TM helices , promoting an increase in receptor phosphorylation and activation .", "Contacts between D4 and D7 persist in the ligand-bound state . DOI: http://dx . doi . org/10 . 7554/eLife . 13876 . 018 The TM domains link the ligand-binding EC domains to the IC domains and are thus expected to play a role in the activation of the intracellular kinase in response to ligand binding .", "Previous NMR studies of in vitro produced RTK TM peptides , inserted into lipid bicelles or detergent micelles , have suggested the existence of two sets of mutually exclusive helix-helix contacts ( Bocharov et al . , 2008; Li et al . , 2010; Fleishman et al . , 2002 ) .", "These observations have been interpreted as evidence that RTK TM domains can form at least two structurally and functionally different dimers .", "Yet , there have been no direct experimental data demonstrating a structural change in the VEGFR-2 TM domain dimer configuration in the plasma membrane , in response to ligand binding , so far .", "Here , we directly observed such a structural change using our quantitative FRET methodology which allowed us to monitor the response of the receptor dimer to ligand binding from the cytoplasmic side of the membrane .", "FRET is extremely sensitive to the distance between the fluorescent proteins , which we measure with 1 Å precision .", "The observed changes in FRET are biologically significant , as the increase in separation between the C-termini of the TM domains correlated with an increase in receptor phosphorylation and thus with receptor activity .", "Our results clearly demonstrate that ligand binding increases VEGFR-2 phosphorylation , which correlates with a conformational change in the TM domain dimer structure .", "A similar mechanism of activation , involving an increase in TM helix C-termini separation , has been proposed for EGFR ( Bocharov et al . , 2008; Arkhipov et al . , 2013; Endres et al . , 2013 ) .", "For the FGF receptors , however , the ligand-induced changes involve a decrease in the separation between the TM domain C-termini ( Sarabipour and Hristova , 2016 ) .", "Decrease in the separation of the TM domains upon ligand binding has also been observed for the constitutively dimeric IGF-1R ( Kavran et al . , 2014 ) .", "Thus , it appears that ligand-induced structural changes are a general feature of RTK activation , but the exact nature of the structural change varies for the different receptors .", "We compared the structural effects of several ligands known to bind to VEGFR-2 , namely three different VEGF-A isoforms ( VEGF-A121 , VEGF-A165a , and VEGF-A165b ) , as well as VEGF-C , VEGF-D , and VEGF-E .", "Our experiments were conducted at very high , saturating ligand concentrations of 3 μg . ml-1 , when ligand concentrations exceeded both the receptor concentrations and the ligand binding coefficients by at least a factor of 100 .", "The FRET efficiency did not depend on receptor concentration , suggesting that all receptors were in the ligand-bound dimeric state .", "We measured the same Intrinsic FRET for EC+TM VEGFR-2 in the presence of all six ligands .", "Furthermore , these ligands gave rise to the same VEGFR-2 activities as determined by Western blotting with phospho-specific antibodies .", "We thus propose that the six ligands induced the same structural changes in the receptor , resulting in a similar VEGFR-2 phosphorylation increase .", "The VEGFR-2 case is thus quite different from the case of the FGFR dimers , which exhibit different structural and biological responses to the ligands fgf1 and fgf2 ( Sarabipour and Hristova , 2016 ) .", "The VEGF ligands are known to have distinct affinities for VEGFR-2 ( Brozzo et al . , 2012; Mac Gabhann et al . , 2010; Qutub et al . , 2009 ) .", "Thus , for VEGFR-2 it is reasonable to assume that binding affinity may determine the specific signal output by VEGFR-2 in response to different ligands .", "Alternatively , different intracellular trafficking of the ligand-bound receptors may dictate signaling specificity ( Clegg et al . , 2015; Ballmer-Hofer et al . , 2011 ) .", "The mutations and substitutions used here to probe VEGFR-2 dimer stability , structure , and activation , promoted significant changes in VEGFR-2 structure and function .", "For instance , the D4 substitution stabilized the EC+TM VEGFR-2 dimer , altered its structure , and abolished ligand binding .", "The D7 substitution , on the other hand , caused receptor oligomerization that rendered the receptor non-responsive to ligand .", "The E764I-T771I-F778I TM domain mutations stabilized the VEGFR-2 dimer in the absence of ligand and changed its structure as reflected by Intrinsic FRET , but did not increase receptor phosphorylation .", "The N762I-V769I-G770I mutations altered the structure of the ligand-bound VEGFR-2 dimer and reduced receptor phosphorylation .", "Quite remarkably , none of the engineered mutations increased VEGFR-2 phosphorylation , suggesting that VEGFR-2 is surprisingly resistant to aberrant activation .", "The only mutation that caused an increase in phosphorylation was the pathogenic C482R mutation , identified in infant hemangiomas ( Boye et al . , 2009 ) .", "Our studies therefore demonstrated that the C482R mutation is a gain-of-function mutation .", "To the best of our knowledge , this is the first report of a pathogenic activating mutation in VEGFR-2 and the first mechanistic study of such a mutation .", "Our results demonstrate that the C482R mutant formed constitutive dimers even in the absence of ligand .", "Western blots performed under reducing and non-reducing conditions revealed that there are no disulfide-linked C482R dimers .", "Instead , quantitative FRET experiments revealed that the constitutive C482R dimers adopt the ligand-bound wild-type dimer conformation , both in the presence and absence of ligand .", "Thus , the C482R mutation increased VEGFR-2 activation by mimicking the structural effect of the ligand .", "Similar behavior has been observed for the pathogenic A391E , R248C , and S249C FGFR3 mutants , linked to skeletal and cranial growth disorders ( Sarabipour and Hristova , 2016; Del Piccolo et al . , 2015 ) .", "Thus , this mechanism of over-activation may apply to RTKs from diverse families ." ], [ "The plasmids encoding for full length wild-type human VEGFR-2 , full length C482R mutant , and full-length VEGFR-2 containing TM mutations , used in the Western Blot experiments , were constructed in the pBE vector .", "The full-length D7 ( VEGFR-2 ) →D6 ( VEGFR-1 ) mutant was in the pcDNA5/FRT vector .", "The full-length D4 ( VEGFR-2 ) →D5 ( VEGFR-1 ) mutant was in the pcDNA3 vector .", "All truncated VEGFR-2 plasmid constructs were engineered in the pcDNA 3 . 1 ( + ) vector .", "All the plasmids contained the N-terminal VEGFR2 signal sequence , which is cleaved off during processing .", "All primers were purchased from Invitrogen ( Calsbad , CA ) .", "The pRSETB-mCherry and pRSETB-YFP plasmids were received from Dr . M . Betenbaugh ( Johns Hopkins University , Baltimore , MD ) and Dr . R . Tsien ( University of California , San Diego ) , respectively .", "Both fluorescent proteins are monomeric and do not drive interactions in the membrane ( King et al . , 2014 ) YFP and mCherry were fused to the C-terminal end of all VEGFR2 constructs .", "To generate the VEGFR-2- ( GGS ) -mCherry and VEGFR-2- ( GGS ) -YFP plasmids , a MluI restriction site was created in the multiple cloning site of the pBE vector , which contained the full length human VEGFR-2 insert .", "The multiple cloning site was located right after the 3’ end of the VEGFR-2 sequence .", "The cDNAs encoding YFP and mCherry were then amplified using Polymerase Chain Reaction ( PCR ) and double digested with MluI and XhoI .", "The double digested YFP and mCherry cDNAs were then ligated with the digested pBE-VEGFR cDNA .", "The sequence encoding for the EC and TM domains of VEGFR-2 were amplified by PCR , double digested using HindIII and EcoRI restriction enzymes , and inserted into a pcDNA3 . 1 ( + ) vector containing the flexible 15 amino acids ( GGS ) 5 linker and YFP or mCherry , producing the VEGFR-2 EC+TM- ( GGS ) 5-YFP and VEGFR-2 EC+TM- ( GGS ) 5-mCherry plasmids .", "The C482R mutant plasmid constructs: VEGFR-2 ( C482R ) , VEGFR2 EC ( C482R ) +TM- ( GGS ) 5-YFP , and VEGFR-2 EC ( C482R ) +TM- ( GGS ) 5-mCherry were created using the QuikChange II XL Site-Directed Mutagenesis Kit ( Stratagene , CA ) from the wild-type constructs .", "The full-length D4 ( VEGFR-2 ) →D5 ( VEGFR-1 ) construct was generated by replacing Ig domain 4 of VEGFR-2 with Ig domain 5 of VEGFR-1 .", "The full-length D7 ( VEGFR-2 ) →D6 ( VEGFR-1 ) construct was created by replacing domain 7 of VEGFR-2 with domain 6 of VEGFR-1 ( detailed in [Hyde et al . , 2012] ) .", "To construct the VEGFR-2 EC ( D4→D5 ) +TM- ( GGS ) 5-YFP and VEGFR-2 EC ( D4→D5 ) +TM- ( GGS ) 5-mCherry plasmids , cDNA encoding EC ( D4→D5 ) +TM was amplified from the full-length VEGFR-2 ( D4→D5 ) plasmid construct .", "This cDNA was double digested with Hind III and EcoRI .", "The digested cDNA was then ligated with pcDNA3 . 1 ( + ) - ( GGS ) 5-YFP or pcDNA3 . 1 ( + ) - ( GGS ) 5-mCherry , doubly digested with Hind III and EcoRI .", "To construct the VEGFR-2 EC ( D7→D6 ) +TM- ( GGS ) 5-YFP and VEGFR-2 EC ( D7→D6 ) +TM- ( GGS ) 5-mCherry plasmids , cDNA encoding EC ( D7→D6 ) +TM was amplified from the VEGFR-2 ( D7→D6 ) plasmid construct .", "This cDNA was double digested with Hind III and EcoRI .", "Finally , the digested cDNA was ligated with pcDNA3 . 1 ( + ) - ( GGS ) 5-YFP or pcDNA3 . 1 ( + ) - ( GGS ) 5-mCherry , doubly digested with Hind III and EcoRI .", "To generate the VEGFR-2 TM- ( GGS ) 5-YFP and VEGFR-2 TM- ( GGS ) 5-mCherry plasmids , the sequence encoding the TM domain of VEGFR-2 ( GAQEKTNLEIIILVGTAVIAMFFWLLLVIILRTVKR ) was amplified using PCR .", "This cDNA was then double digested with HindIII and EcoRI and ligated with digested pcDNA3 . 1 ( + ) - ( GGS ) 5-YFP or pcDNA3 . 1 ( + ) - ( GGS ) 5-mCherry .", "The N762I-V769I-G770I , E764I-T771I-F778I and C482R-N762I-V769I-G770I full length and truncated mutant VEGFR-2 constructs: VEGFR-2 ( N762I-V769I-G770I ) , VEGFR-2 ( E764I-T771I-F778I ) , VEGFR-2 ( C482R-N762I-V769I-G770I ) , VEGFR-2 EC + TM ( N762I-V769I-G770I ) - ( GGS ) 5-YFP , VEGFR-2 EC+TM ( N762I-V769I-G770I ) - ( GGS ) 5-mCherry , VEGFR-2 EC+TM ( E764I-T771I-F778I ) - ( GGS ) 5-YFP , VEGFR-2 EC +TM ( E764I-T771I-F778I ) - ( GGS ) 5-mCherry , VEGFR-2 EC ( C482R ) +TM ( N762I-V769I-G770I ) - ( GGS ) 5-YFP , and VEGFR-2 EC ( C482R ) +TM ( N762I-V769I-G770I ) - ( GGS ) 5-mCherry plasmids were created using the QuikChange II XL Site-Directed Mutagenesis Kit ( Stratagene , CA ) from the wild-type constructs .", "Chinese Hamster Ovary cell ( CHO ) cells were cultures at 37˚C with 5% CO2 for 24 hr .", "Transfection was carried out using either Fugene HD or Lipofectamine , following the manufacturer’s protocol .", "Cells were cotransfected with 3–7 ug of DNA encoding VEGFR2-YFP and VEGFR2-mCherry constructs .", "Cells were vesiculated 24 hr post transfection .", "Vesiculation was performed using a chloride salt vesiculation buffer ( Del Piccolo et al . , 2012 ) consisting of of 200 mM NaCl , 5 mM KCl , 0 . 5 mM MgSO4 , 0 . 75 mM CaCl2 , 100 mM bicine and protease inhibitor cocktail ( Complete mini EDTA-free tabs , Roche Applied Science ) adjusted to pH of 8 . 5 .", "CHO cells were rinsed twice with 30% PBS ( pH 7 . 4 ) , and incubated with 1 mL of chloride salt vesiculation buffer overnight at 37°C .", "Vesicles were produced after 12 hr , and transferred into four-well Nunc Lab-Tek II chambered coverslips for imaging .", "The plasma membrane-derived vesicles produced with this method have been characterized previously ( Sarabipour et al . , 2015 ) .", "They contain other membrane proteins and all the major lipid species found in the plasma membranes of mammalian cells , but lack cytoplasmic content .", "One ml of vesicle solution was collected in a well of a 4 chamber slide and treated with 3000 ng . mL-1 of either human VEGF-A121 ( #8908 , Cell Signaling Technologies ) , human VEGF-A165a ( #8065 , Cell Signaling Technologies ) , human VEGF-C ( #100-20C , PeproTech ) , human VEGF-D ( #100-20D , PeproTech ) , or human VEGF-A165b and VEGF-E , purified as described in Scheidegger et al . ( 1999 ) .", "After 1 hr incubation with ligands at room temperature , the vesicles were imaged .", "The QI-FRET method has been described in detail previously ( Chen et al . , 2010; Del Piccolo et al . , 2015; Sarabipour and Hristova , 2015 ) .", "Vesicles were imaged using a Nikon Eclipse confocal laser scanning microscope using a 60× water immersion objective .", "All the images were collected and stored at a 512 × 512 resolution .", "Three different scans were performed for each vesicle: ( 1 ) excitation at 488 nm , with a 500–530 nm emission filter ( donor scan ) ; ( 2 ) excitation at 488 nm , with a 565–615 nm emission filter ( FRET scan ) ; and ( 3 ) excitation at 543 nm , with a 650-nm longpass filter ( acceptor scan ) .", "Solutions of YFP and mCherry , which served as concentration calibration controls , were purified as described ( Sarabipour et al . , 2014 ) , concentrated in vesiculation buffer , and imaged in the donor , acceptor and FRET channels .", "Using these solution standards , we determined the calibration constants for the donor and the acceptor , iD and iA , and the bleed-through coefficients for the donor and the acceptor , βD and βA as previously described ( Li et al . , 2008 ) .", "Vesicles loaded with a soluble linked YFP-mCherry protein were also imaged in the three channels to obtain the gauge factor GF ( Li et al . , 2008 ) .", "The acceptor concentrations in each vesicle , CA , was calculated according to: ( 1 ) CA=IAiA where IA is the intensity captured in the acceptor channel .", "The sensitized emission of the acceptor in each vesicle was determined as: ( 2 ) ISEN=IFRET−βAIA−βDID Next , we calculate: ( 3 ) ID , corr=ID+GFISEN ( 4 ) CD=ID , corriD where ID , corr is the reconstructed donor intensity in the absence of the acceptor , and CD is the donor concentration .", "The FRET efficiency , E , was calculated according to: ( 5 ) E=1−IDID , corr The FRET efficiency was corrected for the so-called proximity FRET , which occurs when donors and acceptors approach each other by chance within distances of 100 Å or so .", "This correction has been discussed in detail previously ( King et al . , 2014 ) , and is required because the fluorescent proteins are confined to the two-dimensional vesicles .", "The dimeric fraction is calculated from the corrected FRET , ED , and the acceptor fraction , xA , according to: ( 6 ) fD=EDxAE~ Here E~ is the 'Intrinsic FRET' , the FRET efficiency in a dimer containing a donor and an acceptor .", "This is a purely structural parameter , which depends only on the separation and the orientation of the two fluorescent proteins in the dimer , d , but not on the dimerization propensity .", "( 7 ) E˜=11+ ( dR0 ) 6 Here d is the distance between the acceptor and the donor in the dimer , and Rois the Förster radius of the FRET pair ( Chen et al . , 2010 ) .", "For YFP and mCherry , Ro is 53 . 1 Å .", "Based on the law of mass action , the dimeric fraction can be written as a function of the total receptor concentration , T , and the dimerization constant K according to Equation 8: ( 8 ) fD=1T ( T−14K ( 1+8TK−1 ) ) Substituting Equation 8 into 6 , we obtain: ( 9 ) EDxA=1T ( T−14K ( 1+8TK−1 ) ) E~ We use Equation 9 to fit a monomer-dimer model to the measured ED/xA while optimizing for the two adjustable parameters: the dimerization constant K , and the value of structural parameter Intrinsic FRET .", "The dissociation constant Kdiss=1/K is reported in units of receptors .", "μm-2 in Table 1 .", "The free energy of dimerization ( dimer stability ) △G is calculated from the dimerization constant K=1/Kdiss .", "The standard state is defined as nm2 . receptor-1 ( Chen et al . , 2010 ) , and therefore: ( 10 ) ΔG=−RTln ( 106Kdiss ) Chinese Hamster Ovary ( CHO ) cells and Human Embryonic Kidney ( HEK ) 293T cells were cultured at 37˚C with 5% CO2 for 24 hr .", "Transfection was carried out using Fugene HD transfection reagent ( Roche Applied Science ) , following the manufacturer’s protocol .", "Cells were cotransfected with 0 . 1–1 ug of wild type or mutant DNA plasmid constructs encoding VEGFR-2 .", "The cells were starved in medium lacking fetal bovine serum ( FBS ) for 24 hr .", "The cells were then treated with 3000 ng . mL-1 of human VEGF ligands .", "After incubating for 13 min at 37˚C with ligand , the samples were placed on ice prior to lysis .", "The cells were treated with lysis buffer ( 25 mM Tris-HCl , 0 . 5% Triton X-100 , 20 mM NaCl , 2 mM EDTA , phosphatase inhibitor and protease inhibitor , Roche Applied Science ) .", "Lysates were collected following centrifugation at 15 , 000 g for 15 min at 4°C and loaded onto 3–8%NuPAGE Novex Tris–Acetatemini gels or 4–12% NuPAGE Novex Bis-Tris Plus Gels ( Invitrogen , CA ) .", "The proteins were transferred onto a nitrocellulose membrane , and blocked using 5% milk in TBS .", "VEGFR-2 total protein levels were assessed using antibodies against VEGFR-2 ( 55B11; #2479 , Cell Signaling Technologies ) or the HA tag ( 3F10 , Roche Applied Science ) .", "Phosphotyrosine levels were assessed using anti-phospho-VEGFR-2 antibodies ( Tyr1054/1059: D5A6; #3817 or Tyr1175: D5B11; #3770 , Cell Signaling Technologies ) .", "Endogenous VEGF-A levels were detected by staining with anti-VEGF-A antibodies ( PA1080 , Boster Biological Technology Co ) .", "This was followed by anti-rabbit HRP conjugated antibodies ( W4011 , Promega ) or goat anti-rat HRP conjugated antibodies ( sc-2006 , Santa Cruz Biotechnology ) .", "All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:2500 dilution according to the manufacturers' protocols .", "The anti-HA antibodies were used at 1:3000 dilution .", "The proteins were detected using the Amersham ECL detection system ( GE Healthcare ) .", "Following 24 hr starvation , CHO cells expressing full length VEGFR-2 were subjected to cell surface cross-linking using 2 mM BS3 ( Pierce ) , a membrane impermeable crosslinker , for 30 min at room temperature .", "The samples were quenched in 20 mM Tris-HCl for 15 min .", "After one rinse with ice-cold PBS , the cells were lysed and the receptors were detected via Western blotting , using antibodies against VEGFR-2 ( 55B11: #2479 , Cell Signaling Technologies ) , followed by secondary HRP conjugated anti-rabbit antibodies ( W4011 , Promega ) ." ] ]
[ "VEGFR-2 is the primary regulator of angiogenesis , the development of new blood vessels from pre-existing ones .", "VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand , and to undergo dimerization and activation only upon ligand binding .", "Using quantitative FRET and biochemical analysis , we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels , and that these dimers are phosphorylated .", "Ligand binding leads to a change in the TM domain conformation , resulting in increased kinase domain phosphorylation .", "Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure , and for the transition to the ligand-bound active conformation .", "We further show that the pathogenic C482R VEGFR-2 mutant , linked to infantile hemangioma , promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer ." ]
[ "New blood vessels form by growing out from existing vessels .", "A signaling molecule called VEGF is crucial for this process and binds to a receptor protein known as VEGFR-2 .", "This binding activates signaling events within the cells that line the blood vessels to promote the growth of new vessels .", "VEGFR-2 belongs to a family of proteins called receptor tyrosine kinases .", "The receptor has three main parts: one part extends out of the cell and binds to VEGF , another spans the cell’s membrane , while the third part is found inside the cell .", "The current model of VEGFR-2 activation is that VEGF binds to individual VEGFR-2 receptor proteins on the membrane , and brings two of them close enough to form a complex called a dimer .", "The receptor dimer is activated and initiates signaling within the cell .", "Sarabipour et al . tested this current model and revealed that VEGFR-2 could form a dimer even in the absence of VEGF .", "This receptor dimer formed through contacts between parts that are inside the cell as well as the regions embedded in the membrane .", "These VEGFR-2 dimers were active , but only to a low level .", "However , the activity of the receptor was increased in the presence of VEGF .", "Sarabipour et al . found that binding of VEGF promoted VEGFR-2 to change its three-dimensional shape , which made it more active .", "Further work suggested that VEGFR-2 dimers must first correctly assemble in the absence of VEGF so that active VEGFR-2 dimers can form at a later stage .", "Hence a VEGFR-2 dimer on its own is an important intermediate that is needed for full activation of the receptor .", "A mutation in the gene that encodes VEGFR-2 causes a disorder called infantile hemangioma that leads to abnormal vessel formation in young children .", "Sarabipour et al . found that this mutation causes VEGFR-2 to change its shape even when no VEGF is present and thus makes the receptor overly active .", "Finally , tumors need to form new blood vessels to grow and survive .", "These new findings suggest that drugs that interact with VEGFR-2 dimers in the absence of VEGF may be able to prevent the receptor from becoming activated and could thus help cut the blood supply to tumors and treat cancers ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology" ]
Metformin extends C. elegans lifespan through lysosomal pathway
elife-31268-v2
[ [ "With the discovery that aging could be genetically regulated , numerous strategies have been employed to extend lifespan in model organisms , including pharmacologic and dietary interventions ( Longo et al . , 2015 ) .", "Identification of a chemical or pharmacological manipulation that could target human aging and lower the risks associated with age-related diseases becomes a central goal of aging research .", "Administration of metformin , a first-line drug for treatment of type 2 diabetes ( T2D ) , has been shown to extend lifespan in C . elegans and mice ( Anisimov et al . , 2008; Cabreiro et al . , 2013; De Haes et al . , 2014; Martin-Montalvo et al . , 2013; Onken and Driscoll , 2010; Wu et al . , 2016 ) .", "In addition , metformin has been shown to ameliorate diabetic and cardiovascular diseases in patients ( Scarpello , 2003 ) .", "Metformin also lowered the incidence of several other age-related diseases , such as cancer , metabolic syndrome and cognitive disorders ( Foretz et al . , 2014 ) .", "Due to its broad range of health benefits and little side effects , a clinical trial named TAME ( Targeting Aging with Metformin ) was proposed to evaluate metformin’s protective effects against human aging and age-related diseases ( Barzilai et al . , 2016 ) .", "However , despite its intriguing benefits to promote healthy aging , the underlying mode of action of metformin is not well understood and a subject of extensive debate .", "Metformin is generally believed to act through activation of AMP-activated protein kinase ( AMPK ) ( Fryer et al . , 2002; Hawley et al . , 2002; Zhou et al . , 2001 ) , a principal energy sensor that when activated , switches on catabolic pathways such as glycolysis and fatty acid oxidation to produce more ATP ( Burkewitz et al . , 2014; Hardie et al . , 2012 ) .", "It was proposed that metformin might act through inhibition of mitochondrial electron transport chain ( ETC ) Complex I ( El-Mir et al . , 2000; Foretz et al . , 2014; Owen et al . , 2000 ) , resulting in a change of the AMP/ATP ratio and ultimately activating AMPK .", "However , this idea has been challenged recently ( He and Wondisford , 2015 ) , especially when physiological/low concentration ( ~70 uM ) of metformin , which cannot induce AMP/ATP change , is still able to activate AMPK ( Cao et al . , 2014 ) .", "An alternative model , in which metformin activates AMPK through the lysosome-dependent pathway was proposed ( Zhang et al . , 2016 ) .", "In this model , metformin treatment induces lysosomal localization of the scaffold protein AXIN , which brings its associated protein liver kinase B1 ( LKB1 ) to form a complex with v-ATPase-Ragulator on the surface of lysosome ( Zhang et al . , 2016; Zhang et al . , 2013 ) .", "LKB1 then phosphorylates the threonine residue in the activation loop of AMPK , leading to AMPK activation ( Hawley et al . , 1996; Shaw et al . , 2004; Woods et al . , 2003 ) .", "In addition to AMPK activation , metformin administration also inhibit mechanistic target of rapamycin complex 1 ( mTORC1 ) ( Kalender et al . , 2010 ) .", "mTORC1 constitutes another hub for energy and nutrient sensing , and switches on anabolism when activated ( Schmelzle and Hall , 2000 ) .", "The primary pathway for mTORC1 activation requires lysosome-localized Rag GTPases , which form RagA/B–RagC/D heterodimers and recruit mTORC1 to the surface of lysosome through directly binding to the Raptor subunit of mTORC1 ( Kim et al . , 2008; Sancak et al . , 2008 ) .", "v-ATPase-Ragulator complex on lysosomal surface is required for the spatial regulation and activation of mTORC1 by Rag GTPases ( Bar-Peled et al . , 2012; Dibble and Manning , 2013; Jewell et al . , 2013; Sancak et al . , 2010 ) .", "Two distinct mechanisms have been proposed for metformin-induced mTORC1 inhibition: suppression of Rag GTPases ( AMPK-independent mechanism ) or AMPK-mediated phosphorylation of Raptor ( regulatory associated protein of mTORC1 ) ( AMPK-dependent mechanism ) ( Howell et al . , 2017; Kalender et al . , 2010 ) .", "Due to its short lifespan and ease of genetic manipulation , C . elegans becomes a powerful model organism to test the efficacy of metformin in promoting healthspan ( Burkewitz et al . , 2014 ) .", "It has been shown that metformin treatment greatly extends worm lifespan and improves fitness , for example prolonging locomotory ability ( Onken and Driscoll , 2010 ) .", "However , several different mechanisms have been suggested for metformin’s lifespan extension effect in C . elegans .", "For instance , metformin administration may mimic a dietary restriction ( DR ) metabolism ( Onken and Driscoll , 2010 ) .", "In addition , alteration of methionine metabolism in C . elegans has been shown to play a partial role ( Cabreiro et al . , 2013 ) .", "Recently , metformin has been shown to inhibit mTORC1 due to restricted transit of RagA-RagC GTPase through nuclear pore complex ( NPC ) ( Wu et al . , 2016 ) .", "It is possible that these observations reflect downstream consequences of a primary action of metformin .", "Therefore , understanding its direct mechanism of action worth further investigation .", "Elucidating metformin’s mode of action will significantly boost its application to target human aging and prevent age-related diseases ." ], [ "Intrigued by the discoveries that mTORC1 and AMPK share the common activator , v-ATPase-Ragulator complex ( Zhang et al . , 2014 ) , and that metformin may directly act on the lysosomal pathway to promote AMPK activation ( Zhang et al . , 2016 ) , we sought to set up a pure in vitro reconstitution system to explore the mechanism of metformin’s action ( Figure 1 ) .", "We stably expressed LAMP1-RFP-FLAG in HEK293T cells to label lysosomes ( Figure 1A ) and performed FLAG pull-down to enrich lysosomes .", "The purity of lysosome preparation was confirmed by immunoblotting to test for the absence of early endosome , mitochondria , ER or nuclei ( Figure 1B ) .", "Immuno-purified Raptor was then provided to the lysosomes .", "Upon amino acids stimulation , significant portion of Raptor accumulated on the lysosome ( Figure 1C , F ) , indicating the activation of mTORC1 pathway .", "We then supplemented Concanavalin A ( Con A ) , a v-ATPase inhibitor , or Metformin ( Met ) to the reconstitution system .", "Addition of Con A or metformin dissociated Raptor from the lysosome ( Figure 1D , G ) .", "It has been proposed that AMPK might directly phosphorylates Raptor and thus inhibits mTORC1 ( Gwinn et al . , 2008 ) .", "To test if metformin’s effect on mTORC1 inhibition requires AMPK or not , we repeated the in vitro experiments in AMPK knockout MEF cells , and still observed the dissociation of Raptor ( Figure 1—figure supplement 1 ) .", "Taken together , these results suggest that metformin inhibits mTORC1 through the lysosomal pathway independent of AMPK , possibly mimicking Con A to inhibit v-ATPase .", "Lastly , we provided purified AXIN and LKB1 to the in vitro system .", "Immuno-purified LKB1 forms a complex with endogenous STRAD and MO25 , two proteins essential for optimal LKB1 activity ( Figure 1—figure supplement 2 ) .", "Consistent with previous findings , AXIN/LKB1 were recruited to metformin- or Con A-treated lysosomes , and were sufficient to phosphorylate endogenous AMPK in an amino acid-independent manner ( Figure 1E , H ) ( Zhang et al . , 2016; Zhang et al . , 2013 ) .", "It has been proposed that metformin might change the AMP/ATP ratio through inhibition of mitochondrial ETC complex I , ultimately resulting in AMPK activation ( El-Mir et al . , 2000; Foretz et al . , 2014; Owen et al . , 2000 ) .", "To test if metformin could inhibit mitochondrial function and therefore activate mitochondrial stress response , we treated C . elegans hsp-6p::gfp reporter strain with metformin , or rotenone , a well-known mitochondrial ETC complex I inhibitor .", "Unlike rotenone , metformin treatment showed no reporter activation ( Figure 1—figure supplement 3A , B ) .", "It should be noted that others have reported that metformin inhibits ETC complex I through a mechanism distinct from that of rotenone: metformin treatment increases ROS production , whereas rotenone decreases it ( De Haes et al . , 2014 ) .", "Because our in vitro reconstitution system excludes mitochondrial contaminants , it highly suggests that metformin inhibits mTORC1 and activates AMPK mainly through the lysosomal pathway .", "Lastly , we directly measured the metformin’s effect on lysosomal function with the use of a cathepsin assay .", "Cathepsin is a class of cysteine proteinase localized within lysosomes .", "A cathepsin assay is a fluorescence-based assay that cleaves cathepsin’s substrate to release fluorescence .", "Consistent with our idea , metformin administration indeed impaired lysosomal function ( Figure 1—figure supplement 3C , D ) .", "Because our in vitro biochemical results suggested that metformin might act on the v-ATPase-Ragulator complex of the lysosomal pathway to coordinate mTORC1 inhibition and AMPK activation , we next tested if metformin promotes lifespan extension in C . elegans due to similar mechanisms .", "To confirm that metformin also inhibits TORC1 in C . elegans , we first examined phosphorylation status of RSKS-1 , the human ribosomal protein S6 kinase B1 ( S6K ) homolog in C . elegans .", "We used an antibody that specifically detects Thr-389 , a highly conserved phosphorylation site among species ( Figure 2—figure supplement 1A ) .", "This antibody was validated with the used of rsks-1 RNAi ( Figure 2—figure supplement 1B ) .", "Notably , metformin treatment significantly decreased the level of RSKS-1 phosphorylation ( Figure 2A , B ) , suggesting an inhibition of TORC1 pathway .", "We also tested the subcellular localization of HLH-30 , an ortholog of transcription factor EB ( TFEB ) that translocates to the nucleus upon TORC1 inhibition ( Lapierre et al . , 2013; Perera and Zoncu , 2016; Settembre et al . , 2011 ) .", "Indeed , HLH-30 translocalized to the nucleus in the tail region of C . elegans upon metformin administration ( Figure 2C , D ) .", "Once in the nucleus , HLH-30 regulates the transcription of genes related to autophagy , lysosome function and lipid hydrolysis ( Lapierre et al . , 2013; O'Rourke and Ruvkun , 2013 ) .", "Consistently , we also observed up-regulation of transcript levels of hlh-30 downstream targets upon metformin treatment ( Figure 2E ) .", "Taken together , these results indicate that metformin inhibits TORC1 pathway in C . elegans .", "We also tested if metformin’s effect on TORC1 inhibition requires AMPK in C . elegans .", "Consistent with our results in AMPK-/- MEF cells ( Figure 1—figure supplement 1 ) , metformin treatment of aak-2 ( Ce . AMPK ) loss-of-function mutants was still able to drive HLH-30 nuclear accumulation ( Figure 2—figure supplement 2A , B ) and elevate the expression of HLH-30 target genes ( Figure 2—figure supplement 2C ) .", "Inhibition of TORC1 has been shown to result in lifespan extension ( Jia et al . , 2004; Vellai et al . , 2003 ) ; also reviewed by Zoncu et al . , 2011 ) .", "To distinguish if metformin induces lifespan extension solely through Ce . TOR inhibition , or the activation of Ce . AMPK also plays a role , we performed lifespan analysis on control worms or daf-15 heterozygous mutants in the presence or absence of metformin ( Figure 3—figure supplement 1A ) .", "Compared with the control worms , daf-15 heterozygous mutants had a longer lifespan and a decreased level of RSKS-1 phosphorylation ( Figure 3—figure supplement 1B , C ) , indicating that TORC1 inhibition extends C . elegans lifespan .", "More importantly , metformin treatment greatly extended the lifespan of daf-15 heterozygous mutants ( Figure 3A ) .", "To investigate the metabolic effects of metformin on daf-15 heterozygous mutants , we fed L4 worms with metformin and conducted Oil-Red-O ( ORO ) staining to detect neutral fat levels ( Figure 3—figure supplement 1D ) .", "Similar to wild type animals , metformin administration further decreased neutral fat level in daf-15 heterozygous mutants ( Figure 3B , C ) .", "Aged worms start to show muscle deterioration and decrease locomotion rates ( Huang et al . , 2004; Onken and Driscoll , 2010 ) .", "Metformin treatment significantly increased the locomotory ability ( counted by the average bends of worm body per 60 s ) and reduced age pigments of wild type worms and daf-15 heterozygous mutants ( Figure 3—figure supplement 1D , and Figure 3D , E ) , indicating that metformin also improves fitness of daf-15 heterozygous mutants .", "Metformin’s beneficial effect on lifespan and fitness of daf-15 heterozygous mutants could be due to a pathway additive to that of TORC1 inhibition ( e . g . AMPK pathway ) , or simply due to a further suppression of TOR activity in daf-15 heterozygous mutants .", "To test if metformin could further inhibit TORC1 activity in daf-15 heterozygous mutants , we compared HLH-30 nuclear localization between control animals and daf-15 heterozygous mutants in the presence or absence of metformin treatment .", "We found that in daf-15 heterozygous mutants , significant portion of HLH-30 already localized in the nucleus , suggesting an inhibition of TORC1 activity .", "More importantly , metformin administration increased HLH-30 nuclear localization in control worms , but not in daf-15 heterozygous mutants ( Figure 3F , G ) .", "In addition , we performed lifespan experiments with loss-of-function mutants of known downstream targets of TORC1 , which have been reported to mediate TORC1 inhibition-induced longevity ( Robida-Stubbs et al . , 2012 ) .", "We found that metformin could still extend lifespans of hlh-30 , pha-4 or skn-1 mutants ( Figure 3—figure supplement 2 ) .", "These results suggest that metformin may act on a pathway additive to that of TORC1 inhibition to confer fitness benefit .", "Given that metformin activated AMPK in daf-15 heterozygous mutant to the same level as that in the control worms ( Figure 3H , I ) , it is likely that metformin promotes longevity also through activation of AMPK .", "v-ATPase-Ragulator complex may function as lysosomal acceptor for AXIN/LKB1 , which translocate to lysosome and activate AMPK upon metformin treatment ( Figure 1E ) .", "Thus , to test the lysosome-dependent AMPK activation for metformin-induced lifespan extension , we first performed lifespan analysis on vha-3 ( subunit of v-ATPase V0 domain ) , vha-12 ( subunit of v-ATPase V1 domain ) , lmtr-3 ( LAMTOR 3 subunit of Ragulator ) and lmtr-2 ( LAMTOR 2 subunit of Ragulator ) loss-of-function mutants ( Figure 4—figure supplement 1 and Figure 4A ) .", "VHA-3 , VHA-12 , LMTR-3 and LMTR-12 all localized on the lysosomes of C . elegans , as they have similar staining pattern with lysosomal marker LMP-1 ( Figure 4—figure supplement 2 ) .", "All these four mutants had impaired lysosomes , which could be rescued with the expression of corresponding gene ( Figure 4—figure supplement 3 ) .", "vha-3 and vha-12 mutants had longer lifespans compared with wild type animals , whereas lifespan of lmtr-2 or lmtr-3 mutants was comparable to that of the wild type animals ( Figure 4B and C and Figure 4—figure supplement 4A and B ) .", "Lifespan extension in vha-3 and vha-12 mutants might be due to Ce . TOR inhibition , because v-ATPase is required for the spatial regulation and subsequent activation of TORC1 .", "Consistently , failure of lmtr-3 or lmtr-2 to induce lifespan extension suggested that Ce . TOR pathway was not inhibited under lmtr-3 or lmtr-2 deficiency .", "Indeed , RNAi knockdown of vha-3 but not lmtr-3 , was sufficient to induce the nuclear accumulation of HLH-30 and activate HLH-30 downstream targets ( Figure 4—figure supplement 4C and D , and Figure 4D–F ) .", "In addition , knocking down of vha-3 but not lmtr-3 , decreased the level of RSKS-1 phosphorylation ( Figure 4G and H ) .", "More importantly , metformin administration could not further induce the lifespan extension , nor AAK-2 activation in v-ATPase mutants vha-3 and vha-12 , or Ragulator mutants lmtr-3 and lmtr-2 ( Figure 4B–C and I–J and Figure 4—figure supplement 4A , B , E and F ) , suggesting that metformin’s effect on lifespan extension may also depend on the lysosomal pathway of AMPK activation .", "It should be noted that metformin even shortens the lifespan of lmtr-3 or lmtr-2 mutants ( Figure 4C and Figure 4—figure supplement 4B ) .", "However , fractions of censored animals were not increased in metformin-treated lmtr-3 or lmtr-2 mutants ( Figure 4—figure supplement 4G ) , suggesting that metformin did not make the mutants sick .", "AXIN is a scaffold protein required for lysosomal localization of LKB1 and lysosome-dependent activation of AMPK ( Zhang et al . , 2016; Zhang et al . , 2013 ) .", "We could not successfully express AXL-1 ( C . elegans ortholog of AXIN ) with fluorescent tag in worms and test for its subcellular localization .", "Therefore , we expressed EGFP-tagged AXL-1 in mammalian cells and showed that AXL-1 only localized on the lysosome upon metformin treatment ( Figure 5A ) .", "Consistently , without metformin administration , axl-1 mutant alone did not perturb lysosomal function ( Figure 5B ) .", "We then examined the role of AXIN in metformin-induced lifespan extension .", "Metformin treatment failed to activate AAK-2 ( Ce . AMPK ) in axl-1 mutant ( AXIN-deficient animals ) ( Figure 5C , D ) .", "Consistently , metformin was no longer able to extend lifespan of axl-1 mutant animals ( Figure 5E ) .", "Similar to axl-1 , metformin could not extend lifespan of par-4 ( C . elegans ortholog of LKB-1 ) mutants as well ( Figure 5F ) ( Onken and Driscoll , 2010 ) .", "Taken together , these genetic evidences indicate that v-ATPase-Ragulator-AXIN-LKB1-based lysosome pathway is required for the metformin’s effects of lifespan extension .", "It has been shown that metformin triggers a dietary restriction-like state and extends C . elegans lifespan through AMPK pathway ( Onken and Driscoll , 2010 ) .", "Indeed , metformin administration induced a dietary restriction-like state to decrease neutral fat level in wild type animals ( Figure 6A , B ) .", "Conversely , vha-3 ( v-ATPase V0 ) , lmtr-3 ( Ragulator ) and axl-1 ( AXIN ) mutants retained the same levels of ORO staining in the presence or absence of metformin ( Figure 6A , B ) , suggesting that the dietary restriction-like state triggered by metformin requires the lysosome-dependent activation of AMPK .", "Metformin treatment also significantly increased the locomotory ability and lowered age pigments of wild type worms ( Figure 6C and G ) , indicating that metformin promotes youthful physiology and fitness of C . elegans .", "Strikingly , mutation of vha-3 , lmtr-3 or axl-1 impaired metformin's effects on locomotion and age pigments ( Figure 6D–G ) , suggesting that lysosome-dependent activation of AMPK is also required for metformin-attenuated fitness decline in aged animals ." ], [ "Using a pure in vitro reconstitution system that excludes mitochondria , we showed that metformin coordinates mTORC1 inhibition and AMPK activation through lysosomal pathway .", "We further employed genetic manipulation to show that metformin extends C . elegans lifespan and attenuates age-related fitness decline via similar mechanism that requires v-ATPase-Ragulator-AXIN/LKB1 of the lysosomal pathway ( Figure 6H ) .", "Metformin may function by targeting and priming v-ATPase-Ragulator complex on lysosome membrane , which serves as a hub to coordinate mTORC1 and AMPK pathways and govern metabolic programs .", "It is possible that metformin administration might result in a conformational change of v-ATPase-Ragulator complex , which dissociates mTORC1 from lysosome and allows the docking of AXIN/LKB1 for AMPK activation ( Figure 6—figure supplement 1 ) .", "It will be of particular interest in the future to test if v-ATPase is the direct target of metformin .", "Elucidating the molecular mechanism of metformin-mediated lifespan extension will boost its application in the treatment of human aging and age-related diseases ." ], [ "HEK293T cells used for protein purification , virus packaging and stable cell line generation , and AMPKα1/2 double knockout ( DKO ) MEF cells used for stable cell line generation were cultured with DMEM high glucose medium ( HyClone , SH30243 . 01 ) , supplemented with 10% FBS ( Gibico , 10099–141 ) and 1% Penicillin/Streptomycin ( HyClone SV30010 ) .", "293T HEK cells were obtained from ATCC , not authenticated , and mycoplasma negative .", "AMPKα1/2 double knockout ( DKO ) MEF cells were provided by Dr . Benoit Viollet , validated by immunoblotting and determined as mycoplasma negative .", "HEK293T cells and AMPKα1/2 DKO MEF cells stably expressing LAMP1-RFP-FLAG fusion protein , or HEK293T cells stably expressing AXL-EGFP and LAMP1-RFP-FLAG were generated with the lentiviral packaging and infection system .", "0 . 8 × 106 HEK293T cells were seeded into one well of a 6-well-plate and cultured with 2 ml medium .", "After about 20 hr , 0 . 75 ug pMDLg/pRRE , 0 . 3 ug pRSV-Rev , 0 . 45 ug pCI-VSVG and 1 . 5 ug pLJM1-LAMP1-RFP-FLAG ( addgene ) plasmids were co-transfected into cells .", "Medium was changed 8 hr after transfection .", "Cells were then cultured for additional 24 hr to allow virus production , and 2 ml medium containing virus was harvested ( virus packaging ) .", "300 ul virus-containing medium was diluted with 900 ul fresh medium , and added into one well of a 12-well-plate containing HEK293T cells or AMPKα1/2 DKO MEF cells at ~40% confluence ( virus infection ) .", "Several hours after infection , cells stably expressing LAMP1-RFP-FLAG were checked by RFP fluorescence .", "~100% HEK293T cells or ~20% AMPKα1/2 DKO cells contain RFP signal .", "HEK293T LAMP1-RFP-FLAG stable cell line was then infected by virus carrying AXL-EGFP to generate double stable cell line .", "Recombinant proteins Myc-LKB1 ( human ) , Myc-AXIN ( mouse ) , and Myc-Raptor ( human ) were immunopurified from HEK293T cells .", "For purification of each protein , 20 × 15 cm dishes of HEK293T cells were cultured until 80% confluence .", "28 ug of plasmid was transfected to each dish of cells with Lipofectamine 3000 Transfection Reagent .", "Medium was changed 8 hr after transfection .", "After additional 24 hr , medium was carefully discarded and cells were rinsed with PBS .", "1 . 5 ml of TX-100 lysis buffer ( 20 mM Tris-HCl , 150 mM NaCl , 1 mM Na2EDTA , 1 mM EGTA , 2 . 5 mM Sodium pyrophosphate , 1 mM β-glycerophosphate , 1% Triton X-100 , PH = 7 . 4 ) supplemented with proteinase inhibitors was added to each dish of cells .", "Cells were incubated on ice for 10 min , and then scraped off the dishes .", "Cell lysate was sonicated at 60% Ampl for 5 × 30 s ( SONICS VCX 130 PB , UK ) , and centrifuged at 4°C , 10000 rpm for 10 min .", "Supernatant was mixed with 500 ul Myc agarose beads , and rotated overnight at 4°C .", "Beads were washed three times with TX-100 lysis buffer and three times with fractionation buffer ( 50 mM KCl , 90 mM K-Gluconate , 1 mM EGTA , 5 mM MgCl2 , 50 mM Sucrose , 5 mM Glucose , 20 mM HEPES , pH = 7 . 4 , 2 . 5 mM ATP and protease inhibitors were added right before usage ) , and resuspended in 800 ul of fractionation buffer , followed by addition of 10 ul 10 mg/ml Myc peptide .", "After rotating at 4°C for 4 hr , Myc tagged protein in 800 ul fractionation buffer was harvested , followed by addition of 160 ul glycerol , and stored at −20°C .", "LAMP1-RFP-FALG stable cells were cultured until confluence .", "For each sample , 1 × 10 cm dish of HEK293T cells or 5 × 10 cm dishes of MEF AMPKα1/2 DKO cells were scraped off the plate , and pelleted at 1000 rpm , room temperature for 3 min .", "Cell pellet was washed once with fractionation buffer , resuspended in 0 . 8 ml fractionation buffer , and mechanically broken by spraying six times through a 23G needle attached to a 1 ml syringe .", "Broken cells were spun down at 2000 g for 10 min at 4°C to pellet the nuclei , yielding a post-nuclei supernatant ( PNS ) .", "The PNS was diluted to 2 ml with fractionation buffer , and subjected to immunoprecipitation with 50 ul FLAG magnetic beads ( 60% slurry ) for 2 hr at 4°C on a rotator .", "Lysosome on beads was washed three times and resuspended in 300 ul fractionation buffer , supplemented with 1x amino acids ( combining 50x Gln-free MEM amino acids mixture with 100x Glutamine solution from Gibico ) , 250 uM GTP and 100 uM GDP .", "Lysosome was then rotated at 37°C , 650 rpm on a Thermomixer ( Eppendorf , Germany ) for 15 min ( activation step for Raptor binding ) .", "40 ul of purified Myc-Raptor protein was added , and the system was shaken for additional 25 min ( lysosome binding step for Raptor ) .", "10 uM Concanamycin A ( ConA ) or 300 uM Metformin ( Met ) was then provided into the reaction system , and incubated for 30 min ( Raptor off lysosome membrane step ) .", "Lysosome coated magnetic beads were isolated from the reaction system , resuspended with 300 ul fractionation buffer supplemented with 80 ul purified Myc-AXIN and 20 ul purified Myc-LKB1 ( formed a complex with endogenous STRAD and MO25 ) , and then shaken on Thermomixer for 25 min ( AXIN/LKB1 lysosome translocation and AMPK activation step ) .", "For in vitro reconstitution , each sample was prepared with 30 ul 2x laemmli sample buffer .", "Purified lysosomes were checked with LAMP2 ( abcam ab25631 ) , EEA1 ( CST 3288 ) , Prohibitin ( Santa Cruz 28259 ) , PDI ( Santa Cruz 20132 ) , and Histone H3 ( Huaxingbio , HX1850 ) antibodies .", "Myc tagged proteins were detected with anti-Myc antibody ( ImmunoWag YM3203 ) .", "LAMP1-RFP-FLAG labeled lysosomes were probed with Flag antibody ( Sigma F7425 ) .", "T172 site phosphorylated AMPKα subunit and total AMPKα subunit were detected with p-AMPKα ( CST 2535 ) and AMPKα ( CST 2532 ) antibodies respectively .", "STRAD ( Santa Cruz sc-34102 ) and MO25 ( CST 2716 s ) antibodies were used to detect Myc-LKB1-STRAD-MO25 complex .", "For detection of AAK-2 or RSKS-1 phosphorylation , worms at indicated stage were lysed with 2x laemmli sample buffer equal to the volume of worm pellet .", "AAK-2 phosphorylation was probed with human p-AMPKα ( T172 ) antibody ( CST 2535 ) .", "RSKS-1 phosphorylation was probed with human p-S6K1 ( T389 ) antibody ( CST 9205 ) .", "Tubulin ( abcam ab6161 ) was used as loading control .", "Detailed information of Caenorhabditis elegans strains used in this paper was provided in Supplementary file 1–2 .", "Worms were maintained on nematode growth medium ( NGM ) plate with OP50 as standard food .", "All worms , except for pha-4 and par-4 mutants , were cultured at 20°C .", "Metformin was added into molten agar at the concentration of 50 mM while preparing plates .", "Rotenone stock was prepared at 10 ug/ul in DMSO .", "1 ul rotenone stock was diluted with 300ul M9 and added directly onto a 6 cm NGM plate and hood-dried .", "RNA interference ( RNAi ) clones were grown at 37°C overnight in LB containing 50 ug/ml carbenicillin .", "200 ul bacteria were then seeded onto NGM containing 1 mg/ml IPTG and 50 ug/ml carbenicilin , hood-dried and cultured overnight to induce dsRNA expression .", "L1 stage Worms were then seeded onto bacteria lawn , and raised until L4 stage for efficient knockdowns .", "~100 L4 stage ( day 0 for lifespan assays ) worms ( except for lmtr-2 , lmtr-3 , par-4 and pha-4 ) were transferred to plates with or without metformin .", "For lmtr-2 or lmtr-3 mutant strain , ~250 worms were used , due to high percentage of censored worms .", "For par-4 mutant , worms were cultured at 15°C until L4 stage ( day 0 for lifespan assays ) , and ~100 worms were transferred to 25°C for lifespan assay .", "For pha-4 mutant , worms were cultured at 25°C until day 1 adult ( day 0 for lifespan assays ) , and ~100 worms were transferred to 15°C for lifespan assay .", "FUDR was used to prevent reproduction .", "Worms were transferred to new plates and counted every other day .", "FUDR was omitted after day 12 .", "Animals that did not move when gently prodded were scored as dead .", "Animals that crawled off the plate or died from vulva bursting were censored .", "Worms were washed with 1x PBS and resuspended in 120 ul 1x PBS , followed by addition of 120 ul 2x MRWB buffer ( 160 mM KCl , 40 Mm NaCl , 14 mM Na4EGTA , 1 mM Spermidine-HCl , 0 . 4 mM Spermine , 30 mM Na-PIPES , 0 . 2% β-mercaptoethanol , PH = 7 . 4 ) containing 2% PFA .", "Samples were fixed by gently rocking at room temperature for 1 hr .", "Worms were then washed with 1x PBS to remove PFA , and resuspended in 300 ul 60% isopropanol to dehydrate for 15 min at room temperature .", "Worms were pelleted and resuspended with 1 ml 60% ORO solution , and rotated overnight at room temperature ( Preparation of 60% ORO solution: commercial liquid ORO stain was diluted to 60% with H20 , rocked overnight at room temperature , and filtrated with 0 . 22 um filter right before usage ) .", "Worms were settled and ORO staining solution was removed .", "200 ul 1x PBS ( with 0 . 01% triton X-100 ) was added to resuspend the worms .", "Worms were quickly transferred on to a glass slide and photographed with Zeiss Imager M2 microscope .", "Worms were transferred into a drop of M9 on a glass slide , and filmed with Zeiss Imager M2 microscope .", "Body bends were counted by reviewing each frame of the 60 s film .", "Worms were mounted onto an agarose pad attached to a glass slide and photographed with Zeiss Imager M2 microscope .", "Fluorescence intensity was counted by Image J . Magic Red stain ( ImmunoChemistry Technologies #938 , Bloomington , Minnesota ) can be cleaved by cathepsin B , and generate red fluorescent substrate in functional lysosomes .", "Magic Red stain was prepared in 260x DMSO stock following the manufacturer’s instructions .", "3 . 8 ul stock was 5x diluted with M9 , spread onto a well of 24-well-plate containing 1 ml NGM , and dried in hood .", "For metformin’s effect on lysosomal function , wild type L1 stage worms were raised to L4 stage on control or metformin NGM plate containing Magic Red stain .", "For cathepsin assay , L4 stage worms were transferred onto Magic Red containing plate and cultured overnight .", "Worms were photographed with Zeiss Imager M2 microscope and quantified by Image J . The longest CDS sequences of lmp-1 , vha-3/12 and lmtr-2/3 were amplified from worm genomic DNA , and cloned into expression vector containing rpl-28 promoter ( whole body expression ) .", "LMP-1::GFP was initially integrated into C . elegans genome through UV irradiation , and worms were backcrossed for six times .", "mCherry-VHA-3/12 or mCherry-LMTR-2/3 was then injected into worms expressing LMP-1::GFP .", "Worms were photographed by Zeiss Imager M2 microscope .", "The longest CDS sequence of axl-1 was amplified from worm genomic DNA and cloned into mammalian expression construct containing CMV promoter and C-terminal EGFP tag .", "HEK293T cells stably expressing AXL-1-EGFP and LAMP-1-RFP-FLAG were generated as described above .", "Cells were imaged by Zeiss LSM710 confocal microscope .", "hsp-6p::gfp reporter worms were photographed by Zeiss Imager M2 microscope and the fluorescent intensity was quantified by Image J . hlh-30p::hlh-30::gfp reporter worms were photographed by Zeiss Imager M2 microscope and percentage of worms with nuclear localization of HLH-30 was counted .", "To monitor HLH-30 nuclear localization in control or daf-15 heterozygous mutant , we injected hlh-30p::hlh-30::gfp construct into control or daf-15 heterozygous mutant respectively .", "Worms were photographed by Zeiss Imager M2 microscope .", "Percentage of worms with nuclear localization of HLH-30 was counted .", "Worms were washed off plates with M9 and resuspended with TRIzol Reagent and frozen by liquid nitrogen .", "Total RNA was isolated by chloroform extraction and isopropanol precipitation .", "200 ng total RNA was used for reverse transcription with One-Step cDNA Synthesis Kit ( TransGen Biotech , China ) .", "Real-time PCR was carried out using SYBR GREEN PCR Master Mix ( Biorad , Hera Claes , California ) .", "Quantifications of transcripts were normalized to rpl-32 ." ] ]
[ "Metformin , a widely used first-line drug for treatment of type 2 diabetes ( T2D ) , has been shown to extend lifespan and delay the onset of age-related diseases .", "However , its primary locus of action remains unclear .", "Using a pure in vitro reconstitution system , we demonstrate that metformin acts through the v-ATPase-Ragulator lysosomal pathway to coordinate mTORC1 and AMPK , two hubs governing metabolic programs .", "We further show in Caenorhabditis elegans that both v-ATPase-mediated TORC1 inhibition and v-ATPase-AXIN/LKB1-mediated AMPK activation contribute to the lifespan extension effect of metformin .", "Elucidating the molecular mechanism of metformin regulated healthspan extension will boost its therapeutic application in the treatment of human aging and age-related diseases ." ]
[ "As humans are living for longer , age-related diseases – including cancer , diabetes , cardiovascular diseases and cognitive disorders – are becoming more common .", "Many research groups are therefore trying to find drugs that might prevent these diseases or make them less harmful .", "A drug called metformin has been shown to extend the healthy lifespan of animals such as mice and the roundworm Caenorhabditis elegans .", "The drug is also currently used to treat type 2 diabetes in humans and may help to prevent some other age-related diseases .", "However , it is still not clear exactly what effects metformin has on cells .", "Healthy cells need to perform many ‘metabolic’ processes to produce the molecules necessary for survival .", "Cell compartments called lysosomes play a role in many of these processes because they digest unneeded biological molecules .", "Through a combination of biochemical and genetic experiments involving C . elegans and human cells , Chen , Ou et al . found that metformin coordinates two metabolic pathways that both depend on lysosomes .", "Metformin reduces the activity of a pathway ( called mTOR ) that boosts cell growth and the metabolic processes that build complex molecules .", "At the same time , the drug activates a metabolic pathway ( called AMPK ) that breaks down complex molecules .", "Overall , therefore , metformin organizes a switch from a more growth-promoting state to a more growth-restricting state .", "Before metformin can be used more widely to treat human aging and age-related diseases , we need to understand how it works in even more detail .", "Further studies are required to discover which proteins metformin acts on inside cells , and a clinical trial has also been proposed to measure metformin’s effects on healthy human aging and age-related diseases ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "evolutionary biology" ]
Evolution of limb development in cephalopod mollusks
elife-43828-v1
[ [ "Animal appendages have widely varying morphologies and perform a multitude of functions , including locomotion , feeding , and reproduction ( Nielsen , 2012; Ruppert et al . , 2004 ) .", "Limbs evolved on multiple occasions , and the absence of shared ontogenetic or morphological precursors of appendages in many animal lineages is consistent with their independent origins ( Minelli , 2003; Pueyo and Couso , 2005; Shubin et al . , 1997 ) .", "This has led to the view that appendages in different clades of Bilateria are non-homologous morphological innovations that arose by convergent evolution ( Nielsen , 2012; Ruppert et al . , 2004 ) .", "However , despite more than 500 million years of divergence , the independently evolved limbs of arthropods and vertebrates share developmental genetic similarities ( Pueyo and Couso , 2005; Shubin et al . , 1997; Tabin et al . , 1999 ) .", "These discoveries led to debate over whether the genetic program for appendage development evolved in the common ancestor of all bilaterians in the early Cambrian , or whether arthropod and vertebrate appendages have undergone rampant convergence of developmental programs ( Minelli , 2000; Minelli , 2003; Panganiban et al . , 1997; Pueyo and Couso , 2005; Shubin et al . , 1997; Tabin et al . , 1999 ) .", "A major obstacle to resolving this question is that the evidence of a conserved program derives almost exclusively from Ecdysozoa and Deuterostomia ( Pueyo and Couso , 2005; Shubin et al . , 1997 ) , and little is known about molecular mechanisms of limb development in Spiralia , the third major superphylum of Bilateria ( Grimmel et al . , 2016; Prpic , 2008; Winchell and Jacobs , 2013; Winchell et al . , 2010 ) .", "Within spiralians , the phylum Mollusca is the largest lineage , displaying a rich diversity of body plans ( Figure 1A ) dating back to the Cambrian explosion ( Ruppert et al . , 2004; Smith et al . , 2011 ) .", "The evolution of arms and tentacles in cephalopod mollusks contributed to the successful adaptive radiation of these agile marine predators ( Kröger et al . , 2011; Ruppert et al . , 2004 ) .", "Cephalopod limbs are highly muscular appendages that bear cup-shaped suckers on their ventral sides .", "Arms are short and have suckers along the entire ventral surface ( Figure 1B and C ) , whereas tentacles are longer , retractable appendages with suckers restricted to a distal pad ( Figure 1D and E ) .", "Tentacles are thought to be specialized serial homologs of the arms ( Arnold , 1965; Lemaire , 1970; Shigeno et al . , 2008 ) and are present in decapods ( squid and cuttlefish ) but absent in nautilids and octopods .", "Limbs likely evolved de novo in cephalopods ( Figure 1A ) , since no homologous precursor structures have been identified in any other mollusk lineages ( Lee et al . , 2003; Shigeno et al . , 2008 ) .", "To test the hypothesis that cephalopod limbs evolved by recruitment of an ancient gene regulatory network for appendage development that is conserved across Bilateria , we investigated arm and tentacle development in embryos of the cuttlefish , Sepia officinalis ." ], [ "Cuttlefishes are decapod cephalopods that have eight arms and two tentacles ( Figure 1B–E; Figure 1—videos 1 and 2 ) .", "Fertilized cuttlefish eggs undergo superficial cleavage , and scanning electron microscopy and optical projection tomography show that most embryonic development is restricted to the animal pole ( Figure 1H and I ) .", "The first sign of limb formation is observed at stage 16 , when all ten limb primordia ( five on each side ) can be detected as small swellings around the periphery of a flat-shaped embryo , which lies at the top of the large yolk mass ( Figure 1H and M ) .", "Analysis of the mitotic marker phospho-histone H3 ( PHH3 ) at stage 15 revealed localized clusters of PHH3-positive cells in each of the early limb primordia ( Figure 1F and G ) , indicating that initiation of limb outgrowth is caused by localized cell proliferation .", "Discrete limb buds are observed from stage 17 ( Figure 1I and N; Figure 1—video 3 ) .", "As the embryo begins to rise-up on the animal pole around stage 19 , the limb buds start to elongate along the proximodistal axis ( Figure 1J and O; Figure 1—video 4 ) , and by stage 24 , the differential length and morphology of arms relative to tentacles is apparent ( Figure 1L; Figure 1—video 5 ) .", "Analysis of sucker development showed that a sucker field primordium initially forms as a narrow proximodistal ridge along the ventral surface of each limb ( evident by stage 21; Figure 1P ) .", "At later stages , the sucker field ridge cleaves superficially , segregating sucker buds from proximal to distal ( Figure 1Q ) .", "As the arms elongate , the sucker buds are laid down on the entire ventral surface of each arm ( Figure 1L and R; Figure 1—figure supplement 1A and C–G ) , forming four parallel rows across the anteroposterior axis ( Figure 1C; Figure 1—figure supplement 1A ) .", "In the tentacles , the primordial sucker band is restricted to the distal tip , where sucker buds form in eight rows along the anteroposterior axis of the tentacle sucker pads ( Figure 1D; Figure 1—figure supplement 1B ) .", "The full complement of immature sucker bud rows is present on each limb at hatching , and differentiation of the suckers continues during post-hatch development ( Figure 1—figure supplement 1H and I ) .", "To test the hypothesis that cuttlefish limb development is regulated by the same molecular mechanisms that pattern arthropod and vertebrate limbs , despite their independent evolutionary origins , we cloned and characterized cuttlefish orthologs of genes that pattern the three axes of vertebrate and arthropod limbs , and then analyzed their expression patterns during cuttlefish limb development ( Figure 2 and Figure 2—figure supplements 1–10 ) .", "Partial sequences of cuttlefish cDNAs ( Sepia officinalis and Sepia bandensis ) were isolated by rt-PCR , and preliminary identities were determined by comparison with NCBI sequence databases , including the octopus genome .", "Molecular phylogenetic reconstructions were then made by maximum likelihood phylogenetic inference using the best amino acid substitution model for each gene family ( see Materials and methods for details ) .", "Tree topologies with well-supported bootstrap values showed the position of each cuttlefish gene within the targeted gene families , which included Wnt , Tcf/Lef , Frizzled ( Fzd ) , Dachsund ( Dac/Dach ) , Notum , Patched ( Ptc/Ptch ) , Hedgehog ( Hh ) , Bone morphogenetic protein ( Bmp ) , Specificity protein ( Sp ) , and the ANTP and TALE homeobox gene families ( trees are shown in Figure 2—figure supplements 1–10 and are described below; gene accession ID numbers and the data set used in the phylogenetic analyses is provided in Figure 2—source data 1 ) .", "Within the Wnt family of cell signaling proteins , we isolated cuttlefish orthologs of Wnt1 , Wnt2 , Wnt5 , and Wnt7 ( Figure 2—figure supplement 1 ) .", "Phylogenetic analysis of cuttlefish transcription factors identified Tcf3/4 , an ortholog of arthropod Pangolin and a pro-ortholog of vertebrate Tcf3 and Tcf4 ( Figure 2—figure supplement 2 ) , Dac , a pro-ortholog of vertebrate Dach1 and Dach2 ( Figure 2—figure supplement 3 ) , and Sp8/9 , a pro-ortholog of vertebrate Sp8 and Sp9 ( Figure 2—figure supplement 4 ) .", "We also identified numerous homeobox genes , which phylogenetic analyses confirmed to be Dll , a pro-ortholog of vertebrate Dlx genes , Exd , a pro-ortholog of vertebrate Pbx genes , Hth , a pro-ortholog of vertebrate Meis1 and Meis2 , and Engrailed , a pro-ortholog of vertebrate En1 and En2 ( Figure 2—figure supplement 5 ) .", "In addition , we cloned the Wnt extracellular inhibitors Notum and Sfrp-1/2/5 , and the Wnt co-receptor Fzd9/10 ( Figure 2—figure supplements 6 and 7 ) .", "Cuttlefish possess a Bmp-2/4 gene that is an ortholog of arthropod Dpp and a pro-ortholog of vertebrate Bmp2 and Bmp4 ( Figure 2—figure supplement 8 ) , a Hh gene ( Grimaldi et al . , 2008 ) that we show to be a pro-ortholog of the vertebrate hedgehog family ( Figure 2—figure supplement 9 ) , and a gene encoding the Hh receptor Patched , a pro-ortholog of vertebrate Ptch1 and Ptch2 ( Figure 2—figure supplement 10 ) .", "The cuttlefish Sfrp ortholog that we identified as Sfrp1/2/5 was annotated incorrectly in the octopus genome as Frizzled1 ( Figure 2—source data 1 ) .", "We also found two Sp8/9 genes in the octopus genome ( Figure 2—source data 1 ) , and the cuttlefish Sp8/9 gene shows clear orthology to only one of the two octopus genes ( Figure 2—figure supplement 4 ) , suggesting that the Sp8/9 gene underwent a duplication in cephalopod mollusks .", "Therefore , we designate the octopus Sp8/9 paralogs as Sp8/9a and Sp8/9b , and the cuttlefish Sp8/9 gene that we isolated is the ortholog of Sp8/9a .", "We next investigated the spatial and temporal expression patterns of these genes during cuttlefish limb development .", "Genes that pattern the proximodistal axis of arthropod and vertebrate limbs ( Lecuit and Cohen , 1997; Mercader et al . , 1999; Panganiban et al . , 1997; Pueyo and Couso , 2005 ) showed similarly polarized patterns of expression along the proximodistal axis of cuttlefish limb buds , with Exd and Hth restricted proximally ( Figure 2B , F and G; Figure 3A–E; Figure 3—figure supplement 1A and B; and Figure 3—figure supplement 2A , I and J ) and Dll , Dac , Sp8/9a , Wnt1 , Wnt5 , and Wnt7 restricted distally ( Figure 2C , H–J; Figure 3F–I; Figure 3—figure supplement 1C–E and L–N; and Figure 3—figure supplement 2B , C , G , I and J ) .", "At stages 20–21 , the distal expression boundaries of Exd and Hth and the proximal expression boundaries of Dll and Sp8/9a appear to mark the morphological boundary between the proximal sucker-free and the distal sucker-forming regions ( compare right panels in Figure 2F–H and J with Figure 1P ) .", "Indeed , at stages when arms and tentacles begin to develop their distinctive morphologies -- tentacles are longer and have an extensive proximal sucker-free domain -- the Exd/Hth expression domains were found to extend further distally in tentacles ( Figure 3B , D ) compared to arms ( Figure 3A and C ) .", "This distal expansion of the Exd/Hth expression domain matches the expanded sucker-free region and the distal restriction of suckers in tentacles ( Figure 3E ) .", "Our finding that the proximodistal axis of cuttlefish limbs shares patterns of molecular regionalization with arthropod and vertebrate limbs led us to examine whether anteroposterior and dorsoventral axis development are also conserved .", "Posteriorly polarized activation of Hedgehog signaling in arthropod and vertebrate limbs is essential for proper patterning of the anteroposterior axis , and ectopic activation of the Hedgehog pathway induces anterior duplication of posterior structures ( Basler and Struhl , 1994; Kojima et al . , 1994; Riddle et al . , 1993 ) .", "We analyzed Hh expression during cuttlefish limb development at stages 16 to 20 and found that Hh expression is also polarized to one side of cuttlefish limb buds .", "In cuttlefishes , however , Hh expression is restricted to the anterior margin of the limb bud , whereas in arthropods and vertebrates , Hh/Shh is expressed posteriorly ( Figure 2D and K; and Figure 3—figure supplement 2D ) .", "Consistent with the anterior localization of Hh , we detected expression of Patched , which serves as a readout of Hedgehog signal transduction , in an anterior-to-posterior gradient ( Figure 2L ) .", "Thus , anteroposteriorly restricted activation of the Hedgehog pathway is a conserved feature of cephalopod , arthropod , and vertebrate limb development , but the polarity of the signaling center is reversed in cephalopod limbs .", "By stage 21 , the anteriorly restricted Hh domain has diminished and a new , central expression domain appears in the location of the brachial nerve primordia ( Figure 3—figure supplement 1F , K ) .", "We then examined the dorsoventral axis , which is controlled by the antagonistic actions of wg/Wnt and dpp/Bmp signaling in arthropods and vertebrates ( Brook and Cohen , 1996; Cygan et al . , 1997; Diaz-Benjumea et al . , 1994; Jiang and Struhl , 1996; Parr and McMahon , 1995 ) .", "In arthropods , the Wnt ligand wg is expressed ventrally , whereas the Bmp2/4 ortholog dpp is expressed dorsally ( Basler and Struhl , 1994; Diaz-Benjumea et al . , 1994 ) .", "Expression and function of the Wnt-Bmp network is conserved , albeit with inverted polarity , in vertebrate limbs; Wnt7a is expressed dorsally ( Parr and McMahon , 1995 ) and Bmp signaling activates Engrailed1 ( En1 ) ventrally ( Ahn et al . , 2001 ) , and these interactions regulate development of dorsal and ventral limb structures ( Cygan et al . , 1997; Parr and McMahon , 1995 ) .", "During cuttlefish limb development , Bmp2/4 and En show dorsally polarized expression ( Figure 2E , M and N; and Figure 3—figure supplement 2E ) .", "Genes encoding Wnt ligands ( Wnt1 , Wnt5 and Wnt7 ) and cellular components of canonical Wnt signaling cascade ( Tcf3/4 and Frz9/10 ) are expressed broadly throughout the dorsoventral axis of cuttlefish limb buds ( Figure 3F–I and Figure 3—figure supplement 1L–R; and Figure 3—figure supplement 2G , I and J ) ; however , the secreted Wnt antagonists Notum and Sfrp1/2/5 are expressed dorsally in the limb and interlimb regions ( Figure 3J–M ) , with the Sfrp1/2/5 domain extending deeper into the dorsal limb buds ( Figure 2O; Figure 3M ) .", "This dorsal expression of Wnt antagonists suggests a mechanism for restriction of Wnt signaling to the ventral side of the cephalopod limb buds .", "Taken together , these results suggest that the genetic pathways active along the proximodistal , anteroposterior , and dorsoventral axes of cephalopod limbs are homologous ( specifically , orthologous ) to the networks that regulate limb development in arthropods and vertebrates .", "In order to further test this hypothesis , we next performed a series of functional experiments to determine whether polarized expression of these signaling molecules is involved in patterning the anteroposterior and dorsoventral axes of cuttlefish limbs ( described below ) .", "We developed a method for ex-ovo culture of cuttlefish embryos ( see Material and methods ) to allow in vivo manipulations of genetic pathways in early limb buds .", "A hallmark of dorsoventral polarity is the restriction of sucker buds to the ventral surface of the limb ( Figure 1C , D and S ) , and this is preceded by ventral expression of Notum in the sucker-forming region at stage 21 ( Figure 3N–Q ) .", "We asked whether polarized expression of Bmp2/4 on the dorsal side of cuttlefish limb buds is required for the specification of dorsal identity .", "To repress dorsal Bmp activity , we implanted carrier beads loaded with Noggin ( Nog ) , a secreted Bmp inhibitor protein , on the dorsal side of stage 17 limb buds ( Figure 4A ) .", "Implantation of Nog beads on the dorsal side of cuttlefish limb buds resulted in ectopic , dorsal expansion of the Notum mRNA domain ( n = 3/3; control PBS [phosphate buffered saline] beads had no effect on Notum expression [n = 3/3] ) ( Figure 4G , H ) .", "To determine whether inhibition of dorsal Bmp signaling respecifies dorsal cells to form ventral structures , we repeated the experiment and allowed embryos to develop to stage 26–27 .", "Analysis of limb morphology by scanning electron microscopy revealed the presence of ectopic sucker buds on the dorsal surface of Nog-treated limbs ( n = 8/12; Figure 4B; Figure 4—figure supplement 1A and B ) .", "The ectopic dorsal suckers extended around the distal tip of the limb and joined the ventral sucker field .", "By contrast , in limbs that received control PBS beads dorsally , sucker buds were restricted to ventral surface and terminated at the normal dorsal-ventral boundary at the tip of the limb ( n = 15/15; Figure 4C ) .", "Our finding that antagonism of Bmp signaling results in development of ventral structures ( sucker buds ) on the dorsal side of the limb indicates that dorsal Bmp2/4 activity is required for the early specification of dorsal identity in cephalopod limb development .", "We then investigated whether the mechanism of anteroposterior patterning is conserved between cephalopod and vertebrate/arthropod limbs .", "To determine whether the anterior expression of Hh in cuttlefish limb buds controls anteroposterior patterning , we grafted Hh-expressing cells from the thickened funnel epithelium ( Tarazona et al . , 2016 ) to the posterior side of stage 17 limb buds , which created an ectopic source of Hh opposite the endogenous Hh expression domain ( Figure 4D ) .", "We used Hh-expressing cells from the funnel , rather than the anterior side of the limb bud , to exclude the possibility of grafted limb cells undergoing self-differentiation .", "Transplantation of Hh-expressing cells to the posterior side of cuttlefish limb buds resulted in posterior limb duplications ( n = 7/12; Figure 4E and Figure 4—figure supplement 1C , D ) .", "Analysis of morphology and gene expression in host limbs approximately 10 days after receiving the graft revealed that the posterior duplications even contained sucker buds , which were marked by Notum expression ( Figure 4I and J ) .", "By contrast , limbs that received control grafts of stage 24 funnel epithelium that lacks Hh expression ( Tarazona et al . , 2016 ) developed normally ( n = 8/8; Figure 4F ) .", "Although these results suggest that Hh is sufficient to re-specify anteroposterior polarity in cuttlefish limbs , we wanted to exclude the possibility that posterior identity was induced by other factors that could be present in the graft .", "Therefore , we tested whether Hh signaling is necessary for anteroposterior patterning of cephalopod limbs by specifically repressing endogenous Hh signaling .", "A notable morphological feature of cephalopod limbs is the anteroposterior arrangement of parallel sucker rows on the ventral surface ( Figure 1C , D and S ) .", "Based on the results of the transplantation experiments , we reasoned that Hh signaling could regulate the number of sucker rows along the anteroposterior axis of cephalopod limbs , similar to the manner in which Hh specifies digit number along the anteroposterior axis of vertebrate limbs ( Lewis et al . , 2001; Scherz et al . , 2007; Zhu et al . , 2008 ) .", "Transitory treatment ( 2 days ) of cuttlefish embryos at stage 16 , when Hh is first expressed on the anterior side of the early limb bud , with the small molecule cyclopamine , an inhibitor of Smoothened that represses Hh signaling ( Figure 4K ) , disrupted the anteroposterior distribution of sucker rows in arms and tentacles .", "Severity of this phenotype ranged from arms with a reduced number of suckers and sucker rows ( n = 10/10; Figure 4N and O ) to completely sucker-free tentacles ( n = 8/10; Figure 4L ) .", "Control treatments with vehicle only ( DMSO ) did not alter the normal anteroposterior pattern of sucker rows ( n = 8/8; Figure 4M and P ) .", "Finally , to confirm that the phenotype of cyclopamine-treated embryos was not due to failure in brachial nerve differentiation , we examined acetylated tubulin immunofluorescence , which shows that the brachial nerve cords develop in both cyclopamine and DMSO treated embryos ( Figure 4—figure supplement 1E , F ) .", "These results show that Hh signaling is necessary for proper patterning of the anteroposterior axis in cephalopod limb development ." ], [ "Our finding that the proximodistal , dorsoventral , and anteroposterior axes of cuttlefish limb buds are patterned by the same pathways that regulate arthropod and vertebrate limb development suggests that the independent evolution of limbs in cephalopod mollusks involved recruitment of an ancient genetic program for appendage development .", "Discovery of this appendage developmental circuit within Spiralia demonstrates its deep conservation across all three branches of Bilateria ( i . e . , Deuterostomia , Ecdysozoa , and Spiralia ) , suggesting its presence in the common ancestor of all bilaterians ( Figure 5 ) .", "Parallel recruitment of this ancient developmental genetic program may have played a role in the independent evolution of a wide diversity of appendages in many bilaterian lineages ( Moczek and Nagy , 2005; Shubin et al . , 2009 ) .", "The discovery that cephalopod , arthropod , and vertebrate appendages develop using conserved developmental mechanisms does not exclude the possibility that other types of appendages evolved by recruiting a different set of developmental tools ( or by utilizing the same tools but in different patterns ) .", "Examination of gene expression in lateral parapodial appendages of the polychaete worm Neanthes , also a spiralian , led to the suggestion that the molecular mechanisms of polychaete appendage development might not be conserved with ecdysozoans and deuterostomes ( Winchell and Jacobs , 2013; Winchell et al . , 2010 ) .", "However , given that relatively few genes were examined in Neanthes parapodia , it is difficult to conclude whether the reported differences between parapodia and arthropod/vertebrate/cephalopod limbs reflect the unique nature of parapodia or lineage-specific divergences that occurred after recruitment of the core developmental program .", "A study of a different polychaete , Platynereis dumerilii , showed that gene expression is generally conserved in appendages that form during regeneration of caudal trunk segments , although some divergent patterns were observed and these were suggested to reflect taxon-specific differences in appendage morphology ( Grimmel et al . , 2016 ) .", "How parapodia fit into the picture of animal appendage evolution will require additional studies of spiralian appendages to increase the diversity of species , types of appendages , and number of genes/pathways interrogated .", "Nonetheless , our discovery that cephalopod arms and tentacles evolved by parallel recruitment of the same genetic program that orchestrates appendage formation in arthropods and vertebrates suggests that this program was present in the bilaterian common ancestor .", "Activation of this ancient developmental program could also underlie the origin of other morphological innovations , including non-locomotory appendages such as beetle horns ( Moczek and Nagy , 2005; Moczek et al . , 2006 ) and external genital organs of amniote vertebrates ( Cohn , 2011; Gredler et al . , 2014 ) .", "We propose that the genetic program for appendage formation was stabilized in Bilateria , including those lineages that lack limbs , for development of appendage-like structures .", "This hypothesis implies that the ancestral appendage developmental program was not a latent developmental feature that was redeployed each time that limbs evolved , but rather it might have been a continuously activated network that controlled formation of outgrowths in general .", "One of our observations raises the possibility that the gene network that controls appendage formation could be conserved in non-cephalopod mollusks , despite the absence of arms and tentacles in those lineages .", "During cuttlefish funnel/siphon development , we found asymmetric expression of Hh ( Tarazona et al . , 2016 ) and proximodistally polarized expression of Wnt5 and Exd , which partially mirror their expression patterns during arm and tentacle development ( Figure 5—figure supplement 1 ) .", "If this gene network is found to be active in the developing funnel/siphon of non-cephalopod mollusks , then the funnel/siphon would represent a more primitive site of expression in mollusks , given that evolution of the molluscan funnel/siphon predates the origin of cephalopod limbs ( Nielsen , 2012; Ruppert et al . , 2004 ) .", "Further studies of gene expression and function during funnel/siphon development in mollusks will be needed to determine if this clade shows conservation of the appendage development program beyond cephalopod arm and tentacle development .", "Although the bilaterian common ancestor may have used this genetic program to control development of rudimentary outgrowths ( e . g . , appendages , funnel/siphon , genitalia ) , it is also possible that it predates the evolution of locomotory and non- locomotory appendages .", "Studies of cephalic neuroectoderm showed that gene expression patterns controlling the anteroposterior axis of the neuroectoderm mirror the organization of gene expression territories along the proximodistal axis of locomotory appendages , including polarized expression of Sp8 , Dll , Dac and Hth ( Lemons et al . , 2010 ) .", "Similarly , Minelli has suggested that the appendage patterning program could reflect co-option of a more ancient ( pre-bilaterian ) program for patterning the main body axis and , therefore , bilaterian appendages are simply secondary body axes ( Minelli , 2000; Minelli , 2003 ) .", "Cephalopod arms and tentacles have no direct structural homologs in non-cephalopod mollusks; however , they likely formed from the ventral embryonic foot , a morphological and embryological hallmark of the molluscan bodyplan ( Nödl et al . , 2016 ) .", "Therefore , cephalopod arms and tentacles may be considered evolutionary novelties that are derived from a structure that is conserved across Mollusca .", "This raises the question of whether other foot-derived outgrowths/appendages ( e . g . , in sea slugs ) evolved by co-option of the same developmental program that cephalopods , arthropods , and vertebrates use to build appendages .", "Although the results presented here suggest that an ancient and conserved developmental genetic program facilitated the origin of cephalopod limbs , they also indicate that fine-scale regulatory changes may have played a role in the diversification of cephalopod limb morphologies .", "For example , evolution of specialized tentacles from serially homologous arms may have resulted from a distal shift in the expression of proximal identity genes , such as Exd and Hth , which could have extended the proximal sucker-free domain and restricted suckers to a distal pad ( see Figure 3A–E ) .", "Likewise , the results of functional manipulations of Hh signaling in cuttlefish limbs suggests that the diversity in the number of sucker rows in cephalopod limbs ( i . e . four rows in squids and cuttlefishes , two in octopus , and one in vampire squid and glass octopus ) could be explained by modulation of Hh signaling , in the same way that gradual changes to Shh regulation has led to variation in digit number in tetrapod vertebrates ( Scherz et al . , 2007; Shapiro et al . , 2003; Zhu et al . , 2008 ) .", "Finally , we note that while the data presented here point to the existence of a deeply conserved genetic program for appendage development across Bilateria , this does not imply that the limbs of cephalopods , arthropods , and vertebrates are homologous structures , or that limbs were present in the common ancestor .", "Rather , these results show that homologous developmental mechanisms underlie the multiple parallel origins of limbs in bilaterians ." ], [ "Sepia officinalis and Sepia bandensis eggs were purchased from commercial suppliers , incubated until they reached the required stages ( Lemaire , 1970 ) , and prepared for in situ hybridization ( ISH ) and immunohistochemistry as described ( Tarazona et al . , 2016 ) .", "Three-dimensional reconstructions of gene expression in cuttlefish embryos were performed as previously described ( Tarazona et al . , 2016 ) .", "Cuttlefish embryos were fixed in 4% paraformaldehyde in phosphate buffered saline ( PBS ) overnight at 4°C and were washed with PBS the next day .", "Embryos were fixed in 1% osmium tetroxide solution in PBS for 30 min and then washed three times in PBS , dehydrated through a graded ethanol series , critical point dried , and sputter coated with gold .", "Embryonic samples were scanned using a Hitachi SU5000 and Hitachi TM3000 .", "RNA extraction from Sepia officinalis and Sepia bandensis embryos at stages 15–26 was performed using TRIzol reagent ( Ambion ) following the manufacturer’s instructions .", "cDNA synthesis was performed by an AMV reverse transcriptase ( New England Biolabs ) following the manufacturer’s instructions .", "PCR amplification was carried out on Sepia cDNA pools , amplicons were cloned into TA vectors and sequenced .", "We then performed multiple sequence alignments ( MSA ) with ClustalW ( PMID: 7984417 ) using the predicted amino acid sequence of our cuttlefish cDNA fragments , and putative metazoan orthologous genes downloaded from NCBI RefSeq protein databases ( Figure 2—source data 1 ) .", "We performed nine MSA for Wnt , Tcf , Sfrp , Notum , Patch , Hh , Bmp , Sp and Homeodomain families .", "Each of the nine MSA was analyzed by ProtTest ( PMID: 15647292 ) , in order to determine the best combination of amino acid substitution model and other free parameters ( amino acid site frequency , site heterogeneity and invariant sites ) , using Akaike information criterion ( Figure 2—source data 1 ) .", "We applied the best model in RaXML ( PMID: 18853362 ) for each MSA and performed maximum likelihood phylogenetic inference , estimating branch support by bootstrap , and then majority consensus of the trees from all bootstrap partitions was performed to compute the final tree topology .", "All sequences have been deposited in Genbank under accession numbers MK756067-MK756082 ( complete list of entries is provided in Figure 2—source data 1 ) .", "Whole-mount ISH was performed using digoxigenin- and fluorescein-labeled antisense ( or sense control ) RNA probes according to protocols described previously ( Tarazona et al . , 2016 ) .", "Due to limited availability of embryonic material at relevant early developmental stages , only a limited number of S . bandensis embryos were used for ISH .", "Thus , the majority of ISH were performed in S . officinalis embryos using S . officinalis antisense RNA probes , however , some ISH were performed in S . officinalis embryos using S . bandensis antisense RNA probes .", "We validated the specificity of S . bandensis probes in S . officinalis embryos by comparing the gene expression domains marked by these probes in embryonic material from both species at stages 20 and 21 .", "This comparison shows that gene expression territories identified by these probes at these stages were indistinguishable between the two species ( Figure 3—figure supplement 2 ) , consistent with their high level of sequence similarity ( Figure 3—source data 1 ) .", "Excluding the S . bandensis ISH mentioned above , all the experiments described in this work were carried out with S . officinalis embryos .", "Proliferating cells were detected by immunolocalization of Histone H3 Serine 10 phosphorylation using an antibody against H3S10p/PHH3 ( 06–570 , EMD Millipore ) and brachial nerve tissue was detected using an antibody against acetylated alpha tubulin ( ab24610 , Abcam ) .", "A protocol for ex-ovo cuttlefish embryo culture was established for this study , as a modified version of previous descriptions of ex-ovo embryo culture in squid ( Arnold , 1990 ) .", "Briefly , to minimize the problem of bacterial and fungal contamination we started the protocol by taking 10 cuttlefish eggs at the appropriate stage , placing them in a 50 ml tube , and washing them with 0 . 22 μm filtered artificial sea water ( FASW ) five times .", "Eggs were then cleaned with a freshly prepared 5% bleach solution ( 0 . 25% sodium hypochlorite in FASW ) for 5 s and immediately washed with FASW five times .", "The bleaching and washing steps were repeated two to three times .", "Five additional washes with FASW were carried out before incubating the eggs in 2X antibiotic/antimycotic solution ( A5955 , Sigma ) in FASW for 2 hr at ambient temperature .", "Each cuttlefish egg was then transferred to a 50 mm diameter petri dish that was coated with a ~ 5 mm layer of 0 . 5% low melting point agarose ( 16520050 , ThermoFisher ) , and filled with culture medium ( components described below ) .", "The agarose layer had a hemispherical depression in the center of the dish made with a sterile 10 mm acrylic necklace bead before gel solidification .", "The 10 mm hemispherical depression is essential to maintain the normal shape of the yolk mass once the embryos are outside their egg case .", "Embryos were then extracted from their egg cases ( S . officinalis are housed individually , one embryo per egg case ) very slowly and with extreme care to avoid rupturing the yolk mass at the vegetal pole of the egg and were carefully placed in the hemispherical depression in the agarose .", "To extract the embryo , a single 5 mm diameter hole was created in the egg case , which generates a burst of the vitelline liquid and part of the embryo out from the egg case .", "With the hole kept open , the spontaneous shrinkage of the egg case aided in the expelling of the large cuttlefish embryo .", "Of every ten eggs prepared this way , between two and five embryos were damaged and had to be discarded .", "Embryos were cultured at 17°C .", "For protein carrier bead implantation , 150 μm diameter Affi-Gel Blue Gel beads ( 153–7301 , Biorad ) were selected and transferred to 1 mg/ml recombinant human Noggin protein ( 6057 NG , R and D Systems ) in PBS and incubated for 30 min to 1 hr at ambient temperature before being implanted in embryos .", "Control beads were incubated in PBS only .", "Grafts of Hh-expressing tissue were performed using stage 24 donor embryos and carefully dissecting the funnel side of the mantle-funnel locking system , which carries the Hh-expressing thickened funnel epithelium ( Tarazona et al . , 2016 ) .", "The dissected tissue was transferred to 10 mg/ml Dispase II ( D4693 , Sigma ) in cuttlefish culture medium and incubated for 40 min or until the thickened epithelium was easily detaching from the underlying mesenchyme with the aid of forceps .", "Tissue was then transferred to cuttlefish culture medium without Dispase II , where they were washed and then grafted into limb buds of stage 17 host embryos .", "Control grafts were performed using the non-Hh expressing epithelium of the funnel .", "After bead implantation or tissue grafts , embryos were incubated at 17°C until control embryos reached stage 26 , at which point all embryos were collected and prepared for SEM or ISH .", "We used a modified version of a cell culture medium for squid neuron , glia and muscle cells that was previously described ( Rice et al . , 1990 ) .", "Cuttlefish culture medium had no glucose , was buffered with 20 mM HEPES and adjusted the pH to 7 . 6 .", "The medium contained: 430 mM NaCl , 10 mM KCl , 10 mM CaCl2 , 50 mM MgCl2 , 1X MEM Non-Essential Amino Acids Solution ( 11140–076 , Life Technologies ) , 1X MEM Amino Acids Solution ( 11130–051 , Life Technologies ) , 1X MEM Vitamin Solution ( 11120–052 , Life Technologies ) , 2 mM L-Glutamine ( 25030–081 , Life Technologies ) .", "The medium was supplemented with 20% heat inactivated fetal bovine serum ( 16000044 , ThermoFisher ) and 1X antibiotic/antimycotic solution ( A5955 , Sigma ) .", "Cyclopamine treatments were performed as described previously ( Tarazona et al . , 2016 ) with the following modifications; stage 16 embryos were treated with 10 μM cyclopamine ( C988400 , Toronto Research Chemicals ) for 2 days , then washed thoroughly ten times with FASW .", "Embryos were then washed five more times every hour and one time every day before collecting the embryos for SEM .", "Control embryos were treated with 0 . 1% DMSO and then washed as described above ." ] ]
[ "Cephalopod mollusks evolved numerous anatomical novelties , including arms and tentacles , but little is known about the developmental mechanisms underlying cephalopod limb evolution .", "Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods , although they evolved limbs independently .", "In cuttlefish limb buds , Hedgehog is expressed anteriorly .", "Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications .", "Bmp and Wnt signals , which establish dorsoventral polarity in vertebrate and arthropod limbs , are similarly polarized in cuttlefish .", "Inhibition of Bmp2/4 dorsally caused ectopic expression of Notum , which marks the ventral sucker field , and ectopic sucker development .", "Cuttlefish also show proximodistal regionalization of Hth , Exd , Dll , Dac , Sp8/9 , and Wnt expression , which delineates arm and tentacle sucker fields .", "These results suggest that cephalopod limbs evolved by parallel activation of a genetic program for appendage development that was present in the bilaterian common ancestor ." ]
[ "Legs , wings , flippers and tentacles are just some examples of the diverse variety of animal limbs .", "Despite striking differences in form and function , all limbs develop in embryos using similar fundamental processes , like producing an outgrowth from the body and placing structures such as fingers , feathers , or suckers at appropriate positions .", "Animals have solved this problem multiple times during the history of life on Earth , in that limbed animals have arisen from limbless ancestors on many separate occasions .", "It is not clear , however , whether the same genetic instructions shape the developing limbs of all species .", "Species that have limbs fall under three main groups of animals: arthropods , such as insects and crustaceans; vertebrates , like amphibians , reptiles and mammals; and a specialized group of mollusks known as cephalopods , which includes squid , cuttlefish and octopuses .", "It has been over two decades since the discovery that the limbs of vertebrates and insects develop using a similar molecular recipe , but the mechanisms responsible for the limbs of cephalopods had not been determined .", "Tarazona et al . have now established that the genetic mechanisms that control how cuttlefish limbs develop are the same as those used by the limbs of vertebrates and insects .", "These mechanisms are also applied for similar purposes in each animal group .", "Notably , a signaling pathway called hedgehog , which controls the number of fingers that develop on a hand , also dictates the number of suckers on a cuttlefish arm .", "This may mean that an ancient system for creating limbs emerged over 500 million years ago in the earliest animals with bilateral symmetry ( i . e . , animals with mirror image halves ) , and activating this ancient genetic program resulted in the evolution of limbs in different animal lineages .", "The extent of the genetic similarities between cuttlefish , mammals and insects suggests that this mechanism is likely to provide instructions about where cells position themselves in the developing limb .", "The next step is to examine how these common systems are interpreted differently to give arms , legs , wings and other limb forms ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "genetics and genomics", "cancer biology" ]
BRAFV600E induces reversible mitotic arrest in human melanocytes via microRNA-mediated suppression of AURKB
elife-70385-v3
[ [ "Cutaneous melanoma is a potentially deadly skin cancer arising from the pigment-producing melanocytes of the human epidermis .", "An activating mutation in the BRAF proto-oncogene ( BRAFV600E ) drives over half of all cutaneous melanomas ( Garnett and Marais , 2004; Davies et al . , 2002; Pollock et al . , 2003 ) .", "Yet , when a melanocyte acquires a BRAFV600E mutation , the cell does not immediately transform to cancer .", "Instead , it usually undergoes clonal proliferation followed by stable arrest resulting in a benign skin lesion known as a melanocytic nevus or ‘mole’ ( Pollock et al . , 2003; Shain et al . , 2015; Bastian , 2014; Dankort et al . , 2009 ) .", "Despite the continued expression of BRAFV600E , the majority of melanocytic nevi remain innocuous for the lifespan of the individual , suggesting that nevus cells have robust intrinsic defenses against hyperproliferation .", "By inducing hyperproliferation and subsequent arrest , the BRAFV600E mutation elicits divergent , biphasic phenotypes within a single cell—a poorly understood phenomenon .", "Characterization of the mechanisms and environmental factors that distinguish the two phenotypes could illuminate candidate strategies for chemoprevention of melanocytic nevus formation or interception of melanoma initiation .", "The BRAFV600E mutation is clonally present in ~80% of human melanocytic nevi ( Pollock et al . , 2003 ) and melanocyte-specific expression of the oncogene is sufficient to induce nevus formation in mice ( Dankort et al . , 2009; Damsky et al . , 2015; Ruiz-Vega et al . , 2020 ) , suggesting that BRAFV600E drives proliferation arrest in melanocytes .", "The prevailing theory to explain this seemingly paradoxical role for BRAFV600E is oncogene-induced senescence ( OIS ) ( Michaloglou et al . , 2005 ) .", "Cellular senescence is defined as the permanent cell-cycle arrest of previously replication-competent cells ( He and Sharpless , 2017; Roy et al . , 2020 ) .", "Senescence is associated with a variety of molecular hallmarks including elevated expression of p16INK4A and other cyclin-dependent-kinase inhibitors , TP53 , H2AX , lysosomal beta-galactosidase and senescence-associated secretory proteins ( SASPs ) as well as a DNA content profile indicative of arrest in the G0/G1 phases of the cell cycle ( Roy et al . , 2020; Serrano et al . , 1997; Campisi and d’Adda di Fagagna , 2007; Collado and Serrano , 2006; Afshari et al . , 1993; Chatsirisupachai et al . , 2019 ) .", "The concept of OIS as a barrier to tumorigenesis derived from observations of a durable proliferation arrest induced by overexpression of oncogenic RAS or RAF in immortalized human fibroblasts ( Serrano et al . , 1997; Zhu et al . , 1998 ) .", "More recent studies have questioned whether the term ‘senescence’ aptly describes the proliferation arrest of melanocytic nevi .", "Cell cycle re-entry that accompanies nevus recurrence ( King et al . , 2009 ) , eruption ( Burian and Jemec , 2019 ) , or transformation to primary melanoma suggests that the nevus arrest phenotype is reversible rather than permanent .", "Similarly , the expression of senescence markers does not readily distinguish human melanocytic nevi from primary or transformed melanocytes in humans ( 16INK4A , TP53 , H2AX , and beta-galactosidase ) or mice ( any known hallmark of senescence ) ( Ruiz-Vega et al . , 2020; Cotter et al . , 2007; Tran et al . , 2012 ) .", "Setting aside the permanence of OIS , melanocytic nevus arrest was largely thought to be driven by induction of p16INK4A expression from the CDKN2A gene .", "Indeed , early studies demonstrated that BRAFV600E induces p16INK4A expression and that melanocytic nevi frequently express high levels of p16INK4A ( Michaloglou et al . , 2005 ) .", "Despite this , p16INK4A expression is neither ubiquitous across melanocytic nevi , nor uniform within the cells of a single nevus ( Oaxaca et al . , 2020; Zeng et al . , 2018 ) , and neither knockdown of the 16INK4A transcript nor ablation of the CDKN2A locus prevents the onset or rescues BRAFV600E-induced proliferation arrest ( Michaloglou et al . , 2005; Zeng et al . , 2018; Haferkamp et al . , 2009; McNeal et al . , 2015 ) .", "BRAFV600E also stimulates expression of p15INK4B—the translational product of the CDKN2B gene ( McNeal et al . , 2015 ) which is situated adjacent to the CDKN2A locus—suggesting BRAFV600E might orchestrate arrest via multiple cyclin-dependent kinase inhibitors that induce arrest in the G1 phase of the cell cycle , prior to genome synthesis .", "While the loss of the CDKN2A/B locus is a formative event in melanoma progression , genetic evolution studies of melanoma progression suggest that selection against the CDKN2A/B locus occurs after BRAFV600E melanocytes have already escaped nevus-associated arrest ( Zeng et al . , 2018; Shain et al . , 2018 ) .", "Collectively , these data complicate the straightforward model wherein BRAFV600E induces the expression of cell cycle regulators leading to proliferation arrest , which is later circumvented by the genetic loss of those regulators .", "Given these observations , we reasoned that the arrest response of human melanocytes to BRAFV600E might be distinct from previous reports of BRAFV600E-induced OIS in other cell types .", "Specifically , we hypothesized that nevus-associated arrest is conditional and coordinated by reversible changes in gene expression .", "Here , we identify a transcriptional program that is consistently elevated in melanocytic nevi and includes microRNAs ( miRNAs ) previously shown to have diagnostic value ( Babapoor et al . , 2016; Torres et al . , 2020 ) .", "In parallel , we observe that , unlike RAF-induced OIS in fibroblasts , BRAFV600E-induced arrest in human melanocytes is reversible and conditional .", "Merging these two observations , we discover that in melanocytes , regulation of miRNAs coupled to differentiation status permits toggling between BRAFV600E-driven hyperproliferative to arrested phenotypes ." ], [ "Since melanocytic nevi do not consistently express known senescence markers , we sought to characterize the transcripts that are specifically elevated within nevus melanocytes ( Figure 1A ) .", "We analyzed previously published data sets comparing RNA expression between benign and malignant melanocytic lesions ( Shain et al . , 2018; Torres et al . , 2020 ) .", "Each clinical specimen contained matched RNA samples derived from melanocytic nevi and melanomas that arose from those nevi .", "We reasoned that downregulated transcripts within these newly formed melanomas could represent the most immediate barriers to melanocyte growth .", "Of the differentially expressed genes ( adjusted p value<0 . 05 ) , 6 of the top 10 with elevated expression in nevi were non-coding miRNAs ( Figure 1B and Supplementary file 1 ) .", "Among these were miRNAs previously shown as enriched in nevi compared to melanomas , including MIR211-5p , MIR125A-5p , MIR125B-5p , and MIR328-3p ( Torres et al . , 2020; Latchana et al . , 2016 ) .", "To determine whether elevated expression of these miRNAs distinguished nevus melanocytes from normal epidermal melanocytes , we profiled melanocytes from fresh healthy skin and nevi ( Supplementary file 2 ) .", "Three of the miRNAs ( MIR211-5p , MIR328-3p , and MIR125B-5p ) were also more highly expressed in melanocytic nevi as compared to healthy melanocytes or melanomas—an expression pattern consistent with enrichment in arrested melanocytes—whereas one miRNA ( MIR125A-5p ) presented a pattern consistent with lineage-specific expression that is lost upon transformation ( Figure 1C ) .", "We conclude that MIR211-5p , MIR328-3p , and MIR125B-5p are transcripts specifically enriched in arrested melanocytic nevi .", "To investigate whether elevated expression of MIR211-5p , MIR328-3p , or MIR125B-5p induce melanocytic arrest , we introduced synthesized mimetics of the mature miRNAs into primary human melanocytes and tracked proliferation over 7 days in culture .", "The introduction of either MIR211-5p or MIR328-3p resulted in significantly fewer melanocytes compared to a non-targeting control ( Figure 1D ) .", "Increased concentrations of these mimics resulted in a dose-dependent decrease in EdU uptake , consistent with a cell cycle defect ( Figure 1E ) .", "To better characterize the effect of MIR211-5p or MIR328-3p expression on cell cycle and cell viability , we performed time-lapsed quantitative phase imaging ( QPI ) .", "We and others have previously established QPI as a method for adherent cell cytometry that can simultaneously measure melanocyte proliferation , cell death , and cell cycle stage with single-cell resolution ( Hejna et al . , 2017; Barker et al . , 2020; Falck Miniotis et al . , 2014 ) .", "Images were taken every hour for 5 days post-nucleofection ( Figure 1F ) .", "Cells expressing MIR211-5p or MIR328-3p exhibited significantly reduced growth curves ( Figure 1G ) .", "We observed no evidence of increased cell death ( Figure 1H ) , and confirmed this result with Annexin V staining ( Figure 1I ) .", "Experiments performed using three additional melanocyte preparations counted over 7 days post nucleofection yielded similar results ( Figure 1J ) .", "Thus , MIR211-5p and MIR328-3p induce proliferation arrest in human melanocytes .", "Further characterization of the QPI images revealed a distinct increase in intra-cellular dry mass in proliferation-arrested conditions ( Figure 1K ) .", "The dry mass of a cell is a measurement of total biomass ( DNA , RNA , proteins , lipids , etc . ) and is quantitatively measured by QPI .", "Relative dry mass fluctuates with cell cycle stage in a predictable manner ( Falck Miniotis et al . , 2014 ) .", "The 20–40% increase we observed here is characteristic of cells that have doubled their DNA content but failed to complete mitosis .", "We confirmed this finding by measuring DNA content of the arrested populations ( Figure 1L ) .", "We observed significant increases of cells with both 4 N DNA content and greater than 4 N DNA content , indicative of cytokinesis failure following DNA replication ( Figure 1M ) .", "Consistent with this interpretation , time-lapsed imaging revealed incidences of tripolar mitosis and anaphase bridging in miRNA-expression melanocytes—both processes associated with cytokinesis failure ( Normand and King , 2010 ) —which were not observed in control conditions or standard culture ( Video 1 and Figure 1—figure supplement 1 ) .", "We conclude that MIR211-5p and MIR328-3p induce mitotic failure and proliferation arrest when introduced into primary human melanocytes .", "To investigate the mechanism by which MIR211-5p and MIR328-3p induce arrest in primary human melanocytes , we applied our previously established pipeline for identification of relevant miRNA targets ( Figure 2A; Judson et al . , 2013 ) .", "First , we nucleofected MIR211-5p , MIR328-3p , or control RNA mimics into primary human melanocytes freshly isolated from four independent donors and conducted RNA sequencing ( Supplementary file 3 ) .", "We next selected all genes that were significantly downregulated by either miRNA as compared to the control RNA , and further refined the set to the computationally predicted targets of MIR211-5p and/or MIR328-3p .", "Finally , we nucleofected siRNAs targeting the 115 resulting genes and assayed for reduced EdU incorporation that would phenocopy MIR211-5p or MIR328-3p expression ( Supplementary file 4 ) .", "As expected , computationally predicted targets of MIR211-5p and MIR328-3p were enriched in the set of downregulated genes following MIR211-5p or MIR328-3p nucleofection , respectively ( Figure 2B ) .", "AURKB , GPR3 , and MAPKAPK2 showed the greatest magnitude of repression due to miRNA expression with significant EdU reduction upon siRNA-mediated knockdown ( Figure 2C ) .", "Of these , AURKB ( predicted MIR211-5p target ) and GPR3 ( predicted MIR328-3p target ) were significantly inhibited upon nucleofection of either miRNA ( Figure 2D ) .", "We reasoned these genes might represent convergent nodes driving proliferation arrest .", "Further exploration of the AURKB transcript revealed a potential MIR328-3p binding site just before the 3′ untranslated region ( Figure 2E; Dweep et al . , 2014 ) .", "In contrast , we were unable to identify a candidate MIR211-5p binding site in the GPR3 transcript , suggesting indirect inhibition of this gene by MIR211-5p .", "To determine whether decreased AURKB and/or GPR3 expression was associated with melanocytic nevi , we acquired 23 clinical specimens and measured both the transcript and protein expression of these genes ( Figure 2F–H ) .", "The expression of both AURKB and GPR3 protein and mRNA was enriched in melanomas as compared to nevi ( Figure 2G–H ) .", "Taken together , these data demonstrate an inverse expression between two miRNAs ( MIR211-5p and MIR328-3p ) and two targeted mRNAs ( AURKB and GPR3 ) that toggles concurrently with the transition of arrested melanocytic nevi to melanomas .", "To further investigate whether either mRNA was sufficient to rescue miRNA-mediated arrest , primary melanocytes were transduced with constructs expressing either zsGreen ( control ) , AURKB , or GPR3 ( Figure 2I-K ) , then subsequently nucleofected with either MIR211-5p or MIR328-3p .", "Expression of either mRNA partially rescued MIR211-5p-induced arrest , while AURKB , but not GPR3 , partially rescued MIR328-3p-induced arrest ( Figure 2L ) .", "Thus , while other MIR211-5p and MIR328-3p targets , including GPR3 , may play a role in the induction of melanocyte proliferation arrest , these data suggest that AURKB inhibition is a critical convergent node of both miRNAs in facilitating proliferation arrest in nevi .", "This interpretation is further supported by the well-established role of AURKB as an essential orchestrator of successful mitosis ( D’Avino and Capalbo , 2015 ) .", "We hypothesized that AURKB inhibition by MIR211-5p and MIR328-3p may be the underlying mechanism by which BRAFV600E induces proliferation arrest in melanocytes .", "BRAFV600E expression has been demonstrated to result in an accumulation of growth-arrested fibroblasts with 2 N DNA content , presumably in a permanent G0/G1 senescent state ( Michaloglou et al . , 2005; Serrano et al . , 1997; Zhu et al . , 1998 ) .", "In contrast , upon overexpression of MIR211-5p or MIR328-3p , we observed an accumulation of arrested melanocytes with ≥4 N DNA content , and a significant decrease in cells with 2 N DNA content ( Figure 1M ) .", "To determine the DNA content profile of primary human melanocytes under BRAFV600E-induced arrest , we utilized an established lentiviral construct for dose-responsive doxycycline-inducible BRAFV600E expression ( Figure 3A; McNeal et al . , 2015 ) .", "We monitored melanocyte growth under a titration of doxycycline and identified the lowest concentration ( 15 . 6 ng/ml ) that induced sustained arrest after 40 hr exposure ( Figure 3B ) .", "Similar to our observations for miRNA-induced arrest , relative per cell dry mass increased by 50% concurrent with BRAFV600E induced-arrest ( Figure 3C ) .", "Cell cycle profiling confirmed a significant and dose-dependent accumulation of cells with ≥4 N DNA content and a decrease in cells with 2 N DNA content ( Figure 3D–E ) .", "The cell cycle profiles were notably dissimilar to that of melanocytes treated with a pharmacologic CDK4/6 inhibitor ( PD0332991 , Palbociclib ) ( Clark et al . , 2016 ) known to induce G0/G1 arrest .", "Treatment with a pharmacologic AURKB inhibitor ( AZD2811 , Barasertib ) ( Helfrich et al . , 2016 ) that induces G2/M arrest and mitotic failure also resulted in an accumulation of melanocytes with ≥4 N DNA content and a decrease in cells with 2 N DNA content .", "In addition to the accumulation of 2 N cells , previous reports of RAF expression in fibroblasts show that transient expression can induce a durable arrest phenotype ( Zhu et al . , 1998 ) .", "To test the durability of BRAFV600E-induced arrest in human melanocytes , we induced doxycycline-dependent BRAFV600E expression for 48 hr and monitored cell number for an additional 2 days to confirm arrest .", "Media was then replaced to remove doxycycline .", "Within 12 hr of doxycycline removal , the previously arrested melanocytes began to proliferate ( Figure 3F ) , demonstrating that BRAFV600E-induced proliferation arrest in melanocytes is reversible and dependent on continued oncogene expression .", "Nevi recurrence after excision ( King et al . , 2009 ) and eruption upon external stimuli ( Burian and Jemec , 2019 ) , together with our observation that BRAFV600E induced arrest is reversible in melanocytes , suggested that BRAFV600E-induced arrest may depend on secondary signals from the microenvironment .", "Tetradecanoylphorbol acetate ( TPA ) is a protein kinase C ( PKC ) activator commonly used as a mitogen in primary melanocyte media to substitute for endothelin receptor type B activation ( Hsu et al . , 2005 ) .", "In contrast , TPA also inhibits the cell cycle of melanoma cells ( Coppock et al . , 1995; Arita et al . , 1998 ) .", "BRAFV600E expression permits growth of the Melan-A cell line in the absence of TPA ( Wellbrock et al . , 2004 ) .", "We therefore wanted to determine the relative contributions of TPA and BRAFV600E to the BRAFV600E-induced primary melanocyte arrest in vitro .", "We induced BRAFV600E expression in primary human melanocytes in two medias—one containing TPA ( +TPA ) or one containing the endothelin receptor type B ligand , endothelin-1 ( TPA-free ) and observed divergent dose-responsive phenotypes ( Figure 3G ) .", "In the presence of TPA , BRAFV600E expression inhibited cell proliferation .", "By contrast , in TPA-free media , BRAFV600E expression induced proliferation , a phenotype that persisted for the remaining duration of the primary cultures ( 4–6 weeks ) .", "We next subjected melanocytes to the arrest-inducing conditions of doxycycline treatment in +TPA media for 4 days , then replaced with doxycycline-containing , TPA-free media .", "We observed a rapid shift to the proliferative phenotype even in the continued presence of doxycycline ( Figure 3H ) .", "To determine whether the observed reversibility of BRAFV600E-induced growth arrest was due to insufficient duration or degree of expression of the oncogene , we exposed melanocytes to a high concentration of doxycycline ( 60 ng/ml ) for 30 days prior to switching medias ( Figure 3I ) .", "Melanocytes in +TPA media remained arrested for the duration of doxycycline treatment then grew again upon removal of either doxycycline or TPA ( Figure 3J–K ) .", "From these results , we conclude that BRAFV600E expression induces divergent and interconvertible phenotypes—either hyperproliferation or a reversible arrest—in human melanocytes conditional on extrinsic stimuli .", "To investigate the relative contributions of BRAFV600E and TPA to the expression of MIR211-5p and MIR328-3p , we conducted small RNA sequencing of melanocytes grown in +TPA or TPA-free media and with or without BRAFV600E expression .", "The expression of MIR328-3p was relatively stable , demonstrating less than twofold changes across conditions ( Figure 4A–B , Supplementary file 5 ) .", "In contrast , MIR211-5p was one of the most differentially expressed genes in +TPA conditions compared to TPA-free conditions ( adj . p=6 . 8E−23 ) ( Figure 4A–B , Supplementary file 5 ) .", "Induction of BRAFV600E in +TPA conditions did not further alter expression of either miRNA .", "Direct comparison of the arrested melanocytes in +TPA +Dox conditions to the proliferative melanocytes in −TPA +Dox conditions revealed MIR211-5p as the most significantly downregulated miRNA in the proliferative condition ( Figure 4B ) .", "Thus , in comparison to TPA , BRAFV600E induction had minimal effect on the expression of the miRNAs .", "The most notable effect was a decrease of MIR328-3p expression coinciding with the induction of BRAFV600E in TPA-free conditions ( Figure 4A ) .", "While MIR328-3p downregulation likely contributes to increased proliferation in this condition , the more pronounced effect of TPA on MIR211-5p expression suggests that the mechanisms governing whether BRAFV600E induces arrest or proliferation are a function of the cell’s transcriptional state rather than directly downstream of BRAFV600E itself .", "To better characterize the two transcriptional states influencing the BRAFV600E phenotype , we conducted mRNA sequencing of the same specimens .", "Principal component ( PC ) analysis revealed that 97 . 4 % of the variance between samples was explained by two PCs—PC1 ( 81 . 75 variance ) separated transcriptomes based upon exposure to TPA and PC2 ( 15 . 7% variance ) separated transcriptomes based upon BRAFV600E expression ( Figure 4C , Supplementary file 5 ) .", "We conducted differential expression ( DE ) analyses to compare all samples exposed to +TPA versus TPA-free media , regardless of BRAFV600E expression ( Figure 4D , top ) and to compare all samples expressing BRAFV600E , regardless of TPA exposure ( Figure 4D , bottom ) .", "As expected from the PC analysis and similar to the miRNA profiles , most gene expression variance was due to media conditions with a smaller , but consistent , set of genes activated by BRAFV600E .", "One initially surprising result of this analysis was the static expression of both MIR211-5p and MIR328-3p in +TPA conditions , regardless of BRAFV600E expression .", "However , as inhibitors of translation , miRNA function depends on the expression level of its targets ( Mukherji et al . , 2011; Ovando-Vázquez et al . , 2016 ) , effectively functioning as buffers of gene expression that prevent aberrantly high levels of a transcript ( Ebert and Sharp , 2012 ) .", "BRAFV600E has been previously demonstrated as an upstream activator of AURKB transcription in melanoma cells ( Sharma et al . , 2013 ) .", "We therefore reasoned that MIR211-5p expression in +TPA conditions might dampen AURKB activation by BRAFV600E .", "Consistent with this hypothesis , the addition of doxycycline induced AURKB expression in TPA-free/MIR211-5p low conditions , but this effect was not observed in +TPA/MIR211-5p high conditions ( Figure 4E ) .", "We next conducted gene set enrichment analyses ( GSEA ) comparing TPA-regulated genes and BRAFV600E- regulated genes to Molecular Signature Database Hallmark gene sets ( Liberzon et al . , 2015 ) , the KEGG PATHWAY database ( Kanehisa et al . , 2016 ) , and signatures important in BRAFV600E signaling ( Ryu et al . , 2011 ) , human melanocyte differentiation ( Belote et al . , 2021 ) , or melanoma cell state ( Hoek and Goding , 2010; Tirosh et al . , 2016; Tsoi et al . , 2018 ) .", "Among the pathways uniquely enriched with BRAFV600E expression were the expected increases in signatures downstream of BRAFV600E , MAPK ( KRAS ) signaling , and glycolysis ( Haq et al . , 2014 ; Figure 4F and Supplementary file 6 ) .", "Gene sets uniquely enriched in +TPA media included signatures associated with cell cycle regulation , such as the G2-M checkpoint , oxidative phosphorylation , the MITF program , and differentiated melanocytes .", "In contrast , TPA-free media-induced gene sets are associated with cell adhesion and melanocyte progenitor and stem cells .", "Corroborating this observation , melanocytes grown in TPA-free media expressed higher levels of AXL ( Figure 4G–H ) , lower levels of MLANA , a melanocyte differentiation marker , and presented greater activity of aldehyde dehydrogenase , enzymatic activity associated with stemness ( Figure 4I ) .", "These results indicate that exposure to TPA induces a more differentiated melanocyte phenotype , consistent with previous reports ( Prince et al . , 2003; Vidács et al . , 2021; Kormos et al . , 2011 ) .", "To look for synergist effects of both BRAFV600E expression and media conditions , we performed GSEA analyses on each set of pair-wise conditions ( Supplementary file 6 and summarized in Figure 4—figure supplement 1 ) .", "Generally , most gene sets identified in Figure 4F were regulated by only one of the two stimuli .", "For example , genes downstream of BRAFV600E signaling were only influenced by doxycycline exposure .", "Interesting exceptions to this trend were the inflammatory response , which required both stimuli; cell cycle regulation gene sets , which were activated by TPA alone but further elevated when supplemented with doxycycline; and epithelial to mesenchymal transition genes , which were activated by either stimulus .", "A subset of melanocyte dedifferentiation gene sets , specifically neural crest genes , the melanoma invasion program , and the AXL program , but not markers of melanocyte stem cells , were activated by either TPA-free media or BRAFV600E expression , albeit the activation was more pronounced in the former .", "This observation suggests that BRAFV600E expression alone does induce some programs associated with melanocyte dedifferentiation , but with the retention of MIR211-5p expression , the induction is insufficient to overcome growth arrest .", "Collectively , our transcriptomic analyses support a model whereby growth in +TPA conditions induces a more differentiated state in melanocytes coinciding with an increase in MIR211-5p expression ( Figure 4J ) .", "This increased MIR211-5p combined with relatively stable MIR328-3p expression , dampens entry into mitosis and/or successful completion of mitosis at least in part through AURKB inhibition .", "The attenuation of cell division results in an accumulation of cells in G2/M—an effect that is more pronounced in conditions stimulating accelerated growth , such as with BRAFV600E expression .", "To test this model , we transduced doxycycline-inducible BRAFV600E melanocytes in +TPA conditions with lentiviral constructs constitutively expressing either AURKB ( Figure 2I–J ) or miRNA sponges ( Figure 4K ) that inhibit either MIR211-5p or MIR328-3p .", "AURKB expression had no baseline effect on melanocyte growth , but rescued BRAFV600E-induced arrest ( Figure 4L ) , suggesting the inability of the oncogene to induce AURKB in this context serves as a bottleneck in cell division .", "Also consistent with the model , sponges against either MIR211-5p or MIR328-3p increased baseline division rate and rescued BRAFV600E-induced arrest , demonstrating that both miRNAs contribute to inhibition of melanocyte division , with effects that are more pronounced in the presence of growth signals .", "To further investigate whether this mechanism of arrest occurs in clinical nevus specimens , we examined an independent cohort of 15 nevi for evidence of mitotic failure .", "Histopathologic analysis revealed that all nevi within the cohort exhibited binucleation ( Figure 5A ) .", "The percent observed binucleated melanocytes ranged from 3% to 12% ( mean=6 . 3% ) .", "It is important to note that since binucleation can only be observed in the fraction of cells sectioned perpendicular to the plane of the adjacent nuclei , 3–12% is likely an underestimate .", "We also observed giant multinucleated cells in 10 of the 15 nevi , indicative of multiple rounds of DNA synthesis and cytokinetic failure and consistent with previous reports ( Rogers et al . , 2016 ) .", "We sought to determine whether AURKB inhibition was necessary and sufficient for the multi-nucleated arrest we observed in melanocytic nevi .", "We obtained three fresh skin biopsies containing nevi , microdissected the nevus portion , and isolated melanocytes ( Figure 5B ) .", "Nevus-derived melanocytes appeared healthy in culture .", "Consistent with the histopathologic cohort , co-staining with melanocyte marker , MLANA , and Hoechst revealed frequent 4 N and 8 N melanocytes ( Figure 5C ) .", "Transduction of nevus melanocytes with an AURKB and mCherry expressing lentiviral vector at low multiplicity of infection showed that the mCherry positive cells divided significantly more than their mCherry negative counterparts ( Figure 5D–E ) in cells from all three nevus donors .", "This result suggests that expression of AURKB is also rate limiting for the proliferation of human nevus melanocytes , as we observed in primary melanocytes expressing BRAFV600E .", "To test the sufficiency of AURKB inhibition to limit BRAFV600E hyperproliferation in melanoma , we assessed normalized growth rate inhibition of three cultures—primary human melanocytes , an established BRAFV600E human melanoma cell line , and a short-term BRAFV600E human melanoma culture—when exposed to a pharmacologic AURKB inhibitor ( Barasertib ) .", "Normalized growth rate measurements ( such as GR50 ) incorporate baseline cell division rate and are demonstrably more reproducible than the commonly calculated half-maximal inhibitor concentration ( IC50 ) when comparing different cell cultures ( Hafner et al . , 2016 ) .", "An additional advantage of GR50 is the ability to distinguish growth rate inhibition ( GR50 approaches 0 ) versus cell death ( GR50 is negative ) .", "Since GR50 calculations require proliferation curves , we conducted live QPI over 48 hr with a range of Barasertib concentrations ( 0–300 nM ) .", "Primary melanocytes were largely resistant to the compound with reduced growth apparent only at supraphysiological levels ( Figure 5F ) .", "In contrast , both melanoma cultures responded to tenfold lower dosage of the compound with GR50 approaching zero , indicating primarily arrest , not death .", "In these conditions , the per cell dry mass also increased significantly , consistent with a G2/M arrest ( Figure 5G ) .", "Taken together , these data demonstrate that AURKB inhibition limits the rate of BRAFV600E-induced proliferation in human melanocytic nevi and melanomas ." ], [ "Cellular senescence is conventionally defined as a permanent cell-cycle arrest associated with a variety of molecular markers and an irreversible arrest in the G0/G1 phase of the cell cycle ( He and Sharpless , 2017; Roy et al . , 2020; Serrano et al . , 1997 ) .", "The vast majority of melanocytic nevi are stably arrested and harbor BRAFV600E , an oncogenic mutation that induces cellular senescence when expressed in a variety of mammalian cells ( Michaloglou et al . , 2005; Serrano et al . , 1997; Zhu et al . , 1998 ) .", "While these observations seemingly implicate OIS as the mechanism driving nevus-associated proliferation arrest , a substantial body of evidence has challenged this paradigm in recent years ( Ruiz-Vega et al . , 2020; King et al . , 2009; Burian and Jemec , 2019; Cotter et al . , 2007; Tran et al . , 2012; Oaxaca et al . , 2020; Zeng et al . , 2018; Haferkamp et al . , 2009; McNeal et al . , 2015 ) .", "Here , we provide further evidence to challenge the model that acquisition of BRAFV600E induces premature cellular senescence in melanocytes and uncover an alternative mechanism driving nevus formation and stability .", "We propose that acquisition of the BRAFV600E mutation does not necessitate growth arrest , but instead permits melanocytes to toggle between hyperproliferation and mitotic arrest .", "Our transcriptomic analyses demonstrate that external stimuli can modulate the differentiation state of BRAFV600E melanocytes , orchestrate the expression level of MIR211-5p , and inform which phenotype is triggered by the oncogene—before or after the oncogene is introduced .", "The senescence model is largely rooted in seminal work demonstrating that sustained ectopic expression of BRAFV600E induces proliferative arrest in primary human melanocytes ( Michaloglou et al . , 2005 ) —an experiment reproduced by several independent labs ( Haferkamp et al . , 2009; McNeal et al . , 2015; Leikam et al . , 2008; Lu et al . , 2016 ) , including our own ( Zeng et al . , 2018 ) .", "In each of these studies , melanocytes were cultured in TPA-containing media .", "Thus , while observations presented here—that BRAFV600E -induced proliferative arrest is reversible and conditional on the presence of TPA— provide new insights that challenge the senescence model , they do not contradict the data presented in these previous reports .", "One inherent limitation of using any in vitro approach to investigate the mechanisms underlaying clinical phenomena is an inherent incongruity in the magnitude of duration .", "Melanocytic nevi frequently persist for several decades , whereas in vitro experiments are generally conducted on the order of days to weeks .", "In previous in vitro studies , the periods of time melanocytes were monitored subsequent to BRAFV600E introduction ranged from 1 to 3 weeks .", "In studies utilizing an inducible RAF in human fibroblasts , 24 hr of expression was sufficient to drive a durable proliferative arrest , even upon removal of the oncogene ( Zhu et al . , 1998 ) .", "In this study , we induced growth arrest with high levels of BRAFV600E for 30 days and observed the arrest was still reversible .", "It remains possible that an even longer exposure to BRAFV600E would elicit a permanent proliferative arrest consistent with senescence .", "It is noteworthy , however , that the identified mechanisms driving proliferative arrest in vitro were also supported by analyses of clinical nevi specimens .", "While supported by analyses of both primary human melanocytes and clinical specimens , one limitation to this report is the lack of in vivo confirmation using animal models .", "One well-established genetically engineered mouse model of melanoma expresses BRAFV600E from the endogenous locus in tyrosinase-expressing cells with spatiotemporal control .", "The predominant phenotype of BRAFV600E activation in this model is melanocytic hyperplasia , which increases in prominence with BRAFV600E dose ( Dankort et al . , 2009 ) .", "While growth-arrested melanocytic ‘nevus-like’ lesions also form in this model , they are not associated with markers of senescence ( Ruiz-Vega et al . , 2020 ) .", "More recently , an elegant study utilizing a zebrafish model of melanoma demonstrated that driving BRAFV600E expression from promoters associated with melanocyte progenitors , but not differentiated melanocytes , resulted in melanoma initiation ( Arianna et al . , 2021 ) .", "These observations are all consistent with our data and support the conclusion that BRAFV600E-induced proliferation in melanocytes is dependent on differentiation state .", "Further studies will be required to confirm whether the microRNA mechanism reported here is conserved between human and model systems .", "The presented model offers an explanation for several clinical phenomena .", "First , melanocytic nevi form when BRAFV600E drives hyperproliferation followed by proliferation arrest .", "Second , nevus eruption involves the expansion of previously stable nevi over a short period of time .", "Third , incomplete excision of melanocytic nevi can result in subsequent regrowth of the benign lesion ( King et al . , 2009 ) .", "Each event requires interconversion of the BRAFV600E melanocyte between a proliferative and arrested phase .", "Our data demonstrate that BRAFV600E melanocytes can indeed oscillate between proliferative and arrested states and are consistent with each of these phenomena .", "Our interpretation assumes that once acquired , BRAFV600E expression is constant and that the environmental context of the melanocytes is variable , serving as a ‘gatekeeper’ to BRAFV600E-induced proliferation .", "However , if BRAFV600E expression also fluctuates , then an equally plausible interpretation of our data is that BRAFV600E serves as a ‘gatekeeper’ to environmentally induced proliferation .", "Interestingly , nevus eruption has been observed in metastatic melanoma patients treated with single agent selective BRAF inhibitors ( Cohen et al . , 2013; Chen et al . , 2014; Chu et al . , 2012; Dalle et al . , 2013; Zimmer et al . , 2012 ) , consistent with our observation that proliferation arrest in melanocytes requires sustained expression of the oncogene .", "A more thorough understanding of the responsible environmental stimuli in physiologic conditions , discussed in more detail below , would help to clarify which of the mechanisms—variable BRAFV600E , variable environment , or both—underlie nevus formation , eruption , and recurrence .", "It is important to note that while our data support a model of reversible proliferation arrest in nevi , we do not suggest this reversal is commonplace .", "Unquestionably , the vast majority of melanocytic nevi present with sustained arrest .", "However , we do propose that the duration of arrest is dynamic and modulated by exposure to external signals .", "Since our data suggest that loss of MIR211-5p and MIR328-3p expression and restoration of AURKB expression is concurrent with progression to melanoma , identification of the source of cell-extrinsic signals might suggest new strategies for chemoprevention or therapy .", "We have identified TPA , a known activator of PKC signaling , as one extrinsic factor that induces expression of MIR211-5p and a more dedifferentiated state .", "Previous studies have identified TPA as either a mitogen , inhibitor of the G1/S transition , or inhibitor of the G2/M transition , dependent on the context ( Coppock et al . , 1995; Arita et al . , 1998; Arita et al . , 1992; Stavroulaki et al . , 2008; Chao-Hsing and Hsin-Su , 1991 ) .", "We demonstrate that in vitro , TPA activation of MIR211-5p is required for BRAFV600E-induced mitotic failure .", "Yet , the inclusion of TPA in primary melanocyte culture is artificial and the environmental stimulus that regulates MIR211-5p expression in skin remains unknown .", "One hypothesis is that PKC activation from keratinocyte interactions serves as the extrinsic stimulus .", "MIR211-5p is reported to be transcriptionally regulated by both MITF ( Mazar et al . , 2010 ) and UV exposure ( Su et al . , 2020 ) .", "However , TPA-free media containing an endothelin receptor type B ligand , endothelin-1 , did not elicit MIR211-5p expression or BRAFV600E arrest , suggestive that the downstream signaling pathways regulated by the two molecules are not identical .", "PKC activation can also result in increased MAPK signaling ( Herrera et al . , 1998; Ueda et al . , 1996 ) raising the possibility that internal MAPK fluctuations may play a role in toggling between BRAFV600E-induced proliferation versus arrest .", "The term ‘mitogenic window’ describes the specific range of oncogene doses that elicit a hyperproliferative phenotype .", "The mitogenic window for MAPK signaling activation can be remarkably fine .", "Here , we conducted a twofold dilution series of doxycycline but observed no evidence of a mitogenic window .", "However , it remains possible that we simply missed the ‘sweet spot’ in TPA conditions , and that a finer dilution series would reveal a mitogenic window even in the presence of TPA .", "Regardless , our data demonstrate that in TPA conditions the mitogenic window for BRAFV600E expression is , at least , exceedingly narrow , whereas in non-TPA conditions the window is wide open .", "An important future direction will be to identify the nature and physiological source of this signal for melanocytic nevi in human skin .", "Diminished MIR211-5p/MIR328-3p expression concurrent with increased AURKB expression occurs in a substantial portion of melanomas .", "AURKB expression was previously reported as significantly elevated in melanoma metastases ( Sharma et al . , 2013 ) and here we report increased expression in primary melanomas .", "Similarly , MIR211-5p expression is consistently observed as lost in melanoma as compared to melanocytic nevi across independent studies ( Babapoor et al . , 2016; Torres et al . , 2020; Latchana et al . , 2016 ) .", "Indeed , MIR211-5p expression levels classify nevi from melanomas with a high degree of accuracy ( Babapoor et al . , 2016; Torres et al . , 2020 ) .", "We have reported on the diagnostic potential of MIR328-3p expression as well , although multi-study validation was impeded by the absence of hybridization probes in earlier microarray-based profiling data sets ( Torres et al . , 2020 ) .", "In vitro , we have now demonstrated that inhibition of either miRNA is sufficient to block BRAFV600E-induced arrest in melanocytes .", "These data suggest that mitotic failure serves as a barrier to melanoma progression and is consistent with the genome duplication and copy number alterations associated with invasive melanoma ( Shain et al . , 2015; Vergara et al . , 2021 ) .", "In melanoma , the specific cause of this genomic instability is unknown , but reentry of cells into cell cycle after mitotic failure has been shown to cause genome duplication and accumulation of copy number alterations in other cancers ( Telentschak et al . , 2015 ) .", "Further experiments will be necessary to determine whether dysregulation of the spindle checkpoint in nevi contributes directly to the copy number variations present in melanomas .", "In summary , we have identified that BRAFV600E induces a reversible arrest in human melanocytes orchestrated by MIR211-5p/MIR328-3p regulation of AURKB and conditional on the melanocyte differentiation state .", "We present a mechanistic model that allows for melanocytic nevus formation , eruption , and recurrence .", "Moreover , our observations provide an explanation for the genetic duplication and instability inherent to invasive melanomas and open new directions for novel chemopreventative and therapeutic strategies ." ], [ "For FFPE samples , histopathologic review , microdissection , targeted exon sequencing , phylogenetic analysis , and RNA and small RNA sequencing of each tumor area were previously described ( phs001550 . v2 . p1 ) ( Zeng et al . , 2018; Shain et al . , 2018; Torres et al . , 2020 ) .", "For this study , small RNA reads were aligned to human reference ( hg37 ) with Bowtie ( Langmead et al . , 2009 ) and small RNA reference groups ( miRBase21 ) were counted .", "For mRNA sequencing , reads were aligned to human reference ( hg37 ) with Bowtie2 and counted with HTSeq .", "DE analysis was performed using DESeq2 ( Love et al . , 2014 ) with p values adjusted by the Benjamini-Hochberg method ( p-adj ) .", "Previously published RNA sequencing data sets from Shain et al . , 2018 and Torres et al . , 2020 were re-analyzed here with DESeq2 and combined for visualization .", "For RNA profiling from primary melanocytes , total RNA was extracted using TRIzol Reagent ( Thermo Fisher Scientific , 15596-026 ) and further purified with the RNeasy Power Clean Pro Cleanup Kit ( Qiagen , 13997-50 ) to remove melanin .", "For small RNAseq of nevus melanocytes sequencing libraries were constructed with the TailorMix Small RNA Library Preparation Kit ( SeqMatic , CA ) and sequencing was performed on the Illumina HiSeq2500 platform at single-end 50 bp .", "For small RNAseq of cultured BRAFV600E transduced melanocytes , sequencing libraries were constructed with the Qiagen QIAseq miRNA Library Prep Kit , and sequencing was performed on the NovaSeq SP platform at paired-end 50 bp .", "For mRNAseq of microRNA mimic nucleofected melanocytes , 150 bp paired-end sequencing was conducted by GeneWiz .", "For mRNAseq of cultured BRAFV600E transduced melanocytes , sequencing libraries were constructed with the Illumina TruSeq Stranded mRNA Library Prep Kit , and sequencing was performed on the NovaSeq S4 platform at paired-end 150 bp .", "Benign human melanocytic nevi were excised with informed consent from patient donors at the UCSF Dermatology clinic ( San Francisco , CA ) or HCI Dermatology clinic ( Salt Lake City , UT ) according to Institutional Review Board approved protocols .", "Nevus tissue was minced and enzymatically dissociated in 1 mg/ml collagenase ( Sigma-Aldrich , 11213857001 ) and 3 . 3 mg/ml dispase ( Sigma-Aldrich , 4942078001 ) in DMEM ( Thermo Fisher Scientific , 10569044 ) for 1 hr at 37°C .", "Melanocytes were further isolated by 5 days exposure to 10 µg/ml G418 ( InvivoGen , ant-gn-1 ) .", "BRAF status was confirmed via sanger sequencing ( Quintarabio ) using the primer set: BRAF forward: 5′-GCA CGA CAG ACT GCA CAG GG -3′; BRAF reverse: 5′-AGC GGG CCA GCA GCT CAA TAG-3′ .", "BRAF wild-type ( normal ) human melanocytes were isolated from de-identified and IRB consented neonatal foreskins or adult skin .", "Foreskin tissue was incubated overnight at 4°C in dispase and epithelia were mechanically separated from the dermis the following morning .", "Epithelial tissue was minced and incubated in 0 . 25% trypsin ( Gibco 25200056 ) for 4 min at 37°C .", "Trypsin was quenched and tissue centrifuged at 500×g for 5 min at room temperature .", "The cell/tissue pellet was resuspended in Melanocyte medium ( Thermo Fisher Scientific , M254500 ) with HMGS ( Thermo Fisher Scientific , S0025 ) and plated in low volume .", "Melanocytes were expanded in Melanocyte medium with HMGS and assayed in either Melanocyte medium with HMGS ( +TPA ) or Melanocyte medium with HMGS-2 ( TPA-free , Thermo Fisher Scientific , S0165 ) as indicated .", "The 501Mel human melanoma line ( Gift from Dr . Boris Bastian , CVCL_4633 , authenticated via STR profiling ) were cultured in RPMI with 10% fetal bovine serum ( FBS; VWR , S107G ) , 1% penicillin-streptomycin ( Gibco , 15140122 ) , 1% L-glutamine ( Gibco , 25030149 ) , and 1% non-essential amino acids ( Gibco , 11140050 ) .", "HCIMel019 was derived from patient-derived xenograft tumors propagated in mice .", "An HCIMel019 ( P2 ) subcutaneous tumor was resected from mouse , minced in digestion buffer ( 100 μM HEPES ( Gibco , 15630-080 ) , 5% FBS ( DENVILLE , FB5001-H ) , 20 μg/ml gentamicin ( Gibco , 15710-064 ) , 1× insulin ( Gibco , 51500-056 ) , and 1 mg/ml collagenase IV ( Gibco , 17104-019 ) in DMEM ( Gibco , 11965-092 ) ) , and digested overnight at 37°C .", "Cells were filtered through a 100 µm filter ( Falcon , 352360 ) and red blood cells were removed with RBC lysis buffer ( 0 . 5 M EDTA , 0 . 5 M KHCO3 [Sigma-Aldrich , 237205] , and 5 M NH4CL [Sigma-Aldrich , A9434] ) .", "Remaining cells were washed with phosphate-buffered saline ( PBS; Gibco , 10010023 ) , and cultured at 37°C and 5% CO2 in Mel2 media consisting of 80% MCDB153 ( Sigma-Aldrich , M7403 ) , 20% L15 ( Gibco , 11415-064 ) , 2% FBS ( DENVILLE , FB5001-H ) , 1 . 68 mM CaCL ( Sigma-Aldrich , C4901 ) , 1× insulin ( Gibco , 51500-056 ) 5 ng/ml EGF ( Sigma-Aldrich , E9644 ) , 15 μg/ml Bovine Pituitary Extract ( Gibco 13028-014 ) , and 1× Pen/strep ( Gibco , 15070-063 ) .", "Cell cultures were tested monthly for mycoplasma contamination ( ATCC ) .", "Human melanocytes were trypsinized ( Gibco , 25300062 ) , quenched , and centrifuged at 300×g .", "Cells were resuspended in R Buffer ( Thermo Fisher Scientific , MPK1025 ) at 10 , 000 cells/µL .", "About 10 µl of cell slurry was mixed with miRIDIAN microRNA mimics ( Dharmacon: hsa-MIR211-5p C-300566-03-0005 , hsa-MIR328-3p C-300695-03-0005 , MIRControl 1 CN-001000-01-05 , or MIRControl 2 CN-002000-01-05 ) ; or On-TargetPlus siRNA ( Dharmacon , custom library ) ; at 4 µM final concentration and nucleofected using the NEON Transfection System and protocol ( Thermo Fisher Scientific , MPK5000 ) .", "pTRIPZ-diBRAFV600E was a gift from Todd Ridky ( McNeal et al . , 2015 ) .", "pLVX-AURKB ( Addgene #153316 ) and pLVX-GPR3 ( Addgene #153317 ) were generated by subcloning the respective human cDNA ( from Addgene #100142 and #66350 ) into the MluI and BamHI sites of the pLVX-Che-hi3 vector ( a gift of Sanford Simon ) ( Takacs et al . , 2017 ) .", "pLVX-anti-MIR211-5p ( Addgene #153318 ) , pLVX-anti-MIR328-3p ( Addgene #153319 ) , and pLVX-Che-zsGreen ( Addgene #153320 ) were generated by inserting zsGreen with or without a 3′UTR into the MluI and XbaI sites of the pLVX-Che-hi3 vector .", "The 3′UTRs contained seven tandem microRNA binding sites to report and inhibit microRNA function , as previously described ( Judson et al . , 2013 ) .", "ZsGreen was subcloned from pHIV-zsGreen gift from Bryan Welm & Zena Werb ( Addgene #18121 ) ( Welm et al . , 2008 ) .", "2 . 75×106 HEK293T cells were plated on 10 cm tissue culture dishes and grown for ~24 hr in DMEM with 10% FBS .", "For each 10 cm plate , 5 µg lentiviral vector , 3 . 3 µg of pMLV-GagPol , and 1 . 7 µg of pVSV-G packaging plasmids were added to 500 µl of jetPRIME buffer and 20 µl of jetPRIME transfection reagent ( Polyplus , 712-60 ) , and transfected according to the manufacturer’s instructions .", "48 hr post-transfection , viral supernatant was collected and filtered using 0 . 45 µm syringe filters ( Argos 4395-91 ) .", "Human melanocytes seeded at 1 . 0×105–2 . 0×105 cells/well density in six-well plates were incubated in viral supernatant with 10 µg/ml polybrene ( Sigma-Aldrich , TR-1003 ) and centrifuged at 300×g for 60 min at room temperature .", "The viral supernatant was removed and replaced with growth media .", "Transduced cells were either selected with puromycin ( 1 µg/mL for 5 days , Sigma-Aldrich P8833 ) or sorted for mCherry expression using a BD FACSAria II .", "Cells expressing pTRIPZ-diBRAFV600E were treated with doxycycline ( Sigma-Aldrich , D9891 ) at indicated concentrations .", "Live quantitative imaging was performed using either the HoloMonitor M4 imaging cytometer ( Phase Holographic Imaging , Lund , Sweden ) or the Livecyte platform ( Phasefocus , Sheffield , UK ) .", "Analyses of cell proliferation , dry cell mass , and death were acquired with the M4 platform and analyzed using HStudio ( v2 . 6 . 3 ) as previously described ( Hejna et al . , 2017 ) .", "For each experiment , human melanocytes were seeded into six-well plates ( Sarstedt , 83 . 3920 ) at 100 , 000 cells/well and either live-imaged or serially imaged as indicated .", "Analysis of growth rate was acquired with the M4 platform and analyzed using App Suite ( v3 . 2 . 0 . 60 ) as previously described ( Hafner et al . , 2016 ) .", "For each experiment , 100 , 000 melanocytes , 60 , 000 501Mel cells , or 150 , 000 HCIMel019 cells were plated per well and media containing indicated concentrations of barasertib ( AURKB inhibitor; AZD1152-HQPA | AZD2811 , Selleckchem , A1147 ) was added .", "Cells were imaged for 48–72 hr .", "Analyses of fluorescent reporters coupled with QPI were conducted using the Livecyte platform and were analyzed using Analyze ( v3 . 1 ) ( Phasefocus , Sheffield , UK ) .", "Normal human melanocytes were nucleofected with microRNA mimicsor infected with lentiviral vectors ( as above ) and seeded in 48-well plates at a density of 50 , 000 cells/well .", "Four days post-seeding , EdU was added to culture media at a 10 µM final concentration .", "24 hr after EdU addition , media was removed and cells were stained for EdU incorporation and nuclei using the Click-iT EdU Imaging Kit ( Thermo Fisher Scientific , C10337 ) and protocol .", "Images of EdU and nuclei staining were acquired using the Evos FL microscope and quantified with Fiji .", "Protein was collected using RIPA Buffer ( Thermo Fisher Scientific , 89901 ) supplemented with HALT Protease and Phosphatase Inhibitor ( Thermo Fisher Scientific , 78446 ) .", "Immunoblotting was carried out as previously described .", "Membranes were incubated overnight at 4°C with primary antibodies at the following dilutions: anti-HSP90 ( CST , 4874 ) 1:1000 , anti-BRAFV600E ( Spring Bioscience Corp , E19292 ) 1:1000 , anti-phosphoERK1/2 ( CST , 4970 ) 1:1000 , and anti-AURKB ( Abcam , ab2254 ) 1:1000 .", "Membranes were washed 4× with TBS and 0 . 5% Tween20 , incubated with HRP-conjugated secondary antibody ( 1:2000 ) for 30 min at room temperature , and visualized with Lumina Forte Western HRP substrate ( Millipore , WBLUF0500 ) .", "For assessing DNA content , trypsinized melanocytes ( as above ) were resuspended in 400 PBS .", "1 ml –20°C 200 proof ethanol was added dropwise while gently vortexing to achieve 70% final concentration for fixation and incubated overnight at –30°C .", "Cells were centrifuged at 800×g for 5 min , washed with fresh PBS , and incubated in 500 µL FxCycle PI/RNase Staining Solution ( Invitrogen , F10797 ) for 20 min at 37°C .", "Data were collected on the Fortessa ( BD ) at low speed and analyzed with FlowJo v10 . 7 . 1 .", "Annexin V/PI staining was performed by trypsinizing melanocytes as above .", "Cells were washed with PBS and resuspended with Annexin V binding buffer ( BioLegend , 422201 ) at a concentration of 1×106 cell/ml .", "5 µl APC Annexin V antibody ( BioLegend , 640919 ) and 10 µl PI were added .", "Cells were gently vortexed and incubated in the dark at room temperature for 15 min .", "Cells were resuspended in 400 µl of Annexin V Binding Buffer .", "Data were collected on the FACs Verse ( BD ) and were analyzed with FlowJo v10 . 7 . 1 .", "Aldehyde dehydrogenase activity was measured first by trypsinizing melanocytes as above and then using the ALDEFLUOR Kit ( Stemcell Technologies , 01700 ) as described in the manufacture’s protocol .", "The archived clinical specimens used in the study were procured with IRB approval from the UCSF Dermatopathology Pathology Service archives .", "For assessment of AURKB and GPR3 expression , 11 melanoma and 11 melanocytic nevi with previous diagnosis were de-identified and re-verified histologically ( UEL ) .", "For assessment of DNA content , 15 additional melanocytic nevi with previous diagnosis were re-verified histologically ( UEL ) .", "Tissue was fixed in 10% neutral-buffered formalin , processed , embedded in paraffin , and stained with hematoxylin and eosin .", "4 µm FFPE sections were stained with AURKB ( 1:400 dilution , Abcam ab2254 ) or GPR3 ( 1:125 dilution , Abnova H00002827-M01 ) antibodies .", "UEL reviewed immunohistochemical stains with semiquantitative grading for GPR3 ( 0=none; 1=patchy positive; 2=strong positive ) and AURKB ( 0=none; 1=rare positive; 2=scattered positive; 3 = frequent positive; 4=many positive ) .", "For in vitro immunofluorescence , cells were fixed and stained as previously described with AXL ( 1:250 dilution , Cell Signaling Technology #8661 ) or MLANA ( 1:100 , Abcam ab731 ) .", "For differential gene expression , p values were calculated with the DESeq2 ( v1 . 30 . 1 ) default Wald test adjusted by the Benjamini-Hochberg method using a 5% false discovery rate ( FDR ) ( Benjamini et al . , 2001 ) .", "Pathway analyses were analyzed using the fast gene set enrichment package ( Korotkevich et al . , 2016 ) in R with a 10% FDR .", "Gene set enrichment analyses were conducted using GSEA ( v4 . 0 . 3 , Broad Institute ) Preranked tool with 1000 permutations .", "For in vitro experiments , pilot studies were initially conducted in triplicate .", "The required minimal sample size to assess the difference between independent means with independent standard deviations with an alpha error probability of 0 . 05 and a power of 0 . 95 was calculated using G*Power ( v . 3 . 1 . 9 . 4 ) .", "P values were calculated using either paired or unpaired two-tailed t-tests or Wilcoxon tests via Prism 8 ( GraphPad ) as indicated in figure legends .", "For cohorts of clinical specimens , sample number ( n ) refers to the number of specimens , each from a different patient .", "For in vitro experiments , n refers to independent experiments conducted on different days .", "In cases where fresh primary melanocytes were used , the cells in each experiment are derived from an independent donor .", "In cases where cell lines are used , the cells in each experiment represent a different passage and/or day of that line .", "For different conditions within experiments , replicate wells were plated and arbitrarily chosen for each condition ( e . g . , not treated versus treated; different concentrations of a compounds , etc . ) .", "All data were included ." ] ]
[ "Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes .", "Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation , necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi .", "We compared the transcriptomes of melanocytes from healthy human skin , nevi , and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts .", "Two of these microRNAs—MIR211-5p and MIR328-3p—induced mitotic failure , genome duplication , and proliferation arrest in human melanocytes through convergent targeting of AURKB .", "We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional .", "Specifically , BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte .", "We report genome duplication in human melanocytic nevi , reciprocal expression of AURKB and microRNAs in nevi and melanomas , and rescue of arrested human nevus cells with AURKB expression .", "Taken together , our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations ." ]
[ "Lots of people have small dark patches on their skin known as moles .", "Most moles form when individual cells known as melanocytes in the skin acquire a specific genetic mutation in a gene called BRAF .", "This mutation causes the cells to divide rapidly to form the mole .", "After a while , most moles stop growing and remain harmless for the rest of a person’s life .", "Melanoma is a type of skin cancer that develops from damaged melanocytes .", "The same mutation in BRAF that is found in moles is also present in half of all cases of melanoma .", "Unlike in moles , the melanoma-causing mutation makes the melanocytes divide rapidly to form a tumor that keeps on growing indefinitely .", "It remains unclear why the same genetic mutation in the BRAF gene has such different consequences in moles and melanomas .", "To address this question , McNeal et al . used genetic approaches to study melanocytes from moles and melanomas .", "The experiments identified some molecules known as microRNAs that are present at higher levels in moles than in melanomas .", "Increasing the levels of two of these microRNAs in melanocytes from human skin stopped the cells from growing and dividing by inhibiting a gene called AURKB .", "This suggested that these microRNAs are responsible for halting the growth of moles .", "Introducing the mutated form of BRAF into melanocytes also stopped cells from growing and dividing by inhibiting AURKB .", "However , changing the environment surrounding the cells reversed this effect and allowed the melanocytes to resume dividing .", "In this way the mutated form of BRAF acts like a switch that allows melanocytes in skin cancers to start growing again under certain conditions .", "Further experiments found that a drug called barasertib is able to inhibit the growth of melanoma cells with the mutant form of BRAF .", "Future work will investigate whether it is possible to use this drug and other tools to stop skin cancer tumors from growing , and possibly even prevent skin tumors from forming in the first place ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "evolutionary biology", "genetics and genomics" ]
Recurrent evolution of high virulence in isolated populations of a DNA virus
elife-58931-v2
[ [ "Antagonistic coevolution between hosts and their parasites is nearly ubiquitous across the diversity of life ( Burt and Trivers , 2006 ) .", "As a result , genes involved in immune defense are among the fastest evolving genes in host genomes ( Nielsen et al . , 2005; Sackton et al . , 2007; Enard et al . , 2016; Shultz and Sackton , 2019 ) .", "Viruses are a particular fitness burden on hosts; for viruses to persist within populations , they must successfully invade the host organism , contend with the host-immune system , replicate and then transmit the newly produced particles to a new host ( Holmes , 2007; Gifford , 2012 ) .", "Once successfully established in a population , natural selection acts to modulate the rate the virus propagates relative to its virulence , optimizing the ratio of virulence to transmission ( Williams and Nesse , 1991; May and Nowak , 1995; Lipsitch et al . , 1996 ) .", "Due in part to their elevated mutation rate and large population sizes , viruses can accomplish this , and often co-opt or manipulate host-pathways in the process ( Burgyán and Havelda , 2011; Davey et al . , 2011; Palmer et al . , 2019 ) .", "Given the strong selective pressures viruses exert on their hosts ( and hosts on viruses ) , it would be useful to assess how the two players behave in replicate evolutionary experiments .", "Experiments examining the evolution of pathogens and hosts are common in the lab ( Perron et al . , 2006; Paterson et al . , 2010; Bull et al . , 2011; Martins et al . , 2014; Scanlan et al . , 2015 ) and even in patients ( Pennings , 2012; Pennings et al . , 2014; Feder et al . , 2019 ) , but difficult in natural settings ( Souza et al . , 2002; Grubaugh et al . , 2015 ) , so we are rarely able to observe the coevolutionary dynamics between a virus and its host in replicate populations to examine natural coevolution and identify if evolution repeats itself .", "These studies are necessary as they frequently help characterize the pathways viruses have evolved to escape or suppress the host-immune system , and whether the same host pathways are common targets of the same virus ( Sabin et al . , 2010 ) .", "Several studies of viruses in natural populations highlight that the same initial virus can follow the same adaptive path in multiple replicate samples .", "This results in parallel or convergent evolution of a virus better optimized to the host , possible due to the relatively high viral mutation rate and viral population size ( Bull et al . , 1997; Casino et al . , 1999; Crandall et al . , 1999; van Mierlo et al . , 2012 ) .", "However , in other systems , the same initial virus can evolve divergently in a host or location-dependent manner .", "Specifically , virulence and the diversity of mutations can differ dramatically based on differences in the host environment , the extremely strong selective pressures acting on the virus , and the unpredictability of some viruses due to their elevated mutation rate ( Martinez-Picado et al . , 2002; Cuevas et al . , 2003; Real et al . , 2005; Grubaugh et al . , 2015 ) .", "Characterizing how viruses adapt to their long-term hosts in naturally structured populations will help broaden and expand our understanding of how hosts and pathogens evolve in response to each other , and how repeatable evolution is in the face of minor environmental differences .", "We took advantage of a natural host/DNA virus infection model involving populations separated by hundreds of kilometers to study how viruses evolve with their hosts in replicate populations with limited gene flow .", "Drosophila innubila is a mycophagous species that lives in montane forests in the ‘Sky islands’ in southwestern North America .", "They are commonly infected with a double-stranded DNA virus , the Drosophila innubila Nudivirus ( DiNV ) ( Hill and Unckless , 2018 ) .", "A previous study examined the rates of evolution of DiNV ( Hill and Unckless , 2018 ) , finding the envelope and replication machinery to be rapidly evolving in DiNV , suggesting its importance in viral propagation ( Hill and Unckless , 2018 ) .", "Additionally , a viral suppressor of Toll was identified in the genome of a closely related virus and DiNV , suggesting that components of the Toll pathway ( perhaps antimicrobial peptides – AMPs ) interact with the virus , which these viruses have then evolved to suppress ( Palmer et al . , 2019 ) .", "Consistent with this , genes in the Toll pathway and Toll-regulated AMPs are rapidly evolving in D . innubila ( Hill et al . , 2019 ) .", "A more thorough examination of the natural evolution of both the host and virus is necessary to understand how DiNV and D . innubila interact with each other beyond this suppressor protein .", "We surveyed natural genetic variation in DiNV in four replicate populations to infer its co-evolutionary history with D . innubila .", "To this end , we", "( a ) estimated how long DiNV has infected D . innubila ,", "( b ) inferred the evolutionary history of the host and virus among the populations , including signatures of selection and recombination", "( c ) examined host and virus genetic variation associated with viral titer within individual hosts , and", "( d ) characterized gene expression differences associated with that genetic variation .", "We identified two viral multilocus genotypes that differ by 11 focal SNPs and found that these viral types are maintained within the same host population and across multiple isolated host populations .", "These SNPs are tightly linked , likely brought together by a combination of recurrent mutation and recombination , and this linkage appears to be maintained by strong selection .", "One viral type is associated with 100-fold higher viral titer and increased virulence compared to the other .", "Further , we found evidence that the SNPs associated with the high titer type have evolved independently in at least three geographically-isolated viral populations infecting the sympatric D . innubila and D . azteca , and a geographically separate population of viruses infecting D . falleni .", "Together , these results suggest rapid evolutionary dynamics of host–virus interactions , due to recurrent evolution of a highly virulent haplotype that interacts with multiple host pathways ." ], [ "To characterize the evolutionary dynamics of wild Drosophila innubila Nudivirus ( DiNV ) in its host ( D . innubila ) , we sequenced wild-caught individuals from four populations with the expectation that some ( ~40% in previous samples ) individuals would be infected ( Hill and Unckless , 2020 ) .", "We considered strains to be infected with DiNV if they had at least 10x coverage for 95% of the genome .", "We confirmed the infection of a subsample of these using PCR for the presence of a DiNV specific gene ( Supplementary Table 1 ) .", "In total , we used sequencing information for 57 , 92 , 92 , and 92 individuals from the Huachucas ( HU ) , Santa Ritas ( SR ) , Chiricahuas ( CH ) , and Prescott ( PR ) populations with infection rates 26 , 44 , 71 , and 79% , respectively ( Supplementary file 1 - Table 1 ) .", "We also sequenced 35 individual males collected in the Chiricahuas in 2001 ( 52% infected with DiNV ) and 80 individual males collected in the Chiricahua’s in 2018 ( Supplementary file 1 - Table 2 , 40 infected with DiNV and 40 uninfected determined before sequencing using PCR ) .", "We isolated and sequenced DNA from these samples , quality filtered reads , mapped to the genome , and called genetic variation in the viral genomes to assess the extent of adaptation in each viral population .", "Consistent with an arms-race between host and virus , envelope and novel virulence ( GrBNV-like ) genes have a significantly higher proportion of substitutions fixed by adaptive evolution , compared to other viral genes ( McDonald-Kreitman-based statistic Direction of Selection [Stoletzki and Eyre-Walker , 2011] and Selection Effect [Eilertson et al . , 2012; Figure 4—figure supplement 1] , DoS >0 , GLM t-value >1 . 31 , p-value<0 . 05 ) .", "Recurrent adaptive evolution in viral proteins known to interact with the host-immune system suggests an arms-race between D . innubila and DINV .", "To better understand the interactions between host and virus , we performed an association study between viral titer and natural genetic variation in both host and virus .", "We consider viral titer a proxy for virulence .", "For each virus-infected individual , we quantified viral titer ( as the logarithm of the viral genome coverage normalized to host autosomal genome coverage ) and performed an association study across both host and virus variable sites to identify variants significantly associated with viral titer using PLINK ( Purcell et al . , 2007 ) .", "Of 5283 viral SNPs in the 155kbp DiNV genome , 1 , 403 SNPs are segregating in at least five infected host individuals ( five was our minor allele threshold for inclusion in the association study ) .", "Of those 1 , 403 SNPs , 78 are significantly associated with viral titer after multiple testing correction ( FDR < 0 . 01 , Significantly associated SNPs p<0 . 001 over 1000 permutations , Figure 1A ) .", "Of these , 16 are less than 2000 bp upstream of the start site of a gene , 18 are coding nonsynonymous , 11 are coding synonymous and 33 are intergenic .", "The SNP with the absolute lowest p-value for association with viral titer was also the only significantly associated SNP that seemed to segregate within individuals .", "In fact , the frequency of the derived allele in this nonsynonymous polymorphism in the active site of Helicase-2 shows a negative linear relationship with the log of viral titer ( Figure 1—figure Supplement 2 , GLM t-value = −20 . 516 , p-value=5 . 55e-21 ) .", "However , when we ranked samples by viral titer , we found the derived SNP frequency exhibited an almost bell-shaped curve when plotted against rank ( Supplementary Figure 2 ) .", "We also identified a striking association between viral titer and eleven strongly linked polymorphisms found across the DiNV genome ( Figure 1A & D , highlighted SNPs , Table 1 , Figure 1—figure supplements 1–3 , Sig . SNPs ) .", "These SNPs were significantly associated in every population when performing the association study on individual populations , or as a single group with population as a covariate .", "We binned strains with one of the two complete sets of alleles and referred to them as the ‘High Type’ and ‘Low Type’ ( Figure 1B , only contains individuals infected by viruses with a complete set of ‘High’ or ‘Low’ Type SNP variants ) .", "This multilocus genotype includes three non-synonymous SNPs , five SNPs in the UTRs of known virulence factor genes and three intergenic SNPs ( Table 1 ) .", "Viral titer is , on average , 100-fold higher in individuals infected with the full High type virus compared to the ancestral Low type ( Figure 1B ) .", "When comparing the ‘High’ and ‘Low’ viral type to Helicase-2 allele frequency , we found that viral titer increases with Helicase-2 SNP frequency in the Low type ( and some intermediate types ) , while titer decreases with Helicase-2 SNP frequency for most intermediate and High types , suggesting some form of negative interaction between the High type SNP variants and the Helicase-2 SNP ( Supplementary Figure 2 ) .", "Consistent with this , we found a negative interaction between Helicase-2 SNP frequency and the presence of High type on viral titer ( GLM Log10 ( titer ) ~Helicase-2 SNP * Haplotype: t-value = −7 . 815 , p-value=6 . 39e-14 ) .", "Since these 11 SNPs are almost perfectly linked ( Figure 1E ) , if one SNP were a false positive result , all would be false positives .", "We sought to address this in two ways .", "First , these SNPs are significantly associated with viral titer in every population when performing the association test separately or as a group with population as a covariate .", "Therefore , the likelihood of the same SNP ( s ) being false positives in three independent tests is quite small ( p<1E-09 over 3 iterations of 1000 permutations ) .", "Second , we permuted the titer among all individuals within populations and performed the association test ( 100 , 000 total permutations ) .", "For each permutation , we took the SNP with the lowest p-value from each permutation test and the 10 other SNPs most strongly linked to it .", "We then compared the viral titer in those with this permuted ‘High’ haplotype to those with the ‘Low’ haplotype .", "In no case were the permuted differences between High and Low haplotypes as large as those observed from the real data ( Figure 1—figure supplement 2A , 100 , 000 permutations ) , and the distribution of true p-values diverges dramatically from the permuted null expectation ( Figure 1—figure supplement 2B , black ) .", "We found this divergence from the null expectation of p-values disappears when including viral haplotype as a covariate ( Figure 1—figure supplement 2B , red ) , suggesting that the High type drives most of the signal seen in the association study .", "Though we found few strains with an intermediate number of SNPs ( intermediate types ) , viral titer appears to increase as the number of High type SNPs increases ( Figure 1C , GLM t-value = 34 . 971 , p-value=5 . 912e-16 ) , but the rate of increase slows as the number of High type SNPs increases suggesting diminishing returns ( Figure 1C ) .", "Some of these polymorphisms are associated with known virulence factors , or are related to the formation of the viral envelope co-opting the host vesicle trafficking system and are rapidly evolving in nudiviruses ( e . g . 19K , ODV-E56 , PIF-3 ) ( Rohrmann , 2013; Hill and Unckless , 2017; Hill and Unckless , 2018 ) .", "Additionally , several are associated with genes exclusive to a few nudivirus genomes thought to be novel virulence factors , including gp83 , a gene that downregulates Toll-induced antimicrobial peptides ( AMPs ) and upregulates those induced by IMD ( Palmer et al . , 2019 ) .", "Both the Toll and IMD pathway may interact with DNA viruses ( Zambon et al . , 2005; Costa et al . , 2009; Merkling and van Rij , 2013; Ferreira et al . , 2014; Lamiable et al . , 2016; Palmer et al . , 2019 ) .", "Among populations , we found a positive correlation between the frequency of the High type and overall DiNV infection frequency ( Figure 1D , GLM logistic regression z-value = 6 . 104 , p-value=0 . 00883 ) , suggesting that the High type may have a higher transmission rate than the Low type , resulting in a larger number of new individuals infected per DiNV-infected individual .", "We also found both viral types in collections from 2001 and 2017 , with the High type significantly more common in the 2017 collection ( Fisher Exact Test p-value=0 . 0167 , Figure 1D ) .", "It is possible that mutations could segregate within individuals , but this is relatively rare overall , and we found no evidence of the 11 haplotype SNPs segregating within any individual fly ( Supplementary Figure 2 ) .", "In fact , no individual fly contains more than two significantly associated SNPs segregating within the sample , suggesting that hosts are either infected completely with Low type or High type virus particles .", "Given the striking difference in viral titer between High and Low type viruses , we sought to further characterize the differences in infection dynamics between the types , focusing on the differences in expression between viral types .", "We sequenced mRNA from 80 wild D . innubila males collected in 2018 ( Supplementary file 1 - Table 2 , 40 infected with DiNV , 40 uninfected ) and performed a differential expression analysis between infected and uninfected individuals .", "Few genes were differentially expressed ( DE ) between infection states in D . innubila , but these DE genes were enriched for several interesting categories .", "Specifically , we found IMD-induced antimicrobial peptides ( AMPs ) were upregulated upon DiNV infection , while one Toll-induced AMP , and several chorion and heat shock protein genes were downregulated ( Figure 2—figure supplement 1A ) .", "We also compared these results to a laboratory experiment in D . melanogaster of differential expression after infection with a close relative of DiNV ( Palmer et al . , 2018 ) .", "These same genes tend to also be differentially expressed in D . melanogaster .", "Of the 12 genes which are differentially expressed in the same direction in both species , five are AMPs and five are chorion genes ( Figure 2—figure supplement 1B ) .", "This suggests that even with significant evolutionary divergence in both host and virus , the transcriptional response to infection is similar in both hosts .", "It was unexpected that chorion proteins are differentially expressed upon infection by DiNV and Kallithea virus ( Figure 2 , Figure 2—figure supplement 1 ) , especially as DiNV is thought to infect the Drosophila gut ( Unckless , 2011 ) and these samples are male .", "This suggests DiNV may infect more tissues than just the gut , and that chorion proteins are expressed in the germline of both D . innubila sexes , potentially fulfilling different roles in different sexes/species .", "To determine how the High and Low type differ in their ability to infect D . innubila , we compared gene expression between D . innubila infected with the two types from this same collection ( excluding intermediate types , which also showed a significant difference in viral titer between High type and Low type , p-value=0 . 0000143 ) .", "We found 17 host genes and nine viral genes differentially expressed between types ( after we controlled for virus copy number as FPKM/titer for viral genes Figure 2A , FDR-corrected p-value<0 . 01 ) .", "Specifically , three Toll-regulated immune peptides ( IM33 , Bomanins BomBC2 , and BomT2 ) and one JAK-STAT regulated immune peptide ( Listericin ) have reduced expression in High type infected individuals compared to the Low type ( Figure 2 , Figure 2—figure supplement 2 ) .", "Viral genes of interest ( PIF-3 , 19K , gp83 ) have higher expression per viral particle ( FPKM/titer ) in the High type compared to the Low type .", "gp83 also increases in expression per viral particle ( FPKM/titer ) as the number of High type alleles increases ( Figure 2B , t-value = 13 . 732 , p-value=3 . 36e-15 ) .", "Together these results suggest that the High type has increased expression of key virulence factors , which in turn , manipulate the expression of host genes involved in immune defense to result in the observed differences in viral titer .", "Specifically , higher gp83 expression may cause the lower Toll-mediated AMP expression ( Palmer et al . , 2019 ) .", "We reasoned that if the High type is suppressing the host Toll pathway , Myd88 , the Toll signaling protein upstream of AMPs would also have lower expression .", "We did find Myd88 expression is lower in strains infected with the High type ( Figure 2A , though no significantly so ) , which in turn might prevent the host from enacting a proper immune response to DiNV infection ( Figure 2 , Figure 2—figure supplement 1 ) .", "To assess if the virulence differs between virus types , we performed experimental infections of D . innubila males using viral filtrate of strains infected with one of the two types of DiNV .", "Before injections , we used qPCR to identify the viral titer of each sample and dilute all samples to the same viral concentration .", "In these experiments , as viral titer increases , the survival of infected flies decreases , regardless of viral type ( Figure 3—figure supplements 1 and 2 , ANOVA residual deviance = 3 . 536 , p-value=2 . 454e-07 , Cox Hazard Ratio z-value >2 . 227 , p-value<0 . 02592 ) .", "In both types viral titer also increased for the first 3 days of infection ( GLM t-value = 9 . 817 , p-value=3 . 6e-14 ) .", "This established that viral titer is a reasonable proxy for virulence and that DiNV is virulent in D . innubila .", "For a set of four High type and four Low type-infected individuals , we isolated viruses , diluted to roughly equal concentrations of viral particles and performed infections for replicates of 10 males with microneedles dipped in one of the filtrate samples .", "Survival is significantly lower for flies infected with High type viruses when compared to either flies pricked with sterile media ( Figure 3A , Cox Hazard Ratio z-value = 3 . 671 , p-value=0 . 000242 ) or those pricked with Low type virus ( Figure 3A , Cox Hazard Ratio z-value = 4 . 611 , p-value=4 . 02e-06 ) .", "Flies pricked with Low type virus show a non-significant reduction in survival compared to control flies ( Cox Hazard Ratio z-value = 1 . 353 , p-value=0 . 176 ) .", "We also measured viral titer over time using qPCR , and found titer increases through time in flies infected with either type ( Figure 3B , GLM Log10 ( titer ) ~days + type + vial | strain , days t-value = 9 . 912 , p-value=1 . 76e-14 ) .", "Flies infected with High type virus have significantly higher viral titer compared to flies infected with Low type virus ( Figure 3B , GLM Log10 ( titer ) ~days + type + vial | strain , type t-value = 3 . 934 , p-value=0 . 000211 ) .", "These results suggest that the higher viral titer observed in the High Type is also associated with higher virulence .", "We next tested whether genes likely to be involved in host/virus interaction show signs of recurrent natural selection .", "Using McDonald-Kreitman based statistics for estimating the proportion of substitutions fixed by selection ( McDonald and Kreitman , 1991; Stoletzki and Eyre-Walker , 2011; Eilertson et al . , 2012 ) , we tested whether genes that are associated with the High and Low types exhibited different signatures of natural selection compared to other viral genes .", "We calculated the Selection Effect , the proportion of substitutions fixed by adaptive evolution , weighted by the total number of substitutions in the genome ( Eilertson et al . , 2012 ) .", "We found that genes associated with SNPs in the initial association study for viral titer , which defined the High and Low types ( listed in Table 1 and including 19K , PIF-3 and LEF-4 ) have significantly higher rate of substitutions being fixed due to selection than background genes across all categories ( Figure 4¸ type-associated genes versus all other , t-value = 2 . 718 , p-value=0 . 00068 ) .", "When separating the polymorphisms from the substitutions , we found that envelope genes have a significant excess of functional substitutions per site compared to other genes ( Figure 4—figure supplement 2A , GLM t-value = 3 . 62 , p-value=0 . 00107 ) , while genes of unknown function have a significant excess of non-synonymous polymorphisms ( Figure 4—figure supplement 2A , GLM t-value = 2 . 33 , p-value=0 . 02241 ) and a deficit of non-synonymous substitutions ( Figure 4—figure supplement 2A , GLM t-value = −3 . 894 , p-value=9 . 93e-05 ) , which will lower the average selection effect of background genes , suggesting that the relative excess adaptation seen in some genes is in part due to an excess of variation in genes on unknown function .", "We also performed an association study using the host polymorphism and found 13 significantly associated SNPs , after controlling for the viral haplotype ( Figure 4—figure supplement 3 , p<0 . 01 ) , but found no significant enrichments ( p-value>0 . 05 ) or genes of interest ( e . g . those involved in Toll signaling or antiviral pathways ) .", "We therefore looked for enrichments in genes associated with the top 100 significantly associated SNPs versus all other genes and found piRNA genes enriched ( GO enrichment = 3 . 44 , p-value=0 . 035 , FDR-corrected ) .", "Given that siRNA genes are not highly ubiquitously expressed in D . innubila ( Hill et al . , 2019 ) , that DiNV reduces host fecundity ( Unckless , 2011 ) , and that a close relative of DiNV infects the host ovaries ( Palmer et al . , 2018 ) , DiNV could interact with chorion proteins during oogenesis , and piRNAs could be suppressing DiNV ( Lewis et al . , 2018 ) .", "Consistent with the arms race model , host genes we suspect are interacting with DiNV ( such as the association study hits , AMPs , chorion genes , piRNA genes , and extracellular genes ) show elevated levels of substitutions fixed by selection compared to background genes in D . innubila ( Figure 4 and Figure 4—figure supplement 1 , GLM p-value<0 . 05 ) ( Hill and Unckless , 2020 ) .", "Finally , differentially expressed chorion genes , extracellular genes and AMPs have significantly more adaptive substitutions than non-differentially expressed genes in the same categories ( Figure 4 , blue dots , differentially expressed versus all other T-test: D . innubila t-value = 4 . 755 , p-value=0 . 000671 ) .", "When separating the polymorphisms from the substitutions , we found an excess of functional substitutions per site in AMPs ( Figure 4—figure supplement 2B , GLM t-value = 4 . 776 , p-value=1 . 81e-06 ) , driving their excess of adaptive substitutions compared to other genes .", "Overall , these results suggest strong selection is acting on both the host to suppress viral activity and the virus to escape this suppression .", "We were interested in examining the genetic variation of DiNV to determine the relationship of the host and the two putative types of DiNV .", "To determine the appropriate approaches for measuring these patterns , we first need to determine the effective rate of recombination in the virus .", "Though recombination is necessary for proper nudivirus replication ( Kelly , 1982; Kamita et al . , 2003; Rohrmann , 2013 ) , often these recombination events will be between nearly identical viral particles resulting in no detectable signature of recombination across the genome ( Rohrmann , 2013 ) .", "For recombination to leave its signature in genetic variation , two divergent viruses must coinfect the same cell – we refer to this as effective recombination .", "It is unclear how common such recombination is in nudiviruses .", "We used three methods to examine rates of effective recombination across the DiNV genome: First we used GARD to identify the number of recombination breakpoints across samples ( Kosakovsky Pond et al . , 2006 ) .", "Second , we screened for recombination events by finding all four combinations of alleles between two SNPs .", "Third we calculated the linkage disequilibrium pairwise between all SNPs ( Shin et al . , 2006 ) .", "We found recombination is relatively common in DiNV ( Figure 1—figure supplement 3 ) , with 307 potential recombination events genome-wide in recent history ( based on combinations of alleles across our samples ) , and that the genome has relatively low linkage disequilibrium ( Figure 1—figure supplement 3 , Figure 5—figure supplements 1 and 2 ) .", "In DiNV , the eleven SNPs significantly associated with viral titer ( the High and Low types ) , are spread across the ~155 kilobase pair genome , yet are nearly perfectly linked to each other but not to other SNPs ( Figure 1—figure supplement 3 , Figure 5—figure supplements 1 and 2 ) .", "We wanted to examine if it was possible that the High type was generated by recombination of SNPs onto the same background , under the assumption that if the High type was formed via recombination we would expect to find recombination events each side of each significantly associated SNP .", "Using GARD and the four-allele test , six of the significantly associated SNPs show evidence of a recombination event on one side of the SNP , while three have evidence of a recombination event on each side of the significantly associated SNP ( Figure 5—figure supplement 3 , Supplementary Data ) .", "This suggests that recombination could have aided in the formation of the multilocus genotype , by recombination between two intermediate types to form the High type .", "However , we found no evidence of recombination between individuals in different populations ( e . g . no recombinant haplotypes that are ½ CH and ½ PR ) , suggesting little movement of one or more significantly associated SNPs between populations .", "We also found no evidence of recombination between the complete High and complete Low types .", "This suggests that the initial SNPs for the High type have evolved on separate backgrounds in all three populations , though recombination between intermediate strains may have allowed for the formation of the complete High type , or for the generation of other intermediate types .", "For this to happen , each SNP may have recurrently evolved in each population , even if not sequentially .", "We next sought to understand the evolutionary origin of the two putative types .", "Given that both types are found in all populations surveyed ( Figure 1D ) with no evidence of recombination between populations , we hypothesized that this could occur one of three ways: First , the derived haplotype was present ancestrally and has been maintained since before geographic isolation occurred .", "Second , the derived haplotype evolved following geographic isolation and has spread via migration between locations .", "Third , the derived haplotype has recurrently evolved in each location .", "To distinguish between these possibilities and determine the timeframe of divergence , we used the site frequency spectrum of silent DiNV polymorphism to estimate effective population size backwards in time for all populations ( Liu and Fu , 2015 ) .", "We found that the three populations ( CH , HU and SR ) expanded from a single viral particle ( Ne = 1 ) to millions of particles during the last glacial maximum ( 30–100 thousand years ago ) when D . innubila settled its current range ( Figure 5—figure supplement 4; Hill and Unckless , 2020 ) .", "This supports a single invasion event during a host-range change for each location .", "PR appears to expand between 1 and 10 thousand years ago , suggesting a much more recent bottleneck during the range expansion in PR ( Figure 5—figure supplement 4; Hill and Unckless , 2020 ) .", "We aligned genomic regions containing SNPs to two related nudiviruses , Kallithea virus and Oryctes rhinoceros Nudivirus ( OrNV ) ( Wang et al . , 2008; Hill and Unckless , 2018; Palmer et al . , 2018 ) .", "The High type alleles are not present in either Kallithea or OrNV , and are not found in short read information for wild D . melanogaster infected with Kallithea virus ( Webster et al . , 2015 ) , suggesting they are derived in DiNV .", "We generated consensus DiNV sequences for each infected D . innubila individual and created a whole-genome phylogeny to infer geographic diffusion of samples using BEAST2 ( Bouckaert et al . , 2014 ) .", "We then performed ancestral reconstruction of the presence of the High type across the phylogeny using APE ( Paradis et al . , 2004 ) .", "Our samples grouped as three populations ( with HU and SR forming one population ) and , consistent with our expectation , the Low type is the ancestral state ( Figure 5A ) .", "Interestingly we found PR is nested within HU/SR , based on five SNPs which segregate in the Low type HU/SR but are fixed in all PR samples ( Figure 5A ) , consistent with the expansion of DiNV north over time .", "Surprisingly , the High type appears to have evolved repeatedly and convergently within each population , forming separate groups within each population ( Figure 5A ) .", "The High type also clusters within each population in a phylogeny or principal component analysis of all viral SNPs ( Figure 5—figure supplement 1C ) , and when repeating these analyses while excluding the eleven focal SNPs .", "We next surveyed each background SNP ( e . g . SNPs not associated with the High or Low type ) to determine if the general background supports one of the three outlined ways in which the High type evolved and spread in each location .", "We grouped SNPs by their presence in just the High type or Low type ( supporting a single origin and spread by migration ) or if they were unique to a single population but shared between the High and Low types .", "In total , 341 SNPs ( 24% of SNPs surveyed ) are unique to a single population yet are still shared between both High and Low types ( Figure 5 and Figure 5—figure supplement 1 ) , compared to 23 SNPs ( including the 11 High type SNPs ) shared between locations but exclusive to High type samples .", "This is consistent with the lack of recombination between individuals in different populations , suggesting the High type associated SNPs are derived recurrently in each population on different backgrounds .", "Consistent with this , using TreeTime we found that the eleven significantly associated SNPs could have recurrently evolved across our samples , after accounting for recombination and mutation rate ( Kosakovsky Pond et al . , 2006; Sagulenko et al . , 2018 ) .", "We identified 341 SNPs ( 161 for CH , 127 for HU and SR , and 53 for PR ) , are present in all High type samples of a single population but a variable proportion of Low types for that population ( between 19 and 94% ) and are unique to that population .", "This pattern fits with the High type recurrently evolving on a single background ( a different background in each location ) , supporting recurrent evolution of the High type , and against two ancestrally maintained viral types .", "The population-specific background SNPs are spread throughout the DiNV genome , with little evidence of recombination with the High type SNPs , making it unlikely that these SNPs recombined onto different backgrounds , and were instead present when the High type first evolved in each population ( Figure 5—figure supplements 1 and 2 ) .", "We found little evidence of gene conversion producing this background signature ( Figure 5—figure supplement 3 ) .", "While we identified recombination events between background SNPs in the Low type , we found no evidence of this occurring due to their fixed states in the High type , likely because the High type has swept to higher frequencies within individuals before recombination can occur .", "Though there is strong linkage between the High type SNPs , they are not perfectly associated with each other ( Figure 5—figure supplements 1 and 2 ) .", "Using this slight disassociation and APE ( Paradis et al . , 2004 ) , we performed ancestral reconstruction of SNP origins in each population ( assuming recurrent evolution ) and found that , excluding three variable SNPs , the evolution of these SNPs was a similar order in each population ( Figure 5B ) .", "The recurrent evolution of these mutations in a specific order is feasible as the mutation rate is high and the waiting time between mutations should decrease as titer increases .", "Logically , if the wait time for the appearance of a beneficial mutation is 1/ ( 2Ne*μ*s ) , increasing Ne ( with titer ) should decrease the wait time for the appearance of the High type mutations ( Gillespie , 2004 ) .", "To determine if this recurrent evolution is plausible in our estimated timeframe ( ~10 , 000 years ) , we simulated viral populations using a discrete susceptible-infectious model implemented in deSolve ( Soetaert et al . , 2010 ) using estimated baculovirus mutation rates , recombination rates , ranges of viral titer taken our samples and estimated population sizes for each viral population ( parameters described in the methods ) .", "In this model we used an effective mutation rate scaled to viral titer , considering the mutation rate per particle , so total mutations per generation increase with viral titer .", "For simplicity , we limited these simulations to the first five mutations as these are mutations of largest effect and appear to be necessary for the remaining six mutations to appear .", "We also included epistasis which reduces the increase in titer for each subsequent mutation ( titerno . muts ) , as a similar reduction is seen in our samples ( Figure 1C ) .", "The simulations suggest that waiting time for the first mutation that increases titer is highly variable between replicates but usually occurs within 1000 generations ( ~200 years at most , assuming the virus is transmitted from adult hosts to larval hosts via feces , with a 5 host generations per year as a conservative minimum estimate of viral generations per year , in >99 . 93% of replicates , Figure 5C ) .", "The average wait time for each subsequent mutation decreases monotonically ( GLM t-value = -2 . 389 , p-value = 0 . 03686 ) .", "In most cases , the next mutation appears in the background of the previous high titer mutation ( Figure 5C ) due to the elevated effective mutation rate and increased basic reproduction number ( R0 ) .", "In 34 of 1000 simulations , when a mutation does appear on a different background , recombination facilitates the generation of the full complement of mutations .", "Given that effective mutation rate is much higher than the effective recombination rate following the appearance of the first mutation ( as recombination does not scale with titer in nudiviruses and baculoviruses Kamita et al . , 2003 ) , this likely accounts for the mutation rates dominance in our simulations , though recombination likely does play a role in recombination between intermediate strains .", "The accumulation of mutations occurs at close to a geometric ( approximately exponential ) rate .", "Additionally , the standard deviation of time wait times also decreases with each new mutation ( GLM t-value = -2 . 441 , p-value = 0 . 04241 ) , increasing the certainty that the entire multilocus genotype will appear in a population rapidly once the initial mutations appear .", "This chain reaction of adaptation could easily facilitate the repeated evolution of the virulent High type independently in three populations , with all eleven mutations fixing in a population within 6000 generations ( ~1200 years maximum ) in all replicates ( 3372 generations on average , ~675 years maximum ) , a plausible amount of time given our estimated timeframe .", "Since we found two types of DiNV are present in all D . innubila populations , and that other species are infected with DiNV ( Unckless , 2011 ) , we hypothesized that another species could be a reservoir for the less virulent Low type .", "We chose to study D . azteca from the Chiricahuas since it is frequently infected with DiNV ( ~33% infection ) , overlaps with D . innubila , and is genetically divergent ( 40–60 million years ) which could mean a very different genetic interaction between host and virus ( Unckless , 2011 ) .", "We also examined DiNV-infected D . falleni ( a close relative of D . innubila with nonoverlapping geographic range , collected in Athens , Georgia ) as an outgroup .", "We sequenced 36 D . azteca and 56 D . falleni .", "Both viral types are present in both additional species , but the High type is rare in D . azteca ( Figure 6B ) .", "The High type has a significantly higher titer than the Low type in both cases ( Figure 6A , D . azteca GLM t-value = 6 . 71 , p-value=0 . 0056 , D . falleni GLM t-value = 8 . 12 , p-value=0 . 000371 ) .", "Viral titer is not significantly different across species for either High or Low type ( Figure 6A , GLM t-value = −1 . 351 , p-value=0 . 179 ) .", "We also found the D . azteca samples cluster with CH D . innubila samples and contain the CH background SNPs ( Figure 5—figure supplement 1C ) , suggesting no differentiation in the virus infecting different species .", "D . falleni DiNV , on the other hand , clusters completely separately from the other samples , likely due to its geographic separation .", "However , the D . falleni samples still have a derived cluster of High type virus , suggesting a fourth independent evolution of the High type in Georgia .", "Despite the lack of divergence between viruses infecting the two species in Arizona , a lower proportion of the D . azteca population is infected with DiNV , and the High Type is less common than the Low type DiNV ( Figure 6B ) .", "Perhaps , even though the relative differences in titer are preserved between the two species , the Low Type is favored in D . azteca because this reduced virulence leads to a greater basic reproductive rate for the virus in D . azteca .", "Thus , the two types of the virus may be maintained in both host species because though they have become specialized to maximize fitness in one host , transmission between host species could lead to their continued presence in both hosts .", "We repeated our association study in D . azteca , D . falleni , and each D . innubila population separately .", "In all cases we found the 11 High type SNPs are associated with higher viral titer ( GLM t-value >4 . 28 , p-value>0 . 0001 in all cases ) .", "This was not the case for the Helicase-2 SNP ( despite its presence in D . falleni ) nor any other SNPs shared between populations .", "After controlling for the High type , we found no other significant DiNV SNPs in D . azteca associated with viral titer .", "For DiNV infecting D . falleni , we found 478 significantly associated SNPs ( FDR-corrected p-value<0 . 01 ) , though none of them with as large an effect as the High type associated SNPs ." ], [ "Viruses are constantly evolving not just to propagate within a host , but also to optimize their infection across hosts .", "This optimization involves the relationship between their ability to infect an individual and to transmit to others , tempered by the pathogenic effects on infected hosts caused by viral activity ( May and Nowak , 1995; Lipsitch et al . , 1996; Alizon and van Baalen , 2008 ) .", "Since the host is also evolving in response to the virus , an evolutionary arms-race often ensues ( Dawkins and Krebs , 1979; Kaltz and Shykoff , 1998; Daugherty and Malik , 2012 ) .", "DNA viruses have large genomes and often recombination , placing them as a somewhat transitionary pathogen between RNA viruses , bacteria and eukaryotic pathogens and parasites .", "Here , to work towards expanding our understanding of the co-evolution of viruses and their hosts , we examine the population dynamics of Drosophila innubila Nudivirus ( DiNV ) , a DNA virus infecting D . innubila ( Unckless , 2011 ) .", "Within our set of viral samples , we found two DiNV types which differ by 11 SNPs ( Figure 1 , named High and Low types ) .", "One haplotype ( the High type ) is associated with higher viral titer , likely due to an increased manipulation of the host-immune system and increased expression of viral factors ( Figure 2 ) .", "The derived High type has likely recurrently evolved in each population since the last glacial maximum ( ~10 , 000 years ago ) .", "The two types appear to not co-infect individuals , and mutations appear in a similar order as if navigating an epistatic fitness landscape ( Dobzhansky , 1937; Kondrashov et al . , 2002; Gavrilets , 2004 ) .", "Finally , despite the higher titer and transmission rate of the High type compared to the Low type , we found the two types in all sampled populations .", "Thus , a fundamental question is: why are there two haplotypes ?", "Possible explanations are", "( a ) that the two haplotypes are neutral and coexist due to genetic drift ,", "( b ) we caught High type amid a selective sweep , or", "( c ) the two haplotypes are adaptively maintained due to a tradeoff .", "Given the immense differences in titer and survival between High and Low types and the rest of the preponderance of evidence presented above , we find it implausible that the two haplotypes are associated with equal viral fitness and therefore evolving under a model of genetic drift ( Gillespie , 2004 ) .", "It is possible we caught the High type DiNV in the middle of a selective sweep in each population .", "Differing basic reproduction numbers ( R0 ) would result in changes in the ratio of types over time ( as seen between 2001 and 2017 , Figure 1D ) .", "As we only have two time points , we could be witnessing a selective sweep of the High type spreading to fixation ( Nielsen , 2005 ) , with recombination causing the observed differences in the background .", "As we found the High type appears to have evolved recurrently , it would be unlikely that we have caught intermediate sweeps in all four populations sampled ( Figure 1D ) .", "Using the frequency of the High type between 2001 and 2017 CH samples , we can calculate the selection coefficient for the High type if increasing at an exponential rate ( which is likely if mid-selective sweep given the intermediate frequency at both time points ) .", "D . innubila is active during the monsoon season if Arizona ( late July to early September , 10 weeks with a generation time of 2–3 weeks ) ( Patterson and Stone , 1949 ) .", "If we assume five generations per year ( the minimum number of viral generations per year , limited by the host generation time ) and an increase among infected individuals from 52% on 2001 to 71% in 2017 , the selection coefficient = 0 . 0055 , which suggests the High type would take ~350 years maximum to fix in a population once it has arisen ( from a frequency 1/Ne to one in the viral population ) .", "Given the coalescence time of the two types is close to the expansion time of the two viruses ( 2–30 thousand years ) , this does not fit with our results , suggesting the two types are more likely to be adaptively maintained and not amid a selective sweep .", "Further , if the High type was sweeping , it would be remarkable for us to catch these sweeps occurring in all four populations sampled , given the lack of gene flow between populations , our estimated time to fixation of 350 years , and estimated initial infection ~30 thousand years ago .", "Finally , two viral types could be maintained because of a tradeoff between the two virus types .", "We can imagine several scenarios for such a tradeoff .", "First , within a single population of genetically identical hosts , two viral types could coexist if they had equal basic reproduction numbers ( R0 ) ( Nowak and May , 1994; May and Nowak , 1995 ) .", "The basic reproduction number in a simple epidemiology model is the ratio of the transmission rate of the pathogen to its virulence .", "So , while we’ve observed that virulence is higher for the High type virus compared to the Low type , it may be also true that transmission is also higher in the High type ( inferred from Figure 1D ) and those differences are perfectly balanced creating equal R0 .", "We find this unlikely but a useful starting point for considering the adaptive maintenance of the two types .", "A more likely second scenario is one where in a single population of a given species , different host genotypes favor different viral type and that the interaction between optimal R0 among the viral genotypes and susceptibility of the host genotypes creates a stable equilibrium for both host genotypes and viral genotypes ( Anderson and May , 1981 ) .", "These host genotypes could also be different host species .", "In fact , in the Chiricahuas , D . innubila was more likely to be infected with the High type virus and D . azteca was more likely to be infected with the Low type virus ( Figure 6 ) even though the two species are sympatric and the differences in viral titer between the High and Low types were qualitatively similar .", "Therefore , R0 might be higher for the High type in D . innubila and higher for the Low type in D . azteca with some transmission between host species .", "These community dynamics could maintain both viruses in both species .", "The second broad category of tradeoffs involves environmental heterogeneity , which could also create the conditions for the maintenance of both viral types .", "Furthermore , differing environmental conditions ( such as differences in population densities ) could affect the frequencies in each location ( Anderson and May , 1981 ) .", "Finally , different viral types could also preferentially infect different tissues , and have different titers due to limitations of the given infected tissues .", "Previous work in nudiviruses identified nudivurses that infect different tissues including the gut , fat body and ovaries ( Jackson et al . , 2005; Burand et al . , 2012; Palmer et al . , 2018 ) .", "Two DiNV strains infecting different tissues may also help explain the lack of recombination between High and Low types , as viral particles cannot exchange information if they segregate to different tissues .", "However , this would ignore the fact that we found no evidence of coinfection and viruses infecting two tissues could more easily coinfect .", "It is possible that the High type has not recurrently evolved in each population and instead the two viral types are maintained by selection in the face of ongoing gene conversion .", "However , this would not explain the shared background variants which are fixed in the High type and population exclusive , or the evidence of recurrent evolution in the assembled phylogeny ( Figure 5 ) .", "It is entirely possible however that within each population , selection is maintaining the High type due to the fitness deficits of intermediate types , while gene conversion erodes the association of the eleven haplotype SNPs , which is possible given the recombination events found around the significantly associated SNPs ( Figure 3—figure supplement 1 ) .", "Studies of different viruses have found that they can undergo speciation-like events through the accumulation of genetic incompatibilities ( Matsubara and Otsuji , 1978; Rokyta and Wichman , 2009; Meyer et al . , 2016 ) .", "In these cases , the two viral types cannot recombine and produce viable viral progeny .", "This may be occurring in DiNV if infection by one viral type limits the ability of the other type to infect the same cell – akin to prezygotic reproductive isolation ( Coyne and Orr , 2004 ) .", "Alternatively , coinfection could happen but generate inviable recombinant particles ( Meyer et al . , 2016 ) – akin to postzygotic reproductive isolation .", "For example , the fourth mutation might only be favored when in the presence of the derived allele of the third mutation because the third mutation causes a conformational change in protein A that allows the conformational change in protein B caused by the fourth mutation to produce infective viral particles ( Orr , 1995 ) .", "The fact that 19K is known to form a complex with other PIF proteins and other membrane proteins suggests it could play the central role in the formation of this incompatibility system ( Wang et al . , 2007; Rohrmann , 2013 ) .", "If incompatibilities are present between types , they are likely in rapidly evolving vital nudivirus genes ( Hill and Unckless , 2017; Hill and Unckless , 2018 ) .", "As well as being rapidly evolving across baculoviruses and nudivurses , we found these genes also have high rates of adaptive evolution ( Figure 4 ) and are associated with SNPs that distinguish the High and Low DiNV strains ( Figure 1 ) , further supporting the idea that the few genes are key to nudivirus replication are targets of host-suppression and locked in arms-races across the entire nudivirus/baculovirus phylogeny ( Hill and Unckless , 2017 ) .", "DNA viruses such as DiNV have complicated replication cycles and large genomes .", "This makes them a sort of evolutionary intermediate between RNA viruses ( small genomes , high mutation rates ) and eukaryotes ( large genomes , low mutation rates ) and tangential to bacteria and archaea ( intermediate genomes , low recombination rates ) ( Rohrmann , 2013 ) .", "However , adaptation appears to occur through changes in a few key proteins ( Hill and Unckless , 2017; Hill and Unckless , 2018 ) .", "Here we found the evolution of two competing viral types that differ in variants near these few key genes , which cause the strains to differ in virulence and titer .", "Overall our results suggest that the high mutation rates and extremely high levels of selection can result in the repeated and convergent evolution of novel host-virus interactions .", "Additionally , we found that these host-virus interactions for large DNA viruses can be much more complicated than previous models suggest ( Dolan et al . , 2018; Feder et al . , 2019 ) ." ], [ "In this study , we used previously collected and sequenced D . innubila ( Hill and Unckless , 2020 ) .", "Briefly we collected these flies across the four mountainous locations in Arizona between the 22nd of August and the 11th of September 2017 .", "Specifically , we collected at the Southwest research station in the Chiricahua mountains ( ~5400 feet elevation , 31 . 871 latitude −109 . 237 longitude ) , Prescott National Forest ( ~7900 feet elevation , 34 . 540 latitude −112 . 469 longitude ) , Madera Canyon in the Santa Rita mountains ( ~4900 feet elevation , 31 . 729 latitude −110 . 881 longitude ) and Miller Peak in the Huachuca mountains ( ~5900 feet elevation , 31 . 632 latitude −110 . 340 longitude ) .", "Baits consisted of store-bought white button mushrooms ( Agaricus bisporus ) placed in large piles about 30 cm in diameter , at least five baits per location .", "We used a sweep net to collect flies over the baits in either the early morning or late afternoon between one and three days after the bait was set .", "Flies were sorted by sex and species at the University of Arizona and were flash frozen at −80°C before being shipped on dry ice to the University of Kansas in Lawrence , KS .", "During these collections we also obtained D . azteca which we also sorted by species and sex and flash frozen .", "D . falleni were collected using a similar method in the Smoky Mountains ( ~6600 feet elevation ) in Georgia in 2017 by Kelly Dyer , these flies were then sorted at the University of Georgia in Athens GA and shipped on dry ice to the University of Kansas in Lawrence , KS .", "For collected CH D . innubila , D . falleni and D . azteca , we attempted to assess the frequency of DiNV infection using PCR , looking for amplification of the viral gene p47 .", "Using primers from Unckless , 2011 , P47F: 5′–TGAAACCAGAATGACATATATAACGC and P47R: 5′–TCGGTTTCTCAATTAACTTGATAGC .", "We used the following conditions: 95°C 30 s , 55°C 30 s , 72°C 60 s per cycle for 35 cycles .", "We chose the cutoff for viral infection based these PCR infection results .", "We compared the PCR positives with the fold coverage in the genome and found that fly samples with 10x coverage of the viral genome for 90% of the viral genome also had PCR positive results , so we chose this as our cutoff .", "We sorted 343 D . innubila flies , 60 DiNV positive D . falleni and 40 DiNV positive D . azteca which we then homogenized and used to extract DNA using the Qiagen Gentra Puregene Tissue kit ( USA Qiagen Inc , Germantown , MD , USA ) .", "We prepared a genomic DNA library of these 343 DNA samples using a modified version of the Nextera DNA library prep kit ( ~350 bp insert size , Illumina Inc , San Diego , CA , USA ) meant to conserve reagents .", "We sequenced the D . innubila libraries on two lanes of an Illumina HiSeq 4000 run ( 150 bp paired-end ) ( Data to be deposited in the SRA ) .", "We sequenced the D . falleni and D . azteca libraries on a separate run of a lane of an Illumina HiSeq 4000 ( 150bp paired-end ) .", "For 80 male Drosophila innubila collected in 2018 ( indicated in Supplementary file 1 - Table 2 ) , we split the sample homogenate in half , isolated DNA from half as described above and isolating RNA using the Direct-zol RNA Microprep protocol ( R2061 , ZymoResearch , Irvine , CA , USA ) .", "We then polyA-selected on these samples to isolate mRNA and prepared a cDNA library for each of these 80 RNA samples using a modified version of the Nextera TruSeq library prep kit meant to conserve reagents and sequenced these samples on a NovaSeq NS6K SP 100SE ( 100 bp single end ) .", "We also sequenced DNA for these samples , with DNA isolated and prepared as above , also sequenced on a NovaSeq NS6K SP 100SE ( 100 bp single end ) .", "Following sequencing , we removed primer and adapter sequences using cutadapt ( Martin , 2011 ) and Scythe ( Buffalo , 2018 ) and trimmed all data using Sickle ( -t sanger -q 20 l 50 ) ( Joshi and Fass , 2011 ) .", "We masked the D . innubila reference genome ( Hill et al . , 2019 ) , using D . innubila TE sequences and RepeatMasker ( Smit and Hubley , 2008; Smit and Hubley , 2013 ) .", "We then mapped short reads to the masked genome and the Drosophila innubila Nudivirus genome ( DiNV ) ( Hill and Unckless , 2018 ) using BWA MEM ( Li and Durbin , 2009 ) and sorted using SAMtools ( Li et al . , 2009 ) .", "Following this we added read groups , marked and removed sequencing and optical duplicates , and realigned around indels in each mapped BAM file using GATK and Picard , 2020 ( http://broadinstitute . github . io/picard; McKenna et al . , 2010; DePristo et al . , 2011 ) .", "We considered lines to be infected with DiNV if at least 95% of the viral genome is covered to at least 10-fold coverage .", "This most significantly overlaps with our CH D . innubila PCR results ( χ2 = 71 . 791 , p-value=2 . 392e-17 ) .", "Additionally , index-switching at a rate of 0 . 0001 from the highest titer sample to an uninfected sample would still leave the uninfected with ~5 . 2-fold coverage of the viral genome , and so still considered as uninfected .", "We then filtered for low coverage and mis-identified species by removing individuals with low coverage of the D . innubila genome ( less than 5-fold coverage for 80% of the non-repetitive genome ) , and individuals we suspected of being misidentified as D . innubila following collection .", "This left us with 318 D . innubila wild flies with at least 5-fold coverage across at least 80% of the euchromatic genome , of which 254 are infected with DiNV ( Supplementary file 1 - Table 1 ) .", "We also checked for read pairs which were split mapped between the DiNV genome and the D . innubila genome using SAMtools .", "For D . falleni we used a previously generated D . innubila genome with D . falleni variants inserted ( Hill et al . , 2019 ) .", "We masked the genome with Repeatmasker ( Smit and Hubley , 2013 ) and mapped short reads to the masked genome , the repeat sequences and the DiNV genome using BWA MEM and SAMtools ( Li and Durbin , 2009; Li et al . , 2009 ) .", "Then , as with D . innubila we filtered for low coverage and mis-identified species by removing individuals with low coverage ( less than fivefold coverage for 80% of the non-repetitive genome ) leaving us with 56 D . falleni samples infected with DiNV .", "For D . azteca , we downloaded the genome from NCBI ( Accession: GCA_005876895 . 1 ) which we then called repeats from with RepeatModeler ( Smit and Hubley , 2008 ) .", "We masked the genome with Repeatmasker ( Smit and Hubley , 2013 ) and mapped short reads to the masked genome , the repeat sequences and the DiNV genome using BWA MEM and SAMtools ( Li and Durbin , 2009; Li et al . , 2009 ) .", "As with D . innubila we then filtered for low coverage and mis-identified species by removing individuals with low coverage of the D . azteca genome ( less than 5-fold coverage for 80% of the non-repetitive genome ) , which left us with 37 D . azteca samples infected with DiNV .", "We then called DiNV variation using LoFreq ( Wilm et al . , 2012 ) .", "For the 318 sequenced samples with reasonable coverage , for host polymorphism , we used the previously generated multiple strain VCF file , generated using a standard GATK HaplotypeCaller/BCFTools pipeline .", "We used LoFreq ( Wilm et al . , 2012 ) to call polymorphic viral SNPs within each of the 254 DiNV-infected samples , following filtering using BCFtools to remove sites below a quality of 950 and a frequency less than 5% .", "We then merged each VCF to create a multiple strain VCF file , containing 5 , 283 SNPs in the DiNV genome .", "The LoFreq VCF ( Wilm et al . , 2012 ) output contains estimates of the frequency of each SNP in DiNV in each sample , to confirm these frequencies , in SAMtools ( Li et al . , 2009 ) we generated mPileups for each sample and for SNPs of interest ( related to viral titer ) , we counted the number of each nucleotide to confirm the estimated frequencies of these nucleotides at each position in each sample .", "To confirm that there are no coinfections of types , we also subsampled samples and randomly merged Low and High type samples and again generated mPileup files , for SNPs of interest we again counted the number of each nucleotide at each position and confirmed these matched our expected counts in the merged files .", "We then compared these artificial coinfections to actual samples to confirm the presence or absence of coinfections , finding no samples consistent with coinfections .", "We then used SNPeff to identify the annotation of each SNP and label synonymous and non-synonymous ( Cingolani et al . , 2012 ) .", "We extracted the synonymous site frequency spectrum to estimate the effective population size backwards in time using StairwayPlot ( Liu and Fu , 2015 ) .", "Using the viral VCF and the genetics r package we calculated r2 measure of linkage disequilibrium .", "We visualized the linkage between the eleven focal SNPs , and between the eleven focal SNPs and 1000 random SNPs using LDheatmap ( Shin et al . , 2006 ) .", "We also sorted r2 scores by the types of SNPs the measure is between , if it is between the focal type SNPs , the SNPs found on each populations High type background , and other SNPs .", "For 100 , 000 permutations , we randomized the viral titer associated with each strain .", "For each permutation , we binned individuals into high and low artificial haplotypes based on the allele for the SNP at 126118 ( the most significantly associated SNP ) and the 10 SNP most strongly linked to this SNP .", "Finally , we found the difference between the two artificial bins and compared this to the difference between the viral titer of the two haplotypes .", "We then counted the proportion of the 100 , 000 permutations with a difference lower than the true difference .", "We filtered the total viral VCF with annotations by SNPeff and retained only non-synonymous ( replacement ) or synonymous ( silent ) SNPs .", "We also mapped reads from Kallithea virus ( dS ~ 0 . 133 ) and Orcytes rhinoceros Nudivirus ( OrNV , dS ~ 0 . 279 ) to the DiNV genome to identify the ancestral state at each site and polarize SNPs to specific branches ( Hill and Unckless , 2018 ) .", "Specifically , we sought to determine sites which are derived polymorphic in DiNV and which are substitutions fixed on the DiNV branch of the phylogeny .", "After removing singletons , we used the raw counts of fixed and polymorphic silent and replacement sites per gene to estimate McDonald-Kreitman-based statistics , specifically direction of selection ( DoS ) ( McDonald and Kreitman , 1991; Smith and Eyre-Walker , 2002; Stoletzki and Eyre-Walker , 2011 ) .", "We also used these values in SnIPRE ( Eilertson et al . , 2012 ) , which reframes McDonald-Kreitman based statistics as a linear model , taking into account the total number of non-synonymous and synonymous mutations occurring in user defined categories to predict the expected number of these substitutions and calculate a selection effect relative to the observed and expected number of mutations ( Eilertson et al . , 2012 ) .", "We calculated the SnIPRE selection effect for each gene using the total number of mutations on the chromosome of the focal gene .", "For the host , we repeated this process using the SNPeff annotated VCF in the SnIPRE pipeline to identify signatures of adaptation in the host genome .", "We then calculated the difference in each statistic between each gene and the median of all other viral genes , to identify how much that gene deferred from the genomic background .", "We repeated this for the host genome , limiting the comparison to nearby genes ( within 100kbp on the same chromosome ) .", "For 100 male Drosophila innubila collected in 2018 ( indicated in Supplementary file 1 - Table 2 ) , we homogenized each fly separately in 100 µL of PBS .", "We then split the sample homogenate in half , isolated DNA from half as described above and isolating RNA using the Direct-zol RNA Microprep protocol ( R2061 ) .", "Using the isolated DNA , we tested each sample for DiNV using PCR for P47 as described previously , using 40 DiNV-infected samples and 40 uninfected samples .", "We then prepared a cDNA library for each of these 80 RNA samples using a modified version of the Nextera TruSeq library prep kit meant to conserve reagents and sequenced these samples on a NovaSeq NS6K SP 100SE ( 100 bp single end ) .", "We also sequenced DNA for these samples , with DNA isolated and prepared as above , also sequenced on a NovaSeq NS6K SP 100SE ( 100 bp single end ) .", "The DNA sequenced here was mapped as described above , with variation called as described above for other DNA samples .", "Following trimming and filtering the data as described in the methods , we mapped all mRNA sequencing data to a database of rRNA ( Quast et al . , 2013 ) to remove rRNA contaminants .", "Then we mapped the short read data to the masked D . innubila genome and DiNV genome using GSNAP ( -N 1 -o sam ) ( Wu and Nacu , 2010 ) .", "We estimated counts of reads uniquely mapped to D . innubila or DiNV genes using HTSEQ ( Anders et al . , 2015 ) for each sample .", "Using EdgeR ( Robinson et al . , 2010 ) we calculated the counts per million ( CPM ) of each gene in each sample and counted the number of samples with CPM > 1 for each gene .", "We find that over 70 . 3% of genes have a CPM > 1 in at least 70 samples .", "For the remaining genes , we find these genes are expressed in all samples of a subset of the strains ( e . g . DiNV uninfected , DiNV-infected , DiNV-high infected , DiNV-low infected ) .", "This supports the validity of the annotation of D . innubila , given most genes are expressed in some manner , and suggests our RNA sequencing samples show expression results consistent with the original annotation of the D . innubila genome .", "We attempted to improve the annotation of the D . innubila genome to find genes expressed only under infection .", "We extracted reads that mapped to unannotated portions of the genome and combined these for uninfected samples , samples infected with High type DiNV and samples infected with Low type DiNV as three separate samples .", "We then generated a de novo assembly for each of these three groups using Trinity and Velvet ( Schulz et al . , 2012; Haas et al . , 2013 ) .", "We then remapped these assemblies to the genome to identify other transcripts and found the consensus of these two for each sample .", "Using the Cufflinks pipeline ( Ghosh and Chan , 2016 ) , we mapped reads to the D . innubila genome and counted the number of reads mapping to each of these putative novel transcript regions , identifying 15 , 676 regions of at least 100 bp , with at least one read mapping in at least one sample .", "Of these , 717 putative genic regions have at least 1 CPM in all 80 samples , or in all samples of one group ( DiNV uninfected , DiNV-infected , DiNV-low infected , DiNV-high infected ) .", "We next attempted to identify if any of these genes are differentially expressed between types , specifically between uninfected strains and DiNV-infected strains , and between Low-type infected and High-type infected strains .", "Using a matrix of CPM for each putative transcript region in each sample , we calculated the extent of differential expression between each type using EdgeR ( Robinson et al . , 2010 ) , after removing regions that are under expressed , normalizing data and estimating the dispersion of expression .", "We find that 26 putative genes are differentially expressed between infected and uninfected types , and 69 putative genes are differentially expressed between High and Low types .", "We took these regions and identified any homology to D . virilis transcripts using blastn ( Altschul et al . , 1990 ) .", "We find annotations for 37 putative genes are either expressed in all samples , or differentially expressed between samples .", "Of the 14 putative genes expressed in all samples , nine have the closest blast hit to an rRNA gene , and five have hits to unknown genes .", "For 23 differentially expressed putative genes with blast hits , three genes are like antimicrobial peptides ( IM1 , IM14 , IM3 ) , these genes are significantly downregulated upon infection , like other Toll-regulated AMPs , and have significantly lower expression in strains infected with High type DiNV compared to Low types .", "The remaining 20 genes all have similarity to genes associated with cell cycle regulation , actin regulation and tumor suppression genes .", "As the logarithm of viral titer was normally distributed ( Shapiro-Wilk test W = 0 . 05413 , p-value=0 . 342 ) , we used PLINK ( Purcell et al . , 2007 ) to associate nucleotide polymorphism to logarithm of viral titer in infected samples .", "We first generated a relationship matrix for the viral samples using PLINK .", "This kinship matrix allows us control for pairs of individuals who , on average , have similar alleles and which , on average , have dissimilar values .", "We then fit a linear model in PLINK including population , sex , Wolbachia presence , the date of collection and the distance matrix for relationship of each sample ( inferred using PLINK , shown as relationship[strain] ) .", "Before performing the association study , we removed host SNPs found in fewer than five samples and merged SNPs in perfect linkage within 10kbp of each other , filtering down from 5283 viral polymorphisms to 1403 viral polymorphisms .", "We then identified associations between the logarithm of viral titer and the frequency of the viral polymorphism in each individual sample , resulting in the following model:Log10 ( viral titre ) ∼ SNP+hs+w+p+dc+ ( SNP∗hs ) + ( SNP ∗ p ) + ( SNP∗w ) + relationship[strain] Where hs = host sex , p=population of collection , w = Wolbachia presence , dc = date collected , relationship[strain]=distance matrix .", "Following model fitting , performed a posthoc analysis to determine if any clumped SNPs were significant and if they should be assessed separately , we also identified covariates which seemed to show little or no effect on viral titer ( p-value>0 . 1 ) using an ANOVA in R ( R Development Core Team , 2013 ) , and removed these , refitting the model .", "This was done step-wise , resulting in the following model:Log10 ( viral titre ) ∼ SNP+hs+p+ ( SNP ∗hs ) + relationship[strain] Following this we also performed an association study using PLINK ( Purcell et al . , 2007 ) in the host , using previously called host variation , and considering viral haplotype as an additional covariate .", "Before we performed this analysis , we clumped SNPs that are strongly linked ( r2 >0 . 99 ) within 10kbp of each other . Log10 ( viral titre ) ∼ SNP+hs+p+ ( SNP ∗hs ) +vh+ relationship[strain] Whereas previous , and with vh = viral haplotype .", "Using GOrilla ( Eden et al . , 2009 ) , we found no gene categories enriched in the significant SNPs ( Figure 4—figure supplement 3 ) .", "We repeated this analysis for DiNV variants in D . azteca and D . falleni separately .", "We performed the association study twice , first using the original model , then including viral haplotype as an additional covariate .", "Because we are performing multiple correlated tests , we next determined the genome-wide significance threshold for the association between a SNP and the viral titer by permutation .", "We permuted the viral titer information randomly across samples and repeated the genome-wide association studies as described above .", "We recorded the minimum p-value across the genome from 1000 permutations to generate a null distribution .", "To identify if the SNPs involved in the High type are more likely to appear together than expected , we again used a permutation test to identify the frequency that these combinations occur across 1000 permutations , where a P of 0 . 01 = 7 . 987812e-10 .", "Following the identification of the viral haplotype associated with viral titer we sought to determine the effect of viral haplotypes in actual infections .", "For 20 samples with fly homogenate , we determine the viral titer and haplotype following filtration with a 0 . 22 µM filter .", "We performed qPCR for the viral gene p47 ( Forward 5-TCGTGCCGCTAAGCATATAG-3 , Reverse 5-AAAGCTACATCTGTGCGAGG-3 ) on 1 µL of fly filtrate per sample and compared the estimated Cq values across three replicates to estimated viral copy number to confirm viral concentration ( protocol: 2 min at 95°C , 40 cycles of 95°C for 30 s and 59°C for 20 s , followed by 2 min at 72°C ) .", "Following this we diluted samples to similar Cq values , relative to the sample with the highest Cq value .", "We confirmed this by repeating qPCR with p47 primers of 1 µL of each sample .", "For each filtrate sample we performed infections on 30 D . innubila males 4–5 days following emergence using pricks with sterile needles dipped in viral filtrate .", "We recorded survival of each fly each day and removed dead flies .", "Finally , we took samples 1 , 3 , and 5 days post infection and measured viral copies of p47 relative to tpi at each time point .", "For each DiNV-infected D . innubila sample , we reconstructed the consensus DiNV genome infecting them using GATK AlternateReferenceMaker and the VCF generated for each strain ( McKenna et al . , 2010; DePristo et al . , 2011 ) .", "We used BEAST2 with the SkyLine package ( Stadler et al . , 2013 ) to build the phylogeny of DiNV genomes using 100 million iterations with a burn in of 1 million , sampling every 1000 trees ( Bouckaert et al . , 2014 ) .", "After generating a set of phylogenies in BEAST2 , we checked that convergence had occurred across the samples using Tracer and generated a final consensus phylogeny using TreeAnnotator ( Bouckaert et al . , 2014 ) .", "For each High type SNP , we reconstructed the evolution of the SNP as character states across the BEAST2 phylogenies to infer the SNP appearance order .", "We used the all different rates ( ARD ) discrete model in APE ( Paradis et al . , 2004 ) on 1000 randomly sampled converged BEAST2 phylogenies to infer SNP states at each branch and calculated the bootstrap support for the order of appearance for each mutation in each population .", "To independently identify recurrent mutations across the DiNV phylogeny , we also used TreeTime ( Sagulenko et al . , 2018 ) .", "Finally , we created a matrix of SNPs present in at least two viral samples and used this matrix in a principal component analysis in R ( R Development Core Team , 2013 ) , labeling each sample by their viral type in the PCA ( based on the presence or absence of the significant 19K SNP ) .", "We sought to simulate the infection of DiNV in D . innubila when considering the evolution of a high titer viral haplotype , specifically if two viral types can be maintained against each other at stable frequencies , and if the high viral haplotype with five shared mutations could evolve recurrently in the given time period given realistic parameters .", "We used the R package DeSolve ( Soetaert et al . , 2010 ) to simulate infection dynamics in an SI model .", "We did not include the resistance class , under the assumption that flies won’t live long enough to clear the infection .", "Therefore , the proportion of population infected per generation is as follows:dInfecteddTime=Susceptible*Infected* β- ( Infected* γ ) Where β = infection parameter ( e . g . the increased likelihood an infected individual spreading its infection before dying ) and γ = virulence parameter ( e . g . the increased likelihood an infected individual has of dying before it can spread its infection ) .", "Infected = The proportion of the population infected with DiNV .", "Susceptible = the proportion of the uninfected population .", "We attempted to assess if the ‘high titer’ viral haplotype could possibly evolve recurrently in each population in the time scale seen in our findings .", "We again used the SI model , this time discrete with population sizes set to 1 million individuals , based on StairwayPlot estimates ( Liu and Fu 2015 ) , starting with 1 infected individual ( 1/Ne ) .", "We reasoned that if the wait time between beneficial mutations is 1/ ( 2Ne*u*s ) , and increasing viral titer also increases Ne , then the increased titer also decreases the wait time between the appearance of mutations .", "For each infected individual in the population , we tracked the viral titer and also recorded the presence of absence of five mutations , with each mutation increasing the titer of infection , but each further mutation having successively smaller increases in viral titer ( titerno . muts ) , representing the epistatic interaction of high titer associated mutations seen in the viral haplotype .", "Viral titer did not increase if a mutation occurs before the preceding mutation was present occurred , to matching the order of appearance of mutations seen in DiNV .", "In all simulations , we assumed 5 viral generations per year , based on the assumption of 5 host generations per year being the minimum number of viral generations ( likely much higher ) , and so useful to conservatively estimate the amount of time required for the recurrent evolution of the High type .", "In the simulations , we multiplied the infection parameter , mutation parameter and virulence parameter by viral titer , under the assumption that viral titer increases both infection and death rate , and the mutation rate is per viral particle ( Maeda et al . , 1993; Kamita et al . , 2003 ) .", "We considered a per site mutation rate of 10−6 mutations per generation , based on estimated baculovirus mutation rate ( Rohrmann , 2013; Chateigner et al . , 2015 ) , with a specific mutation rate for each of the high titer variants of 3 . 3e-7 ( 1e-6 mutations per base per generation * 1/3 for the correct mutation ) * viral titer , where each mutation occurs independently ( so all five could arise in one generation at a rate of ( 3 . 3e-7 ) 5 = 4 . 12e-33 ) .", "We then simulated populations in replicate 1000 times for 100 , 000 generations with a starting infection frequency of 10% for the ‘low titer’ haplotype , recording the frequency of the virus in a population , the frequency of the haplotype and the time that each ‘high titer’ mutation reaches high enough frequency to escape stochastic behavior and behave deterministically under selection ( Gillespie , 2004 ) .", "We also factored in recombination between each variant site a rate of 2% per site-window per generation ( Kamita et al . , 2003 ) .", "Specifically , we randomly paired viral genomes and in 2% of pairs we randomly recombined the variant combinations .", "We did not increase recombination rate as titer increased as this was not observed in nature ( Kamita et al . , 2003 ) .", "To estimate the possible selection coefficient for DiNV in the CH population , we assumed an exponential distribution and five host generations per year ( 80 host generations between 2001 and 2017 ) .", "We then solved the following equation:P2017=P2001* ( 1+s ) t Where P2017 = the frequency of the High type among viral samples in 2017 ( 71% ) , P2001 = the frequency of the High type among viral samples in 2001 ( 52% ) , s = the selection coefficient and t = the number of generations ( 80 ) .", "We then used this estimated selection coefficient in the same equation to find the number of generations ( t ) to go from 1/2Nes ( 0 . 0000714 , assuming an Ne of 1000000 ) to fixation ( 0 . 99 ) :0 . 99=0 . 0000714* ( 1+0 . 0055 ) t We chose D . innubila samples infected with DiNV and with sequenced genomes , four infected with the High type DiNV and four infected with the Low type .", "For these samples we estimated their viral copy number per host genome as described previously .", "We used qPCR on p47 and tpi to find the differences in Cq values to calculate the concentration of each sample relative to the lowest concentration sample and diluted 50 µL of filtrate for each sample to match the concentration of each sample to the samples with the lowest titer ( IPR07 ) .", "For a separate 50 µL of the IPR01 sample , we performed 1 in 10 serial dilutions to give 45 µL of filtrate at full concentration , 1 in 10 concentration , 1 in 100 concentration and 1 in 1000 concentration .", "Using these sets of samples ( matched titer and serial dilutions ) we next performed experimental infections .", "We transferred 50 D . innubila ( of roughly equal sex ratio ) to new food and let them lay eggs for 1 week , following this we collected male offspring aged 2–5 days for experimental infections .", "Across four separate days in the mornings ( between 9am and 11am ) , we infected the collected male flies with each sample .", "For flies in batches of 10 , we performed pricks with microneedles dipped in the prepared viral filtrate .", "For each day we also had two control replicates of 10 flies pricked with microneedles dipped in sterile media .", "Following infections , we checked on each vial of 10 flies one-hour post infection and removed dead flies ( likely killed by the needle instead of the virus ) .", "We also checked each vial each morning for 15 days , removing dead flies ( freezing to determine the viral titer ) , and flipping flies to new food every 3–4 days .", "Checking at 10am each day , we recorded the day that each fly died , what filtrate they had been infected with , and what replicate/infection day set they belonged to .", "We next looked for differences in survival over time compared to sterile wound controls using a Fit proportional hazards regression model in R ( R Development Core Team , 2013; Kassambara et al . , 2017 ) , considering titer , viral isolate ( nested in haplotype ) and replicate/vial as co-variates ( day of death ~ [titer or haplotype | strain] + infection date ) .", "For a second set of experimental infections ( performed as described above , stabbed with diluted filtrate from different strains ) , we also removed three living flies 1 hr , 1 day , and 5 days post infection .", "Using qPCR , we found the difference in p47 log-Cq and tpi log-Cq to estimate the viral copy number for each sample over time ." ] ]
[ "Hosts and viruses are constantly evolving in response to each other: as a host attempts to suppress a virus , the virus attempts to evade and suppress the host’s immune system .", "Here , we describe the recurrent evolution of a virulent strain of a DNA virus , which infects multiple Drosophila species .", "Specifically , we identified two distinct viral types that differ 100-fold in viral titer in infected individuals , with similar differences observed in multiple species .", "Our analysis suggests that one of the viral types recurrently evolved at least four times in the past ~30 , 000 years , three times in Arizona and once in another geographically distinct species .", "This recurrent evolution may be facilitated by an effective mutation rate which increases as each prior mutation increases viral titer and effective population size .", "The higher titer viral type suppresses the host-immune system and an increased virulence compared to the low viral titer type ." ]
[ "Animals constantly evolve to protect themselves against viruses , and in turn , viruses evolve to escape their host’s new defenses .", "As a result , genes involved in this arms’ race are some of the fastest evolving in nature .", "A better understanding of how host-virus evolution works could help in the search for treatments for many human and animal diseases .", "Repetition is one of the gold standard requirements for biological experiments .", "Watching different groups of animals and viruses evolve under the same conditions makes it possible for researchers to work out whether certain changes are more likely than others .", "This is easy to do in the laboratory , where conditions can be controlled , but much more complicated to accomplish in the wild .", "Wild populations are rarely completely isolated , and often face different environmental conditions .", "One animal-virus pair for which this is not the case is made up of the fly Drosophila innubila , and its virus Drosophila innubila nudivirus .", "They live in the 'sky islands' of North America , patches of forests surrounded by hundreds of kilometers of desert .", "These islands are like natural test tubes , isolated ecosystems each with its own separate fly and virus populations and limited gene flow between populations .", "To understand how this virus-host pair evolves , Hill and Unckless sequenced the genomes of flies and viruses from four different populations .", "While the fly genomes did not show evidence of strong differences between populations , the virus genomes did .", "There were two distinct types of virus , one of which was a lot more effective than the other at infecting flies , possibly because it was better at blocking the fly's immune defenses .", "Unexpectedly , this virus type had evolved more than once , emerging separately on at least four different occasions .", "Hill and Unckless suggest that the natural interactions between flies with similar genomes and the virus guide evolution down the same path time and time again .", "This work on wild populations contributes to the understanding of the evolution of viruses and their hosts .", "One question left unanswered is why both types of virus ( one more effective at infecting the flies and the other less so ) persist in each population when one is better at blocking the fly's immune response ?", "Future work using isolated populations like these could shed more light on the pressures that shape the evolution of viruses and their hosts , potentially helping in the study of human viruses , like HIV ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Neural ensemble dynamics in dorsal motor cortex during speech in people with paralysis
elife-46015-v2
[ [ "Speaking requires coordinating numerous articulator muscles with exquisite timing and precision .", "Understanding how the sensorimotor system accomplishes this behavioral feat requires studying its neural underpinnings , which are critical for identifying ( Tankus and Fried , 2018 ) and treating the causes of speech disorders and for building brain-computer interfaces ( BCIs ) to restore lost speech ( Guenther et al . , 2009; Herff and Schultz , 2016 ) .", "Speaking is also a uniquely human behavior , which presents a high barrier to electrophysiological investigations .", "Previous direct neural recordings during speaking have come from electrocorticography ( ECoG ) ( Bouchard and Chang , 2014; Cheung et al . , 2016; Mugler et al . , 2014 ) or single-unit ( SUA ) recordings from penetrating electrodes during the course of clinical treatment for epilepsy ( Chan et al . , 2014; Creutzfeldt et al . , 1989; Tankus et al . , 2012 ) or deep brain stimulation for Parkinson’s disease ( Lipski et al . , 2018; Tankus and Fried , 2018 ) .", "Such studies have begun to characterize motor cortical population dynamics underlying speech ( Bouchard et al . , 2013; Chartier et al . , 2018; Pei et al . , 2011 ) , but not at the finer spatial scale ( compared to ECoG ) or across larger neural ensembles ( compared to single electrodes ) afforded by the high-density intracortical recordings widely used in animal studies ( Allen et al . , 2019; Cohen and Maunsell , 2009; Kiani et al . , 2014; Smith and Kohn , 2008 ) , including those examining arm reaching ( Carmena et al . , 2003; Churchland et al . , 2012; Kaufman et al . , 2016; Maynard et al . , 1999 ) .", "We studied speech production at this resolution by recording from multielectrode arrays previously placed in human motor cortex as part of the BrainGate2 BCI clinical trial ( Hochberg et al . , 2006 ) .", "This research context dictated two important elements of the present study’s design .", "First , both participants had tetraplegia due to spinal-cord injury but were able to speak; this enabled observing motor cortical spiking activity during overt speaking , in contrast to earlier studies of attempted speech by participants unable to speak ( Brumberg et al . , 2011; Guenther et al . , 2009 ) .", "However , these participants’ long-term paralysis means that their neurophysiology may differ from that of people who are able-bodied; we will discuss the need for interpretation caution in the Discussion .", "Second , the electrode arrays were in dorsal ‘hand knob’ area of motor cortex , which we previously found to strongly modulate to these participants’ attempted movement of their arm and hand ( Ajiboye et al . , 2017; Brandman et al . , 2018; Pandarinath et al . , 2017 ) .", "Speech-related activity has not previously been reported in this cortical area , but there are several hints in the literature that dorsal motor cortex may have speech-related activity .", "Although imaging experiments consistently identify ventral cortical activation during speaking tasks , a meta-analysis of such studies ( Guenther , 2016 ) indicates that responses are occasionally seen ( though not , to our knowledge , explicitly called out ) in dorsal motor cortex .", "Additionally , behavioral ( Gentilucci and Campione , 2011; Vainio et al . , 2013 ) , transcranial magnetic stimulation studies ( Devlin and Watkins , 2007; Meister et al . , 2003 ) , and electrical stimulation mapping studies ( Breshears et al . , 2018 ) have reported interactions ( and interference ) between motor control of the hand and mouth .", "This close linkage between hand and speech networks has been hypothesized to be due to a need for hand-mouth coordination and an evolutionary relationship between manual and articulatory gestures ( Gentilucci and Stefani , 2012; Rizzolatti and Arbib , 1998 ) .", "Here , we explicitly set out to test whether neuronal firing rates in this dorsal motor cortical area modulated when participants produced speech and orofacial movements ." ], [ "We recorded neural activity during speaking from participants ‘T5’ and ‘T8’ , who previously had two arrays each consisting of 96 electrodes placed in the ‘hand knob’ area of motor cortex ( Figure 1A , B ) .", "The participants performed a task in which on each trial they heard one of 10 different syllables or one of 10 short words , and then spoke the prompted sound after hearing a go cue ( Figure 1—figure supplement 1 shows audio spectrograms and reaction times for these tasks ) .", "We analyzed both sortable SUA that could be attributed to an individual neuron’s action potentials ( Figure 1C , D ) , and ‘threshold crossing’ spikes ( TCs ) that might come from one or several neurons ( Figure 1—figure supplement 2 ) .", "Firing rates showed robust changes during speaking of syllables ( Figure 1 , Figure 1—figure supplement 2 , Video 1 ) and words ( Figure 1—figure supplement 3 ) .", "Significant modulation was found during speaking at least one syllable ( p<0 . 05 compared to during silence ) in 73/104 T5 electrodes’ TCs ( 13/22 SUA ) and 47/101 T8 electrodes ( 12/25 SUA ) .", "Active neurons were distributed throughout the area sampled by the arrays , and most modulated to speaking multiple syllables ( Figure 1B and Figure 1—figure supplement 4 ) , suggesting a broadly distributed coding scheme .", "This is consistent with previous single neuron recordings in the temporal lobe ( Creutzfeldt et al . , 1989; Tankus et al . , 2012 ) .", "Three observations lead us to believe that this neural activity is related to motor cortical control of the speech articulators ( Chartier et al . , 2018; Conant et al . , 2018; Mugler et al . , 2018 ) rather than perception or language .", "First , modulation was significantly stronger when speaking compared to after hearing the auditory prompts: the neural population firing rate change compared to the silent condition was 4 . 03 times higher after the go cue compared to after the audio prompt for the T5-syllables dataset , 2 . 90x for the T8-syllables dataset ( Figure 1E ) , 6 . 71x higher for the T5-words dataset , and 2 . 12x for T8-words ( Figure 1—figure supplement 3 ) .", "Modulation following the audio prompt , although small , was significant when compared to a 1 s epoch just prior to the prompt ( p<0 . 01 , sign-rank test , all four datasets ) .", "In this study , we are unable to disambiguate whether this prompt-related response reflects auditory perception , movement preparation , or small overt movements preceding vocalization .", "We will primarily focus on the larger , later neural modulation putatively related to speech production .", "Second , analysis of an additional dataset in which participant T5 spoke 41 different phonemes revealed that neural population activity showed phonemic structure ( Figure 1—figure supplement 5 ) : for example , when phonemes were grouped by place of articulation ( Bouchard et al . , 2013; Lotte et al . , 2015; Moses et al . , 2019 ) , population firing rate vectors were significantly more similar between phonemes within the same group than between phonemes in different groups ( p<0 . 001 , shuffle test ) .", "Third , in both participants , 99 of 120 electrodes that were active during speaking syllables ( 24 out of 25 sorted neurons ) also were active during production of at least one of seven non-speech orofacial movements ( Figure 2 and Figure 2—figure supplement 1 ) .", "We also observed weak but significant firing rate correlations with breathing ( Figure 2—figure supplement 2 ) .", "Modulation for speaking was stronger than for unattended breathing ( ~4 . 7 x ) and instructed breathing ( ~2 . 6 x ) , and modulation for attempted arm movements was stronger than for speaking and orofacial movements ( ~2 . 8 x , Figure 2—figure supplement 3 ) .", "We next performed a decoding analysis to quantify how much information about the spoken syllable or word was present in the time-varying neural activity .", "Multi-class support vector machines were used to predict the spoken sound ( or silence ) from single trial TCs and high-frequency LFP power ( Figure 3 ) .", "Cross-validated prediction accuracies for syllables were 84 . 6% for T5 ( 10 classes , mean chance accuracy was 10 . 1% across shuffle controls ) and 54 . 7% for T8 ( 11 classes , chance was 8 . 6% ) .", "Word decoding accuracies were 83 . 5% for T5 ( 11 classes , chance was 9 . 1% ) and 61 . 5% for T8 ( 11 classes , chance was 9 . 3% ) .", "We also used the same method to decode neural activity from 0 to 500 ms after the speech prompt and found that classification accuracies were only marginally better than chance ( overall accuracies between 11 . 1% and 16 . 6% across the four datasets , p<0 . 05 versus shuffle controls in three of the four datasets; Figure 3C gray bars ) .", "The much higher neural discriminability of syllables and words during speaking rather than after hearing the audio prompt is consistent with the previously enumerated evidence that modulation in this cortical area is related to speech production .", "During speaking , decoding accuracies for all individual sounds were above chance ( p<0 . 01 , shuffle test ) .", "Decoding mistakes ( Figure 3B ) and low-dimensional representations ( Figure 3A ) tended to follow phonetic similarities ( e . g . ba and ga , a and ae ) .", "This observation is consistent with previous ECoG studies ( Bouchard et al . , 2013; Cheung et al . , 2016; Livezey et al . , 2019; Moses et al . , 2019; Mugler et al . , 2014 ) , although the larger neural differences we observed between unvoiced k and p and the beginning of their voiced counterparts at the start of ga and ba suggests strong laryngeal tuning ( Dichter et al . , 2018 ) .", "These neural correlate similarities may reflect similarities in the underlying articulator movements ( Chartier et al . , 2018; Lotte et al . , 2015; Mugler et al . , 2018 ) .", "These multielectrode recordings enabled us to observe motor cortical dynamics during speech at their fundamental spatiotemporal scale: neuron spiking activity .", "Specifically , we examined whether two known key dynamical features of motor cortex firing rates during arm reaching were also present during speaking .", "Importantly , both of these features were revealed when looking not at individual neurons’ firing rates , but rather were seen when examining the time courses of population activity ‘components’ that act as lower dimensional building blocks ( or condensed summaries ) of the many individual neurons’ activities ( Gallego et al . , 2017; Pandarinath et al . , 2018; Saxena and Cunningham , 2019 ) .", "The first prominent neural population dynamics feature ( ‘dynamical motif’ ) we tested for is inspired by previous nonhuman primate ( NHP ) experiments showing that the neural state undergoes a rapid change during movement initiation which is dominated by a condition-invariant signal ( CIS ) ( Kaufman et al . , 2016 ) .", "In that study , Kaufman and colleagues provide a comprehensive exposition on why a large neural component that is highly invariant across many different arm reaches is a non-trivial feature of neural population data and could , despite its non-specificity , be important to the overall computations being performed .", "A similar CIS was recently also reported during NHP grasping ( Intveld et al . , 2018 ) .", "The second dynamical motif we tested for follows studies of NHP arm reaches ( Churchland et al . , 2012; Kaufman et al . , 2016 ) and human point-to-point hand movements ( Pandarinath et al . , 2015 ) , which showed that subsequent peri-movement neural ensemble activity is characterized by orderly rotatory dynamics .", "That is , a substantial portion of moment-by-moment firing rate changes can be explained by a simple rotation of the neural state in a plane that summarizes the correlated activity of groups of neurons .", "These observations , in concert with neural network modeling ( Kaufman et al . , 2016 ) , have led to a model of motor control in which , prior to movement , inputs specifying the movement goal create attractor dynamics toward an advantageous initial condition ( Shenoy et al . , 2013 ) .", "During movement initiation , a large transient input ‘kicks’ the network into a different state from which activity evolves according to rotatory dynamics such that muscle activity is constructed from an oscillatory basis set ( akin to composing an arbitrary signal from a Fourier basis set ) ( Churchland et al . , 2012; Sussillo et al . , 2015 ) .", "We tested whether motor cortical activity during speaking also exhibits these dynamics by applying the analytical methods of Churchland et al . ( 2012 ) and Kaufman et al . ( 2016 ) .", "These analyses used two different dimensionality reduction techniques ( Cunningham and Yu , 2014 ) to reveal latent low-dimensional structure in the trial-averaged firing rates for different conditions ( here , speaking different words ) .", "Both methods sought to find a modest number of linear weightings of different electrodes’ firing rates ( forming the aforementioned neural population activity ‘components’ ) that capture a large fraction of the overall variance .", "This is akin to principal components analysis ( PCA ) , but unlike PCA , each method also looks for a specific form of neural population structure: jPCA ( Churchland et al . , 2012 ) seeks components with rotatory dynamics , whereas dPCA ( Kaufman et al . , 2016; Kobak et al . , 2016 ) decomposes neural activity into CI and condition-dependent ( CD ) components .", "Importantly , these methods do not spuriously find the sought dynamical structure when it is not present in the data ( Churchland et al . , 2012; Elsayed and Cunningham , 2017; Kaufman et al . , 2016; Kobak et al . , 2016; Pandarinath et al . , 2015 ) .", "We found that these two prominent population dynamics motifs were indeed also present during speaking .", "Like in Kaufman et al . ( 2016 ) , the largest dPCA component summarizing each participants’ neural activity during movement initiation was largely CI: this component was 98 . 7% CI in participant T5 , and 87 . 3% CI in participant T8 ( Figure 4A ) .", "Figure 4B shows that in T5 , this ‘CIS1’ component , which rapidly increased after the go cue , was essentially identical regardless of which word was spoken .", "In T8 , the CIS1 was not as cleanly condition-invariant , but nonetheless showed a similar increase following the go cue for each word .", "We also found this condition-invariant neural population activity component in all four additional datasets that we examined: T5’s and T8’s syllables task datasets , as well as two additional replication datasets in which participant T5 spoke just five of the words ( Figure 4—figure supplement 1 ) .", "These results were also robust across different choices of how many dPCs to summarize the neural population activity with ( Figure 4—figure supplement 2 ) .", "We attribute the difference in how condition-invariant the CIS1 component was between the two participants to the much smaller speech task-related neural modulation recorded in participant T8 compared to in T5 , as demonstrated in Figure 1—figure supplement 3B and the lower classification accuracies of Figure 3 .", "The practical consequence of T8’s substantially weaker speech-related modulation is that much more of the neural population activity that dimensionality reduction tries to summarize was not task-relevant ( i . e . is ‘noise’ for the purpose of these analyses ) .", "This lower signal-to-noise ratio can also be appreciated in how the ‘elbow’ of T8’s cumulative neural variance explained by PCA or dPCA components ( Figure 4—figure supplement 1A , B ) occurs after fewer components and explains far less overall variance .", "Lastly , we looked for rotatory population dynamics around the time of acoustic onset .", "Figure 5A shows ensemble firing rates projected into the top jPCA plane ( i . e . the subspace defined by jPC1 and jPC2 ) .", "In participant T5 , all conditions’ neural state trajectories rotated in the same direction ( similarly to Churchland et al . , 2012; Pandarinath et al . , 2015 ) , and rotatory dynamics could explain substantial variance in how population activity evolved moment-by-moment during speaking .", "Application of a recent population dynamics hypothesis testing method ( Elsayed and Cunningham , 2017 ) revealed that this rotatory structure was significantly stronger than expected by chance in T5’s speaking data , but not in T8’s speaking data ( Figure 5B ) or when this analysis was applied to neural activity following the audio prompt ( Figure 4—figure supplement 1H ) .", "As was the case for the condition-invariant dynamics , these results were also consistent across additional datasets ( Figure 4—figure supplement 1E–H ) and across the choice of how many PCA dimensions in which to look for rotatory dynamics ( Figure 4—figure supplement 2B ) .", "We again attribute the observed between-participants difference to T8’s smaller measured neural responses during speech , which likely reflect his older arrays’ lower signal quality .", "Consistent with this , T8’s BCI computer cursor control performance was also substantially worse than T5’s ( Pandarinath et al . , 2017 ) .", "Other factors that could also have contributed to T8’s reduced speech-related neural activity include his tendency to speak quietly and with less clear enunciation ( consistent with Jiang et al . , 2016 ) , array placement differences , and differences in cortical maps between individuals ( Farrell et al . , 2007 ) .", "Videos 2 and 3 show the temporal relationship between these two dynamical motifs – an initial condition-invariant neural state shift , followed by rotatory dynamics .", "Neural state rotations occurred after the condition invariant translation; by comparison , in Kaufman et al . ( 2016 ) the neural rotations also lagged the CIS shift , but in the monkey arm reaching data these rotations either partially overlapped with , or more immediately followed , the CIS shift .", "We note that existing models of how a condition-invariant signal ‘kicks’ dynamics into a different state space region where rotatory dynamics unfold ( Kaufman et al . , 2016; Sussillo et al . , 2015 ) do not require that the CIS and rotatory dynamics must be orthogonal , but in these data we did observed that the CIS1 and jPCA dimensions were largely orthogonal ( Figure 4—figure supplement 1E ) ." ], [ "There are three main findings from this study .", "First , these data suggest that ‘hand knob’ motor cortex , an area not previously known to be active during speaking ( Breshears et al . , 2015; Dichter et al . , 2018; Leuthardt et al . , 2011; Lotte et al . , 2015 ) , may in fact participate , or at least receive correlates of , neural computations underlying speech production .", "Speech-related single-neuron modulation might have been missed by previous studies due to the coarser resolution of ECoG ( Chan et al . , 2014 ) .", "If this finding holds true in the wider population , this would underscore that the familiar ‘motor homunculus’ ( Penfield and Boldrey , 1937 ) is overly simplistic .", "It is generally recognized that motor cortex does not rigidly follow a sequential point-to-point somatotopy , and indeed , Penfield and colleagues were aware of this and intended for their diagram to be a simplified summary of results showing partially overlapping motor fields that also varied substantially across individuals ( Catani , 2017 ) .", "However , the patchy mosaicism amongst nearby body parts in the current view of precentral gyrus organization still features a dorsal-to-ventral progression and separation of the major body regions ( leg , arm , head ) ( Farrell et al . , 2007; Schieber , 2001 ) .", "The presence of neurons responding to mouth and tongue movements in the dorsal ‘arm and hand’ area of motor cortex indicates that sensorimotor maps for different body parts are even more widespread and overlapping than previously thought .", "Given our previous finding that activity from these same arrays encodes intended arm and hand movements ( Pandarinath et al . , 2017 ) , these observations are consistent with the hypothesis that the systems for speech and manual gestures are interlocked ( Gentilucci and Stefani , 2012; Rizzolatti and Arbib , 1998; Vainio et al . , 2013 ) .", "However , emerging work from our group showing that neurons in this area also modulate during attempted movements of the neck and legs ( Willett et al . , 2019 ) suggests that much of the body is represented ( to varying strengths ) in dorsal motor cortex .", "Thus , the observed neural overlap between hand and speech articulators may be a consequence of distributed whole-body coding , rather than a privileged speech-manual linkage .", "Our data suggest that the observed neural activity reflects movements of the speech articulators ( the tongue , lips , jaw , and larynx ) : modulation was greater during speaking than after hearing the prompt; the same neural population modulated during non-speech orofacial movements; and in T5 , the neural correlates of producing different phonemes grouped according to these phonemes’ place of articulation .", "We also found that firing rates showed modest correlation with T5’s unattended and instructed breathing , which invites the question of how this activity relates to the precise control of breathing necessary for speaking and whether breath-related activity differs depending on behavioral context .", "A deeper understanding of how motor cortical spiking activity relates to complex speaking behavior will require future work connecting it to continuous articulatory ( Chartier et al . , 2018; Conant et al . , 2018; Mugler et al . , 2018 ) and respiratory kinematics and , ideally , the underlying muscle activations .", "An important unanswered question , however , is to what extent these results were potentially influenced by cortical remapping due to tetraplegia .", "While we cannot rule this out , we believe that remapping of face representation to the hand knob area is unlikely .", "Despite these participants’ many years of paralysis , the sites we recorded from still strongly modulate during attempted hand and arm movements ( Ajiboye et al . , 2017; Brandman et al . , 2018; Pandarinath et al . , 2017 ) .", "We also verified in participant T5 that modulation during attempted arm movements was stronger than during speech production .", "Our ongoing work also indicates that this area modulates during attempts to move other body parts ( e . g . the leg ) which , like the arm , are also paralyzed ( Willett et al . , 2019 ) .", "Taken together , these results are inconsistent with this area being ‘taken over’ by functions related to the participants’ remaining capability to make orofacial movements .", "Furthermore , motor cortical remapping following arm amputation was recently shown to be smaller than previously thought ( Wesselink et al . , 2019 ) , and in particular much smaller than what would be needed to move lip representations to hand cortex ( Makin et al . , 2015 ) .", "On the sensory side , emerging evidence suggests that cortical reorganization following injury in adults is more limited than previously thought ( Makin and Bensmaia , 2017 ) , and a recent microstimulation study in the hand somatosensory cortex of a person with tetraplegia did not find functional reorganization ( Flesher et al . , 2016 ) .", "While these threads of evidence argue against remapping , definitively resolving this ambiguity would require intracortical recording from this eloquent brain area in able-bodied people .", "Assuming that these results are not due to injury-related remapping , we are left with the question of why this speech-related activity is found in dorsal ‘arm and hand’ motor cortex .", "Speech is spared following lesions in this area ( Chen et al . , 2006; Tei , 1999 ) , indicating that it is not necessary for speech production .", "Nonetheless , it is possible that dorsal motor cortex plays some supporting role in speaking , perhaps contributing to more demanding speaking tasks , or that this activity reflects speech efference copy for coordinating orofacial and upper extremity movements .", "This would be in line with theoretical arguments that high dimensional representations resulting from mixed selectivity – in this case , both within major body regions ( a given neuron being tuned for multiple arm movements or for multiple orofacial movements ) and across major body regions ( neurons being tuned for both arm and face movements ) – enable more complex computations ( Fusi et al . , 2016 ) such as coordinating movements across the body .", "We anticipate that it will require substantial future work to understand why speech-related activity co-occurs in the same motor cortical area as arm and hand movement activity , but that this line of inquiry may reveal important principles of how sensorimotor control is distributed across the brain ( Musall et al . , 2019; Stringer et al . , 2019 ) .", "Our second main finding is that , based on offline decoding results , intracortical recordings show promise as signal sources for BCIs to restore speech to people with some forms of anarthria .", "Decoding the neural correlates of attempted speech production ( Brumberg et al . , 2011 ) into audible sounds or text may be more desirable than approaches that decode covert internal speech ( Leuthardt et al . , 2011; Martin et al . , 2016 ) or more abstract elements of language ( Chan et al . , 2011; Yang et al . , 2017 ) because decoding attempted movements leverages existing neural machinery that separates internal monologue and speech preparation from intentional speaking .", "The present results compare favorably to previously published decoding accuracies using ECoG ( Mugler et al . , 2014; Ramsey et al . , 2018 ) despite our dorsal recording locations likely being suboptimal for decoding speech .", "Multi-electrode arrays placed in ventral motor cortex would be expected to yield even better decoding accuracies .", "Furthermore , recent order-of-magnitude advances in the number of recording sites on intracortical probes ( Jun et al . , 2017 ) point to a path that stretches far forward in terms of scaling the number of distinct sources of information ( neurons ) for speech BCIs .", "That said , these results are only a first step in establishing the feasibility of speech BCIs using intracortical electrode arrays .", "We decoded amongst a limited set of discrete syllables and words in participants who are able to speak; future studies will be needed to assess how well intracortical signals can be used to discriminate between a wider set of phonemes ( Brumberg et al . , 2011; Mugler et al . , 2014 ) , in the absence of overt speech ( Brumberg et al . , 2011; Martin et al . , 2016 ) , and to synthesize continuous speech ( Akbari et al . , 2019; Anumanchipalli et al . , 2019; Makin et al . , 2019 ) .", "We also observed worse decoding performance in participant T8 , highlighting the need for future studies in additional participants to sample the distribution of how much speech-related neural modulation can be expected , and what speech BCI performance these signals can support .", "Our third main finding is that two motor cortical population dynamical motifs present during arm movements were also significant features of speech activity .", "We observed a large condition-invariant change at movement initiation in both participants , and rotatory dynamics during movement generation in the one of two participants whose arrays recorded substantially more modulation .", "We speculate that these neural state rotations are well-suited for generating descending muscle commands driving the out-and-back articulator movements that form the kinematic building blocks of speech ( Chartier et al . , 2018; Mugler et al . , 2018 ) .", "The presence of these dynamics during both reaching and speaking could indicate a conserved computational mechanism that is ubiquitously deployed across multiple behaviors to shift the circuit dynamics from withholding movement to generating the appropriate muscle commands from an oscillatory basis set .", "Testing and refining this hypothesis calls for examining whether these two dynamical motifs are present across an even wider range of behaviors and body parts .", "For instance , there is emerging evidence that rotatory dynamics may be absent in movements with a greater role of sensory feedback , such as hand grasping ( Suresh et al . , 2019 ) .", "This interpretation should also be tempered by the major unresolved question of whether these dynamics in dorsal motor cortex play a causal role in speaking and/or echo similar dynamics in other areas , such as ventral motor cortex , which are more directly involved in speech ( Bouchard et al . , 2013 ) .", "An alternative interpretation is that if dorsal motor cortex merely receives an efference copy or ‘coordination’ signal about speech articulator movements , its dynamics may resemble those during arm reaching because this is what the inherent properties of the local circuit are set up to generate – even if in the speech case , this activity is not helping construct muscle activities .", "Testing these hypotheses will require future research involving recording from the speech articulator muscles ( analogous to recording from arm muscles in Churchland et al . , 2012 ) , causally stimulating the circuit ( Dichter et al . , 2018 ) , and examining whether these neural ensemble dynamical motifs are present during speech production in ventral ( speech ) motor cortex ." ], [ "The two participants in this study were enrolled in the BrainGate2 Neural Interface System pilot clinical trial ( ClinicalTrials . gov Identifier: NCT00912041 ) .", "The overall purpose of the study is to obtain preliminary safety information and demonstrate proof of principle that an intracortical brain-computer interface can enable people with tetraplegia to communicate and control external devices .", "Permission for the study was granted by the U . S . Food and Drug Administration under an Investigational Device Exemption ( Caution: Investigational device . Limited by federal law to investigational use ) .", "The study was also approved by the Institutional Review Boards of Stanford University Medical Center ( protocol #20804 ) , Brown University ( #0809992560 ) , University Hospitals of Cleveland Medical Center ( #04-12-17 ) , Partners HealthCare and Massachusetts General Hospital  ( #2011P001036 ) , and the Providence VA Medical Center  ( #2011–009 ) .", "Both participants gave informed consent to the study and publications resulting from the research , including consent to publish photographs and audiovisual recordings of them .", "Participant ‘T5’ ( male , right-handed , 64 years old at the time of the study ) was diagnosed with C4 AIS-C spinal cord injury 10 years prior to these research sessions .", "He retained the ability to weakly flex his left elbow and fingers and some slight and inconsistent residual movement of both the upper and lower extremities .", "T5 was able to speak normally and converse naturally without hearing assistance , but had some trouble hearing from his left ear .", "Participant ‘T8’ ( male , right-handed , 56 years old at the time of the study ) was diagnosed with C4 AIS-A spinal cord injury 11 years prior to these sessions .", "He retained restricted and non-functional voluntary shoulder girdle motion on both sides , and non-functional voluntary finger extension on his left side .", "He had no sensation below the shoulder .", "T8 was able to speak normally and converse naturally with the assistance of hearing aids in both his ears .", "Participants performed a syllables task consisting of discrete trials in which they spoke out loud one of 10 different phonemes or consonant-vowel syllables in response to an auditory prompt .", "These prompts were i ( as in ‘beet’ ) ; ae ( as in ‘bat’ ) ; a ( as in ‘bot’ ) ; u ( as in ‘boot’ ) ; ba; da; ga; sh ( as in the start of ‘shot’ ) , and the unvoiced k and p .", "All pronunciations were American English .", "Video 1 provides a continuous audio recording of one set of each type of syllables task trial .", "Participants sat comfortably in a chair facing a microphone in a quiet room .", "They were instructed to refrain from attempting movements or speaking during trials except when prompted to speak by a custom experiment control software written in MATLAB ( The Mathworks ) .", "During trials , they were also asked to fixate on the same object in front of them .", "Each trial began with two beeps to alert the participant that the trial was starting .", "Approximately 1 s after the start of the second beep , a pre-recorded syllable prompt was played via computer speakers .", "Two clicks played ~2 s after the start of the prompt served as the go cue that instructed the participant to speak back the prompted sound .", "The next trial started 2 . 8 s after the start of the second click .", "There was also an eleventh ‘silent’ condition which was identical to the spoken syllables trials , except that instead of playing a syllable prompt , the speakers played a nearly-silent audio file consisting of ambient background noise recorded in the same environment as the syllable prompts .", "The participants had been previously instructed not to say anything in response to this silent prompt .", "The task was performed in blocks consisting of 10 trial sets .", "Each set contained 11 trials: one trial of each syllable , plus silence , presented in a randomized order .", "After the task was explained to each participant , he was given time to practice a few sets of the task until he indicated that he was ready to begin data collection .", "At the end of each set , we paused the task until the participant indicated that he was ready to continue .", "These inter-set pauses typically lasted less than 10 s .", "Participants performed three consecutive blocks of the task during a research session , with longer pauses of several minutes between blocks during which we encouraged the participant to rest , adjust his posture for comfort , and take a drink of water .", "Both the audio prompts played by the experiment control computer , and the participant’s voice , were recorded by the microphone ( Shure SM-58 ) .", "This audio signal was recorded via the analog input port of the electrophysiology data acquisition system and digitized at 30 ksps together with the raw neural data ( see Neural Recording section ) .", "Each trial’s acoustic onset time ( AO ) was manually determined by visual and auditory inspection of the recorded audio data .", "During this review , we also excluded infrequent trials where the participant spoke at the wrong time or when the trial was interrupted ( for example , if a caregiver entered the room ) .", "Isolated sounds can be difficult to discriminate , and our participants sometimes misheard a syllable prompt as a phonetically similar prompt .", "In particular , T5 misheard the majority of da prompts as ga ( or occasionally as ba ) .", "Both participants made a few other substitutions between similar syllables .", "In this study , we were interested in the neural correlates of preparing and then generating speech , which should reflect the syllable that the participant perceived .", "We therefore labeled these misheard trials based on the spoken , rather than prompted , syllable for subsequent analyses .", "This left an insufficient number of T5 da trials for subsequent neural analyses; thus , there are 11 conditions shown in T8’s Figure 1 firing rate plots and Figure 3 confusion matrices , but only 10 conditions for T5 .", "The number of trials analyzed for each participant , after excluding trials and re-labeling misheard trials as described above , were: silent ( 30 trials for T5 , 30 trials for T8 ) ; i ( 30 , 28 ) ; u ( 30 , 31 ) ; ae ( 28 , 30 ) ; a ( 30 , 30 ) ; ba ( 31 , 29 ) ; ga ( 50 , 34 ) ; da ( 0 , 27 ) ; k ( 30 , 27 ) ; p ( 30 , 33 ) ; sh ( 30 , 30 ) .", "We refer to these datasets as ‘T5-syllables’ and ‘T8-syllables’ .", "Participants also performed a words task which was identical to the syllables task except that they heard and repeated back one of 10 short words , rather than syllables , in response to the auditory prompt .", "Each participant performed three blocks of ten repetitions of each word during one research session .", "We refer to these datasets as ‘T5-words’ and ‘T8-words’ .", "Two consecutive trials were excluded from the T8-words dataset because of a large electrical noise artifact across almost all electrodes .", "The specific words , and the number of trials analyzed for each participant , were: ‘beet’ ( 30 T5 trials , 29 T8 trials ) ; ‘bat’ ( 30 , 29 ) ; ‘bot’ ( 30 , 28 ) ; ‘boot’ ( 30 , 30 ) ; ‘dot’ ( 30 , 29 ) ; ‘got’ ( 29 , 29 ) ; ‘shot’ ( 29 , 28 ) ; ‘keep’ ( 30 , 30 ) ; ‘seal’ ( 30 , 30 ) ; ‘more’ ( 30 , 30 ) .", "As with the syllables task , there was also a silent condition ( 30 T5 trials , 30 T8 trials ) .", "During two additional research sessions ( as part of a follow-up study ) , participant T5 performed the words task with only five of the 10 words .", "The conditions and trial counts in these two replication datasets , which we refer to as ‘T5-5words-A’ and ‘T5-5words-B’ , were: ‘seal’ ( 33 trials in T5-5-words-A , 34 trials in T5-5words-B ) ; ‘shot’ ( 34 , 34 ) ; ‘more’ ( 33 , 34 ) ; ‘bat’ ( 34 , 33 ) ; beet’ ( 34 , 34 ) ; and a silent condition ( 34 , 34 ) .", "Silent condition trials were assigned a ‘faux AO’ so that neural data from comparable epochs of silent and spoken trials could be visualized and analyzed ( for example , for generating trial-averaged , AO-aligned firing rates in Figure 1 or for decoding silent trials’ neural activity in Figure 3 ) .", "Specifically , each silent trial’s AO was set to equal the mean AO ( relative to the go cue ) for all the spoken syllables or words during the same block .", "Participants also performed an orofacial movement task with a similar trial structure as the syllables and words tasks .", "Seven different movement conditions were instructed with auditory prompts: ‘mouth open’ , ‘lips forward’ , ‘lips back’ , ‘tongue right’ , ‘tongue down’ , ‘tongue up’ , and ‘tongue left’ .", "An additional ‘stay still’ condition was analogous to the silent condition of the syllables and words tasks .", "Prior to the first block of the orofacial task , a researcher explained the prompts to the participant , demonstrated the movements , and ran the participant through a few practice sets .", "Due to clinical trial protocols , we did not collect kinematic tracking data such as electromagnetic midsagittal articulography ( Chartier et al . , 2018 ) or ultrasound recordings ( Conant et al . , 2018 ) .", "A video recording of the participants’ faces ( without markers ) did allow the researchers to confirm that the participants were making the instructed movement with acceptable timing precision .", "Given this limitation , we limited our use of these data to broadly testing for neural responses during orofacial movements , rather than quantifying precise moment-by-moment relationships between neural activity and kinematics .", "Similar to the syllables and words task , an orofacial movement trial began with two ready beeps , after which the computer speaker played a movement prompt ( e . g . ‘lips forward’ ) .", "This was followed by the pair of go clicks; the participants were previously informed that they should begin moving after the second click .", "Approximately 1 . 9 s after the go cue click , the experiment control system played the verbal command ‘return’ , which instructed the participant to return to a neutral orofacial posture ( e . g . close the mouth after ‘mouth open’ , move the tongue left after ‘tongue right’ ) .", "The trial ended ~1 . 9 s after the start of ‘return’ .", "The purpose of using a return cue was so that there was a known epoch after the movement go cue during which we knew that the participant was not yet returning .", "The return cue also provided the participant with dedicated time to return to a neutral orofacial position , so that all trials would start from roughly the same posture .", "For T8 , the ‘return’ instruction was immediately followed by a go click .", "However , we observed that T8 started the return movement upon hearing ‘return’ rather than waiting for the go click .", "We therefore removed the return go click prior to T5’s research sessions , and instead instructed T5 to start the return movement when he heard ‘return’ .", "In the present study , we did not examine the return portion of the orofacial movement task .", "Each participant’s orofacial movements and syllables datasets were collected on the same day during the same research session; three blocks of the orofacial movement task immediately followed three blocks of the syllables task .", "We will refer to these orofacial movements task datasets as ‘T5-movements’ and ‘T8-movements’ .", "No trials were excluded from these datasets; thus , there were 30 trials of each condition for each participant .", "During an additional research session , participant T5 performed a many words task in which he spoke 420 unique words ( from Angrick et al . , 2019 ) designed to broadly sample American English phonemes .", "These words were visually prompted , with one word appearing per trial .", "Each trial started with an instruction period in which a red square appeared in the center of a computer screen facing the participant .", "White text above the square instructed what word the participant should say once given a go cue ( e . g . ‘Prepare: ‘Dog’’ ) .", "This instruction period lasted 1 . 2 to 1 . 8 s ( mean 1 . 4 s , exponential distribution ) after which the square turned green , the text changed to ‘Go’ , and an audible beep was played .", "This served as the go cue for T5 to speak out loud the instructed word .", "A second beep occurred 1 . 5 s later , which marked the end of the trial .", "The next trial began 1 s later .", "The 420 words were divided into four sets , with each set spoken during a continuous block of trials with short breaks between blocks .", "Each word set was repeated three times during this research session , with a given set’s words appearing in a different random order during each block .", "We call this the ‘T5-phonemes’ dataset .", "T5’s breath-related abdomen movements were measured with a piezo respiratory belt transducer ( model MLT1132 , ADIntruments ) .", "The stretch sensor was wrapped around his abdomen at the point where it maximally expanded during breathing .", "Analog voltage signals from the belt were input to the neural signal processor via one of its analog input channels .", "These data were digitized at 30 ksps along with the neural data .", "Our goal was to test whether there is breath-correlated neural activity during ‘unattended’ breathing ( i . e . natural ‘background’ breathing , when the participant was not consciously attending to his breath ) and during consciously attended ‘instructed’ breathing .", "Both of the unattended and instructed conditions were collected during the same research session , and we refer to this as the ‘T5-breathing’ dataset .", "For the unattended breathing condition , we recorded neural and breath proxy measurements while T5 performed a BCI computer cursor task as part of a different study , and during an interval where he was resting quietly after completing the BCI task .", "For the instructed breathing task , we recorded neural and breath proxy measurements while T5 performed a cued breathing task that followed a similar structure as the many words task described in the previous section .", "On each trial , the on-screen instruction text was either ‘Prepare: Breathe in’ or ‘Prepare: Breathe out’ .", "The order of these two trial types was randomized within consecutive two-trial sets , such that breaths in and breaths out were counterbalanced and no more than two out breaths or two in breaths could be prompted in a row .", "After a random delay of 1 . 2 to 1 . 6 s ( mean 1 . 4 s , exponential distribution ) , the go cue instructed the participant to breathe in or out according to the instruction .", "After 1 . 5 s , an audible beep and the on-screen text changing to ‘Return’ instructed the participant to return to a neural lung inflation position .", "‘Return’ stayed on screen for 1 . 5 s , after which the inter-trial interval was 1 s .", "A block consisted of 12 trials , after which the participant was given a chance to take a break , relax , and breathe naturally before the next block .", "The participant reported that this task was comfortable and that he was able to match his breaths to the instructions without difficulty .", "The purpose of this task , which was performed on a separate day from the other datasets , was to compare the neural modulation when making orofacial movements and speaking , versus when attempting to make arm and hand movements .", "The task had a similar visually instructed structure to the instructed breathing task .", "During the instructed delay period , text displayed the upcoming movement , for example , ‘Prepare: Say Ba’ , or ‘Prepare: Open Hand’ .", "There was also a ‘Prepare: Do Nothing’ instruction , which otherwise had the same trial structure as the instructed movements .", "After a random delay period of between 1400 and 1800 ms , the go cue appeared .", "During this epoch , T5 attempted to make the instructed movement as best as he could .", "This resulted in complete movements for all the orofacial and speaking movements and ‘shoulder shrug’ , partial movements for some of the arm movements ( e . g . ‘flex elbow in’ ) , and no overt movement for the other arm movements ( e . g . ‘close hand’ , ‘thumb up’ ) .", "We analyzed neural data from 200 ms to 600 ms after the go cue .", "We note that insofar as there was somatosensory and proprioceptive feedback only during the actualized movements , this would be expected to increase the observed neural modulation to orofacial movements and speaking , and decrease the modulation to attempted arm and hand movements .", "The go cue stayed on for 1500 ms . This was followed by a return period in which the text changed to ‘Return’; during this epoch , the participant was instructed to return his body to a neutral posture .", "Thirty-two trials were collected for each movement type .", "We refer to this as the ‘T5-comparisons’ dataset .", "Both participants had two 96-electrode Utah arrays ( 1 . 5 mm electrode length , Blackrock Microsystems ) neurosurgically placed in dorsal ‘hand knob’ area of the left ( motor dominant ) hemisphere’s motor cortex .", "Surgical targeting was stereotactically guided based on prior functional and structural imaging ( Yousry et al . , 1997 ) , and subsequently confirmed by review of intra-operative photographs .", "T5 and T8 had arrays placed 14 and 34 months , respectively , prior to the present study’s prompted words , syllables , and orofacial movements tasks .", "The T5-breathing and T5-comparisons datasets were recorded 26 months after array placement , the T5-5words-A and T5-5words-B datasets were recorded 28 months after array placement , and the T5-phonemes dataset was recorded 29 months after array placement .", "Arrays were placed in areas anticipated to have arm movement-related activity because two goals of the clinical trial are", "1 ) testing the feasibility of intracortical BCI-based communication using point-and-click keyboards and", "2 ) restoration of reach and grasp function via control of a robotic arm or functional electrical stimulation .", "We note that these implant sites are distinct from the closest known speech area , which is the dorsal laryngeal motor cortex ( Bouchard et al . , 2013; Dichter et al . , 2018 ) .", "In this study , we looked for neural correlates of speaking in dorsal motor cortex .", "To help contextualize the results , here we summarize the other behaviors associated with modulation of the neural activity recorded by these same arrays .", "Our previous studies have reported that T5 and T8 controlled BCI computer cursors by attempting movements of their arm and hand ( Brandman et al . , 2018; Pandarinath et al . , 2017 ) .", "T8 was also able to use intended arm movements to command movements of his own paralyzed arm via functional electrical stimulation ( Ajiboye et al . , 2017 ) .", "We also recorded movement task outcome error signals from T5’s arrays; these signals indicated whether the participant succeeded or failed at acquiring a target using a BCI-controlled cursor ( Even-Chen et al . , 2018 ) .", "Neural signals were recorded from the arrays using the NeuroPort system ( Blackrock Microsystems ) .", "Voltage was measured between each of the 96 electrodes’ uninsulated tips and that array’s reference wire .", "Wire bundles ran from each array to cranially-implanted connector pedestals .", "During research sessions , a ‘patient cable’ with a unity gain pre-amplifier was connected to each array’s corresponding pedestal and carried signals to an isolated unity gain front-end amplifier .", "These signals were analog filtered from 0 . 3 Hz to 7 . 5 kHz , digitized at 30 kHz ( 250 nV resolution ) , and sent to the neural signal processor via fiber-optic link .", "As mentioned earlier , amplified analog voltage data from the microphone were input to the neural signal processor and were digitized time-locked with the neural signals .", "All these digitized data were sent over a local network to a connected PC where they were recorded to disk for subsequent analysis .", "The naming scheme for neurons or electrodes in figures is <participant>_<array #> .", "<electrode #> .", "For example , 'neuron T5_2 . 4' in Figure 1 refers to a participant T5 neuron identified on the second array ( which is the more medial of each participant’s two arrays ) on electrode #4 ( according to the manufacturer’s electrode numbering scheme ) .", "For both participants , we did not observe major differences between the two arrays , and we confirmed that the neural population analyses results ( ensemble modulation to speech/movements/breathing , phoneme neural correlate similarities , speech decoding , condition-invariant and rotatory population dynamics ) were similar when data from each array were analyzed separately .", "We therefore pool together data from both arrays in all the presented results .", "Neuronal action potentials ( spikes ) were detected as follows .", "We first applied a common average re-referencing to each electrode within an array by subtracting , at each time sample , the mean voltage across all electrodes on that array .", "These voltage signals were then filtered with a 250 Hz asymmetric FIR high-pass filter designed to extract spike activity from this type of array ( Masse et al . , 2014 ) .", "To measure single unit activity ( SUA ) , time-varying voltages were manually ‘spike sorted’ by an experienced neurophysiologist using Plexon Offline Spike Sorter v3 .", "This process identified action potentials belonging to putative individual neurons amongst the high amplitude voltage deviation events .", "Occasionally , the same action potential can be recorded on multiple electrodes ( this could happen if a neuron is very large , if an axon passes multiple electrodes , or if there is some electrical cross-talk in the recording hardware ) .", "To prevent creating duplicate single neuron units , we excluded ‘cross-talk units’ if their spike time series ( using 1 ms binning ) had greater than 0 . 5 correlation with another unit’s .", "When this happened , we kept the unit with the better spike sorting isolation .", "Unless otherwise stated , time-varying firing rate plots , also known as peristimulus time histograms ( such as in Figure 1D ) were constructed by smoothing spike trains with a 25 ms s . d . Gaussian kernel and averaging continuous-valued firing rates across trials of the same behavioral condition .", "Spike sorting allows us to make statements about the properties of individual motor cortical neurons ( for example , how many syllables they modulate to , as in Figure 1—figure supplement 4B ) .", "However , a limitation of spike sorting is that action potential event ‘clusters’ with insufficient isolation from other clusters are discarded .", "For chronic multielectrode array recordings , this can mean that activity recorded from the majority of electrodes is not analyzed , despite these neural signals having a strong relationship with the behavior of interest .", "This problem is particularly acute in human neuroscience , where replacing arrays , or using newer methods that provide a higher SUA yield ( for example high-density probes or optical imaging ) , is not currently possible .", "Relaxing the constraint that action potential events must be unambiguously from the same neuron and instead analyzing voltage threshold crossings ( TCs ) is an effective way to substantially increase the information yield of chronic electrode arrays .", "In this study , we examined TCs in a number of analyses .", "Decoding TCs or other non-SUA signals has become standard practice in the intracortical BCI field ( e . g . Ajiboye et al . , 2017; Brandman et al . , 2018; Collinger et al . , 2013; Even-Chen et al . , 2018; Pandarinath et al . , 2017 ) .", "This method also provides information about the dynamics of the neural state ( i . e . it can be used to make scientific statements about ensemble activity under many conditions ) despite combining spikes that may arise from one or more neurons; we provide empirical and theoretical justifications in Trautmann et al . ( 2019 ) .", "In the present study , when we refer to an ‘electrode’s’ firing rate , we mean TCs recorded from that electrode .", "When we refer to a neuron’s firing rate , we mean sorted single unit activity .", "Figure 1—figure supplement 2 shows example TCs firing rates , including from the same electrodes that the example neurons in Figure 1 were sorted from .", "A threshold of −4 . 5 × root mean square ( RMS ) voltage was used for all analyses and visualizations except for the t-SNE visualization and decoding analyses shown in Figure 3 .", "This threshold choice is somewhat arbitrary but is conservative; it accepts large voltage deviations indicative of action potentials from one or a few neurons near the electrode tip .", "For the Figure 3 analyses , we used a more relaxed threshold of −3 . 5 × RMS because we found that this led to slightly better classification performance in a separate pilot dataset ( consisting of T5 speaking five words and syllables , collected a month prior to the datasets reported here ) which we used for choosing hyperparameters .", "The better performance of a less restrictive voltage threshold is consistent with collecting more information by accepting spikes from a potentially larger pool of neurons ( Oby et al . , 2016 ) .", "This trade-off was acceptable because for these engineering-minded decoding analyses , we were less concerned about the possibility of missing tuning selectivity or fast firing rate details due to combining spikes from more neurons .", "Electrodes with TCs firing rates of less than 1 Hz ( at a −4 . 5 × RMS threshold ) were considered non-functioning and were excluded from analyses unless there was well-isolated SUA on the electrode .", "This electrode exclusion applied to both spikes and the local field potential signal described below .", "Electrodes having TCs time series with greater than 0 . 5 correlation with another electrode’s were marked for cross-talk de-duplication .", "To determine which electrode to keep , we chose the one that had the fewest spikes co-occurring ( 1 ms bins ) with the other electrode ( s ) ’ ( i . e . we kept the electrode with putatively more unique information ) .", "For the neural decoding analyses ( Figure 3 ) , we also extracted a high-frequency local field potential ( HLFP ) feature from each electrode by taking the power of the voltage after filtering from 125 to 5000 Hz ( third-order bandpass Butterworth causal filtering forward in time ) .", "HLFP is believed to contain substantial power from action potentials ( Waldert et al . , 2013 ) ; we view this feature as capturing spiking ‘hash’ , that is multiunit activity local to the electrode with contributions from smaller-amplitude and more distant action potentials than TCs .", "Our previous study found that this signal is highly informative about hand movement intentions and is useful for real-time BCI applications ( Pandarinath et al . , 2017 ) .", "This feature has some similarities to the ‘high gamma’ activity examined by ECoG studies; the definition of high gamma varies in exact frequency from study to study , but generally has a lower cutoff between 65 and 85 Hz and an upper cutoff between 125 and 250 Hz ( Bouchard et al . , 2013; Chartier et al . , 2018; Cheung et al . , 2016; Dichter et al . , 2018; Martin et al . , 2014; Mugler et al . , 2014; Ramsey et al . , 2018 ) .", "However , the intracortical HLFP in this study should not be viewed as being the exact same as ECoG high gamma activity due to differences in electrode location , electrode geometry , and HLFP’s higher frequency range .", "To quantify which electrodes’ spiking activity changed during speaking ( Figure 1B insets , Figure 1—figure supplement 4 ) , we calculated each electrode’s mean firing rate from 0 . 5 s before to 0 . 5 s after AO , yielding one datum per electrode , per trial .", "For each syllable , a rank-sum test was then used to determine whether there was a significant change in the distribution of single-trial firing rates when speaking the syllable compared to the silent condition ( p<0 . 05 , Bonferroni corrected for the number of syllables ) .", "To identify which electrodes responded to orofacial movements ( Figure 2 , Figure 2—figure supplement", "1 ) we performed a similar analysis , except that the analysis epoch was from 0 . 5 s before to 0 . 5 s after the go cue .", "This epoch captures strong modulation , as can be seen by the example firing rate plots in Figure 2 .", "We note that firing rate changes preceding the go cue indicate either substantial movement preparation activity , or that the participants were ‘jumping the gun’ and started moving in anticipation of the go cue; either way , this response indicates modulation related to making orofacial movements .", "In lieu of a silent condition , the movement conditions’ firing rate distributions were compared to that of the ‘stay still’ condition .", "The same methods were used to quantify which single neurons’ activities changed during speaking or orofacial movements; for this , we analyzed SUA rather than electrodes’ −4 . 5 × RMS TCs .", "To measure the differences in neural modulation across the recorded population following the audio prompt and following the go cue ( ‘population modulation’ in Figure 1E , Figure 1—figure supplement 3B ) , at each time point ( aligned to either the audio prompt or the go cue ) we quantified the differences between the firing rate vector for a given spoken condition yspeak ( for example , the vector of firing rates across the ga syllable trials , where each element of the vector is the firing rate for one electrode ) and ysilent , the firing rate vector for the silent condition .", "Importantly , however , we did not simply use ||yspeak — ysilent|| , the Euclidean norm of the vector difference between these two conditions’ trial-averaged firing rates .", "The problem with that approach is that a vector norm always yields a non-negative value , meaning that if it is used to measure neural activity differences , the metric will be upwardly biased: it will return a positive value instead of 0 even when population firing rates for the two conditions are essentially the same .", "This is because estimates of firing rates for two sets of trials , even if they are drawn from the same underlying distribution ( i . e . from the same behavioral context ) will inevitably differ , even just slightly , resulting in a positive vector difference norm .", "This problem becomes worse when dealing with lower trial counts and low firing rates , and makes it difficult to distinguish weak population modulation from noise .", "To avoid this issue and better estimate neural population activity differences , we used a cross-validated variant of the vector difference norm; we will refer to this metric as the ‘neural distance’ .", "For N1 trials from condition 1 ( for example , saying ga ) and N2 trials from condition 2 ( for example , silent trials ) , we calculate a less biased estimate of the squared vector norm of the difference in the two conditions’ mean firing rates using: ( 1 ) D=1N11N2∑i=1N1∑j=1N2[y1i−y2j]T⋅[y1{1:N1}/i−y2{1:N2}/j]which leaves one trial out from each condition when calculating the differences in means .", "Critically , the dot product is taken between firing rates computed from fully non-overlapping sets of trials , and can be negative .", "To convert this to a signed distance more analogous to a Euclidean vector norm , we define the final neural distance metric as d=sign ( D ) D .", "This cross-validated neural distance has units of Hz; much like with a standard Euclidean vector norm , having more electrodes , and these electrodes having larger firing rate differences between the two conditions , will both result in larger overall distances .", "Unlike a Euclidean vector norm , our population neural distance metric can produce negative values .", "This is required for the metric to be unbiased and should be interpreted as evidence that the true distance between the two distributions’ population firing rates is near zero .", "A benefit of allowing negative values is that time-averaging across an epoch of essentially no underlying firing rate differences will give a mean distance close to zero .", "The derivation of this metric is described in detail in Willett et al . ( 2019 ) , and a software implementation is available at https://github . com/fwillett/cvVectorStats .", "For statistical testing , we compared the time-average of this neural distance across two comparison epochs: a prompt epoch ( 0 to 1 s after the audio prompt ) and a speaking epoch ( 0 to 1 . 75 s after the go cue for T5 , 0 . 5 to 1 . 75 s after go for T8 ) .", "We chose a later speaking epoch start for T8 to better match this participant’s delayed speech-related modulation , which could reflect less anticipatory preparation prior to the ( predictable ) go cue time , and/or the reduced speech-related modulation recorded on T8’s arrays .", "This resulted in one datum for each epoch per speech condition , for example 10 pairs of ( prompt , speech ) value pairs corresponding to each syllable .", "We compared the resulting prompt and speech epoch distributions with a Wilcoxon signed-rank test .", "The same procedure was used to compare the prompt epoch neural population modulation to a ‘baseline’ epoch consisting of the 1 s leading up to the audio prompt .", "When we report the ratio between population modulation during the go epoch and during the prompt epoch , this ratio was computed after taking the mean modulation across all syllables/words for each epoch .", "To generate Figure 1—figure supplement 5 , we first manually segmented each word spoken in the T5-phonemes dataset into its constituent phonemes using the Praat software package ( Boersma and Weenink , 2019 ) .", "This resulted in 3892 total phonemes .", "The number of occurrences across the 41 unique phonemes ranged between 14 ( /ɔ/ ) and 239 ( /t/ ) , with a median of 80 occurrences .", "For each unique phoneme , we isolated a 150 ms window of TCs centered around the onset of each instance of that phoneme .", "This produced an ( # instances ) × electrodes firing rate matrix for each phoneme .", "We used these data matrices to calculate the neural population activity difference between all pairs of phonemes using the cross-validated neural distance metric described in the ‘Neural population modulation’ section .", "This resulted in the matrix of phoneme pair neural distances in Figure 1—figure supplement 5A .", "Within-phoneme neural distances ( the diagonal elements of the distance matrix ) were calculated by comparing half of the instances of a given phoneme with the other half; the distances shown are the mean distances across 20 such random splits of each phoneme .", "To relate these neural distances to known differences in the speech articulator movements required to produce the phonemes , we grouped phonemes by their place of articulation as in Moses et al . ( 2019 ) .", "We then compared within-group neural distances to between-groups neural distances ( Figure 1—figure supplement 5B ) .", "Every pair of phonemes in the Figure 1—figure supplement 5A neural distance matrix contributes one datum to either the red distribution in that figure’s panel B ( if the two phonemes are in the same articulatory grouping ) or to the black distribution ( if the two phonemes are in different groups ) .", "The exception to this is that the three phonemes that are sole members of their own lonely groups were not included in this analysis .", "The summary statistic of this comparison was the difference between the mean of within-group neural distances and the mean of between-groups neural distances .", "This statistic was compared against a null distribution built by taking the same summary metric after shuffling neural distance matrix rows and columns , repeated 10 , 000 times .", "This null distribution assumes that the phonemes are grouped arbitrarily ( but with the same number and sizes of groups ) , and not according to place of articulation .", "Comparing the true within-group versus between-groups difference to this null distribution ( Figure 1—figure supplement 5C ) provides a p-value for rejecting the null hypothesis that phoneme neural distances are no more correlated with articulatory grouping than expected by chance .", "The dendrogram shown in Figure 1—figure supplement 5D was generated by applying the widely used ‘unweighted pair group method with arithmetic mean’ ( UPGMA ) hierarchical clustering algorithm ( Sokal and Michener , 1958 ) to the phoneme neural distance matrix .", "To generate breath-triggered firing rates ( Figure 2—figure supplement 2 ) , we first identified breath peak times from the breath belt stretch transducer measurements .", "The belt signals were pre-processed by removing rare outlier values ( >50 μV difference between consecutive samples ) and then low-pass filtering ( 3 Hz pass-band ) the signal both forwards and backwards in time to avoid introducing a phase shift .", "An example of this filtered signal is shown in Figure 2—figure supplement 2A .", "Breath peaks were then found using the MATLAB findpeaks function , with key parameters of MinPeakDistance = 1 s , and MinPeakProminence = 0 . 3⋅B , where B is the median of all peak prominences found by first running findpeaks using MinPeakDistance = 5 s ( in other words , we required a peak to be at least 30% of the prominence of the ‘big’ peaks in the data ) .", "Breath peak-aligned firing rates were calculated by treating each identified breath peak as one trial , and trial averaging across neural snippets aligned to each breath peak time .", "Each TCs’ or SUA’s breath-related modulation depth was defined as the maximum – minimum firing rate observed in the interval from 2 s before the breath peak to 1 . 5 s after the breath peak .", "To calculate whether a given modulation depth was statistically significant , we used a shuffle control in which we compared the true data’s modulation depth to the distribution of modulation depths observed over 1001 random shuffles in which faux peak breath times were uniformly drawn from the data duration .", "For comparing breath-related and speaking-related modulation depths ( Figure 2—figure supplement 2F ) , we defined a given electrode’s speech modulation depth in the T5-syllables dataset as its maximum – minimum firing rate from 2 . 5 s before acoustic onset to 1 s after acoustic onset .", "The neural ensemble modulation comparisons presented in Figure 2—figure supplement 3 were calculated as follows: mean TCs firing rates for each T5-comparisons dataset instructed movement condition were calculated for each electrode from 200 to 600 ms after the go cue .", "The resulting firing rate vectors were compared to firing rate vectors similarly constructed from the ‘do nothing’ condition .", "Modulation was calculated by taking the unbiased neural distance between these firing rate vectors as described above in the ‘Neural population modulation’ section .", "To visualize single-trial high dimensional neural data ( Figure 3A ) , we used t-distributed stochastic neighbor embedding ( tSNE ) , a dimensionally reduction technique which seeks to represent high-dimensional vectors ( such as our time-varying , multielectrode neural data ) in a low-dimensional space ( such as a 2D plot that can be easily visualized ) .", "The tSNE algorithm finds a nonlinear mapping such that similar high-dimensional feature vectors end up close together in the low-dimensional view , while dissimilar vectors end up far apart ( Van Der Maaten and Hinton , 2008 ) .", "A neural feature vector was constructed for each trial as follows: for each functioning electrode , spike rates and HLFP power were calculated in ten 100 ms bins that spanned from 0 . 5 s before to 0 . 5 s after AO .", "These features were concatenated into a vector; for example , for the T5-syllables dataset , a single trial’s neural data were represented as a 104 electrodes × 2 features per electrode ×10 time bins = 2080 dimensional vector .", "All trials’ feature vectors were then projected into a 2D space using the tsne function in MATLAB R2017b’s Statistics and Machine Learning Toolbox with NumDimensions = 2; Perplexity = 15 ( this is the number of local neighbors examined for each datum ) ; Algorithm = exact ( suitable for our relatively small dataset ) ; and Standardize = true ( this z-scores the input data , which was desirable due to the variability between different electrodes and the vastly different scales between spike rates and HLFP power ) .", "All other algorithm parameters were set to their defaults .", "Figure 3A does not have axis labels because t-SNE does not return meaningful axes or units; only the relative distances between points have meaning .", "We evaluated how well the identity of the syllable or word being spoken could be decoded from neural data by classifying single trial neural data .", "Neural feature vectors were constructed for each trial as described above .", "These vectors were then associated with a class label , which was the sound being spoken ( i . e . word , syllable , or silence ) .", "We trained support vector machines ( SVMs ) , a standard classification tool , to predict the class label from a ‘new’ neural feature vector which the classifier had not been trained on .", "Prediction accuracies were cross-validated using a leave-one-trial-out paradigm in which the classifier was trained on all trials except the trial being classified , and this was repeated for all trials in a dataset .", "Multiclass classification was achieved using the error-correcting output code ( ECOC ) technique , which trains multiple binary SVMs between all pairs of labels , that is a one-versus-one coding design .", "When classifying new input data , the ECOC technique picks the class that minimizes the sum of losses over the set of binary SVM classifiers .", "Specifically , we used MATLAB R2017b’s implementation: a multiclass model object was fit ( fitcecoc ) using the SVM template ( templateSVM ) .", "Key parameters were to use a linear kernel; OutlierFraction = 0 . 05 ( expecting 5% of data points to be outliers ) ; and Standardize = true ( which z-scores the neural features based on the training data ) .", "All other parameters were set to their default values .", "We note that we did not heavily optimize our classification method; rather , our goal here was to use a standard tool to gauge the classification performance that these intracortical neural signals support .", "More sophisticated machine learning techniques ( e . g . Angrick et al . , 2019; Livezey et al . , 2019 ) are likely to provide additional improvements .", "To measure chance prediction performance , we used a shuffle test in which we randomly permuted the class labels associated with all trials’ neural data .", "The same classifier training and leave-one-out prediction process was then repeated on these shuffled data 101 times .", "An underlying motivation for the neural population dynamics analyses described in the next several sections is the idea that the activity of many thousands or millions of neurons in a circuit ( of which we can only measure on the order of 100 neurons in humans with current technology ) can be summarized by the time-varying activity of a handful of latent ‘components’ .", "In this framing , individual neurons’ firing rates reflect various mixtures of these underlying components; in all the analyses we used , this mapping from components to firing rates is assumed to be linear .", "These components are not meant as discrete physical ‘things’ in the brain , but rather are mathematical abstractions which capture meaningful patterns in the activities of networks of neurons .", "They are useful insofar as they can help generate hypotheses about the computations neural populations are performing by describing their prominent activity patterns .", "To this end , not only can latent components succinctly describe the ‘neural state’ ( i . e . the firing rate of all neurons at a given moment in time ) , but furthermore , the time evolution of these components is often more conducive to interpretation and understanding than more complex descriptions of all the individual neurons’ firing rates .", "Here , we built on previous studies showing that these components’ changes over time can be effectively modeled as a lawful time-varying oscillatory dynamical system ( Churchland et al . , 2012; Pandarinath et al . , 2015 ) , and that they reveal a simple population-level pattern in which there is a stereotyped response at the initiation of many different movements ( Kaufman et al . , 2016 ) .", "This ‘dynamical system’ framework is extensively reviewed in Shenoy et al . ( 2013 ) as well in the two key studies that inspire the neural population dynamics analyses of the present study ( Churchland et al . , 2012; Kaufman et al . , 2016 ) .", "We looked for the aforementioned dynamical motifs using two different dimensionality reduction techniques that were specifically designed to reveal the presence ( or absence ) of these population dynamics features .", "For these analyses , we primarily examined the prompted word speaking task datasets because this was a more naturalistic behavior than the prompted syllables speaking task .", "Participants reported that it was more difficult to discriminate syllables than words , and that speaking stand-alone syllables felt somewhat awkward , whereas saying words was easy .", "Consequently , a practical benefit of the words task over the syllables task is that behavior was more stereotyped across trials , which facilitates precise trial-averaging , and there were very few mis-heard or mis-spoken words .", "Results for the same analyses applied to the syllables task data are shown in Figure 4—figure supplement 1 .", "Both of these neural population state analyses were performed on TCs , which contained more information about the neural population state than the more limited number of recorded SUA .", "All electrodes with TCs firing rates greater than 1 Hz were included .", "The Churchland-Cunningham and Kaufman studies analyzed a combination of both SUA from single-electrode recordings and TCs from multielectrode recordings , depending on the dataset , while Pandarinath et al . ( 2015 ) also analyzed just TCs .", "To avoid cumbersome switching of terms when describing our methods and comparing them to those of these previous studies , we will use the generic term ‘unit’ to refer to a single channel of neural information , whether it be SUA or TCs .", "The first population dynamics motif we tested for was a specific form of population-level structure at the initiation of movement: a large condition-invariant signal , previously described in Kaufman et al . ( 2016 ) .", "We closely followed Kaufman and colleagues’ analysis methods , adapting them as necessary for these human speaking datasets .", "As in Kaufman et al . ( 2016 ) , spike trains were trial-averaged within a behavioral condition ( in our case , speaking one of the 10 different words ) , smoothed with a 28 ms s . d . Gaussian , and ‘soft normalized’ with a 5 Hz offset .", "Normalization means that each unit’s firing rate was normalized by its range across all times and conditions .", "This prevents units with very high firing rates from dominating the estimate of neural population state ( Pandarinath et al . , 2018 ) .", "The ‘soft’ refers to adding an offset ( 5 Hz in these analyses ) to the denominator to reduce the influence of units with very small modulation .", "Trial-averaged firing rates were calculated from a speech initiation epoch of 200 ms before go cue to 400 ms after the go cue for T5 , and 100 ms to 700 ms after the go cue for T8 .", "T8’s epoch was shifted later relative to T5’s to account for T8’s later neural population activity divergence from the silent condition ( Figure 1—figure supplement 3B ) .", "This yields a N × C × T data tensor , where N is the number of units , C is the number of word conditions ( 10 ) , and T is the number of time samples ( 600 , using 1 ms sliding bins ) .", "We used demixed principal components analysis ( dPCA ) , a dimensionality-reduction technique developed by Kobak et al . ( 2016 ) , to look for condition-invariant activity patterns in these high-dimensional neural recordings .", "This dimensionality reduction method is conceptually similar to PCA , in that it finds a specified number of dPC ‘components’ that can be thought of as ‘building blocks’ from which the responses of individual units can be composed .", "As with PCA , dPCA attempts to compress the data by identifying dimensions that capture a large fraction of the variance .", "This takes advantage of the fact that unless the responses of neurons are all independent from one another ( which in practice is not the case ) , then most of the variance of the full population response can be accurately reconstructed as a weighted sum of a smaller number of dPC components .", "Where dPCA differs from PCA is that it can explicitly attempt to find components that marginalize variance attributable to different parameters of the experiment ( such as time or task variables ) .", "This is possible because dPCA is a supervised method that trades off finding dimensions that maximize variance in favor of finding dimensions that partition the variance based on labeled properties of the data .", "In our case , this ‘demixing’ was attempted between:", "1 ) condition and condition + time interactions , which together form the condition-dependent ( CD ) components of the neural population activity; and", "2 ) time only , which forms condition-invariant ( CI ) components .", "In other words , dPCA sought a set of components of the population activity for which the time-varying neural responses during producing different words look the same , and also for another set of components which vary across speaking conditions ( i . e . are ‘tuned’ for what word is being spoken ) .", "Importantly , such variance marginalization ( i . e . demixing the parameters ) may not be achievable; it depends on the structure of the data itself .", "Each component that dPCA returns is associated both with how much overall neural variance it captures ( the lengths of the bars in Figure 4A ) , and how much of this variance is CI or CD ( red and blue fraction of each bar , respectively ) .", "Thus , the success of this demixing can be examined based on how purely CI or CD each component is .", "This in turn reveals whether there exists a large and almost completely condition-invariant component of the population neural activity .", "Kaufman and colleagues used an earlier version of the dPCA method and code package , called ‘dPCA-2011’ ( Brendel et al . , 2011 ) .", "We used the MATLAB implementation of ‘dPCA-2015’ ( Kobak et al . , 2016 ) , downloaded from https://github . com/machenslab/dPCA .", "This is an updated , improved , and widely adopted version of the technique which was not yet available at the time when the Kaufman et al . ( 2016 ) analyses were performed .", "We specified that dPCA should return eight total components , which was less than then 10 to 12 used in Kaufman et al . ( 2016 ) .", "This reflects the reduced complexity of our datasets , in the sense that they had fewer conditions ( 10 versus 27–108 ) and fewer units ( 96–106 versus 116–213 ) .", "We also repeated the analyses using 2 to 12 dPCs and observed very similar results .", "Default dpca function parameters were used , with parameters numRep = 10 ( repetitions for regularization cross-validation ) and simultaneous = true ( indicating that the single-trial neural data were simultaneously recorded across electrodes ) for the dpca_optimizeLambda and dpca_getNoiseCovariance functions .", "Unlike the dPCA-2011 used by Kaufman et al . ( 2016 ) , dPCA-2015 does not enforce that the neural dimensions found for capturing variance attributable to different parameters ( here , the CI and CD components ) be orthogonal .", "For example , while the first three ( largely CI ) components for T5 in Figure 4A are orthogonal by construction ( as are the five largely CD components ) , these CI and CD components need not be orthogonal .", "We quantified the angles between the demixed principal axes ( the dPCA encoder dimensions ) , and the ( related but distinct degree of correlation between the resulting dPCA components , using the methods described in Kobak et al . ( 2016 ) and implemented in the dPCA code pack .", "Unlike Kobak et al . ( 2016 ) , we used a p-value threshold of 0 . 01 rather than 0 . 001 for the Kendall rank correlation coefficient test between each pair of dimensions’ electrode weightings vectors .", "This means that we were more conservative in the sense that we were more likely to flag neural dimensions as non-orthogonal .", "For measuring the angle between the CIS1 dimension and the first jPC plane ( Figure 4—figure supplement 1E ) , we used the subspacea package for MATLAB , downloaded from https://www . mathworks . com/matlabcentral/fileexchange/55-subspacea-m ( Knyazev and Argentati , 2002 ) .", "To test whether the CIS1 was significantly non-orthogonal to each of the jPCA dimensions individually , we used the same Kendall rank correlation test as described above .", "The second form of neural population structure we tested for was rotatory ( i . e . oscillatory ) low-dimensional dynamics .", "We applied methods previously developed to identify and quantify rotatory dynamics in motor cortex during NHP arm reaching ( Churchland et al . , 2012 ) .", "These methods were also recently applied to show rotatory dynamics during hand movements of BrainGate2 study participants ( Pandarinath et al . , 2015 ) .", "Churchland , Cunningham and colleagues introduced the jPCA dimensionality reduction technique for this purpose; we employed their MATLAB analysis package , downloaded from https://churchland . zuckermaninstitute . columbia . edu/content/code .", "Trial-averaged firing rates for each word speaking condition were generated from 150 ms before to 100 ms after acoustic onset to capture an epoch when speech-producing articulator movements were being produced .", "Following Churchland et al . ( 2012 ) and Pandarinath et al . ( 2015 ) , these firing rates were soft-normalized with a 10 Hz offset and smoothed with a Gaussian kernel; we used a 30 ms s . d . kernel as in Pandarinath et al . ( 2015 ) .", "These firing rates were ‘centered’ by subtracting the across-condition mean firing rate of each unit at each time point , and then sampled every 10 ms . The dimensionality of these data was reduced via PCA to six; this ensured that rotatory dynamics would be sought within population activity components that were strongly present in the data .", "jPCA was then used to find planes with rotatory structure within this six-dimensional subspace .", "The jPCs are found by fitting the following linear dynamical system: ( 2 ) x˙=Mskewxwhere x is the neural state ( i . e . the PCA dimensionality-reduced population firing rate ) at a given time , x˙ is its time derivative , and Mskew is constrained to be a skew-symmetric matrix .", "The first jPCA plane , which has the strongest rotatory dynamics , is defined by the two complex eigenvectors of Mskew with the largest eigenvalues .", "The choice of real vectors jPC1 and jPC2 within this plane is arbitrary and , following convention , were chosen such that conditions’ activities are maximally spread along jPC1 at the start of the analysis epoch .", "Figure 5A plots the trial-averaged population activity during speaking each word ( after subtracting the across-conditions mean ) in this top jPCA plane .", "The red/black/green color of each word condition’s neural trajectory corresponds to its projection along jPC1 at the start of the epoch; this display style is intended to assist in observing that amplitude and phase tend to unfold lawfully from the initial neural state .", "It is worth emphasizing that each jPC is simply a linear weighting of different units’ firing rates , and that the six jPCs form an orthonormal basis set that spans the same subspace as the top six PCs .", "The strength of rotatory dynamics was quantified as the goodness of fit for Equation 2 for a 2 × 2 Mskew in the first jPCA plane , and for a 6 × 6 Mskew in the 6-dimensional subspace defined by the top 6 PCs of the data .", "Figure 5B reports this 6D fit quality .", "To calculate the statistical significance of rotatory population dynamics structure in our data , we applied the ‘neural population control’ approach developed by Elsayed and Cunningham ( Elsayed and Cunningham , 2017 ) .", "This method was developed to address a potential concern that many specific phenomena that an experimenter could test for ( such as fitting low-dimensional rotatory dynamics to neural data ) can be found ‘by chance’ in a sufficiently high-dimensional , complex dataset such as the time-varying firing rates of many neurons .", "To address this , the method tests whether an observed feature of the population activity is ‘novel’ in the sense that it cannot be trivially predicted from known simpler features in the data .", "This is achieved by constructing surrogate datasets with simple population structure ( in the form of means and correlations across time , neurons , and behavioral conditions ) matched to the real data .", "If the neural recordings contain population-level structure that is coordinated above and beyond these first- and second-order features , then the quantification method used to describe this structure should return a stronger read-out when applied to the original dataset than to the surrogate datasets .", "In our case , we used this approach to test whether it is ‘surprising’ to see rotatory dynamics in neural population data , given the particular smoothness across time , units , and word speaking conditions present in these data .", "A similar approach was used in Elsayed and Cunningham ( 2017 ) to further validate the original rotatory dynamics finding of Churchland et al . ( 2012 ) .", "We used the MATLAB code associated with Elsayed and Cunningham ( 2017 ) from https://github . com/gamaleldin/TME to generate 1000 surrogate datasets with time , neuron , and condition means and covariance matched to the real data using the tensor maximum entropy algorithm ( ‘surrogate-TNC’ flag in fitMaxEntropy ) .", "We then ran the same jPCA analyses described above on these surrogate datasets and recorded the rotation dynamics goodness of fit for the best Mskew matrix found for each surrogate dataset .", "This distribution of surrogate dataset R2 values serves as a null distribution for significance testing: we calculated a p-value by counting how many of the surrogate datasets’ R2 exceeded that of the true original dataset .", "The goal of Videos 2 and 3 is to visualize how participant T5’s neural population activity undergoes a condition-invariant ‘kick’ after the go cue ( Figure", "4 ) followed by rotatory dynamics around acoustic onset ( Figure 5 ) .", "To do so , we projected the ensemble neural activity during speaking short words into a lower dimensional neural state space designed to capture both the prominent condition-invariant component ( hence , CIS1 is one of the three projection dimensions ) and rotatory dynamics ( hence , the remaining two dimensions are the top jPCA plane ) .", "Plotting the word conditions’ neural state trajectories in the same state space required harmonizing the slightly different pre-processing used in the dPCA ( Figure", "4 ) and jPCA ( Figure", "5 ) analyses .", "Specifically , the trial-averaged neural trajectories in these videos were generated using the 30 ms s . d . Gaussian smoothing and 5 Hz soft-normalization parameters from the dPCA analysis .", "The CIS1 dimension was found by applying dPCA to the same time epoch as in Figure 4 ( 200 ms before go to 400 ms after go ) , and the jPC1 and jPC2 dimensions were found by applying jPCA to the same time epoch as in Figure 5 ( 150 ms before to 100 ms after acoustic on ) .", "To facilitate viewing the neural state trajectories in three ( orthogonal ) dimensions consisting of [CIS1 , jPC1 , jPC2] , for these videos only we enforced that jPC1 and jPC2 be orthogonal to CIS1 ( empirically , without this constraint the top jPCA plane was 75° from the CIS1 , as shown in Figure 4—figure supplement 1E ) .", "To do so , prior to running jPCA , the trial-averaged firing rates were projected into the null space of the CIS1 ( the orthogonal complement of the first column of the encoder matrix returned by dPCA ) .", "That is , instead of jPCA operating on the E = 96 electrodes firing rates , it operated on a 96−1 = 95 dimensional projection of the firing rates .", "The overall consequence of these decisions is that in these videos , the neural state is projected onto the exact same CIS1 dimension as in Figure 4 , whereas the jPC1 and jPC2 dimensions differ slightly from Figure 5 due to the aforementioned spike train pre-processing differences and CIS1 orthogonalization ." ] ]
[ "Speaking is a sensorimotor behavior whose neural basis is difficult to study with single neuron resolution due to the scarcity of human intracortical measurements .", "We used electrode arrays to record from the motor cortex ‘hand knob’ in two people with tetraplegia , an area not previously implicated in speech .", "Neurons modulated during speaking and during non-speaking movements of the tongue , lips , and jaw .", "This challenges whether the conventional model of a ‘motor homunculus’ division by major body regions extends to the single-neuron scale .", "Spoken words and syllables could be decoded from single trials , demonstrating the potential of intracortical recordings for brain-computer interfaces to restore speech .", "Two neural population dynamics features previously reported for arm movements were also present during speaking: a component that was mostly invariant across initiating different words , followed by rotatory dynamics during speaking .", "This suggests that common neural dynamical motifs may underlie movement of arm and speech articulators ." ]
[ "Speaking involves some of the most precise and coordinated movements humans make .", "Learning how the brain produces speech could lead to better treatments for speech disorders .", "But it can be challenging to study .", "Human speech is unique , limiting what can be learned from animal studies .", "There also are few opportunities where it would be safe or ethical to take measurements from inside a person’s brain while they talk .", "Most previous studies have recorded brain activity during speech in patients who have had electrodes placed in the brain for epilepsy or Parkinson’s disease treatment .", "Now , Stavisky et al . show that brain cells that control hand and arm movements are also active during speech .", "Two patients who had lost the use of their arms and legs but were able to speak participated in the study .", "The two individuals were already enrolled in a pilot clinical trial of a brain-computer interface to help them control prosthetic devices .", "As part of this trial , the volunteer participants had two 100-electrode arrays surgically placed in the part of the brain that controls the movement of the arms and hands .", "This study made the unexpected discovery that brain cells multitask controlling not just arm and hand movements , but also carry information about movements of the lips , tongue and mouth necessary for speech .", "Stavisky et al . also found similarities in the patterns of brain activity during hand and arm movements and speech .", "By analyzing the activity in these brain cells as the two individuals recited words and syllables , Stavisky et al . were also able to train computers to identify which sound the person spoke from the brain activity alone .", "This is a first step towards developing a technology that could synthesize speech from a person’s brain activity as they try to speak .", "Much more work is needed to synthesize continuous speech .", "But the study provides initial evidence that it might be possible to use recordings from inside the brain to one day restore speech to individuals who have lost it ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "chromosomes and gene expression", "biochemistry and chemical biology" ]
A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
elife-64131-v1
[ [ "Exogenous factors such as ionizing radiation and genotoxic chemicals can cause DNA damage .", "However , endogenous processes such as DNA replication and cellular metabolism can also damage DNA ( Lambert and Carr , 2013 ) .", "A particularly severe form of DNA damage is a DNA double-strand break ( DSB ) , in which a single normal chromosome is separated into two pathological chromosomes .", "Homologous recombination ( HR ) is a major mechanism responsible for accurately repairing DSBs .", "HR is also critically important for DNA replication ( Ait Saada et al . , 2018 ) .", "Accordingly , defects in HR lead to genome instability , which drives human diseases such as cancer ( Prakash et al . , 2015 ) .", "During HR , the DNA ends that are exposed at a DSB are resected to form 3’ overhangs , which are immediately coated by the single-stranded DNA ( ssDNA ) binding protein RPA ( Symington and Gautier , 2011 ) .", "The RecA-family recombinase Rad51 then displaces RPA to form a helical nucleoprotein filament known as the presynaptic filament ( Sun et al . , 2020 ) .", "This filament can locate a segment of double-stranded DNA ( dsDNA ) with substantial sequence similarity ( i . e . , homology ) to the ssDNA ( Greene , 2016 ) .", "Upon identifying a homologous region , the Rad51 filament invades the intact dsDNA , displacing the non-complementary strand and forming base pairs with the complementary strand .", "Within the context of this displacement loop ( D-loop ) recombination intermediate , the 3’-end of the invading strand can be extended by utilizing the complementary strand as a template for DNA synthesis , allowing for the recovery of lost genetic information ( McVey et al . , 2016 ) .", "The D-loop can also be expanded by Rad51-driven DNA strand exchange , which increases the extent of base pairing between the two DNA molecules .", "Consequently , D-loops can be processed to form Holliday junctions , which may be resolved as either crossover or non-crossover products , or they may be disassembled prior to Holliday junction formation , resulting exclusively in non-crossover outcomes ( Mehta and Haber , 2014 ) .", "As the entity capable of identifying homology and driving DNA strand exchange , Rad51 is integral to DNA repair by HR ( Shinohara et al . , 1992; Muris et al . , 1993; Sung , 1994 ) .", "However , Rad51 does not function alone in vivo .", "Several other proteins that are required for HR have been identified in the fission yeast Schizosaccharomyces pombe including Rad52 , Rad54 , the Rad51 paralogs Rad55-Rad57 , Swi5-Sfr1 , and the lesser studied Shu complex ( Ostermann et al . , 1993; Muris et al . , 1996; Khasanov et al . , 1999; Tsutsui et al . , 2000; Akamatsu et al . , 2003; Khasanov et al . , 2004; Martín et al . , 2006 ) .", "These factors are mostly conserved in the budding yeast Saccharomyces cerevisiae despite the large evolutionary distance separating the two yeasts , although it should be noted that the S . cerevisiae homolog of Swi5-Sfr1 ( Mei5-Sae3 ) is only involved in meiotic HR ( San Filippo et al . , 2008; Hoffman et al . , 2015; Argunhan et al . , 2017a ) .", "This suggests that the requirement for a diverse array of auxiliary factors to promote recombinational DNA repair has been conserved throughout evolution , highlighting its importance .", "However , our understanding of how auxiliary factors promote Rad51 activity remains incomplete , although they seem to perform largely non-overlapping roles ( Zelensky et al . , 2014 ) .", "Sfr1 was first identified in S . pombe as an interactor of Rad51 that forms a complex with Swi5 specifically involved in promoting Rad51-dependent DNA repair ( Akamatsu et al . , 2003 ) .", "The Swi5-Sfr1 heterodimer stimulates DNA strand exchange by potentiating Rad51’s ATPase activity and stabilizing Rad51 filaments ( Haruta et al . , 2006; Kurokawa et al . , 2008 ) .", "In addition to being widely conserved among eukaryotes , the mechanisms through which Swi5-Sfr1 promotes HR appear to be highly similar in yeasts and mammals ( Tsai et al . , 2012; Su et al . , 2014; Su et al . , 2016; Argunhan et al . , 2017a; Lu et al . , 2018 ) .", "The Rad51 paralogs Rad55-Rad57 are another group of evolutionarily conserved auxiliary factors .", "Rad55 and Rad57 were identified in S . pombe based on sequence homology and genetic screening , respectively ( Khasanov et al . , 1999; Tsutsui et al . , 2000 ) .", "Relatively little is known about the molecular function of Rad55-Rad57 due to the biochemical intractability of the complex , although much like Swi5-Sfr1 , it is thought to be an obligate heterodimer .", "The biochemical analysis that has been performed with S . cerevisiae proteins suggests that Rad55-Rad57 promotes Rad51 filament formation on RPA-coated ssDNA and protects the Rad51 filament from disruption by the Srs2 anti-recombinase ( Sung , 1997; Liu et al . , 2011 ) .", "This is consistent with cytological observations in both S . cerevisiae and S . pombe indicating that the number of DNA damage-induced Rad51 foci , which represent Rad51 filaments at sites of ongoing DNA repair , are reduced in the absence of Rad55/Rad57 ( Gasior et al . , 1998; Gasior et al . , 2001; Akamatsu et al . , 2007 ) .", "Among recombination auxiliary factors , the absence of Rad52 results in the most severe phenotype , with deletion mutants displaying DNA damage sensitivity exceeding the rad51∆ single mutant; this has been attributed to the absolute dependency of Rad51 on Rad52 , as well as Rad51-independent functions of Rad52 ( Doe et al . , 2004 ) .", "The rad54∆ mutant also shows severe DNA damage sensitivity that is indistinguishable from rad51∆ ( Muris et al . , 1997 ) , highlighting the absolute requirement for Rad54 in Rad51-dependent DNA repair .", "By contrast , the rad57Δ and sfr1Δ mutants show only moderate sensitivity to DNA damage , while the rad57Δ sfr1Δ double mutant is as sensitive as the rad51Δ single mutant .", "Based on this additivity , it was proposed that Rad55-Rad57 and Swi5-Sfr1 comprise independent sub-pathways of HR that function in parallel to promote Rad51-dependent DNA repair ( Akamatsu et al . , 2003; Akamatsu et al . , 2007 ) , although recent evidence has evoked a re-examination of this model ( Argunhan et al . , 2020 ) .", "To learn more about the relationship between Rad51 and its auxiliary factors , we sought to identify regions of Rad51 that are important for interactions with auxiliary factors .", "This led to the identification of an evolutionarily conserved acidic patch comprised of three residues: E205 , E206 , and D209 .", "Mutation of all three residues to Ala completely ablates Rad51-dependent DNA repair , as does a single charge-reversal mutation , indicating that the negative character of this patch is critical for DNA repair .", "Mechanistically , these defects in DNA repair stem from abrogation of the interaction with both Rad55-Rad57 and Rad52 , leading to impaired recruitment of Rad51 to sites of DNA damage .", "Remarkably , biochemical reconstitutions indicate that neutralization of the acidic patch also impairs the interaction with Rad54 , demonstrating that a single motif of Rad51 is important for its interaction with Rad55-Rad57 , Rad52 , and Rad54 .", "We propose that this acidic patch of Rad51 comprises a fundamental motif that is essential for interactions with auxiliary factors and therefore recombinational DNA repair ." ], [ "Several motifs important for the enzymatic activity of Rad51 are located in the highly conserved ATPase core domain , which is characterized by a β-sheet consisting of mixed parallel and antiparallel β-strands ( Story et al . , 1992; Pellegrini et al . , 2002; Shin et al . , 2003; Conway et al . , 2004 ) .", "These include the Walker A and B motifs , which are important for ATP binding and hydrolysis ( Saraste et al . , 1990; Story and Steitz , 1992 ) , and two DNA binding sites: Site 1 , which is comprised of Loop 1 and Loop 2 , and Site 2 ( Howard-Flanders et al . , 1984; Story et al . , 1992 ) .", "Examination of a homology ( i . e . , computational ) model of S . pombe Rad51 ( SpRad51 ) revealed that the surface of these regions is enriched in positive charge , consistent with roles in the binding of ATP and DNA ( Figure 1A , left ) .", "By contrast , mostly negatively charged regions were found on the opposite face of SpRad51 , including a protruding acidic patch , which we refer to as the PAP hereafter ( Figure 1A , right ) .", "The PAP is among the most negatively charged regions on the surface of SpRad51 ( Figure 1—figure supplement 1A ) and is situated on a short α-helix preceding the outermost β-strand of the central β-sheet ( Figure 1B and Video 1 ) .", "Three acidic residues were seen to project out from this α-helix: E205 , E206 , and D209 ( Figure 1C , D and Video 2 ) .", "These residues were also examined in the context of a previously published homology model of the SpRad51 presynaptic filament ( Ito et al . , 2020 ) and found to form acidic patches constituting dense negatively charged regions on the exterior of the filament ( Figure 1E and Video 3 ) .", "The equivalent α-helix in the human Rad51 ( HsRad51 ) presynaptic filament—the structure of which was determined by cryo-electron microscopy ( Xu et al . , 2017 ) —also had a negative surface charge ( Figure 1—figure supplement 1B ) , as did the corresponding α-helix in the S . cerevisiae Rad51 ( ScRad51 ) presynaptic filament ( Figure 1—figure supplement 1C ) —the structure of which was determined by X-ray crystallography ( Conway et al . , 2004 ) .", "While E205 was replaced with a conservative Asp residue in ScRad51 and D209 was conserved in HsRad51 , E206 is the only PAP residue that showed conservation in both ScRad51 and HsRad51 ( Figure 1D ) .", "Thus , we initially focused on E206 .", "HR plays a particularly important role in the repair of ultraviolet light ( UV ) -induced DNA damage in S . pombe due to the existence of a UV damage endonuclease pathway ( McCready et al . , 2000 ) .", "To examine whether E206 is important for DNA repair , it was mutated to Ala and a strain containing this mutation at the native locus was constructed .", "rad51-E206A showed the same resistance to UV-induced DNA damage as wild type in a clonogenic survival assay ( Figure 2A ) , suggesting that this mutation does not affect the intrinsic ability of Rad51 to repair DNA .", "In the rad57∆/rad55∆ background , Rad51-mediated HR is reduced but not abolished , and this remaining recombinational DNA repair is dependent on Swi5-Sfr1 .", "There is a similar reduction in recombinational DNA repair in the sfr1∆/swi5∆ background , where the remaining Rad51-mediated HR is dependent on Rad55-Rad57 .", "However , the rad57∆ sfr1∆ double mutant displays a complete loss of Rad51-mediated DNA repair , phenocopying rad51∆ .", "Thus , employing the rad57∆ and sfr1∆ backgrounds allows for the evaluation of DNA repair promoted by Swi5-Sfr1 and Rad55-Rad57 , respectively ( Akamatsu et al . , 2003 ) .", "The rad51-E206A rad57∆ strain was no more sensitive to UV than rad57∆ ( Figure 2B ) , suggesting that Rad51-E206A is as proficient as wild-type Rad51 in Swi5-Sfr1–dependent DNA repair .", "By contrast , rad51-E206A was as sensitive as rad51∆ in the absence of Sfr1 ( Figure 2C ) , indicating that the recombinational DNA repair promoted solely by Rad55-Rad57 is ablated by the Rad51-E206A mutation .", "The generality of these findings was confirmed by performing spot tests with several different genotoxins that induce replication fork stalling and a variety of lesions in DNA ( Figure 2—figure supplement 1A–C ) .", "The DNA damage sensitivity of sfr1∆ rad51-E206A is comparable in severity to rad51∆ , which itself shows similar sensitivity to rad57∆ sfr1∆ and rad54∆ ( Muris et al . , 1997; Akamatsu et al . , 2003 ) .", "The rad52∆ mutant is even more sensitive to DNA damage , although this added sensitivity stems from Rad51-independent roles of Rad52 ( Doe et al . , 2004 ) ; a mutation that abolishes the interaction between Rad51 and Rad52 would be expected to phenocopy rad51∆ , not rad52∆ .", "Thus , it is possible that the DNA damage sensitivity associated with rad51-E206A , which manifests in the sfr1∆ background , reflects a defect in the interaction of Rad51 with Rad52 or Rad54 , rather than with Rad55-Rad57 .", "We sought to distinguish between these possibilities genetically .", "Rqh1 is a RecQ-family helicase ( homologous to Sgs1 in S . cerevisiae and the BLM and WRN helicases in humans ) that functions in S phase to prevent the accumulation of toxic recombination intermediates that arise during DNA replication ( Murray et al . , 1997; Stewart et al . , 1997 ) .", "Accordingly , rqh1∆ cells are sensitive to the ribonucleotide reductase inhibitor hydroxyurea ( HU ) .", "It was previously shown that rad57∆ and sfr1∆ robustly suppress the HU sensitivity of rqh1∆ whereas rad52∆ and rad54∆ do not , presumably because the former mutations reduce HR while the latter mutations eliminate it completely ( Hope et al . , 2005 ) .", "If Rad51-E206A is defective in the interaction with Rad55-Rad57 rather than Rad52/Rad54 , then the rqh1∆ rad51-E206A strain should phenocopy rqh1∆ rad57∆ rather than rqh1∆ rad52∆/rad54∆ .", "Consistently , rad51-E206A suppressed the HU sensitivity of rqh1∆ to almost the same degree as rad57∆ ( Figure 2D ) .", "Furthermore , the suppression conferred by sfr1∆ , which is dependent on Rad55-Rad57 ( Hope et al . , 2005 ) , was ablated by rad51-E206A .", "By contrast , the suppression imparted by rad57∆ was epistatic to rad51-E206A , suggesting that they involve the same mechanism .", "At 5 mM HU , rad57∆ sfr1∆ could partially suppress rqh1∆ sensitivity , whereas rad51∆ and rad54∆ could not .", "Importantly , the sfr1∆ rad51-E206A strain was still similar to rad57∆ sfr1∆ , strongly suggesting that the defect associated with rad51-E206A is related to Rad55-Rad57 .", "Immunoblotting experiments revealed that the level of Rad51-E206A was comparable to wild-type Rad51 ( Figure 2—figure supplement 1D ) , indicating that the DNA repair defect of rad51-E206A is not caused by reduced protein stability .", "Previous yeast two-hybrid ( Y2H ) analysis suggested that Rad51 physically interacts with Rad55-Rad57 ( Hays et al . , 1995; Johnson and Symington , 1995; Tsutsui et al . , 2001 ) .", "Thus , a feasible explanation for the DNA damage sensitivity of rad51-E206A is that the E206A mutation disrupts the physical interaction between Rad51 and Rad55-Rad57 .", "To test this , a sequence encoding 12 copies of the V5 epitope was fused to rad55+ at its native locus , yielding the rad55-12xV5 strain .", "This strain was as resistant to DNA damage as the untagged wild type ( rad55+; Figure 2—figure supplement 1E ) , indicating that the tag does not interfere with the function of Rad55-Rad57 in DNA repair .", "In vivo co-immunoprecipitation ( co-IP ) experiments were performed in both sfr1+ and sfr1∆ backgrounds .", "While robust signal was observed for wild-type Rad51 , substantially less Rad51-E206A was seen to co-IP with Rad55 ( Figure 2E ) , indicating that the E206A mutation impairs Rad51–Rad55-Rad57 complex formation .", "The presence of Sfr1 did not have an effect on complex formation , irrespective of the E206A mutation .", "These results suggest that the DNA repair defect associated with rad51-E206A is related to impaired Rad51–Rad55-Rad57 complex formation and that the suppression of this DNA damage sensitivity by Swi5-Sfr1 is not through enhancing physical binding .", "Although E206 does not belong to a canonical motif involved in ATP hydrolysis or DNA binding , it remained formally possible that the E206A mutation impaired the enzymatic activity of Rad51 .", "Rad51-E206A was therefore purified to homogeneity from Escherichia coli to investigate its biochemical properties ( Figure 3—figure supplement 1 ) .", "Rad51-E206A shifted both ssDNA and dsDNA to a similar extent as wild-type Rad51 in electrophoretic mobility shift assays ( EMSAs; Figure 3A , B ) , suggesting that the E206A mutation does not affect the ability of Rad51 to bind DNA .", "In addition to DNA binding , ATP hydrolysis by Rad51 is important for the DNA strand exchange reaction ( Ito et al . , 2018 ) .", "Furthermore , Swi5-Sfr1 stimulates the ATPase activity of Rad51 ( Haruta et al . , 2006 ) .", "We therefore examined whether Rad51-E206A is proficient for ATP hydrolysis , both with and without Swi5-Sfr1 .", "In the absence of Swi5-Sfr1 , the ATP turnover number ( kcat ) of both Rad51 and Rad51-E206A was ~0 . 2 min−1 ( Figure 3C ) .", "The inclusion of Swi5-Sfr1 elicited an approximately twofold increase in ATP hydrolysis by both proteins , demonstrating that Rad51-E206A can hydrolyze ATP like wild type and is proficient for the ATPase stimulation imparted by Swi5-Sfr1 .", "Next , an assay with plasmid-sized DNA substrates was employed to examine the strand exchange activity of Rad51-E206A ( Figure 3D ) .", "Rad51 drives the pairing of circular ssDNA ( css ) with homologous linear dsDNA ( lds ) to yield joint molecule intermediates ( JMs ) .", "Following strand transfer over the length of the dsDNA substrate , JMs are converted into nicked-circular dsDNA molecules ( NCs ) , which are the products in this assay .", "The different DNA species can be separated by agarose gel electrophoresis and visualized .", "Under our standard reaction conditions , wild-type Rad51 cannot promote JM or NC formation in the absence of auxiliary factors ( Haruta et al . , 2006; Kurokawa et al . , 2008 ) .", "However , when reaction conditions were modified by increasing the concentrations of Rad51 and DNA substrates , both Rad51 and Rad51-E206A were seen to drive the efficient pairing of css and lds to produce JMs in the absence of auxiliary factors ( Figure 3E ) .", "This allowed us to evaluate whether the E206A mutation impaired the intrinsic strand exchange activity of Rad51 .", "Although neither protein was able to drive the robust accumulation of NC , we nevertheless quantified the total yield ( JM + NC ) at each time point and found them to be comparable for both Rad51 and Rad51-E206A .", "Our genetic analysis suggested that Rad51-E206A is proficient for the DNA repair promoted by Swi5-Sfr1 .", "To corroborate these findings , strand exchange reactions were supplemented with Swi5-Sfr1 .", "Note that under these standard assay conditions , which differ from those employed above , Rad51 alone cannot promote JM or NC formation , thus allowing us to better examine the effect of Swi5-Sfr1 ( Haruta et al . , 2006; Kurokawa et al . , 2008 ) .", "The inclusion of Swi5-Sfr1 efficiently stimulated both Rad51 and Rad51-E206A , with a similar accumulation of JMs and NC observed in both cases ( Figure 3F ) .", "Taken together , these results indicate that Rad51-E206A retains intrinsic recombinase activity and a functional interaction with Swi5-Sfr1 .", "Our results with Rad51-E206A suggested that the PAP is required specifically for the interaction with Rad55-Rad57 .", "We sought to test whether other mutations in the PAP also affect the interaction with Rad55-Rad57 .", "Strains were constructed in which the rad51+ gene at its native locus was replaced with rad51-E205A , rad51-D209A , or rad51-EED ( E205A , E206A , D209A ) .", "Structural models revealed that the negative surface charge of the PAP is completely neutralized by the EED mutation without affecting its protruding nature ( Figure 4—figure supplement 1A , B ) .", "The DNA damage sensitivity of these mutant strains was assessed by spot-test .", "Like rad51-E206A , the rad51-E205A strain did not show any DNA damage sensitivity in the presence of both Rad55-Rad57 and Swi5-Sfr1 ( Figure 4A ) .", "By contrast , rad51-D209A showed marked sensitivity to DNA damage , although it was still significantly more resistant than rad51∆ .", "Strikingly , the rad51-EED mutant was as sensitive to DNA damage as rad51∆ .", "Since the E205A and E206A mutations alone did not sensitize cells to DNA damage , but combining them with the partially functional D209A completely incapacitated Rad51 , we also examined the rad51-EE strain , in which both Glu residues are mutated to Ala .", "Unlike rad51-E205A and rad51-E206A , rad51-EE showed moderate sensitivity to DNA damage , although this was milder than the sensitivity of rad51-D209A .", "These results indicate that neutralization of the PAP completely abolishes Rad51-dependent DNA repair , and while all three residues are important , D209 plays a more prominent role than E205 and E206 .", "Because our data suggested that the E206A mutation impairs the interaction of Rad51 with Rad55-Rad57 , in vivo co-IP experiments were performed to examine how other mutations in the PAP affect complex formation with Rad55-Rad57 .", "A reduction in the amount of Rad51 that co-IPs with Rad55-12xV5 was observed in the rad51-EE , rad51-D209A , and rad51-EED strains ( Figure 4B ) , and this reduction roughly correlated with the DNA damage sensitivity of the corresponding mutants .", "This result suggests that the DNA damage sensitivity of PAP mutants is partly due to reduced complex formation with Rad55-Rad57 .", "However , given that some complex formation was still observed in the rad51-EED strain , we infer that the PAP is important but not essential for complex formation with Rad55-Rad57 .", "In contrast to what was observed in the presence of Sfr1 , rad51-E205A showed mild DNA damage sensitivity in the sfr1∆ background ( Figure 4C ) and this modest difference became more obvious at higher doses of UV irradiation ( Figure 4—figure supplement 1C ) .", "Both the rad51-EED and rad51-EE mutants showed comparable sensitivity to rad51∆ in this background , as was expected from the phenotype of rad51-E206A .", "Notably , the rad51-D209A strain was also as sensitive to DNA damage as rad51∆ in the absence of Sfr1 .", "These results are consistent with the notion that the PAP is important for DNA repair promoted by Rad55-Rad57 .", "However , because the DNA damage sensitivity of rad51-EED is similar to that of rad51∆ ( Figure 4A ) , which clearly exceeds that of rad57∆ , it seemed likely that the function of the PAP is not restricted to Rad55-Rad57–dependent DNA repair .", "To directly test if the PAP is involved in Rad55-Rad57–independent DNA repair , PAP mutants were introduced into the rad57∆ background .", "The rad51-E205A mutant was as resistant to DNA damage as rad51+ in the absence of Rad57 , suggesting that Rad55-Rad57–independent DNA repair mechanisms are not impaired in this mutant ( Figure 4D ) .", "By contrast , rad51-EE showed a modest increase in sensitivity compared to rad51+ .", "rad51-D209A was even more sensitive than rad51-EE , although this sensitivity was still not as severe as rad51-EED and rad51∆ .", "Consistently , rad51-EED and rad51∆ were the only strains among this set that showed a clear slow-growth phenotype ( note the small colony size on the ‘No treatment’ plate in Figure 4D ) .", "The fact that the D209A and EE mutations further sensitized rad57∆ cells to DNA damage , combined with the severe sensitivity of rad51-EED , suggests that Rad55-Rad57–independent DNA repair is also impaired in these mutants .", "These results imply that the PAP is also relevant to the function of auxiliary factors other than Rad55-Rad57 ( explored below ) .", "If the negativity of the PAP is important for Rad51-dependent DNA repair , we reasoned that a charge-reversal mutation would be more disruptive than the Asp/Glu-to-Ala mutations employed thus far .", "The E205A and E206A mutations did not sensitize cells to DNA damage in the presence of both Rad55-Rad57 and Swi5-Sfr1 ( Figure 4A ) .", "However , because E206 shows a higher degree of conservation than E205 , it was mutated to Lys , yielding the rad51-E206K strain .", "In stark contrast to the rad51-E206A strain , rad51-E206K showed a clear slow-growth phenotype and was as sensitive to DNA damage as rad51∆ in the presence of both Rad55-Rad57 and Swi5-Sfr1 ( Figure 4—figure supplement 1D ) , just like rad51-EED .", "Taken together , these results show that the negative character of the PAP is integral to recombinational DNA repair .", "To elucidate the mechanistic defects in DNA repair associated with neutralization of the PAP , previous immunostaining protocols for the visualization of Rad51 foci were adopted ( Loidl and Lorenz , 2009; Argunhan et al . , 2017b ) .", "Surface-spread nuclei were prepared from log-phase cultures , with or without prior UV irradiation , and immunostained with a polyclonal anti-Rad51 antibody ( Figure 5A ) .", "Nuclei were then picked manually and foci were quantified by an automated approach using FIJI software ( see Materials and methods for more details; Schindelin et al . , 2012 ) .", "Examples of nuclei scored by this method are shown in Figure 5B .", "In the absence of UV irradiation , the vast majority of nuclei in all strains lacked Rad51 foci ( Figure 5—figure supplement 1 ) .", "By contrast , UV-irradiated nuclei contained substantially more Rad51 foci , and this was not seen in the rad51∆ strain , confirming the DNA damage-dependence and specificity of these cytological entities ( Figure 5C ) .", "Nuclei from wild-type and rad52∆ strains formed an average of 8 . 6 and 1 . 7 foci , respectively , in close agreement with a previous report ( Lorenz et al . , 2009 ) .", "As expected , both sfr1∆ and rad57∆ strains formed fewer foci than wild type , and rad57∆ sfr1∆ formed even fewer foci still ( Akamatsu et al . , 2007 ) .", "Strikingly , nuclei from rad51-EED showed a drastic reduction in the number of foci , much like the rad57∆ sfr1∆ double mutant and the rad52∆ single mutant .", "These results indicate that the severe DNA damage sensitivity of rad51-EED stems from defects in the mechanisms promoting the recruitment of Rad51 to sites of DNA damage .", "The DNA damage sensitivity and impaired Rad51 recruitment phenotypes of rad51-EED are clearly more severe than rad57∆ , pointing towards additional roles of the PAP beyond facilitating the interaction with Rad55-Rad57 .", "This could reflect an impairment in the intrinsic activity of Rad51-EED .", "Alternatively , the EED mutation could impair the interaction with Swi5-Sfr1 , since the phenotypes of rad51-EED are comparable to rad57∆ sfr1∆ .", "To test these two possibilities , Rad51-EED was purified to homogeneity ( Figure 6—figure supplement 1A ) .", "Rad51-EED shifted ssDNA and dsDNA comparably to wild-type Rad51 in EMSAs ( Figure 6—figure supplement 1B , C ) , suggesting that the EED mutation does not affect the ability of Rad51 to bind DNA .", "Because reaction products ( NCs ) were not observed in the assay that was previously employed to monitor the intrinsic strand exchange activity of Rad51 ( Figure 3D , E ) , a shorter lds substrate was utilized with the reasoning that this might prove a less challenging substrate ( Figure 6A ) .", "Both Rad51 and Rad51-E206A generated similar amounts of partial duplex molecules ( PD ) , which are the reaction products in this assay ( Figure 6B ) .", "Unexpectedly , Rad51-EED consistently displayed increased strand exchange activity .", "While the reason for this is unclear , these results at least indicate that neutralization of the PAP does not impair the intrinsic activity of Rad51 .", "If the EED mutation impairs the interaction with Swi5-Sfr1 , then the binding of Swi5-Sfr1 to Rad51-EED should be abrogated .", "Co-IP experiments with purified proteins revealed reproducible differences in the amount of Rad51-E206A and Rad51-EED that co-IP’d with Sfr1 , but these differences were relatively subtle ( Figure 6C ) .", "Moreover , in our canonical strand exchange assay ( Figure 3D ) , these differences in physical binding did not affect the stimulation of Rad51 by Swi5-Sfr1 ( Figure 6D ) , suggesting that they are not of functional significance .", "We also examined the interaction of Swi5-Sfr1 with Rad51-E206K .", "Similarly to Rad51-EED , we saw a reproducible reduction in the co-IP of Rad51-E206K with Sfr1 , but this difference is relatively subtle ( less than twofold; Figure 6—figure supplement 1D ) .", "These results indicate that PAP mutations do not impair the potentiation of Rad51 by Swi5-Sfr1 , leading us to conclude that the severe DNA damage sensitivity of the rad51-EED strain is unlikely to be due to a defect in the interaction with Swi5-Sfr1 .", "While a defect in the interaction with Rad54 could explain the severity of DNA damage sensitivity observed for rad51-EED , this is unlikely to be the cause of the sensitivity as rad54∆ is not defective in the recruitment of Rad51 to sites of DNA damage ( Figure 5C ) .", "Thus , the remaining possibility that could account for the phenotypes of rad51-EED is that the EED mutation impairs the interaction of Rad51 with Rad52 .", "To examine whether Rad51-Rad52 complex formation was affected by PAP mutations , in vivo co-IP experiments were performed .", "Comparable amounts of Rad52 were seen to co-IP with Rad51 , Rad51-E206A , and Rad51-EE , whereas a clear reduction was observed for Rad51-D209A ( Figure 7A ) .", "Strikingly , Rad52 was completely undetectable in the Rad51-EED immunoprecipitate , indicating that the EED mutation ablates Rad51-Rad52 complex formation in vivo .", "These results suggest that the exquisite DNA damage sensitivity of rad51-EED is due to defects in the interactions with both Rad55-Rad57 and Rad52 .", "The identity of the band indicated as Rad52 was verified by using a rad52∆ strain ( Figure 7—figure supplement 1A ) .", "To directly test whether the EED mutation disrupts the binding of Rad51 to Rad52 , a co-IP experiment was conducted with purified proteins .", "Comparable amounts of Rad52 co-IP’d with Rad51 and Rad51-E206A ( Figure 7B ) .", "By stark contrast , only background levels of Rad52 were seen to co-IP with Rad51-EED .", "Similar results were obtained in the reciprocal co-IP experiment ( Figure 7—figure supplement 1B ) .", "These results clearly indicate that the PAP is essential for the Rad51-Rad52 interaction .", "Our results demonstrated that the PAP is involved in the interaction of Rad51 with both Rad55-Rad57 and Rad52 .", "We therefore considered the possibility that the PAP is commonly utilized by multiple auxiliary factors and sought to examine the interaction of Rad51 with Rad54 .", "Unlike Rad55-Rad57 and Rad52 , Rad54 functions primarily in the later stages of HR ( Sugawara et al . , 2003; Lisby et al . , 2004 ) .", "Defects in the recruitment of Rad51 to DNA damage sites—as seen in rad51-EED—would block downstream events , obscuring analysis of the Rad51-Rad54 interaction .", "To circumvent this , Rad54 with an N-terminal FLAG tag was purified to near-homogeneity from E . coli ( Figure 7—figure supplement 1C ) and its physical interaction with Rad51-EED was examined in vitro .", "Similar amounts of Rad51 and Rad51-E206A were seen to co-IP with Rad54 , whereas an approximately threefold reduction in the co-IP of Rad51-EED was observed ( Figure 7C ) .", "To examine whether this difference is of functional significance , strand exchange reactions were conducted under conditions where wild-type Rad51 alone did not produce JM or PD molecules ( Rad51-EED nevertheless showed increased intrinsic strand exchange activity [Figure 7D] , as expected from our previous experiments [Figure 6A , B] ) .", "Remarkably , the EED mutation rendered Rad51 less sensitive to the stimulatory effect of Rad54 , with approximately fourfold more Rad54 required to achieve maximal activity .", "These results indicate that , in addition to facilitating the interaction of Rad51 with Rad55-Rad57 and Rad52 , the PAP is also important for the interaction with Rad54 ." ], [ "E205 , E206 , and D209 were found to form the PAP on the exterior of the Rad51-ssDNA filament ( Figure 1A–C ) .", "Individual mutations within the PAP impaired DNA repair to differing extents .", "D209A had the highest penetrance , whereas E206A , and to a much lesser extent E205A , conferred sensitivity only in the sfr1∆ background , where DNA repair is strictly dependent on Rad55-Rad57 ( Figure 4A and C and Figure 4—figure supplement 1C ) .", "These genetic data are consistent with the notion that the PAP is more relevant to the function of Rad55-Rad57 than Swi5-Sfr1 .", "When both Glu residues were mutated to Ala ( rad51-EE ) , moderate DNA damage sensitivity was observed even in the presence of both Rad55-Rad57 and Swi5-Sfr1; this synergism implies that the functions of E205 and E206 are related , and that the ability of Sfr1 to suppress the DNA damage sensitivity conferred by E206A is partially dependent on E205 .", "Moreover , mutating all three residues to Ala ( rad51-EED ) completely abolished Rad51-dependent DNA repair .", "The additive effect of combining mutations suggests that the overall negativity of the PAP is important for its function .", "This is bolstered by the finding that the replacement of E206 with a positively charged Lys residue is highly disruptive , phenocopying the neutralization of all three residues via Ala mutations ( Figure 4—figure supplement 1D ) .", "Thus , while all three residues are relevant to PAP function , there appears to be a hierarchy: D209A is more important than E206 , which itself is more important than E205 .", "This suggests that the closer a residue is to the C-terminal end of the α-helix ( top of the helix in our structural models ) , the more important it is for PAP function ( discussed below ) .", "Negatively charged regions were seen in the equivalent α-helices of both ScRad51 and HsRad51 ( Figure 1—figure supplement 1B , C ) , indicating structural conservation of the PAP .", "Furthermore , sequence alignments suggest that the PAP is widely conserved among eukaryotes , with residues E206 and D209 each showing conservation in seven-out-of-eight eukaryotic Rad51 orthologues ( Figure 7—figure supplement 2A ) .", "Interestingly , examination of a structural model revealed conservation of the PAP in Dmc1 , the meiosis-specific RecA-family recombinase ( Figure 7—figure supplement 2B ) .", "The archaeal RadA polymer also exhibited substantial structural conservation of the PAP ( Figure 7—figure supplement 2C ) .", "By contrast , this region showed poor sequence conservation in bacterial RecA , and although a short α-helix at the equivalent position appeared to exist in the RecA monomer ( Figure 7—figure supplement 3A ) , a loop was seen instead in the RecA-ssDNA filament structure ( Figure 7—figure supplement 3B ) .", "Furthermore , despite this region being negatively charged , this was partially due to the close proximity of residues E32 and D33 from the adjacent RecA monomer .", "We interpret these analyses to mean that the PAP is conserved in eukaryotic Rad51/Dmc1 and archaeal RadA , but not in bacterial RecA .", "While the mediator and ssDNA annealing activities of Rad52 are provided by RecFOR in bacteria , only eukaryotes and archaea also possess recombinase paralogs and Rad54 ( Zelensky et al . , 2014 ) .", "We postulate that the PAP may have evolved in organisms where the RecA-family recombinase is potentiated by multiple distinct auxiliary factors .", "Consistently , it is notable that Dmc1 is subjected to extensive regulation by auxiliary factors: Swi5-Sfr1 , Hop2-Mnd1 , and Rdh54 simulate Dmc1 ( Haruta et al . , 2006; Chi et al . , 2009; Tsubouchi et al . , 2020 ) ; and Rad52 has been proposed to inhibit Dmc1 ( Murayama et al . , 2013 ) .", "The PAP-containing α-helix precedes a β-strand that is critically important for Rad51 polymerization ( Pellegrini et al . , 2002; Shin et al . , 2003; Conway et al . , 2004 ) .", "This β-strand is involved in facilitating interactions with the FxxA consensus sequence within the inter-domain linker of the adjacent monomer ( corresponding to 108FTTA111 in SpRad51 ) .", "The Phe residue of the adjacent monomer inserts into a hydrophobic pocket above the β-strand , which stabilizes inter-subunit contacts .", "Because some auxiliary factors that contain the FxxA motif employ Rad51 mimicry to interact with Rad51 , FxxA has been proposed to function as a Rad51 interaction motif ( Pellegrini et al . , 2002; Shin et al . , 2003 ) .", "Neither Swi5 nor Sfr1 contain the FxxA consensus sequence and we recently identified two non-FxxA sites within the intrinsically disordered N-terminal half of Sfr1 that are responsible for the binding of Swi5-Sfr1 to Rad51 ( Argunhan et al . , 2020 ) .", "By contrast , both Rad57 ( 145FELA148 ) and Rad52 ( 17FNTA20 and 338FISA341 ) contain the FxxA consensus sequence , as does Rad54 ( 804FIRA807 ) .", "Rad55 does not contain an FxxA motif , and notably , Y2H analysis suggested that Rad51 interacts with Rad57 but not Rad55 ( Tsutsui et al . , 2001 ) .", "Furthermore , Y2H analysis mapped the minimal SpRad51-interacting domain of Rad52 to a C-terminal fragment ( 310-469 ) containing the 338FISA341 sequence ( Kim et al . , 2002 ) , and mutation of the corresponding Phe residue to Ala in ScRad52 ( F349A ) completely abrogated its binding to Rad51 ( Kagawa et al . , 2014 ) .", "Taken together , these observations strongly suggest that Rad52 employs the FxxA motif to bind Rad51 .", "Our results demonstrate that neutralization of the PAP impairs the interaction with Rad55-Rad57 , Rad52 , and Rad54 , while the interaction with Swi5-Sfr1 is mostly unaffected ( Figures 4B , 6C , D and 7A–D and Figure 7—figure supplement 1B ) .", "We cannot exclude the possibility that the PAP has some role in facilitating the interaction with Swi5-Sfr1 , but a simple inference that can be drawn from these results is that the PAP is particularly important for auxiliary factors that employ Rad51 mimicry to modulate Rad51 .", "The close proximity of the PAP to the Phe residue within the FxxA motif of the adjacent monomer—Cα-Cα distances for PAP residues and SpRad51-F108 are predicted to be 13 . 0 Å ( D209 ) , 17 . 3 Å ( E206 ) , and 17 . 5 Å ( E205 ) —means it would be well-placed to influence interactions with auxiliary factors that employ Rad51 mimicry ( Figure 7E ) .", "We speculate that this may be the mechanism through which the PAP exerts its effects on Rad51 potentiation .", "Since the negative charge of the PAP is important for recombinational DNA repair ( Figure 4A and Figure 4—figure supplement 1D ) , it is likely that the PAP participates in electrostatic interactions that are important for the modulation of Rad51 by these auxiliary factors , which may utilize the PAP as a landing pad to facilitate FxxA insertion .", "We anticipate that a basic patch may exist that is close in three-dimensional space to the FxxA motif of each auxiliary factor , and this basic patch may be critically important for interacting with the PAP and facilitating the binding to Rad51 .", "Interestingly , Y2H analysis by Kim et al . , 2002 isolated several other mutations in SpRad51 ( G177S , C179F , G282D , and L274P ) that disrupt the interaction with Rad52 , and these residues are also in reasonably close proximity to F108 ( Figure 7—figure supplement 3C ) .", "Notably , the G177S and C179F mutations also impaired the interaction with Rad54 .", "Moreover , the C179F mutation even abrogated the interaction with Rad57 and the self-association of Rad51 .", "Given the close proximity of G177 and C179 to the PAP ( Cα-Cα9–12 Å ) , it is possible that mutation of these residues affects PAP function and/or F108 insertion .", "These Y2H results are consistent with our proposal that multiple auxiliary factors interact with Rad51 via a mechanism involving the PAP .", "PAP mutations impaired complex formation with Rad55-Rad57 in vivo ( Figures 2E , 4B and 7A ) , but even in rad51-EED , complex formation was not completely abolished .", "By contrast , while formation of the Rad51-Rad52 complex was mostly unaffected by mutation of only the Glu residues within the PAP , the D209A mutation led to a marked reduction in complex formation ( Figure 7A ) .", "Consistent with the notion that the PAP as a whole is important , the EED mutation completely abrogated Rad51-Rad52 complex formation .", "Co-IP experiments with purified proteins directly demonstrated that the interaction of Rad51 with both Rad52 and Rad54 is facilitated by the PAP ( Figure 7B–D and Figure 7—figure supplement 1B ) .", "Performing such experiments with Rad55-Rad57 awaits the purification of this biochemically intractable complex .", "If multiple auxiliary factors utilize the same motif to potentiate Rad51 , they would be expected to compete for Rad51 binding .", "However , because auxiliary factors perform non-overlapping roles in HR , the requirement for binding Rad51 is likely to be temporally distinct , with Rad52 being the most upstream binding partner , followed by Rad55-Rad57 , then Rad54 ( Sugawara et al . , 2003; Lisby et al . , 2004 ) .", "Furthermore , the Rad51 nucleoprotein filament contains as many PAP motifs as there are monomers in the filament , providing ample opportunities for auxiliary factors to bind Rad51 in a non-competitive manner .", "Notably , the PAP is important for interactions with auxiliary factors involved in both the early ( Rad52 and Rad55-Rad57 ) and late ( Rad54 ) stages of DNA strand exchange , indicating that the PAP is integral to the role of Rad51 in HR .", "Further research is needed to examine the temporospatial coordination of Rad51 binding , especially in light of the finding that some auxiliary factors such as Rad55-Rad57 and Swi5-Sfr1 interact with each other ( Argunhan et al . , 2020 ) .", "Substantial genetic analysis suggests that the defects of rad51-E206A stem specifically from an impairment in the interaction with Rad55-Rad57 .", "This is supported by biochemical analysis demonstrating that the intrinsic strand exchange activity of Rad51-E206A is comparable to wild type , and its interaction with Swi5-Sfr1 , Rad52 , and Rad54 is not grossly impaired .", "Interestingly , the rad51-E206A mutant was proficient for DNA repair in the presence of Sfr1 , but completely defective in its absence ( Figure 2A–C and Figure 2—figure supplement 1A–C ) .", "This suppression did not occur through enhancing Rad51–Rad55-Rad57 complex formation ( Figure 2E ) , suggesting that Swi5-Sfr1 can functionally compensate for defects in the physical association of Rad51 with Rad55-Rad57 .", "Swi5-Sfr1 may function in a contingency capacity for Rad55-Rad57 such that a moderate reduction in Rad51–Rad55-Rad57 complex formation does not affect DNA repair as long as Swi5-Sfr1 is present .", "Suppression of the DNA damage sensitivity associated with rad51-E206A by Sfr1 contradicts the commonly accepted model wherein Rad55-Rad57 and Swi5-Sfr1 comprise independent sub-pathways of HR ( Akamatsu et al . , 2003; Akamatsu et al . , 2007 ) .", "We recently showed that Rad55-Rad57 can suppress defects in the interaction between Swi5-Sfr1 and Rad51 , and that Swi5-Sfr1 physically interacts with Rad55-Rad57 ( Argunhan et al . , 2020 ) .", "Thus , there is increasing evidence to suggest that Rad55-Rad57 and Swi5-Sfr1 , while capable of functioning independently of each other , collaboratively promote Rad51-dependent DNA repair .", "In summation , we have characterized an acidic patch of Rad51 that comprises an evolutionarily conserved motif important for the interaction of Rad51 with its major auxiliary factors: the Rad51 paralogs Rad55-Rad57 , Rad52 , and Rad54 .", "While this motif is essential for HR in S . pombe , the extent to which it is required in other organisms remains to be determined and will likely be a focal point of future research ." ], [ "Sequence alignments were prepared using Clustal Omega ( UniProt identifiers are: S . pombe , P36601; S . cerevisiae , P25454; U . maydis , Q99133; C . elegans , G5EGG8; D . melanogaster , Q27297; G . gallus , P37383; M . musculus , Q08297; H . sapiens , Q06609 ) .", "All structural depictions were prepared using UCSF Chimera ( Pettersen et al . , 2004 ) .", "The Coulombic Surface Coloring feature in UCSF Chimera was employed to depict surface charge .", "The model of an SpRad51 monomer was generated by Phyre2 ( intensive mode; Kelley et al . , 2015 ) based on two known structures of ScRad51 ( Conway et al . , 2004; Chen et al . , 2010 ) , with Protein databank ( PDB ) identifiers 1SZP and 3LDA; and one known structure of HsRad51 ( Short et al . , 2016 ) with PDB identifier 5JZC .", "46 residues ( 1–41 and 361–365 ) of the SpRad51 monomer were modeled ab initio and these low confidence regions were omitted from the model .", "The SpRad51-ssDNA filament model consisting of three monomers is a homology model that was described previously by Ito et al . , 2020 .", "This structure was previously deposited in the Biological Structure Model Archive ( identification code BSM00017 ) .", "The structure of Rad51-EED was analyzed by substituting E205 , E206 , and D209 to Ala through the Rotamer feature .", "The model of an SpDmc1 monomer was also generated by Phyre2 ( intensive mode; Kelley et al . , 2015 ) based on three known structures of RadA ( Shin et al . , 2003; Wu et al . , 2004; Chen et al . , 2007 ) , with PDB identifiers 1PZN , 1T4G , and 2DFL; two known structures of ScRad51 ( Conway et al . , 2004; Chen et al . , 2010 ) , with PDB identifiers 1SZP and 3LDA; and one known structure of HsRad51 ( Short et al . , 2016 ) with PDB identifier 5JZC .", "Residues 1–14 were modeled ab initio and were therefore omitted from the model .", "The HsRad51-ssDNA filament shown is the structure resolved by cryo-electron microscopy ( Xu et al . , 2017; PDB 5H1B ) , whereas the ScRad51-ssDNA filament shown is the structure solved by X-ray crystallography ( Conway et al . , 2004; PDB 1SZP , chains E and F ) .", "Note that ssDNA density is missing from the structure of the ScRad51-ssDNA filament model , despite the formation of crystals in the presence of ssDNA ( Conway et al . , 2004 ) .", "The structure of the Pyrococcus furiosus RadA polymer was solved by X-ray crystallography ( Shin et al . , 2003; PDB 1PZN , chains G , A , and B ) .", "The RecA monomer ( Story et al . , 1992; PDB 2REB ) and ssDNA filament ( Chen et al . , 2008; 3CMU ) structures were solved by X-ray crystallography .", "S . pombe strains used in this study are listed in the Key Resources Table .", "All strains are isogenic derivatives of strain YA119 ( Akamatsu et al . , 2003 ) .", "For most assays , strains of the h minus mating type were employed ( Msmt-0 ) , except in Figure 1D ( mat1PD17::LEU2 ) .", "Standard media was used for growth ( YES ) , selection ( YES with drugs or EMM ) , and sporulation ( SPA ) , as described previously ( Hentges et al . , 2005 ) .", "All reasonable requests for strains will be fulfilled by the co-corresponding authors ( B . Argunhan and H . Iwasaki ) .", "Primers used in this study are listed in the Key Resources Table .", "In order to introduce the rad51-E206A mutation at the native rad51+ locus , plasmid p6 ( pET11b-rad51+; Haruta et al . , 2006 ) was amplified with mutagenic primers 398–400 and 399–405 to produce amplicons 1 and 2 , respectively .", "Amplicon 3 , containing the ADH1 terminator ( TADH1 ) and kanMX6 cassette , was amplified from the C-terminal epitope tagging plasmid p51 ( pFA6a-13xMYC-kanMX6 ) using primers 350–351 .", "Regions upstream and downstream of the rad51+ locus were amplified with primers 409–412 and 417–419 to yield amplicons 4 and 5 , respectively .", "All five amplicons were cloned onto the pBlueScript II SK ( + ) vector using the In-Fusion Cloning Kit ( Takara ) to generate plasmid pNA46 .", "pNA46 was then cut with restriction enzymes MscI and XhoI ( NEB ) and the fragment containing rad51-E206A-TADH1-kanMX6 was gel extracted and transformed into strain NA310 ( wild type ) .", "rad51-E205A , rad51-EE , rad51-D209A , rad51-EED , and rad51-E206K were made in exactly the same way using plasmids pBA144 , pBA146 , pBA145 , pBA148 , and pBA152 , respectively .", "To generate the rad55-12xV5 strain , plasmid p85 ( pNX3c-PK12; Amelina et al . , 2016 ) was PCR-amplified with primers 200–201 and the wild-type strain ( NA310 ) was transformed with this amplicon .", "All cloning and genetic manipulations were confirmed by DNA sequencing .", "Sequence alignments were prepared with Clustal Omega .", "A single colony was streaked as a patch onto a YES plate and grown at 30°C for 1–2 days .", "This patch was then resuspended in 2 mL of YES and grown with shaking at 30°C for ~24 hr .", "Cells were seeded into 2 mL of fresh YES ( 0 . 25 × 106 cells/mL for rad+; 0 . 5 × 106 cells/mL for rad- ) and grown with shaking at 30°C until they reached log phase .", "For spot tests , cell density was adjusted to 2 × 107 cells/mL and tenfold serial dilutions were made .", "5 µL of each dilution was spotted onto YES plates without drugs and YES plates containing DNA damaging agents .", "For UV treatment , a YES plate without drugs was UV-irradiated .", "Cells were incubated at 30°C or 21°C , as indicated .", "For clonogenic survival assays , cells were prepared as described above and spread onto YES plates , which were then exposed to acute UV irradiation of the specified dose .", "Colonies were counted after 3–4 days of incubation at 30°C and survival percentage was expressed relative to the number of colonies on the untreated plate .", "Statistical analysis was by unpaired two-tailed t-test using GraphPad Prism ( version 8 ) .", "A single colony was streaked as a patch on a YES plate and grown for 1–2 days at 30°C .", "This patch was resuspended in 2 mL of YES and grown with shaking at 30°C for 24 hr .", "Cells were seeded into 70 mL of fresh YES ( 0 . 15 × 106 cells/mL for rad+; 0 . 30 × 106 cells/mL for rad- ) and grown in 500 mL baffled flasks with shaking at 30°C until they reached log phase .", "Cells were harvested and cell pellets were resuspended in 35 mL of sterile water and divided into two equal aliquots ( ±UV ) .", "+UV aliquots were transferred into a petri dish at a depth of ~0 . 5 cm and exposed to UV ( 200 J/m2 ) , while -UV aliquots were mock treated .", "Each aliquot was then resuspended in 35 mL fresh YES and allowed to recover at 30°C for 3 hr with shaking .", "Cells were then harvested and treated as previously described ( Argunhan et al . , 2020 ) .", "Briefly , 1 × 108 cells were resuspended in 1 mL of ice-cold water and mixed with 150 µL of 1 . 85 M NaOH 7 . 5% β-mercaptoethanol on ice for 15 min . 150 µL of 55% TCA was then added , followed by mixing and a further 10 min incubation on ice .", "Precipitated proteins were pelleted by sequential centrifugation ( 20000 g , 10 min , 2°C; then 20000 g , 1 min , 2°C ) .", "Pellets were resuspended in 100 µL of urea buffer ( 8 M urea , 5% SDS , 200 mM Tris-Cl pH 6 . 8 , 1 mM EDTA , 0 . 01% BPB ) freshly supplemented with 0 . 1 M DTT and 0 . 2 M Tris and dissolved on a thermomixer ( 65°C 10 min 1300 RPM ) .", "Proteins were then separated by SDS-PAGE and transferred to PVDF membranes .", "Antibodies against Rad51 ( 1:10 , 000; Argunhan et al . , 2020 ) and Tubulin ( 1:10 , 000; Sigma T5168 ) were employed .", "Horseradish peroxidase ( HRP ) -conjugated secondary antibodies were purchased from GE Healthcare ( mouse , 1:5 , 000 , NA931; rabbit , 1:5 , 000 , NA934 ) or Jackson Immunoresearch Labs ( rat , 1:10000 , 712-035-153 ) .", "A single colony was resuspended in 20 mL YES and grown with shaking at 30°C ( 24 hr for rad+; 48 hr for rad- ) .", "Cells were seeded into 1 L of fresh YES ( 0 . 2 × 106 cells/mL for rad+; 0 . 4 × 106 cells/mL for rad- ) in 5 L baffled flasks and grown with shaking at 30°C until they reached log phase .", "Cultures were then supplemented with 0 . 5 mM PMSF and harvested by centrifugation ( 6000 g , 15 min , 2°C ) , and pelleted cells were washed with 20 mL of yeast wash solution ( 50 mM HEPES [pH 7 . 5] , 70 mM KOAc , 1 mM PMSF ) .", "Aliquots of 2 × 109 cells were pelleted , frozen in liquid nitrogen , and stored at −80°C until use .", "IP experiments were then carried out as previously described ( Argunhan et al . , 2017b ) .", "Briefly , a single cell pellet was resuspended in 400 µL of KA50 buffer ( 50 mM HEPES-KOH [pH 7 . 5] , 50 mM KOAc , 5 mM MgOAc , 0 . 05% Igepal CA-630 , 10% glycerol , 0 . 25 mM TCEP , 0 . 5 mM PMSF , 2x cOmplete protease inhibitor cocktail [Roche] , 10 mM β-glycerophosphate , 10 mM NaF , 1 mM sodium orthovanadate ) and mixed with an equal volume of glass beads ( 500 micron ) on ice .", "Cells were then broken using a Yasui Kikai Multi-beads Shocker ( 2700 RPM , 30 s on/off , 12 cycles , 2°C ) .", "The lysate was recovered , and the glass beads were washed with 200 µL of KA50 buffer and combined with the lysate .", "The lysate was then treated with 250 units of TurboNuclease ( Accelagen ) for 30 min at 4°C with mixing .", "The lysate was then sequentially cleared ( 20 , 000 g , 10 min , 2°C; then 20 , 000 g , 5 min , 2°C ) , a sample was taken for immunoblotting ( input ) and mixed with an equal volume of 2x SDS loading buffer ( 120 mM Tris-HCl [pH 6 . 8] , 4% SDS , 20% glycerol , 0 . 02% BPB , 200 mM DTT ) , and the remaining soluble cell extract was divided into three equal aliquots in protein Lo-Bind tubes ( Eppendorf ) .", "Anti-V5 ( mouse; MCA1360 Bio-Rad ) , anti-Rad51 ( rabbit; Haruta et al . , 2006 ) , or ChromPure Human IgG ( mock antibody; 009-000-003 Jackson Immunoresearch Laboratories ) antibodies were premixed with Dynabeads Protein A ( Thermo Fisher Scientific ) and incubated with each aliquot of soluble cell extract with gentle mixing ( 3 hr , 4°C ) .", "Aqueous fractions were separated using a magnetic stand and the beads were briefly washed with buffer KA50 ( 300 µL , x3 ) .", "The beads were then resuspended in 75 µL of 1x SDS loading buffer and proteins were eluted using a thermomixer ( IP; 65°C 10 min 1300 RPM ) .", "Samples were separated by SDS-PAGE , transferred to PVDF membranes and detected with the following antibodies: anti-V5 , mouse ( 1:10 , 000; Bio-Rad ) ; anti-Rad51 , rat ( 1:10 , 000; Argunhan et al . , 2020 ) ; anti-Rad52 , rabbit ( 1:5 , 000; Kurokawa et al . , 2008 ) ; and anti-Tubulin , mouse ( 1:10 , 000 , T5168 Sigma-Aldrich ) .", "HRP-conjugated secondary antibodies were purchased from GE Healthcare ( mouse , 1:5000 , NA931; rabbit , 1:5 , 000 , NA934 ) or Jackson Immunoresearch Labs ( rat , 1:10 , 000 , 712-035-153 ) .", "FIJI software ( Schindelin et al . , 2012 ) was used for quantification of in vivo IPs .", "Briefly , background subtraction was performed by the rolling ball method and the signal for IP’d and co-IP’d protein was quantified .", "The co-IP’d signal was then divided by the IP’d signal and expressed relative to wild type .", "Graphs were prepared using GraphPad Prism ( version 8 ) .", "E . coli BL21 ( DE3 ) RIPL strain was used for purification of S . pombe Rad51 , RPA , Swi5-Sfr1 , and Rad52 .", "Rad51 ( plasmid p6; pET11b-rad51+ ) , Rad51-E206A ( plasmid pNA42; pET11b-rad51-E206A ) , and Rad51-EED ( plasmid pBA161; pET11b-rad51-E205A-E206A-D209A ) were expressed with 1 mM IPTG at 18°C for ~14 hr and purified exactly as previously described ( Kurokawa et al . , 2008 ) .", "Rad51 mutants behaved similarly to wild-type Rad51 throughout the purification process .", "S . pombe RPA was expressed from plasmid p69 ( pET11b-ssb2-ssb3-ssb1 ) and Swi5-Sfr1 was expressed from plasmid p71 ( pBKN220-sfr1-swi5 ) as for Rad51 , and purified exactly as previously described ( Haruta et al . , 2006; Kurokawa et al . , 2008 ) .", "Rad52 was expressed from plasmid p76 ( pET11b-rad52+ ) with 1 mM IPTG at 30°C for 3 hr and purified exactly as previously described ( Kurokawa et al . , 2008 ) .", "S . pombe Rad54 fused to an N-terminal tag—containing a hexahistidine tag , the Fh8 fusion partner ( Costa et al . , 2013 ) , and a FLAG epitope , with a PreScission protease recognition site between the Fh8 and FLAG components ( pBA110; pET15b-6xHis-Fh8-PreScission-1xFLAG-rad54+ ) —was expressed in 20 L of E . coli strain BL21 ( DE3 ) Star at an OD of ~0 . 45 with 1 mM IPTG at 18°C for ~14 hr .", "Cells were harvested by centrifugation , washed with E . coli wash solution ( 50 mM Tris-Cl [pH 7 . 5] , 150 mM NaCl , 1 mM PMSF ) , and stored at −80°C until required .", "Cell pellets ( ~53 g ) were then resuspended in 250 mL of R buffer ( 20 mM Tris-Cl [pH 7 . 5] , 10% glycerol , 1 mM EDTA ) containing 500 mM NaCl , 5 mM MgOAc , 0 . 5 mM ATP , 2 mM imidazole , 1 mM DTT , 0 . 5 mM PMSF , and 0 . 01% igepal CA-630 .", "Cells were then disrupted by sonication and the lysate was cleared by ultracentrifugation ( 70 , 000 g 1 hr 2°C ) .", "The clarified lysate was incubated with 10 mL of cOmplete His-Tag Purification Resin ( Roche ) with gentle mixing ( 1 hr 4°C ) .", "The resin was poured into a glass column ( Bio-Rad Econo-column , 2 . 5 cm x 10 cm ) and washed sequentially with the same buffer used for lysis ( 50 mL x4 ) and R buffer containing 500 mM NaCl , 5 mM imidazole , 1 mM DTT , and 0 . 5 mM PMSF ( 50 mL x8 ) .", "Proteins were then eluted in R buffer containing 300 mM NaCl , 300 mM imidazole , and 0 . 5 mM TCEP ( 2 mL x4 ) .", "Eluates were combined , supplemented with PreScission protease ( GE Healthcare ) , and dialyzed against 1 L of the same buffer without imidazole ( overnight 4°C ) .", "The protein sample was diluted in 2x volumes of R buffer containing 0 . 5 mM TCEP and applied to a 1 mL HiTrap Q column equilibrated with R buffer containing 100 mM NaCl and 0 . 5 mM TCEP .", "Rad54 , which was found in the flow-through , was then applied to a 1 mL HiTrap Heparin column equilibrated with R buffer containing 100 mM NaCl and 0 . 5 mM TCEP .", "Proteins were then eluted with a linear gradient ( 20 mL 0 . 1–0 . 6 M NaCl ) .", "Peak fractions containing Rad54 were combined , diluted with 9x volumes of R buffer containing 0 . 5 mM TCEP , then applied to a 1 mL Resource S column equilibrated with R buffer containing 50 mM NaCl and 0 . 5 mM TCEP .", "Proteins were eluted with a linear gradient ( 20 mL 0 . 05–0 . 7M NaCl ) , then peak fractions were pooled and developed in a 16/60 Superdex 200 PG gel filtration column in R buffer containing 300 mM NaCl and 0 . 5 mM TCEP .", "Peak fractions were pooled , diluted with 2x volumes of R buffer containing 0 . 5 mM TCEP , and applied to a 1 mL Resource S column .", "Proteins were eluted with a linear gradient ( 40 mL 0 . 1–0 . 5 M NaCl ) .", "Rad54 eluted at ~220 mM NaCl and was subsequently concentrated using a Vivaspin six centrifugal concentrator ( MWCO 30 kDa ) .", "The concentration was estimated by measuring the A280 with a molar extinction coefficient of 72530 M−1 cm−1 .", "The concentrated protein was frozen in small aliquots using liquid nitrogen and stored at −80°C until required .", "The yield of highly purified Rad54 was ~0 . 5 mg .", "Chromatography columns were purchased from GE Healthcare .", "Rad51 ( wild type , Rad51-E206A , or Rad51-EED ) was incubated with 30 micromolar nucleotide ( µM nt ) of PhiX174 virion DNA or 20 µM nt of ApaLI-linearized PhiX RFI DNA in EMSA buffer ( 30 mM HEPES-KOH [pH 7 . 5] , 150 mM KCl , 3 mM MgCl2 , 2 mM ATP , 1 mM DTT , 5% glycerol ) .", "The 10 µL reaction was incubated at 37°C for 15 min . 1 . 1 µL of 2% glutaraldehyde was then added and incubation was continued for a further 5 min at 37°C .", "2 . 5 µL of loading dye was added and 3 µL of the reaction was loaded onto a 0 . 8% agarose gel in TAE buffer ( 50 V 120 min ) .", "The gel was then stained with SYBR Gold ( Thermo Fisher Scientific ) and imaged using a LAS4000 mini ( GE Healthcare ) .", "The ATPase assay was conducted exactly as previously described ( Argunhan et al . , 2020 ) .", "Briefly , 5 µM of Rad51 or Rad51-E206A was mixed with 10 µM nt of PhiX174 virion DNA on ice in ATPase buffer ( 30 mM Tris-Cl [pH 7 . 5] , 100 mM KCl , 3 . 5 mM MgCl2 , 1 mM DTT , 5% glycerol ) , and either 0 . 5 µM Swi5-Sfr1 or the equivalent volume of protein storage buffer was added .", "Reactions were then initiated through the addition of 0 . 5 mM ATP and 10 µL aliquots were withdrawn at multiple timepoints spanning the initial reaction rate , immediately mixed with 2 µL of 120 mM EDTA and stored at room temperature .", "The concentration of inorganic phosphate was then measured with a commercial malachite green phosphate detection kit ( BioAssay Systems ) according to the manufacturer’s instructions .", "To examine the intrinsic strand exchange activity of Rad51 , 15 µM of Rad51 ( wild type , Rad51-E206A , or Rad51-EED ) was incubated with 30 µM nt PhiX174 virion DNA ( NEB ) for 5 min at 37°C in strand exchange buffer ( 30 mM Tris-HCl [pH7 . 5] , 1 mM DTT , 150 mM KCl , 3 . 5 mM MgCl2 , 2 mM ATP , 8 mM phosphocreatine , 8 units/mL creatine phosphokinase and 2 . 5% glycerol ) .", "Next , 1 µM of RPA was added to the reaction and after a 5 min incubation , the reaction was initiated with 20 µM nt of PhiX RFI DNA ( NEB ) linearized with ApaLI .", "In the case of the shortened lds substrate , a 1 . 6 kb dsDNA fragment was employed instead , and this was prepared by digesting PhiX RFI DNA with both MfeI and XhoI .", "10 µL was collected immediately after the addition of dsDNA ( 0 min ) and the remaining reaction was then incubated at 37°C .", "10 µL aliquots were subsequently withdrawn at the indicated timepoints ( 15 , 30 , 60 , and 120 min ) .", "At each timepoint , samples were supplemented with 1 µL of psoralen ( 200 µg/mL ) and subjected to psoralen-UV crosslinking to capture labile DNA structures .", "Reactions were then deproteinized through addition of 1 . 8 µL of stop solution ( 6 . 6 mg/mL proteinase K and 2 . 65% SDS ) and incubation at 37°C for 30 min . 2 . 5 µL of loading dye ( 15% w/v Ficoll , 0 . 25% Bromophenol blue , 0 . 25% Xylene cyanol and 20 mM Tris [pH 7 . 5] ) was added to the reactions , and following mixing , 4 µL of the reaction was loaded and resolved in a 0 . 8% agarose gel in TAE buffer ( 50 V 120 min ) .", "The gel was then stained with SYBR Gold ( Thermo Fisher Scientific ) and imaged using a LAS4000 mini ( GE Healthcare ) .", "In strand exchange assays containing Swi5-Sfr1 , 5 µM of Rad51 ( wild type , Rad51-E206A , or Rad51-EED ) was incubated with 10 µM nt PhiX174 virion DNA ( NEB ) for 5 min at 37°C in strand exchange buffer ( same as above except the total salt concentration was 100 mM KCl ) .", "Next , 0 . 5 µM of Swi5-Sfr1 ( or the indicated concentration ) was added and following a 5 min incubation at 37°C , 1 µM of RPA was included .", "After a further 5 min at 37°C , the reaction was initiated with 10 µM nt of PhiX RFI DNA ( NEB ) linearized with ApaLI and incubated for 120 min at 37°C .", "Subsequent procedures were carried out as described above .", "In strand exchange assays containing Rad54 , 7 µM of Rad51 ( wild type , Rad51-E206A , or Rad51-EED ) was incubated with 7 µM nt PhiX174 virion DNA ( NEB ) for 8 min at 37°C in strand exchange buffer ( same as above except the total salt concentration was 100 mM KCl ) .", "Next , 0 . 7 µM RPA was added and incubation was continued at 37°C for 8 min .", "The reaction was then initiated with 14 µM nt of the shortened lds ( described above ) and immediately supplemented with the indicated concentration of Rad54 .", "Incubation was continued for 120 min at 37°C .", "Subsequent procedures were carried out as described above .", "Quantification was performed as previously described ( Haruta et al . , 2006 ) except that FIJI software was employed ( Schindelin et al . , 2012 ) .", "Briefly , background was subtracted using the rolling ball method , the signal corresponding to lds , NC/PD , and ( JM/1 . 5 ) in a given lane was summed and set equal to 100% , and the signal for NC/PD and/or JM was expressed as a percentage of this .", "Strains were cultured and treated ( ±UV ) exactly as described in Extraction and detection of cellular proteins , and nuclear spreads were prepared as described ( Loidl and Lorenz , 2009; Argunhan et al . , 2017b ) with minor modifications .", "10 mL of cell culture ( ~1×108 cells ) was harvested by centrifugation ( 2300 g 2 min ) , washed with 1 mL of 1 M sorbitol , and then resuspended in 1 mL of lysing solution ( 1 M sorbitol , 1 mM DTT , 0 . 2 mg/mL zymolyase , 12 mg/mL lysing enzyme [Sigma-Aldrich L1412] ) and incubated at 30°C for 30 min with gentle mixing .", "Spheroplasts were washed with 1 mL of 1 M sorbitol-1xMES ( 100 mM 2-[N-morpholino] ethanesulfonic acid , 1 mM EDTA , 0 . 5 mM MgCl2 ) , then resuspended with 100 µL of 1x MES premixed with 350 µL of 3 . 3% of paraformaldehyde and immediately spread onto glass slides .", "Once the slides had half-dried ( ~10 min ) , they were washed with Photo-Flo 200 Solution ( 1 mL x2; 146 4510 Kodak ) and left to fully air-dry in the dark ( ~1 hr ) before storing at −20°C .", "For immunostaining , the slides were first washed in a Coplin jar with PBS and blocked using PBS-5% BSA solution for 30 min at room temperature in a moisture chamber .", "100 µL of PBS-5% BSA supplemented with affinity purified anti-Rad51 antibody ( 1:300 , provided by Hiroshi Iwasaki ) was spread onto the slides with a cover slip and incubated at 4°C overnight in a moisture chamber .", "Slides were washed with PBS ( 5 min x3 ) and subsequent steps were carried out with minimal exposure to ambient light .", "100 µL of PBS-5% BSA supplemented with anti-rabbit Alexa fluor 488 antibody ( 1:300; A11034 Thermo Fisher Scientific ) was spread onto the slide with a cover slip , followed by a 3 hr incubation in a moisture chamber at room temperature .", "Slides were then washed with PBS ( 5 min x3 ) and left to air-dry ( ~30 min ) .", "Mounting media ( 90% glycerol , 0 . 1xPBS , 1 mg/mL p-Phenylenediamine [Sigma P6001] , 1 µg/mL 4’ , 6-diamidino-2-phenylindole [DAPI; Sigma D9542] ) was added in a dropwise fashion onto each slide , spread with a coverslip , and then sealed .", "Images of nuclei were captured using a wide field fluorescent microscope ( Nikon Eclipse 80i ) with a 100x objective fitted with an sCMOS camera ( C13440 Hamamatsu ) .", "The number of foci for each spread nucleus was quantified using FIJI ( Schindelin et al . , 2012 ) according to the following procedure .", "Following the application of a Gaussian blur ( sigma = 2 ) to the Rad51 ( green ) channel , the DAPI ( blue ) and Rad51 channels were subjected to Auto Thresholding ( MaxEntropy ) and Auto Local Thresholding ( Bernsen , radius = 13 ) , respectively .", "The Rad51 channel was then subjected to the Watershed and Ultimate Points processes , followed by the Find Maxima function .", "The DAPI channel was subjected to the Analyze Particles process ( ≥200 pixels2 ) to add the areas of nuclei to the Region-Of-Interest ( ROI ) Manager .", "These ROIs were then overlayed onto the Rad51 channel and the measure function of the ROI Manager was employed .", "The resulting RawIntDen value for each nucleus was then divided by 255 to yield the number of foci .", "A graph portraying the results was prepared using GraphPad Prism ( version 8 ) , which was also used for statistical analysis ( Wilcoxon ranked sum test ) .", "Purified proteins ( 250 nM each ) were mixed together on ice in IP buffer ( 30 mM Tris-Cl [pH 7 . 5] , 150 mM NaCl , 3 . 5 mM MgCl2 , 0 . 1% igepal CA-630 , 0 . 25 mM TCEP , 1 mM ATP , 5% glycerol; input sample ) and incubated at 30°C for 15 min .", "Reactions were then incubated on ice for 5 min and supplemented with either anti-FLAG M2 affinity agarose gel ( Sigma-Aldrich ) for IP of Rad54 or Dynabeads Protein A ( ThermoFisher Scientific ) preincubated with anti-Sfr1 , anti-Rad51 , or anti-Rad52 antibodies ( Haruta et al . , 2006; Kurokawa et al . , 2008 ) .", "Following mixing at 4°C for 1 . 5 hr , the beads were washed with IP buffer ( 400 µL x1 ) and eluted in 1x SDS loading buffer ( IP sample ) .", "Proteins were then separated by SDS-PAGE and detected by immunoblotting with anti-Rad51 ( rat 1:10 , 000; Argunhan et al . , 2020 ) , anti-Sfr1 ( mouse 1:200; Argunhan et al . , 2020 ) , anti-FLAG ( mouse 1:10 , 000; Sigma-Aldrich F3165 ) , or anti-Rad52 ( rabbit 1:10 , 000; Kurokawa et al . , 2008 ) antibodies .", "FIJI software ( Schindelin et al . , 2012 ) was used for quantification of in vitro IPs .", "Briefly , background subtraction was performed by the rolling ball method and the signal for IP’d and co-IP’d protein was quantified .", "The co-IP’d signal was then divided by the IP’d signal and expressed relative to wild type .", "Graphs were prepared using GraphPad Prism ( version 8 ) ." ] ]
[ "Homologous recombination ( HR ) is essential for maintaining genome stability .", "Although Rad51 is the key protein that drives HR , multiple auxiliary factors interact with Rad51 to potentiate its activity .", "Here , we present an interdisciplinary characterization of the interactions between Rad51 and these factors .", "Through structural analysis , we identified an evolutionarily conserved acidic patch of Rad51 .", "The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites .", "This acidic patch was found to be important for the interaction with Rad55-Rad57 and essential for the interaction with Rad52 .", "Furthermore , biochemical reconstitutions demonstrated that neutralization of this acidic patch also impaired the interaction with Rad54 , indicating that a single motif is important for the interaction with multiple auxiliary factors .", "We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair ." ]
[ "The DNA molecule contains the chemical instructions necessary for life .", "Its physical integrity is therefore vital , yet it is also under constant threat from external and internal factors .", "As a result , organisms have evolved an arsenal of mechanisms to repair damaged DNA .", "For instance , when the two complementary strands that form the DNA molecule are broken at the same location , the cell triggers a mechanism known as homologous recombination .", "A protein known as Rad51 orchestrates this process , helped by an array of other proteins that include Rad55-Rad57 , Rad52 , and Rad54 .", "These physically bind to Rad51 and activate it in different ways .", "However , exactly how these interactions take place remained unclear .", "To find out more , Afshar et al . examined models of the structure of Rad51 , revealing that three of the protein’s building blocks create a prominent , negatively charged patch that could be important for DNA repair .", "Yeast cells were then genetically manipulated to produce a modified version of Rad51 in which the three building blocks were neutralised .", "These organisms were unable to repair their DNA .", "Further biochemical tests showed that the modified protein could no longer attach well to Rad55-Rad57 or Rad54 , and could not stick to Rad52 at all .", "In fact , without its negatively charged patch , Rad51 could not find the ends of broken DNA strands , a process which is normally aided by Rad55-Rad57 and Rad52 .", "Taken together , these results suggest that the helper proteins all interact with Rad51 in the same place , even though they play different roles .", "Faulty DNA repair processes have been linked to devastating consequences such as cell death or cancer .", "Understanding the details of DNA repair in yeast can serve as a template for research in more complex organisms , opening the possibility of applications for human health ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "neuroscience" ]
Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis
elife-30203-v2
[ [ "Signal transduction between cells depends on the regulated exocytosis of vesicles to liberate neurotransmitters , neuropeptides and hormones .", "Neuronal exocytosis is driven by an evolutionarily conserved machinery that targets vesicles to the plasma membrane , attaches them to the membrane ( sometimes referred to as vesicle ‘docking’ ) , and molecularly matures ( ‘primes’ ) them to a fusion-competent state .", "Primed vesicles reside in the so-called Readily Releasable Pool ( RRP ) whose fusion triggering leads to transmitter/hormone release .", "The assembly of the thermodynamically stable neuronal SNARE complex formed by the vesicular SNARE protein VAMP2/synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1 and SNAP-25 provides energy needed for vesicle fusion ( Jahn and Fasshauer , 2012 ) .", "Indeed , SNARE proteins are already required for membrane attachment and priming ( de Wit et al . , 2009; Imig et al . , 2014; Walter et al . , 2010 ) , and during fusion they continue to influence fusion pore properties through their transmembrane domains ( Chang et al . , 2015; Dhara et al . , 2016; Fang and Lindau , 2014 ) .", "The protein families Unc18 ( sec-1 ) and Unc13 interact with the neuronal SNAREs and are required for membrane attachment , priming and fusion ( Rizo and Südhof , 2012 ) .", "The Ca2+-dependent activator protein for secretion ( CAPS ) is another priming factor in PC12-cells , chromaffin cells and neurons ( Jockusch et al . , 2007; Liu et al . , 2008; Grishanin et al . , 2004 ) , which interacts with SNAREs to stimulate their assembly ( James et al . , 2009 ) .", "A function of CAPS in membrane attachment of vesicles was also described in synapses of C . elegans , hippocampal neurons and PC12 cells ( Imig et al . , 2014; Kabachinski et al . , 2016; Zhou et al . , 2007 ) , but not in mouse chromaffin cells ( Liu et al . , 2008 ) .", "Vesicle fusion is temporally linked to electrical stimulation of the cell by voltage gated Ca2+ channels that activate upon depolarization .", "In mouse chromaffin cells , the resulting increase in intracellular Ca2+ concentration is sensed by the vesicular Ca2+ binding proteins synaptotagmin-1 , and −7 ( Schonn et al . , 2008 ) , which interact with the SNAREs ( Zhou et al . , 2015; Schupp et al . , 2016 ) , and trigger vesicle fusion ( Südhof , 2013 ) .", "The lipid bilayer of the plasma membrane – apart from taking part in the vesicle to plasma membrane merger - contains a variety of signaling lipids that can regulate exocytosis , most notably phosphatidylinositols ( PIs ) and diacylglycerols ( DAGs ) .", "Phosphatidylinositols constitute a family of lipids , which can be phosphorylated on one or more positions of the inositol headgroup , giving rise to specific signals on cell membrane compartments ( Di Paolo and De Camilli , 2006; Balla , 2013 ) .", "PI ( 4 , 5 ) P2 is the major phosphatidylinositol in the inner leaflet of the plasma membrane where it plays multiple essential roles in cell motility , actin cytoskeleton organization , ion channel activity , and vesicle exocytosis ( Di Paolo and De Camilli , 2006; Martin , 2012 ) .", "Signaling lipids are recognized by specific protein motifs , particularly C1 , C2 and pleckstrin homology ( PH ) domains ( Martin , 2015 ) .", "One of the best characterized signaling lipid interactions with exocytosis proteins is that of the synaptotagmin-1 ( syt-1 ) C2B-domain with PI ( 4 , 5 ) P2 ( Schiavo et al . , 1996; Honigmann et al . , 2013 ) .", "The syt-1 C2B domain binds Ca2+ , and is essential for triggering of vesicle fusion ( Mackler et al . , 2002 ) .", "In vitro , PI ( 4 , 5 ) P2 binding to the C2B domain markedly increases the affinity for Ca2+ ( van den Bogaart et al . , 2012; Li et al . , 2006 ) .", "Thus , interactions of syt-1 with synaptic PI ( 4 , 5 ) P2 ensure that the C2B domain interacts with the plasma membrane ( Bai et al . , 2004 ) and aid the triggering of vesicle fusion by bringing its Ca2+-affinity into the physiological range .", "All known Munc13 isoforms contain C1- and C2 domains that regulate exocytosis .", "The DAG analog phorbolester binds to the Munc13 C1 domain to strongly enhance exocytosis ( Rhee et al . , 2002; Basu et al . , 2007 ) .", "Membrane binding of Munc13 can be further augmented by PI ( 4 , 5 ) P2 binding to the neighboring C2B domain , which also influences the fusion probability of synaptic vesicles ( Shin et al . , 2010 ) .", "CAPS contains a PH domain which binds PI ( 4 , 5 ) P2 and is essential for vesicle priming ( Nguyen Truong et al . , 2014; Loyet et al . , 1998; Kabachinski et al . , 2014 ) .", "Because different species of signaling lipids may regulate different essential exocytosis proteins , systematic investigation of the relevant interactions for exocytosis is needed .", "The most successful approaches to tease apart molecular components relevant for neurosecretion have been to mutate proteins of the release machinery or to regulate their expression .", "This is not directly possible for signaling lipids; instead , enzymes of lipid metabolism have been targeted .", "Early experiments in permeabilized bovine adrenal chromaffin cells using bacterial phospholipase C ( PLC ) showed that secretion depends on PI ( 4 , 5 ) P2 at an upstream ATP-dependent priming step ( Eberhard et al . , 1990 ) .", "Exocytosis was further found to require the PI ( 4 , 5 ) P2 synthesizing enzyme phosphatidylinositol-4-phosphate 5-kinase ( Hay et al . , 1995; Gong et al . , 2005 ) .", "Moreover , experiments using fast capacitance measurements showed that overexpression of PI ( 4 , 5 ) P2 generating or degrading enzymes increased or decreased the RRP , respectively ( Gong et al . , 2005; Milosevic et al . , 2005 ) .", "Even though the initial experiments indicated that PLC , which produces DAG at the expense of PI ( 4 , 5 ) P2 , inhibits rather than stimulates secretion ( Eberhard et al . , 1990; Hay et al . , 1995 ) , later experiments showed that phorbolesters – assumed to mimic DAG – strongly stimulate secretion when added to naïve cells ( Smith et al . , 1998 ) .", "The phorbol ester effect has since been studied in a number of cell types , and has been found to rely on the activation of two priming factors Munc13 ( via its C1 domain ) ( Rhee et al . , 2002; Betz et al . , 1998 ) and Munc18-1 ( via protein kinase C phosphorylation ) ( Wierda et al . , 2007; Genc et al . , 2014; Nili et al . , 2006 ) .", "In the presence of PLC activity , experiments to increase or decrease the levels of PI ( 4 , 5 ) P2 might cause correlative changes in DAG .", "The same concern exists for the conversion of PI ( 4 , 5 ) P2 to PI ( 3 , 4 , 5 ) P3 , which might have profound effects on exocytosis in spite of being present in low abundance ( Khuong et al . , 2013 ) .", "Even the fastest existing techniques to manipulate PI ( 4 , 5 ) P2 levels ( by voltage , light , or chemical dimerization ) ( Murata et al . , 2005; Suh et al . , 2010; Idevall-Hagren et al . , 2012 ) operate on similar speeds as PI ( 4 , 5 ) P2 metabolism and vesicle priming ( i . e . tens of seconds ) ( Voets et al . , 1999 ) , making it a general concern how to tease apart the effect of PI ( 4 , 5 ) P2 from that of its metabolites including DAG and other phosphatidylinositols .", "To manipulate cellular PI ( 4 , 5 ) P2 levels rapidly and distinguish its function from those of its metabolites in fast cellular reactions we here developed and characterized a new chemical tool: caged , membrane permeant PI ( 4 , 5 ) P2 .", "We show that our compound is taken up into living cells and verify that its UV-uncaging generates physiologically active PI ( 4 , 5 ) P2 with sub-second temporal precision .", "Uncaging induced the re-distribution of proteins containing PI ( 4 , 5 ) P2 binding motifs and locally increased actin-levels .", "Capacitance measurements in chromaffin cells showed that following PI ( 4 , 5 ) P2 uncaging exocytosis is enhanced , and the RRP increased , which we demonstrate is specific to PI ( 4 , 5 ) P2 by contrasting the effects of DAG-uncaging .", "Systematic investigation of the relevant effector proteins revealed a requirement for the potentiation on syt-1 and Munc13-2 , but not on CAPS .", "These results suggest two distinguishable types of PI ( 4 , 5 ) P2 effector proteins: ones that require stoichiometric PI ( 4 , 5 ) P2-binding to exert their function , and ones that function in the local enrichment of PI ( 4 , 5 ) P2 at the vesicle fusion site .", "Finally , making full use of the rapid uncaging kinetics , we investigate the immediate effects of increasing the levels of signaling lipids on exocytosis and discover that PI ( 4 , 5 ) P2 but not DAG uncaging induces the rapid exocytosis of few vesicles from the RRP .", "Our data provide an example of how caged lipid compounds can be used to tease apart relevant interactions in fast biological reactions like neurosecretion ." ], [ "To achieve fast elevation of PI ( 4 , 5 ) P2 levels on the relevant timescale for exocytosis we developed photoactivatable ( caged ) , membrane-permeant PI ( 4 , 5 ) P2 derivatives ( Figure 1 ) .", "Optical uncaging is uniquely suited to increase the levels of signaling molecules non-invasively with high temporal precision ( Höglinger et al . , 2014 ) .", "Synthesis was based on a commercially available enantiomerically pure precursor ( Figure 1a ) .", "Because the hydroxyl and phosphate groups on the inositol ring make PI ( 4 , 5 ) P2 a highly charged molecule it cannot pass across the cellular plasma membrane .", "To make the molecule membrane permeant , these groups were equipped with protective groups of acetoxymethyl ( AM ) esters and butyrates ( Bt ) , respectively ( as detailed in Figure 1a legend and Methods ) .", "Once inside cells , these protective groups are removed by endogenous carboxyesterases ( Schultz , 2003 ) .", "Similar approaches were successfully applied to other phosphoinositides previously ( Laketa et al . , 2014; Mentel et al . , 2011 ) .", "Photosensitivity was achieved by the addition of a photocleavable cage designed to interfere with biological functions at the phosphate residues in positions 4 or 5 of the inositol ring .", "The coumarin caging group was chosen for its extraordinarily fast release kinetics as well as its intrinsic fluorescence , which allows verification of cellular uptake .", "The resulting intermediates 4a and 4b ( Figure 1a ) were subsequently coupled to either a dioctanoylglycerol ( compound 11 , legend to Figure 1 ) or a stearoyl-arachidonoylglycerol ( compound 14 , legend to Figure 1 ) bearing phosphoramidite reagent to form the fully protected caged PI ( 4 , 5 ) P2 intermediates ( Figure 1 ) .", "One-pot deprotection and alkylation with AM bromide gave the caged , membrane-permeant PI ( 4 , 5 ) P2 derivatives ( 1a , b; 2a , b ) in 12% and 10% overall yield , respectively ( Figure 1a–b ) .", "We first validated that UV uncaging activated lipid-protein interactions with known PI ( 4 , 5 ) P2 binding domains .", "For this , we directly diluted our compound in an imaging buffer ( to a final concentration of 20 µM ) and made use of the fact that if the solution was not heavily mixed by vortexing , some of the lipid formed micelles clearly visible on the bottom of the coverslip in the light microscope .", "The solution furthermore contained a reconstituted fusion protein of the PI ( 4 , 5 ) P2-binding PH domain of PLC-δ1 linked to EGFP .", "Illumination in the TIRF field was used to limit light excitation to the surface of the glass coverslip .", "When EGFP was excited , the presence of PH-EGFP was visible as background in the solution of the TIRF field and some of the PH-EGFP was enriched on the micelles .", "Following UV-uncaging with a 405 nm laser in the TIRF field , the EGFP signal at these positions was greatly increased , indicative of PH-EGFP recruitment to the micelles from the surrounding solution ( Figure 2a ) .", "To confirm that the micelles were indeed composed of cg-PI ( 4 , 5 ) P2 , we investigated the images during the irradiation with 405 nm light , which revealed their fluorescence , confirming the presence of the coumarin group ( Figure 2a ) .", "These results demonstrate that UV-cleavage of the coumarin cage activates the compound for interactions with proteins bearing PI ( 4 , 5 ) P2 binding motifs .", "To verify that the protective groups synthesized on our compound enabled cellular uptake , we investigated its cellular distribution making use of the intrinsic fluorescence of the coumarin cage .", "Human Embryonic Kidney ( HEK ) cells were first loaded with a membrane labelling dye excitable with infrared light ( CellMask ) .", "Cells were then loaded with caged ( cg ) PI ( 4 , 5 ) P2 by incubation for 30 min at 37°C with 20 µM of our compound ( 2a , b , diluted from a 20 mM DMSO-stock ) in the presence of 0 . 02% Pluronic ( prepared with heavy vortexing ) to facilitate membrane passage .", "Cells receiving identical treatment but without the compound served as controls .", "To assess the localization of cg-PI ( 4 , 5 ) P2 quantitatively , cells were imaged on a spinning disc confocal microscope and fluorescence line profiles obtained from many cells that bordered open extracellular space ( as opposed to ones in contact with other HEK cells ) .", "The line profiles were then aligned to local fluorescence maxima of the CellMask signal indicating the position of the plasma membrane .", "Subsequently , line profiles from all cells were averaged ( Figure 2b ) .", "We saw that average coumarin fluorescence increased inside the plasma membrane , demonstrating cellular uptake .", "Moreover , coumarin fluorescence was clearly observed at the position of the plasma membrane; however , it was also present inside the cell , possibly on endosomes .", "This is not surprising , as the coumarin group is expected ( and , indeed , intended ) to block interactions with PI ( 4 , 5 ) P2-binding proteins .", "This includes those proteins that usually establish a strict pattern of phosphoinositide composition on distinct cellular organelles .", "Therefore , it is an unavoidable side effect of using caged lipids that their distribution will be broader than the native lipid .", "Inevitably , visualization of the coumarin fluorescence by its excitation leads to its uncaging .", "This could be shown by continuous imaging which significantly reduced the coumarin fluorescence at the location of the plasma membrane ( bar graph Figure 2b ) .", "As expected , neither the fluorescence gradient across the membrane nor the decrease in intensity at the plasma membrane was observed in control cells ( Figure 2b ) , indicating that our compound is taken up into cells , present at the plasma membrane and uncaged there .", "We next validated that UV uncaging liberated physiologically active PI ( 4 , 5 ) P2 in living cells .", "For this , we transfected HEK cells with the PLC-δ1-PH-EGFP construct and looked for a possible recruitment of EGFP fluorescence to the plasma membrane by TIRF microscopy upon UV-uncaging .", "However , we found that even before uncaging , EGFP fluorescence intensities were very high and did not increase further upon UV-uncaging , which we attribute to saturation of the sensor due to relatively high plasma membrane levels of PI ( 4 , 5 ) P2 already at rest ( data not shown ) .", "To circumvent this problem , we co-transfected COS-7 cells with a plasma membrane targeted 5-phosphatase which degrades PI ( 4 , 5 ) P2 ( Posor et al . , 2013 ) .", "Upon UV-uncaging in cells loaded with cg-PI ( 4 , 5 ) P2 we found a small , but highly significant increase of EGFP fluorescence at the cell’s footprint in line with the liberation of PI ( 4 , 5 ) P2 at the plasma membrane , while no such effect was observed in control cells , which were also subjected to UV-light , but not loaded with cg-PI ( 4 , 5 ) P2 ( Figure 2c ) .", "The relatively small effect size may be caused by the 5-phosphatase activity which likely rapidly degrades uncaged PI ( 4 , 5 ) P2 at the plasma membrane before it can be detected by the PLC-δ1-PH-EGFP sensor which is why this experiment may underestimate the amount of liberated PI ( 4 , 5 ) P2 .", "We also investigated the behavior of our compound in cells were PI ( 4 , 5 ) P2 was not constitutively depleted , but where degradation was acutely induced pharmacologically .", "Endogenous PI ( 4 , 5 ) P2 cause the quantitative plasma membrane binding of the high-affinity PLC-δ1-PH-RFP sensor in tsA-201 cells ( Figure 2d ) .", "By simultaneously expressing M1 muscarinic receptors in these cells , we could acutely degrade PI ( 4 , 5 ) P2 by the application of oxotremorine-M ( Oxo-M ) which activates M1 receptors to stimulate PLC .", "This rapidly decreases PI ( 4 , 5 ) P2 levels selectively at the plasma membrane and reliably induced the relocalization of PLC-δ1-PH-RFP sensor from the plasma membrane to the cytosol in control cells ( Figure 2d ) , indicating near complete plasma membrane PI ( 4 , 5 ) P2 breakdown .", "Because this assay monitors the sensor’s dissociation it may be better suited to monitor PI ( 4 , 5 ) P2 liberation close to the sensor’s initial location .", "We therefore combined OxoM-treatment with PI ( 4 , 5 ) P2-uncaging , which prevented PLC-δ1-PH-RFP membrane dissociation ( Figure 2d ) , in line with substantial PI ( 4 , 5 ) P2 release at the plasma membrane overruling PLC activity .", "Uncaging itself did not interfere with PI ( 4 , 5 ) P2 breakdown , because DAG was still produced ( validated by parallel imaging with a DAG biosensor ) ( Figure 2—figure supplement 1b , c ) .", "To verify that UV-uncaging of our compound activated PI ( 4 , 5 ) P2-dependent cellular responses , we investigated effects of cg-PI ( 4 , 5 ) P2 uncaging on actin bundles , for whose polymerization a pivotal role of PI ( 4 , 5 ) P2 is firmly established ( Di Paolo and De Camilli , 2006; Rohatgi et al . , 1999 ) .", "Actin bundles were visualized by TIRF microscopy in the footprints of HEK cells expressing the actin marker Lifeact-RFP ( Figure 3a ) .", "HEK cells loaded with cg-PI ( 4 , 5 ) P2 were compared to non-loaded cells .", "Following the measurement of baseline fluorescence ( five frames ) in the RFP channel , cells were exposed to TIRF illumination with UV light ( 405 nm laser ) .", "This lead to a significant and specific increase in lifeact-RFP in cells loaded with cg-PI ( 4 , 5 ) P2 ( Figure 3a ) , in line with PI ( 4 , 5 ) P2 uncaging causing actin accumulation near the plasma membrane .", "Because we eventually wanted to investigate the physiological effects of PI ( 4 , 5 ) P2 in adrenal chromaffin cells , we next studied whether PI ( 4 , 5 ) P2 uncaging would also increase PI ( 4 , 5 ) P2 levels at their plasma membrane .", "As it is a distinct advantage of our compound that cellular PI ( 4 , 5 ) P2 can be rapidly and specifically increased by light without interfering with basic PI ( 4 , 5 ) P2 metabolism , we wanted to verify PI ( 4 , 5 ) P2 uncaging without the prior manipulation of its resting levels .", "For this we employed a different biosensor harboring the lower affinity PH-domain of PLCδ4 ( Kabachinski et al . , 2014; Lee et al . , 2004 ) .", "Chromaffin cells were infected with a virus expressing PLCδ4-PH-EGFP , and incubated with caged PI ( 4 , 5 ) P2 ( 2a , b ) .", "Experiments were performed on the same microscope setup later used for electrophysiological recordings .", "Uncaging cg-PI ( 4 , 5 ) P2 by a 1–2 ms light pulse from a Xenon flash bulb caused rapid translocation of the sensor towards the periphery of the cell , in line with PI ( 4 , 5 ) P2 release in the plasma membrane ( Figure 3b ) .", "In cells not incubated with cg-PI ( 4 , 5 ) P2 , no translocation was observed .", "Ongoing imaging of the PLCδ4-PH-EGFP allowed us to estimate the time constant of the [PI ( 4 , 5 ) P2] relaxation to ~25 s ( Figure 3b ) .", "A second flash caused markedly less translocation , suggesting that most of the cg-PI ( 4 , 5 ) P2 had already been uncaged .", "In this experiment , the relatively modest recruitment amplitude of to the plasma membrane is probably affected by the widespread intracellular localization of cg-PI ( 4 , 5 ) P2 ( Figure 2b ) , because uncaging likely also increases PI ( 4 , 5 ) P2 on intracellular membranes .", "However , the fact that PH-domains overall relocalize to the plasma membrane ( Figures 2c and 3b ) indicate that PI ( 4 , 5 ) P2 is uncaged there .", "The essential role of PI ( 4 , 5 ) P2 for exocytosis was realized more than 25 years ago , through experiments showing that PI ( 4 , 5 ) P2 depletion inhibits exocytosis from cracked-open adrenal chromaffin cells and PC12 cells ( Eberhard et al . , 1990; Hay et al . , 1995 ) .", "Later experiments showed that PI ( 4 , 5 ) P2 delivery to the intracellular compartment via the patch pipette increased the RRP ( Milosevic et al . , 2005 ) .", "To investigate the physiological effects of PI ( 4 , 5 ) P2 uncaging , cg-PI ( 4 , 5 ) P2 was loaded into the cells ( see Materials and methods ) and exocytosis was induced with a depolarization protocol to allow Ca2+ influx ( Voets et al . , 1999 ) .", "Exocytosis was monitored using patch-clamp capacitance measurements , which report on increased plasma membrane area upon vesicle fusion .", "After a pre-pulse , which elicited indistinguishable exocytosis in both groups ( Figure 4a ) , cells were either subjected to UV-light flashes ( PI ( 4 , 5 ) P2 uncaging group ) or not ( control group ) , and a second depolarization protocol was used to assess the effect of uncaging .", "PI ( 4 , 5 ) P2 uncaging significantly augmented exocytosis in wildtype cells ( Figure 4b , ci ) .", "The overall level of exocytosis found in these experiments is similar to previous findings from wild type Bl6 mice ( Voets et al . , 2001a; Voets et al . , 2001b ) .", "Note that in these experiments , to protect against any effect of the loading protocol or the cg-PI ( 4 , 5 ) P2 compound itself , both control and uncaging groups were loaded with the cg-PI ( 4 , 5 ) P2 , but only the uncaging group was exposed to UV-light .", "Nevertheless , in separate experiments we compared cells loaded with cg-PI ( 4 , 5 ) P2 to cells exposed to the same loading protocol , but without cg-PI ( 4 , 5 ) P2 , and found that without UV light cg-PI ( 4 , 5 ) P2 had no effect on depolarization induced exocytosis ( Figure 4—figure supplement 1 ) .", "We specifically investigated the role of the fatty acid tail in exocytosis using two different compounds , one containing a short DOG-analog ( 1a , b ) and one containing the natural SAG-chain ( 2a , b ) .", "However , we found similar potentiation in both cases , showing that the inositol headgroup is responsible for the enhanced exocytosis ( Figure 4b , ci ) .", "Importantly , the augmentation of exocytosis was not due to changes in Ca2+ influx during membrane depolarization , as revealed by similar Ca2+ currents during the depolarizations ( Figure 4cii , Figure 4—figure supplement 2 ) , and unchanged intracellular Ca2+ concentration immediately after PI ( 4 , 5 ) P2 uncaging ( Figure 4—figure supplement 3a ) .", "Because the compounds 1a , b and 2a , b elicited similar effects , we pooled both datasets for further analysis .", "The depolarization protocol included steps of varying duration which can be used to quantify the release of different populations of vesicles undergoing fusion .", "The first six 10 ms depolarizations release vesicles positioned close to Ca2+ channels in the so-called Immediately Releasable Pool ( IRP ) ( Voets et al . , 1999 ) , while the following two longer ( 100 ms ) depolarizations ( a total of four were given ) are assumed to deplete the full Readily Releasable Pool ( RRP; the IRP is part of the RRP [Voets et al . , 1999] ) .", "We found that PI ( 4 , 5 ) P2 uncaging did not influence the release of the IRP .", "However , the RRP was approximately doubled by PI ( 4 , 5 ) P2 uncaging ( Figure 5a , b ) , confirming that increasing PI ( 4 , 5 ) P2 enhances priming of vesicles into the RRP ( Milosevic et al . , 2005 ) .", "In addition , secretion elicited by residual Ca2+ in-between depolarizations was enhanced ( as seen by steeper slopes of the capacitance increase , Figure 5a , right-hand panels ) ; this could be due to faster priming followed by fusion or increased fusion probability of the remaining vesicles .", "Augmentation of the RRP was noted before in adrenal chromaffin cells following longer-term elevation of PI ( 4 , 5 ) P2 ( Milosevic et al . , 2005 ) , but those manipulations might also have elevated the levels of the downstream metabolite DAG .", "Indeed , Phorbol esters , which are assumed to act as DAG analogues , augment the RRP size in chromaffin cells ( Smith et al . , 1998 ) .", "Therefore , we wanted to distinguish between PI ( 4 , 5 ) P2 vs . DAG requirements for rapid exocytosis augmentation in chromaffin cells .", "To this end we performed DAG uncaging .", "Coumarin-caged DAG ( Nadler et al . , 2013 ) ( cg-DAG , Figure 5b ) was infused into cells via the patch pipette .", "Because both cg-DAG and cg-PI ( 4 , 5 ) P2 bear the same fluorescent coumarin cage , titration of coumarin fluorescence at different ( known ) DAG concentrations in the patch pipette allowed us to estimate the cellular concentration of cg-PI ( 4 , 5 ) P2 in the experiments above .", "This concentration was not known , since AM-ester loading allows progressive accumulation in the cell with time .", "Based on comparable fluorescence values in the titration , we estimated that the final intracellular concentration of the caged PI ( 4 , 5 ) P2 corresponded to ~29 μM ( Figure 5c ) and therefore performed DAG uncaging using 30 or 45 μM DAG .", "However , UV-induced DAG uncaging ( unlike PI ( 4 , 5 ) P2 uncaging ) failed to potentiate secretion in adrenal chromaffin cells ( Figure 5d ) .", "Next , we sought an independent method to confirm that refilling of the primed vesicle pool ( RRP ) depends on PI ( 4 , 5 ) P2 , and not on DAG in a similar concentration regime .", "To this end , we performed double Ca2+-uncaging experiments ( Figure 5—figure supplement 1 ) .", "In these experiments no caged lipids were present , but UV uncaging of a photolysable Ca2+ chelator allowed the direct triggering of vesicles without voltage depolarization of the cell .", "Ca2+ uncaging increases intracellular Ca2+ concentrations to the tens-of micromolar range , which is sufficient to deplete the entire RRP and expected to activate endogenous PLC , leading to PI ( 4 , 5 ) P2 degradation .", "Two sequential Ca2+-uncaging stimuli were used to assess RRP sizes and thus refilling in cells incubated with a PLC inhibitor , or an inactive control compound .", "Recovery of the RRP was significantly enhanced by inhibiting PLC ( Figure 5—figure supplement 1 ) , indicating that preventing PI ( 4 , 5 ) P2 degradation enhances vesicle priming .", "Thus , conversion of endogenous PI ( 4 , 5 ) P2 to DAG is overall negative for refilling of the RRP , which confirms our findings using caged lipid compounds .", "We next sought to identify relevant PI ( 4 , 5 ) P2 effectors among the molecular release machinery .", "Syt-1 , the Ca2+ sensor for rapid exocytosis in chromaffin cells ( Voets et al . , 2001a ) , was among the first PI ( 4 , 5 ) P2-binding presynaptic proteins to be identified ( Schiavo et al . , 1996; Honigmann et al . , 2013; van den Bogaart et al . , 2012; Bai et al . , 2004 ) .", "The relevance of PI ( 4 , 5 ) P2-binding was indicated by mutation , which increased the Ca2+ requirements for exocytosis .", "However , mutations can have other effects than those intended in the experiment .", "For instance , the same residues in the syt-1 C2B domain interacting with PI ( 4 , 5 ) P2 were also shown to interact with the neuronal SNARE complex ( Zhou et al . , 2015 ) .", "Therefore , and to complement those experiments , we here uncaged PI ( 4 , 5 ) P2 in syt-1 knockout mice , and found that uncaging did not potentiate exocytosis ( Figure 6a ) .", "Proper loading of the compound was ensured after the experiment by the intrinsic fluorescence of the coumarin group ( Figure 6—figure supplement 1a ) .", "Exocytosis from syt-1 KO cells is reduced compared to wild type cells ( Voets et al . , 2001a ) , although sizable release – including a small RRP ( Mohrmann et al . , 2013 ) – remains .", "We asked whether the lack of PI ( 4 , 5 ) P2 augmentation was due to the smaller exocytosis amplitude , rather than the lack of syt-1 .", "To this end , we reanalyzed data , identifying wild type cells with intrinsically low exocytosis amplitude , and syt-1 KO cells with high exocytosis amplitude .", "However , we still found significant potentiation in WT cells , but not in syt-1 KO cells ( Figure 6—figure supplement 1d ) , suggesting a molecular requirement for syt-1 .", "Neurosecretion is known to depend on the key vesicle priming factor Munc13 , and the relevant isoform in chromaffin cells , Munc13-2 , harbors a C2-domain ( C2B ) , which displays a strong PI ( 4 , 5 ) P2-dependence ( Shin et al . , 2010; Kabachinski et al . , 2014 ) .", "Adrenal chromaffin cells isolated from Munc13-2 knockout mice lacked the capacity of PI ( 4 , 5 ) P2 uncaging to potentiate exocytosis ( Figure 6b ) .", "Thus , PI ( 4 , 5 ) P2 potentiation in chromaffin cells occurs via specific activation of the vesicular release machinery and requires syt-1 and Munc13-2 .", "To identify additional molecular targets , we repeated experiments in knockout mouse cells for the major PI ( 4 , 5 ) P2 binding proteins CAPS1 and −2 .", "CAPS interacts with PI ( 4 , 5 ) P2 via a pleckstrin homology domain and loss of this interaction impedes vesicle exocytosis by reducing the number of releasable vesicles ( Nguyen Truong et al . , 2014 ) .", "However , uncaging PI ( 4 , 5 ) P2 in CAPS1 and −2 double knockout mice revealed a similar enhancement of release upon PI ( 4 , 5 ) P2 uncaging as in wild type cells ( Figure 6c ) , arguing that augmentation of exocytosis observed here occurs independently of CAPS , or bypasses CAPS ( see Discussion ) .", "Surprisingly , the IRP size was actually reduced by PI ( 4 , 5 ) P2-uncaging in the Munc13-1 KO , whereas it was ( nonsignificantly , p<0 . 08 ) increased in the CAPS-1/2 DKO ( Figure 6 ) .", "The implication of this finding is unclear , but Munc13 and CAPS-proteins play distinct roles during priming ( Kabachinski et al . , 2014; Liu et al . , 2010 ) , and if they are both required for the formation of the IRP-vesicles , then the elimination of one or the other might create IRPs with distinct properties , including PI ( 4 , 5 ) P2-dependence .", "We conclude that PI ( 4 , 5 ) P2-dependent activation of exocytosis operates via Munc13-2 and syt-1 to potentiate RRP size .", "Use of cg-PI ( 4 , 5 ) P2 for the first time allowed investigating the consequences of an abrupt increase in PI ( 4 , 5 ) P2 abundance on a subsecond timescale .", "When inspecting the capacitance trace around the first uncaging flash ( see Figure 4a for stimulation protocol ) , we found an abrupt jump in the capacitance , indicating fast fusion of a few ( 5-10 ) vesicles ( Figure 7a ) .", "This jump was observed only with the first uncaging flash , indicating that it is unlikely to be a photo-artifact ( Figure 7—figure supplement 1 ) .", "The release appears specific , because the size of the response strongly correlated with the RRP sizes in these cells ( Figure 7b ) .", "Furthermore , uncaging of the PI ( 4 , 5 ) P2 downstream metabolite DAG , bearing the same photolysable coumarin group as PI ( 4 , 5 ) P2 did not induce any capacitance increase ( Figure 7a–c ) .", "Finally , the jump was reduced in size – or absent – in cells from Munc13-2 , CAPS-1/2 and syt-1 knockout mice , which all have smaller RRP sizes ( Liu et al . , 2008; Mohrmann et al . , 2013; Man et al . , 2015 ) ( Figures 5 , 6 and 7b ) .", "Thus , rapidly increasing PI ( 4 , 5 ) P2 levels can fuse vesicles .", "The release of a fraction of the RRP is consistent with the stimulation of some vesicles close to fusion threshold ( Yang et al . , 2002 ) whose Ca2+-sensitivity may increase further due to the interaction of syt-1 with PI ( 4 , 5 ) P2 ( van den Bogaart et al . , 2012;Li et al . , 2006 ) , leading to increased release probability ." ], [ "Here , we developed a photocaged membrane-permeant PI ( 4 , 5 ) P2 and combined this compound with high time-resolution electrophysiology and genetic manipulations to identify relevant PI ( 4 , 5 ) P2 effectors in neuroendocrine chromaffin cells .", "The main PI ( 4 , 5 ) P2-binding proteins in the secretory pathway are syt-1 ( Schiavo et al . , 1996; Honigmann et al . , 2013; van den Bogaart et al . , 2012; Bai et al . , 2004 ) , the Ca2+-sensor for exocytosis , and Munc13-2 ( Shin et al . , 2010 ) and CAPS ( Loyet et al . , 1998; Kabachinski et al . , 2014 ) , two priming proteins , which are responsible for establishing and replenishing the RRP ( Man et al . , 2015; Liu et al . , 2010 ) .", "Possible effects of PI ( 4 , 5 ) P2-binding to these proteins in living cells were previously not investigated by altering PI ( 4 , 5 ) P2 levels , but by using correlative analyses following protein mutation ( Li et al . , 2006; Shin et al . , 2010 ) .", "However , exactly which of these proteins are acutely activated by PI ( 4 , 5 ) P2 to facilitate secretion was not clear .", "By uncaging our compound we could now verify that PI ( 4 , 5 ) P2 enhances exocytosis and by studying mouse knockouts we could provide mechanistic insight into distinct PI ( 4 , 5 ) P2-dependent processes in exocytosis: PI ( 4 , 5 ) P2 uncaging specifically potentiated RRP size , but not the size of the IRP , which forms a subpool of the RRP , consisting of vesicles co-localized with Ca2+-channels ( Voets et al . , 1999 ) .", "One interpretation is that IRP-vesicles are already saturated with PI ( 4 , 5 ) P2; another possible explanation is that the IRP is limited in size by additional factors ( for instance the availability of Ca2+-channels ) , and therefore cannot be further augmented .", "Based on the different types of effects we observe in our experiments ( loss of PI ( 4 , 5 ) P2-dependent augmentation in syt-1 KO and Munc13-2 KO vs . no effect in CAPS DKO ) , two different mechanisms of PI ( 4 , 5 ) P2-binding proteins can be envisioned ( Figure 8 ) : the first is specific , and probably stoichiometric , PI ( 4 , 5 ) P2-binding to support protein function , for instance the ability of Munc13 to stimulate SNARE-complex formation , or to increase the Ca2+ binding affinity of syt-1 during secretion triggering .", "The other potential function is to co-localize the fusion machinery with PI ( 4 , 5 ) P2-patches in the plasma membrane .", "The latter , but not the former , function might be bypassed by PI ( 4 , 5 ) P2-uncaging , which uncovers PI ( 4 , 5 ) P2 under the vesicle ( Figure 8 ) .", "Thus , lipid uncaging can serve as an exquisite tool to distinguish between these two different functions of PI ( 4 , 5 ) P2-binding proteins , just as Ca2+ uncaging has been instrumental in distinguishing between effects on Ca2+-binding to the release machinery itself , and effects on colocalizing vesicles with Ca2+ channels ( Voets et al . , 1999; Wadel et al . , 2007 ) .", "Therefore , the established essential requirement of the CAPS PH-domain for its function in vesicle priming ( Nguyen Truong et al . , 2014; Kabachinski et al . , 2014 ) can be reconciled with our findings here , if the function of CAPS is to cause local enrichment of PI ( 4 , 5 ) P2 at the sites of vesicle priming , where it will interact with other exocytotic proteins .", "The use of uncaging made it possible for the first time to investigate the consequences of an acute , millisecond , increase in PI ( 4 , 5 ) P2 and DAG abundance .", "We found that PI ( 4 , 5 ) P2 , but not DAG , uncaging caused the rapid fusion of vesicles ( Figure 7a , c ) .", "The amount of fusion correlated with the RRP size , and this correlation extended to the knockouts tested ( Figure 7b ) .", "Thus , the acutely fusing vesicles probably constitute a fraction of the RRP .", "The fact that rapid fusion was only seen when uncaging PI ( 4 , 5 ) P2 , but not DAG , argue that PI ( 4 , 5 ) P2 is more directly linked to exocytosis triggering in adrenal chromaffin cells , possibly because PI ( 4 , 5 ) P2 binding to the C2-domains in Munc13-2 and syt-1 directly change the Ca2+ affinities of those domains .", "The fusing vesicles might be members of the ‘Highly Calcium Sensitive Pool’ ( HCSP ) , which fuse at lower Ca2+ concentrations than the rest of the RRP vesicles ( Yang et al . , 2002 ) .", "Since these vesicles are close to fusion threshold , rapid binding of PI ( 4 , 5 ) P2 might increase the Ca2+-affinity of syt-1 enough that the vesicles fuse due to a rapid increase in Ca2+-affinity rather than a rapid increase in Ca2+ concentration as would normally be the case .", "A definite advantage of the photocaged approach is that it allows inducing sub-second increases in the phospholipid composition of membranes , which can be used to identify direct effects of a phospholipid before its metabolism takes place .", "A possible complication is that by shielding the head group , the lipid will no longer be recognized by proteins ( enzymes , lipid-shuttling proteins ) that establish the cellular pattern of lipid composition between different organelles .", "Thus , the localization of the caged lipid will likely be broader than for the native lipid .", "Indeed , investigating the sub-cellular distribution of our cg-PI ( 4 , 5 ) P2 revealed the uptake in compartments other than the plasma membrane ( Figure 2b ) .", "However , our data also clearly show its specific uncaging at the plasma membrane , making it a suitable tool to address reactions at the latter ( Figures 2 and 3 ) .", "Moreover , uncaging PI ( 4 , 5 ) P2 in chromaffin cells led to a specific potentiation of vesicle priming ( Figures 4–6 ) , which is consistent with previous findings using enzymatic over/underexpression ( Gong et al . , 2005; Milosevic et al . , 2005 ) , and with the use of a PLC-inhibitor to prevent PI ( 4 , 5 ) P2 breakdown ( Figure 5—figure supplement 1 ) .", "Furthermore , the effect depended on known PI ( 4 , 5 ) P2-binding proteins .", "Thus , although we cannot rule out that PI ( 4 , 5 ) P2 is liberated elsewhere in the cell , PI ( 4 , 5 ) P2 uncaging results in valid and specific effects on exocytosis .", "The wide-spread distribution of our caged compound may also be considered a distinct advantage , because this allows to study the consequences of its focal liberation in regions where PI ( 4 , 5 ) P2 is sparse .", "Collectively , our data demonstrate the power of caged phospholipids to dissect physiological functions of different , but interconvertible , phospholipids .", "The main power of the approach is that it outpaces the rate of metabolism/interconversion of one lipid into another .", "Using this method we have dissected the molecular requirement for the potentiating effect of PI ( 4 , 5 ) P2 on exocytosis , and we have demonstrated a novel , acute effect of uncaged PI ( 4 , 5 ) P2: to trigger rapid exocytosis .", "We anticipate that caged lipid second messengers will serve as valuable experimental tools to uncover mechanistic details of fast cellular processes ." ], [ "Conditions:", "( a ) DMF-DMA , 140°C , 22 hr , 70%;", "( b ) NaIO4 , THF:H2O 1:1 , 25°C , 2 hr , 97%;", "( c ) NaBH4 , MeOH , 25°C , 60% .", "Abbreviations: DMF N , N-dimethylformamide , DMF-DMA dimethylformamide dimethylacetal , THF tetrahydrofuran , MeOH methanol , R 7-diethylamino-2-oxo-2H-chromen-4-yl .", "The following plasmids were generously given to us: Human mRFP-PH ( PLCδ1 ) from Ken Mackie ( The Gill Center for Biomolecular Science , Bloomington , Indiana ) ; GFP-PKD-C1ab from Tamas Balla ( National Institutes of Health , Bethesda , MD ) , M1R from Neil Nathanson ( University of Washington , Seattle , WA ) , Lifeact-RFP ( pmRFPruby-N1*Lifeact ( GB lab plasmid nr 28 ) in pmRFPruby-N1 ) from Geerd van den Gogaard ( Radboud University Medical Center , Nijmegen , the Netherlands ) , and the PLCδ4-PH-mKate2 plasmid from Thomas F . J . Martin ( Department of Biochemistry , University of Wisconsin ) and the EGFP-PH-PH ( PLCδ1 ) plasmid from Michael Krauss ( Leibniz-Forschungsinstitut für Molekulare Pharmakologie , Berlin , Germany ) .", "The PLCδ4-PH was fused to an EGFP lentiviral plasmid under the control of CMV promotor and lentiviral particles were produced following standard protocols .", "HEK and COS-7 cells were transfected using Lipofectamine 2000 ( Life Technologies ) or Lipofectamine LTX ( in the case of Lifeact-RFP , Thermo Fisher ) according to manufacturer’s protocol using a total amount of 1 . 8 µg of DNA per well ( 6-well plate ) for HEK cells and 2 µg EGFP-PH-PH together with 1 µg mCherry-INPP5E-CAAX for COS-7 cells .", "For imaging of PI ( 4 , 5 ) P2 on glass , cg-PI ( 4 , 5 ) P2 was added to imaging buffer ( HBSS with 5% FCS ) to a final concentration of 20 µM .", "The high-affinity PI ( 4 , 5 ) P2-sensor PLCδ1-PH-GFP was stored in a 1 . 8 mg/ml PBS/20% Glycerol stock .", "This was added 1:20 to the cg-PI ( 4 , 5 ) P2 solution ( e . g . 5 µl in 100 µl ) .", "The solution was pipetted onto a glass coverslip and imaged using a TIRF microscope ( Nikon Ti Eclipse ) , equipped with an incubation chamber ( 37°C ) , a x60 TIRF objective ( Apo TIRF 1 . 49NA , Nikon ) , a sCMOS camera ( Neo , Andor ) , four excitation laser lines: ( 405 , 488 nm , 568 nm , 647 nm ) an appropriate dicroic mirror ( Di01-R405/488/561/635 ) , filter ( FF01-446/523/600/677 ) .", "The TIRF microscope was operated by open-source ImageJ-based micromanager software ( https://micro-manager . org/ ) .", "Images were captured at 1 s intervals using a 488 nm laser ( 200 ms exposure ) at 50% power ( 30 MW ) .", "Image analysis was performed with Fiji ( ImageJ ) .", "Each 488 nm excitation frame was immediately followed by an uncaging frame , performed using a 405 nm laser ( 200 ms exposure ) at 100% power ( 60 MW ) .", "ROIs of cg-PI ( 4 , 5 ) P2 on glass were selected in the 405 nm channel and the fluorescence intensities of the PLCδ1-PH-EGFP sensor in the same ROIs in response to uncaging over time measured in the 488 nm channel HEK 293T and COS-7 cells used for experiments depicted in Figure 2b and c were purchased from ATCC ( https://www . lgcstandards-atcc . org ) ; the identity of the cells has been confirmed by STR profiling performed by ATCC .", "Cell lines were tested for mycoplasma contaminations on a monthly basis .", "Cells were cultured in DMEM medium ( Lonza ) supplied with 10% fetal bovine serum ( FBS , Gibco 10270–106 ) and 1% penicillin/streptomycin .", "Cells were not used beyond passage 30 from original .", "Preparation of cg-PI ( 4 , 5 ) P2 was performed in the dark under red light .", "Loading solution was prepared by adding cg-PI ( 4 , 5 ) P2 to imaging buffer ( HBSS with 5% FCS ) to a final concentration of 20 µM ( from a 20 mM DMSO-stock ) .", "An equal volume of Pluronic F-127 ( Thermo Fisher Scientific , 20% in DMSO ) was added ( final concentration: 0 . 02% Pluronic F-127 ) .", "The final DMSO concentration was 0 . 2% .", "The loading solution was thoroughly vortexed for 3 min .", "Cell medium was removed from the cells and cg-PI ( 4 , 5 ) P2 loading solution was pipetted gently in at the edge of the well .", "Cells were incubated with the loading solution for 30 min in a CO2-incubator at 37°C .", "Loading solution was removed and cells were gently washed twice with imaging buffer .", "Control loading solutions contained DMSO in place of cg-PI ( 4 , 5 ) P2 .", "CellMask Deep Red plasma membrane stain was stored in the dark at room temperature in a 5 mg/ml stock in DMSO ( Thermo Fisher Scientific ) was applied to HEK 293 T cells after loading with cg-PI ( 4 , 5 ) P2 .", "Cells were incubated in CellMask ( 1:1000 dilution of stock in imaging buffer ) for 5 min .", "Cells were washed twice in imaging buffer and imaged immediately .", "The experiments depicted in Figure 2b were performed on a Spinning Disk Confocal Microscope ( Nikon TI-Eclipse ) equipped with an incubation chamber ( 37°C ) , a x60 objective ( P-Apo NA 1 . 40 , Nikon ) , Yokogawa spinning disk ( CSU-X1 ) , an EMCCD camera ( AU-888 Andor ) , four excitation laser lines: ( 405 , 488 nm , 561 nm , 638 nm ) , an Borealis unit ( Andor ) , an appropriate dicroic mirror ( Di01-R405/488/561/635 ) and specific filter ( BP450/50 and BP700/75 for coumarin and CellMask , respectively ) .", "The microscope was operated by NIS Elements ( Nikon ) .", "Images were captured at 0 . 5 s intervals ( 200 ms exposure ) using a 638 nm laser at 20% power ( 100 mW ) and a 405 nm laser at 30% power ( 100 mW ) .", "Images were analysed with Fiji ( ImageJ 1 . 50 g ) .", "Line profile ROIs used to investigate fluorescence intensities across the cell membrane were placed in the CellMask imaging channel ( excitation 638 nm ) .", "ROIs were selected such that they crossed the plasma membrane from the extracellular space into nucleus-free cytosol at a 90° angle in relation to the visible cell membrane .", "In each frame , a 3 µm long sub-region of each line profile ROI was selected and aligned such that the mid-point of the line coincided with the position of the plasma membrane ( recognized as a local maximum in the intensity value of the CellMask staining ) .", "This position was found using the second output parameter of the built-in MatLab function ‘max’ ( MatLab vers . 7 . 12 . 0 R2011a ) .", "The intensity values along the line profile at 15 positions preceding and 15 positions succeeding the mid-point were read out .", "The exact same line positions were considered for the images containing the coumarin fluorescence ( 405 nm excitation ) .", "In both channels , the pixel intensity value of the 1 st position on each line ( i . e . 1 . 5 µm extracellular to the plasma membrane ) was subtracted from values at all other positions to obtain background subtracted line profiles .", "Line profiles were then averaged across cells .", "For experiments depicted in Figure 2c , COS-7 cells were transfected with EGFP-PH-PH ( PLCδ1 ) and mChINPP5E-CAAX ( Posor et al . , 2013 ) .", "Cells were loaded with cg-PI ( 4 , 5 ) P2 and Pluronic F-127 as described above and imaged on the TIRF setup as described for the in vitro imaging .", "Images were captured at 1 s intervals with 200 ms exposure using a 488 nm laser at 50% power ( 30 MW ) , immediately followed by a 561 nm laser at 100% power ( 50 MW ) .", "Between the 10th and 11th loop ( 10–11 s ) , UV uncaging was performed with a single 400 ms exposure frame using a 405 nm laser at 100% power ( 60 MW ) .", "COS-7 cells expressing the constitutive phosphatase and lipid sensor were analysed by selecting circular ROIs of plasma membrane in the 488 nm channel only and measuring mean intensities over time .", "A ratio of fluorescence intensity in these ROIs was calculating by dividing intensities after the UV uncaging frame by the corresponding intensities prior to the UV uncaging frame .", "tsA201 cells were purchased from Sigma-Adrich ( St . Louis , MO , USA ) , and the identity of the cells has been confirmed by STR profiling performed by Sigma-Aldrich .", "The cells have been eradicated from mycoplasma at the European Collection of Cell Cultures ( ECACC ) .", "Cells were cultured at 37°C and 5% CO2 in DMEM-medium ( Invitrogen Inc , Carlsbad , USA ) supplemented with 10% FBS ( PAA , Pasching , Austria ) and 0 . 2% penicillin/streptomycin ( Invitrogen Inc . , USA ) .", "Transfection was performed with Lipofectamine 2000 ( Invitrogen Inc . , USA ) according to the manufacturer’s specifications .", "Cells were plated onto poly-D-lysine coated glass chips 16–20 hr before experiments .", "The tsA201 cell experiments were carried out at room temperature on a Zeiss LSM710 laser confocal microscope ( Zeiss LLC , Thornwood , NY ) .", "Cells were superfused with Ringer’s solution ( 160 mM NaCl , 2 . 5 mM KCl , 2 mM CaCl2 , 1 mM MgCl2 , 8 mM glucose , and 10 mM HEPES at pH 7 . 4 ) throughout the experiments .", "Uncaging of PI ( 4 , 5 ) P2 was achieved on the microscope by a combined 5 s light pulse of both a 405 nm diode and a 451 nm laser line at 50% intensity of the light sources .", "The Lifeact-RFP experiments depicted in Figure 3a were performed in HEK 293T cells provided by Dr Therese Schaub and Victor Tarabykin ( Institute of Cell Biology and Neurobiology , Charité Berlin ) .", "These were cultured in DMEM GlutaMAX ( Thermo Fisher/Gibco ) supplied with 10% fetal bovine serum ( FBS , Gibco 16140063 ) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere ( 5% CO2 ) .", "Cells were not used beyond passage 40 from original .", "This cell line was not tested for mycoplasm contaminations .", "The cells density was between 0 , 25–1 × 106 plated on 24 mm glass coverslips .", "18–24 hr following transfection with Lifeact-RFP , loading solution was prepared by adding cg-PI ( 4 , 5 ) P2 .", "to culture medium removed from cells , to a final concentration of 20 µM ( from a 20 mM DMSO-stock ) .", "An equal volume of Pluronic F-127 ( Thermo Fisher Scientific , 20% in DMSO ) was added ( final concentration: 0 . 02% Pluronic F-127 ) .", "The final DMSO concentration was 0 . 2% .", "The loading solution was thoroughly vortexed for 3 min .", "Loading was performed as described above ( 37°C , 30 min ) .", "Cells were washed twice and imaged in a solution containing ( in mM ) 145 NaCl , 3 KCl , 10 HEPES , 1 CaCl2 , 1 MgCl2 and 6 Glucose at pH 7 . 4 and osmolarity , 290 mOsm/l .", "Imaging was performed on a Nikon Ti eclipse TIRF microscope equipped with an incubation chamber ( 37°C ) , a x100 objective ( Apo TIRF 1 . 49NA , Nikon ) , an EMCCD camera ( iXon 888 Andor , EM gain set to 300 ) , and suitable filtersets .", "Image acquisition was controlled by the Nikon NIS-Elements AR Software ( vers . 4 . 51 . 01 ) .", "Frames were collected at 2 Hz , images in the RFP channel were acquired by excitation with a 561 nm laser ( 2% intensity ) and an exposure time of 100–200 ms . Following the acquisition of five frames in the RFP-channel , three consecutive UV frames were acquired at 2 Hz by excitation with a 405 nm laser at 25% laser intensity .", "Images during UV light were captured on the same camera with an exposure time of 100 ms . Imaging was then immediately resumed at 2 Hz in the RFP channel with the laser and camera settings mentioned above .", "Image analysis was performed offline in Fiji ( ImageJ 1 . 50 g ) .", "Several equally sized circular ROIs were placed in the RFP images on filamentous structures presumed to be actin bundles ( white circles in the left-hand images depicted in Figure 3a ) .", "The mean intensity value per ROI was calculated and corrected for background signal by subtraction of the mean intensity within one equally sized ROI placed in a background region outside the cell ( yellow circle in the left-hand images depicted in Figure 3b ) .", "Background subtraction was performed in each frame .", "The intensity values of all ROIs within one cell were then averaged frame-wise and normalized by dividing the mean intensity values of all frames by that of the first frame .", "These normalized intensities were then averaged frame-wise across all investigated cells .", "Wildtype chromaffin cells were prepared as described previously ( Sørensen et al . , 2003 ) and used for experiments after 3–5 days .", "Cells were loaded with AM-ester coupled caged lipid compounds for varying durations .", "All lipid compounds were kept in 20–25 mM stock solutions in DMSO and stored at −20°C .", "Stock solutions were diluted in the cellular medium and Pluronic was added to facilitate uptake of the compound .", "The solution was heavily vortexed to avoid the generation of micelles before placing it onto the cells at a final lipid concentration of 20 µM with 0 . 02% Pluronic .", "Cells were kept in a CO2-incubator at 37°C for 30 to 45 min .", "In order to document successful loading of the caged compounds , cells were checked after recordings for fluorescence levels .", "For recordings , cells were transferred to a recording chamber and superfused with external recording solution containing ( in mM ) : 145 NaCl , 2 . 8 KCl , 2 CaCl2 , 1 MgCl2 , 10 HEPES , 11 . 1 glucose , adjusted to pH 7 . 2 with NaOH .", "The solution had an osmolarity of approximately 305 mOsm .", "The patch pipette solution contained ( in mM ) : 100 Cs-glutamate , 8 NaCl , 32 Cs-HEPES , 2 Mg-ATP , 0 . 3 NaGTP , one ascorbic acid , 0 . 4 Fura-4f ( Invitrogen ) , 0 . 4 furaptra ( Invitrogen ) , adjusted to pH 7 . 2 with CsOH .", "For DAG-uncaging experiment , the coumarin-caged DAG ( cg-DAG ) was loaded into the cells through the patch pipette for 60–100 s prior to stimulation .", "The patch pipette solution contained ( in mM ) : 125 Cs-glutamate , 40 Cs-HEPES , 2 Mg-ATP , 0 . 3 NaGTP , 0 . 5 EGTA , 0 . 030 or 0 . 045 cg-DAG , adjusted to pH 7 . 2 with CsOH .", "The setup used for patching and uncaging of lipids consisted of a Zeiss inverted microscope ( Axiovert 10 ) equipped with a specialized flash lamp ( Rapp Optoelectronic , JML-C2 ) .", "The light passed through a 395 nm low-pass filter , a light guide and a TILL Photonics dual port condensor before being focused on the sample through a Fluar 40X/N . A . 1 . 30 oil objective for maximal UV transmittance .", "For the composition of the filter cube , see below .", "Cells were voltage clamped to −70 mV ( liquid junction potential was not corrected for ) .", "After 1 min at rest , the cellular membrane was depolarized by stepping the voltage six times to +20 mV for 10 ms at 300 ms intervals followed by four 100 ms depolarizations at 400 ms intervals .", "The cell membrane capacitance was measured before , after , and in-between depolarizations ( Voets et al . , 1999 ) .", "8 . 5 s after the final depolarization , a strong flash of UV-light ( 1–2 ms duration , JML-C2 , setting around 300V on the third capacitor bank ) was triggered to uncage the lipid while the cellular capacitance was measured .", "Uncaging was repeated four times at the same power at 15 s intervals , after which another round of voltage depolarizations was initiated .", "All capacitance measurements were performed using the Lindau-Neher technique ( Lindau and Neher , 1988 ) .", "Amperometry measurements were performed with 5-μm-diameter polyethylene-insulated carbon fibers ( Thornel P-650/42 , Cytec [Bruns , 2004] ) .", "The voltage was clamped at 700 mV via an EPC-7 using an external power supply .", "Currents were filtered at 3 kHz and sampled at 12 kHz .", "For analysis , amperometric traces were filtered off-line at 1 kHz .", "To quantify the IRP size , the cellular capacitance 2 s into the recording ( after the last 10 ms depolarization pulse ) was subtracted by the cellular capacitance at the beginning of the recording .", "The RRP size was quantified as the cellular capacitance at 2 . 8 s ( after the second 100 ms depolarization ) subtracted by the cellular capacitance at the beginning of the recording .", "The IRP size was subtracted from this value in each cell to isolate the release elicited by the first two 100 ms depolarizations ( RRP-IRP ) .", "The total capacitance increase was measured 4 s after the beginning of the recording .", "From this the RRP size was subtracted in each cell to quantify the exocytosis increase caused by the last two 100 ms depolarizations ( total-RRP ) .", "The step size elicited by the lipid-uncaging was measured in each cell by calculating the difference between the cellular capacitance 100 ms before and 300 ms after the UV-flash , which was elicited 500 ms after the beginning of the recording .", "Traces in Figure 4a were filtered with a binomial Gaussian filter using the ‘smooth’ function ( window of 23 points ) in IGOR Pro ( vers . 6 . 22A ) for clarity .", "In experiments where Ca2+ was uncaged the patch pipette solution contained the following ( in mM ) 100 Cs-glutamate , 8 NaCl , 4 CaCl2 , 32 HEPES , 2 Mg-ATP , 0 . 3 NaGTP , five nitrophenyl-EGTA , one ascorbic acid ( to prevent photo damage to the Ca2+-dyes ) , 0 . 4 fura-4f ( Invitrogen ) , 0 . 4 furaptra ( Invitrogen ) , adjusted to pH 7 . 2 with CsOH .", "Ca2+ uncaging experiments and Ca2 +microfluorimetry were performed as described previously ( Walter et al . , 2014 ) .", "Loading of the caged compound was evaluated semi quantitatively , using the fluorescence of the compound .", "To quantify the fluorescence , cells were imaged on a Zeiss Axiovert 200 equipped with a TILL Monochromator V and a 25X/N . A . 0 . 8 LD LCI Plan-Apo oil/water/air objective with 405 nm excitation light and an EM-CCD camera ( Andor 885 , gain 1 ) .", "Images were exposed for 500 ms . Fluorescence intensities were quantified using Image J software ( version 1 . 46 r ) by integrating the fluorescence in a square region ( 61 × 61 pixel ) containing the cell , subtracted by the integrated intensity of the same size of background .", "For live-cell imaging using lentivirally encoded low-affinity PI ( 4 , 5 ) P2-sensor EGFP-PLCδ4-PH , chromaffin cells were transduced with lentivirus 24 hr after seeding and allowed to express for 24–48 hr .", "Imaging was carried on a Zeiss Axiovert 200 equipped with a TILL Monochromator V and a flash lamp from Rapp Optoelectronic ( JML-C2 ) ; both were coupled through the epifluorescence port using a 2-way splitter from TILL Photonics .", "An F-Fluar 40X/1 . 30 oil objective , and a CCD camera ( PCO sensicam ) .", "To enable UV flashing and imaging of EGFP we used a dichroic mirror with efficient reflection from the near-UV range up to 488 nm ( Chroma 495dcxru ) , together with a long-pass emission filter ( Chroma et500 LP ) .", "Imaging was carried out using 488 nm excitation light and 80 ms exposure times .", "A single image was acquired prior to the first UV-flash ( pre-flash ) followed by subsequent images at 1 Hz .", "The second UV-flash was applied 38 . 5 s after the first flash .", "To quantify the redistribution of PLCδ4PH-EGFP the fluorescence background was removed from images by subtracting the mean intensity of the background .", "Unusually bright spots on the PM , visible on some of the images , were cut out and excluded from analysis .", "Integrated fluorescence density of a circular region of interest ( ROI1 ) containing the entire cell was calculated .", "For each cell another , smaller , ROI – ROI2 – was defined as the interior of the cell , excluding the periphery .", "The content of the inner ROI was subtracted from the other ROI to isolate the intensity of the periphery of the cell ( which includes the plasma membrane ) , and the ratio of periphery to inner ROI was calculated , i . e . ( ROI1-ROI2 ) /ROI2 .", "This ratio was calculated as a function of time and normalized to the pre-flash ratio .", "These normalized values were then plotted as a function of time .", "Results are shown as average ±s . e . m . unless otherwise indicated , with n referring to the number of cells for each group .", "Two-tailed paired or unpaired t-tests or Mann-Whitney U-test ( if data were heteroscedastic ) were used to compare between two groups , as indicated in figure legends .", "Significance was assumed when p<0 . 05 .", "Statistical testing was performed using SigmaPlot 12 . 3 ( Systat Software Inc ) .", "In figures , the significance levels are indicated by asterisks; *p<0 . 05; **p<0 . 01; ***p<0 . 001 ." ] ]
[ "Phosphatidylinositol-4 , 5-bisphosphate [PI ( 4 , 5 ) P2] is essential for exocytosis .", "Classical ways of manipulating PI ( 4 , 5 ) P2 levels are slower than its metabolism , making it difficult to distinguish effects of PI ( 4 , 5 ) P2 from those of its metabolites .", "We developed a membrane-permeant , photoactivatable PI ( 4 , 5 ) P2 , which is loaded into cells in an inactive form and activated by light , allowing sub-second increases in PI ( 4 , 5 ) P2 levels .", "By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells , we show that PI ( 4 , 5 ) P2 uncaging potentiates exocytosis and identify synaptotagmin-1 ( the Ca2+ sensor for exocytosis ) and Munc13-2 ( a vesicle priming protein ) as the relevant effector proteins .", "PI ( 4 , 5 ) P2 activation of exocytosis did not depend on the PI ( 4 , 5 ) P2-binding CAPS-proteins , suggesting that PI ( 4 , 5 ) P2 uncaging may bypass CAPS-function .", "Finally , PI ( 4 , 5 ) P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles , revealing a rapid role of PI ( 4 , 5 ) P2 in fusion triggering .", "Thus , optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors ." ]
[ "Cells in our body communicate by releasing compounds called transmitters that carry signals from one cell to the next .", "Packages called vesicles store transmitters within the signaling cell .", "When the cell needs to send a signal , the vesicles fuse with the cell's membrane and release their cargo .", "For many signaling processes , such as those used by neurons , this fusion is regulated , fast , and coupled to the signal that the cell receives to activate release .", "Specialized molecular machines made up of proteins and fatty acid molecules called signaling lipids enable this to happen .", "One signaling lipid called PI ( 4 , 5 ) P2 ( short for phosphatidylinositol 4 , 5-bisphosphate ) is essential for vesicle fusion as well as for other processes in cells .", "It interacts with several proteins that help it control fusion and the release of transmitter .", "While it is possible to study the role of these proteins using genetic tools to inactivate them , the signaling lipids are more difficult to manipulate .", "Existing methods result in slow changes in PI ( 4 , 5 ) P2 levels , making it hard to directly attribute later changes to PI ( 4 , 5 ) P2 .", "Walter , Müller , Tawfik et al . developed a new method to measure how PI ( 4 , 5 ) P2 affects transmitter release in living mammalian cells , which causes a rapid increase in PI ( 4 , 5 ) P2 levels .", "The method uses a chemical compound called “caged PI ( 4 , 5 ) P2” that can be loaded into cells but remains undetected until ultraviolet light is shone on it .", "The ultraviolet light uncages the compound , generating active PI ( 4 , 5 ) P2 in less than one second .", "Walter et al . found that when they uncaged PI ( 4 , 5 ) P2 in this way , the amount of transmitter released by cells increased .", "Combining this with genetic tools , it was possible to investigate which proteins of the release machinery were required for this effect .", "The results suggest that two different types of proteins that interact with PI ( 4 , 5 ) P2 are needed: one must bind PI ( 4 , 5 ) P2 to carry out its role and the other helps PI ( 4 , 5 ) P2 accumulate at the site of vesicle fusion .", "The new method also allowed Walter et al . to show that a fast increase in PI ( 4 , 5 ) P2 triggers a subset of vesicles to fuse very rapidly .", "This shows that PI ( 4 , 5 ) P2 rapidly regulates the release of transmitter .", "Caged PI ( 4 , 5 ) P2 will be useful to study other processes in cells that need PI ( 4 , 5 ) P2 , helping scientists understand more about how signaling lipids control many different events at cellular membranes ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells
elife-07687-v2
[ [ "The life of a cell is characterized by homeostasis , where a constant input of energy is required to balance the synthesis and degradation of the molecules that are essential for viability .", "This is a difficult balancing act under ideal conditions , but cells are often not so lucky .", "Rather , sources of stress ranging from radiation to exogenous chemical and biological agents and even chemical byproducts of primary metabolism can cause damage to critical molecules ( Balaban et al . , 2005 ) .", "To counteract these insults , cells have evolved surveillance mechanisms to detect damage , allowing them time to repair the damage or to otherwise adapt to stress ( Stoecklin and Bukau , 2013 ) .", "For example , the integrity of the genome is important for cellular viability , and it is closely monitored ( Ciccia and Elledge , 2010 ) .", "When DNA damage is detected , one or more cellular checkpoints are activated to allow the cell time to repair the damage , or in the case of multicellular organisms , initiate apoptosis if the damage is too severe to repair ( Jackson and Bartek , 2009 ) .", "Similarly , the spindle assembly checkpoint ensures that mitosis does not proceed until each sister chromatid is productively engaged by microtubules at its kinetochore ( Foley and Kapoor , 2013 ) .", "Defects in either of these quality control ( QC ) surveillance mechanisms lead to genomic instability and frequently drive human pathologies such as cancer ( Sperka et al . , 2012 ) .", "Proteins are equally important to cellular viability .", "In the past several decades , studies in organisms from bacteria to humans have revealed that cells broadly monitor protein QC during translation as well as after mature proteins have entered the proteome ( Chen et al . , 2011; Wolff et al . , 2014 ) .", "Damaged proteins are typically degraded by either the ubiquitin-proteasome system ( UPS ) or through autophagy , although we still lack a clear understanding of how cells detect damaged proteins and how they decide to either refold or degrade these substrates if refolding is not productive ( Wong and Cuervo , 2010 ) .", "However , it is clear that disruptions to the protein-folding environment trigger cellular stress responses that mitigate the effects of the misfolded or unfolded proteins ( Vabulas et al . , 2010; Walter and Ron , 2011; Nargund et al . , 2012 ) .", "Not surprisingly , the inability to mount these stress responses is correlated with decreased cellular fitness , especially under conditions of protein-folding stress ( Morimoto , 2011 ) .", "Failure to refold or degrade unfolded or misfolded proteins is detrimental to the health of cells and organisms and is linked to a multitude of human diseases ( Morimoto , 2008; Knowles et al . , 2014 ) .", "The first of the protein stress responses to be described was the heat shock response ( HSR ) in 1962 , although it was another 20 years before the sensor , the transcription factor HSF1 , was identified ( Ritossa , 1962; Parker and Topol , 1984; Wu , 1984 ) .", "The HSR provided the first molecular mechanism for a cellular response to proteotoxic stress , in this instance presumably triggered by thermally unfolded proteins .", "A decade passed before Walter and colleagues discovered a second unfolded protein response activated by misfolded proteins in the secretory pathway ( Cox et al . , 1993 ) .", "This unfolded protein response , called the erUPR ( Unfolded Protein Response ) , induces the transcription of genes involved in helping proteins fold or otherwise restoring the secretory pathway to a state that is conducive to protein homeostasis ( Walter and Ron , 2011 ) .", "The erUPR also resets protein translation , reducing the synthesis of additional proteins when the environment is not conducive to proper folding ( Harding et al . , 2000 ) .", "More recently , Ron and Haynes have characterized a conceptually similar unfolded protein response in mitochondria ( mitoUPR ) ( Haynes et al . , 2007 ) .", "One common feature of these three protein-folding stress responses is that they induce the transcription of genes involved in mitigating the effects of stress .", "The HSR induces protein chaperones to assist in refolding ( Vabulas et al . , 2010 ) .", "The erUPR induces the production of ER-resident chaperones as well as biosynthetic genes that lead to the expansion of the ER , and the mitoUPR induces the transcription of Hsp60 and other nucleus-encoded mitochondrial genes to maintain homeostasis ( Walter and Ron , 2011; Pellegrino et al . , 2014 ) .", "A second common feature of these stress response pathways is that they are typically studied using extreme , often non-physiological perturbations such as transferring a plate of cultured cells to a higher temperature or treating cells with high concentrations of Dithiothreitol ( DTT ) or other drugs that dramatically disrupt the folding environment in the secretory pathway ( e . g . , tunicamycin or thapsigargin ) or in the mitochondria ( e . g . , ethidium bromide ) ( Oslowski and Urano , 2011; Nargund et al . , 2012 ) .", "All of these perturbations result in the misfolding of hundreds or thousands of different proteins and likely other biomolecules as well .", "Nevertheless , despite the indiscriminate nature of these perturbants , they have been extremely useful in allowing us to discover and study these fundamental stress response pathways .", "Our interest in this area was sparked by a simple question: are mammalian cells sensitive to the appearance of one , specific unfolded protein ?", "And if so , does the materialization of this unfolded protein elicit a response that helps cells eliminate or adapt to this stressor ?", "We used engineered proteins whose folding state can be controlled using a cell-permeable small molecule to rapidly create a single unfolded protein in mammalian cells , and herein , we report that the appearance of unfolded protein elicits a co-ordinated transcriptional response that is distinct from the HSR and the other UPRs .", "Cells that trigger this response are more resistant than unstressed cells when challenged with other protein-folding stressors .", "Finally , the creation of unfolded protein in the nucleus elicits a response that is distinct from the response to unfolded cytosolic protein ." ], [ "We previously developed a technique to regulate the expression level of any protein-of-interest in living cells using a cell-permeable small molecule ( Banaszynski et al . , 2006 ) .", "We started by screening a library of genes encoding mutants of the human FKBP12 protein to identify mutants whose metabolic stability could be regulated by a high-affinity ligand called Shield-1 ( S1 ) .", "These mutants are called destabilizing domains ( DDs ) .", "In the absence of S1 , the DDs are ubiquitylated and degraded by the proteasome , whose processivity ensures the degradation of any fused partner proteins ( Egeler et al . , 2011 ) .", "We have similarly engineered Escherichia coli dihydrofolate reductase ( ecDHFR ) and the ligand-binding domain of the human estrogen receptor to behave as DDs using ligands such as trimethoprim and tamoxifen to regulate protein stability ( Iwamoto et al . , 2010; Miyazaki et al . , 2012 ) .", "Others have begun to apply these tools to study a wide range of biological processes ( Raj et al . , 2013 , 2014; Beck et al . , 2014; Razooky et al . , 2015 ) .", "The DDs are not well folded in the absence of their stabilizing ligands ( Egeler et al . , 2011 ) .", "NMR spectroscopy of several FKBP-derived DDs revealed that their capacity to induce degradation in cells correlates with their degree of unfolding in vitro .", "Complementary urea denaturation studies revealed that DDs sample unfolded conformations to different extents , but all of the DDs are strongly stabilized by the addition of S1 .", "These mechanistic studies suggest that DDs extensively sample an unfolded conformational state when expressed in cells .", "Importantly , this unfolded conformation does not irreversibly aggregate , but rather equilibrates between unfolded and folded states , permitting S1 to stabilize the folded conformation .", "Unfolded DDs are recognized by the cellular QC machinery and targeted for degradation through ubiquitylation ( Egeler et al . , 2011; Chu et al . , 2013 ) .", "However , S1 binding prevents the DDs from sampling the unfolded state , thus , rescuing DDs from degradation .", "Therefore , we use S1 as a small molecule switch to toggle DDs expressed in cells between folded and unfolded states with a high degree of temporal control .", "We took advantage of this unique conditional behavior to characterize the response mounted by mammalian cells to a single unfolded protein species .", "We stably introduced cDNA encoding the FKBP-derived L106P DD fused to superfolder Green Fluorescent Protein ( GFP ) ( DD-GFP ) fusion protein into NIH3T3 fibroblasts ( Pédelacq et al . , 2006 ) .", "Fluorescence-activated cell sorting ( FACS ) was used to select those cells expressing high amounts of DD-GFP fusion protein .", "We maintained the stabilizing S1 ligand in culture media at all times to avoid either stressing the cells with unfolded protein or forcing the cells to adapt to the presence of unfolded protein .", "To initiate the stress , S1 was withdrawn to create the unfolded DD ( Figure 1A ) .", "We harvested cells 45 , 135 , and 405 min following S1 withdrawal , isolated mRNA , and analyzed changes in the transcriptome using mRNA-seq .", "Changes in transcription are a hallmark of the known protein homeostatic stress responses ( de Nadal et al . , 2011 ) . 10 . 7554/eLife . 07687 . 003Figure 1 . Unfolded DD induces transcriptional response .", "( A ) Schematic representation of the strategy used to create a unfolded protein in cells using the destabilizing domain ( DD ) .", "( B–D )", "Changes in transcript levels measured by mRNA-seq are shown for genes that respond strongly to heat shock ( HS ) ( panel B ) , tunicamycin ( panel C ) , and the appearance of the unfolded DD ( panel D ) .", "Transcript levels are shown relative to unperturbed cells .", "FC = fold-change . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 00310 . 7554/eLife . 07687 . 004Figure 1—figure supplement 1 . Unfolded DD responsive genes from mRNA-seq . Changes in transcript levels measured by mRNA-seq are shown for genes that respond strongly to HS ( red ) , tunicamycin ( green ) , and the appearance of the unfolded DD ( black ) .", "Transcript levels are shown relative to unperturbed cells .", "FC = fold-change . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 004 As an experimental control , samples of the DD-GFP-expressing cells were treated with tunicamycin to initiate the UPR in the secretory pathway ( erUPR ) , and cells were harvested at the same timepoints for mRNA-seq analysis .", "As an additional control for the HSR , DD-GFP-expressing cells were shifted from 37°C to 42°C and analyzed by mRNA-seq at 45 and 135 min .", "Heat-shocked cells were not analyzed at 405 min due to significant lethality .", "For both erUPR- and HSR-control samples , S1 was maintained throughout the experiment .", "The DD-GFP-expressing cells were exposed to the three conditions ( unfolded DD , heat shock ( HS ) , and tunicamycin ) , and mRNA-seq was used to quantify changes in transcript levels relative to the reference sample of DD-GFP-expressing cells in which the S1 ligand was not withdrawn .", "To identify any spurious effects that might arise from treating cells with S1 , NIH3T3 cells were transduced with cDNA encoding superfolder GFP alone ( Maynard-Smith et al . , 2007 ) .", "These cells were treated with S1 in the same manner as the DD-GFP-expressing cells , and mRNA-seq was performed at the same timepoints .", "Cells incubated at 42°C showed strong induction of canonical heat-shock genes such as Hsph1 and Hspa1b ( Figure 1B ) , confirming that this exposure causes them to mount an HSR ( Murray et al . , 2004 ) .", "Similarly , treatment with tunicamycin induced expression of known erUPR genes such as Hspa5/Bip and Ddit3/Chop ( Figure 1C ) ( Murray et al . , 2004 ) .", "Because DD degradation depends on ubiquitin , we examined changes in the mRNA levels of the four mammalian ubiquitin genes .", "Unfolded DD caused increased transcript levels of two ubiquitin genes , Ubc and Ubb , whereas expression of the other two ubiquitin-encoding genes was unchanged ( Figure 1D and Figure 1—figure supplement 1 ) .", "HS also led to increased levels of the Ubc mRNA , and all three perturbations caused increases in Ubb transcript levels .", "The mRNA-seq analysis also revealed genes whose mRNA levels were induced exclusively by the unfolded DD and not by the other two perturbations , including Mdm2 and Psmb3 ( Figure 1D ) .", "A complete list of differentially expressed genes is provided in Supplementary file 1 and Supplementary file 2 .", "Quantitative real-time PCR ( RT-qPCR ) was used to validate the results from the mRNA-seq analysis as shown in Figure 2 and Figure 2—figure supplement 1A . 10 . 7554/eLife . 07687 . 005Figure 2 . Cellular transcriptional response is dose-dependent . Unfolded DD was created in two cell lines , which express DD-GFP levels that differ by a factor of three ( Figure 2B ) .", "Cells were harvested at the indicated times following withdrawal of Shield-1 ( S1 ) , and transcript levels were quantified using real-time PCR ( RT-qPCR ) .", "Transcript levels were normalized to GAPDH and expressed relative to unperturbed samples . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 00510 . 7554/eLife . 07687 . 006Figure 2—figure supplement 1 . Lower levels of unfolded DD do not induce the stress response , but a different unfolded protein does .", "( A ) Unfolded DD was created in two NIH3T3 cell lines , which express DD-GFP levels that differ by a factor of three ( Figure 2B ) .", "Cells were harvested 45 , 135 , and 405 min following withdrawal of S1 , and transcript levels were quantified using RT-qPCR .", "Transcript levels were normalized to GAPDH and expressed relative to unperturbed samples .", "( B ) Comparison of fluorescent level between DD-GFP cells that were used for the mRNA-seq analysis in Figure 1 and a cell line expressing lower levels of DD-GFP as quantified by analytical flow cytometry .", "( C ) An NIH3T3 cell line was made expressing the DD derived from Escherichia coli DHFR .", "Unfolded DD was induced by withdrawal of trimethoprim , and transcript levels were quantified using RT-qPCR .", "Transcript levels were normalized to GAPDH and expressed relative to unperturbed samples . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 006 To determine how different doses of unfolded protein impacted the induction of this cellular response , we created a second NIH3T3 cell line expressing the FKBP-derived DD fused to superfolder GFP , but with protein levels approximately threefold lower than the original level ( Figure 2—figure supplement 1B ) .", "We also created a control cell line expressing unfused GFP in which GFP levels were similar to the DD-GFP cell line when stabilized by ligand .", "We cultured both the lower-expressing DD-GFP and the GFP-only cell lines in the presence of S1 and analyzed transcript levels using RT-qPCR following withdrawal of the stabilizing ligand .", "The control cell line expressing only GFP showed no significant changes in transcript levels .", "In contrast , cells expressing lower levels of DD-GFP displayed changes in mRNA levels of the same genes that were affected by higher levels of unfolded DD .", "Moreover , the degree of mRNA induction correlated with the level of protein expression ( Figure 2 , Figure 2—figure supplement 1A ) , establishing that lower levels of unfolded DD induced less pronounced changes in mRNA levels .", "In order to test whether this transcriptional response is unique to the unfolded FKBP protein , we created a cell line expressing a DD derived from the E . coli DHFR protein fused to superfolder GFP ( Iwamoto et al . , 2010 ) .", "Upon withdrawal of the stabilizing ligand , trimethoprim for this DD , we characterized the cellular response to the unfolded ecDHFR-derived DD using RT-qPCR ( Figure 2—figure supplement 1C ) .", "The unfolded ecDHFR-derived DD elicited changes in mRNA levels similar to the changes induced by unfolded FKBP , although the extent of the changes was less pronounced .", "We used hierarchical clustering to compare the cellular responses to all three perturbations ( Figure 3A ) , revealing one cluster of genes whose transcript levels are induced by the unfolded DD but not by the HSR or the erUPR ( asterisk in Figure 3A ) .", "Analysis of this group revealed enrichment in genes involved in ‘intracellular protein traffic’ ( p-value = 3 . 7e-6 ) and ‘cell cycle’ ( p-value = 8 . 4e-6 ) when analyzed for biological process GO terms ( Huang et al . , 2008 , 2009 ) .", "Pathway analysis of the same gene cohort identified the ‘ubiquitin-proteasome pathway’ ( p-value = 0 . 025 ) and the ‘p53 pathway’ ( p-value = 0 . 041 ) as being enriched ( Supplementary file 3 ) . 10 . 7554/eLife . 07687 . 007Figure 3 . Transcriptome profiles of unfolded DD compared to HS and ER stress .", "( A ) NIH3T3 cells expressing DD-GFP in the presence of S1 were perturbed by HS ( 42°C ) , treatment with 5 μM tunicamycin , or by withdrawal of S1 to create unfolded DD .", "Cells were harvested 45 , 135 , and 405 min following these perturbations , and transcript levels were quantified using mRNA-seq .", "Genes responding to any of the three perturbations ( false discovery rate [FDR] <0 . 05 ) were hierarchically clustered to examine the relationships between the expression patterns .", "Each row represents a gene and each column is an experimental replicate .", "Transcript changes are represented as log2 fold-changes relative to unperturbed samples .", "( B ) Venn diagram depicting significantly induced and repressed genes ( FDR <0 . 05 ) from each of the three treatments: unfolded DD , HS , and tunicamycin treatment ( Tu ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 007 In response to the unfolded DD , NIH3T3 cells up-regulated the expression of 0 , 63 , and 759 genes 45 , 135 , and 405 min , respectively , following withdrawal of S1 ( false discovery rate [FDR] <0 . 05 ) .", "The mRNA levels of 0 , 69 , and 909 genes were significantly reduced following S1 withdrawal ( Supplementary file 1 and Supplementary file 2 ) .", "To put the magnitude of this response in perspective , NIH3T3 cells responded to HS by up-regulating the mRNA levels of 186 and 791 genes and down-regulating 26 and 579 genes at 45 and 135 min , respectively , following application of stress ( Supplementary file 1 and Supplementary file 2 ) .", "Using tunicamycin to induce the erUPR , we observed increases in 586 , 211 , and 2403 and decreases in 349 , 184 , and 2659 transcript levels 45 , 135 , and 405 min following treatment ( Supplementary file 1 and Supplementary file 2 ) .", "Given the potential for temporal differences in the cellular response to the different sources of proteotoxic stress , genes from all timepoints for each stress were grouped as either up-regulated or down-regulated , and the relationships among the three data sets are shown in Venn diagram format in Figure 3B .", "Focusing on the 534 genes that are up-regulated in response to the unfolded DD but not the other two perturbations , gene enrichment analysis revealed that ‘proteolysis’ ( p-value = 2 . 1e-4 ) and ‘protein metabolism/modification’ ( p-value = 1 . 4e-3 ) are over-represented in the category of biological process .", "Pathway analysis revealed that the ‘ubiquitin-proteasome pathway’ ( p-value = 9 . 3e-6 ) and the ‘p53 pathway’ ( p-value = 0 . 021 ) are over-represented ( Supplementary file 3 ) .", "Looking more closely at the responses of individual genes to the perturbations , we noticed that some mRNA levels rise rapidly whereas others are induced later .", "We then performed gene enrichment analysis for the unfolded DD samples using the list of earlier ( mRNA levels rise significantly by 135 min ) and later ( 405 min ) responders .", "Analysis of the 63 early-response genes highlighted ‘protein folding’ ( p-value = 7 . 5e-4 ) and ‘stress response’ ( p-value = 2 . 1e-3 ) as the biological processes most significantly over-represented ( Supplementary file 4 ) .", "The same analysis for the late-response genes revealed ‘protein folding’ , ‘stress response’ , ‘protein metabolism and modification’ , and ‘proteolysis’ as over-represented biological processes ( p-values = 5 . 7e-6 , 8 . 0e-6 , 9 . 4e-6 , and 4 . 9e-4 , respectively , Supplementary file 4 ) .", "When we analyzed these two gene groups for biological pathways , the early-response group was over-represented for the ‘p53 pathway’ and ‘apoptosis-signaling pathway’ ( p-values = 1 . 2e-6 and 1 . 1e-3 , respectively , Supplementary file 4 ) .", "The late-response genes were enriched in ‘ubiquitin-proteasome pathway’ ( p-value = 1 . 2e-4 , Supplementary file 4 ) .", "When analyzed for annotations related to molecular function , both groups revealed enrichment of ‘Hsp70 family chaperone’ , ‘chaperone’ , and ‘microtubule family cytoskeletal protein’ ( p-values = 5 . 8e-4 , 7 . 8e-4 , and 3 . 3e-5 , respectively , Supplementary file 4 ) .", "We next compared the list of all genes induced by the unfolded DD against the list of all genes induced by either HS or by tunicamycin ( Figure 4A ) .", "Only 105 genes overlapped between the 787 induced by unfolded DD and the 826 induced by HS ( p-value = 3 . 00e-7 by hypergeometric comparison ) , suggesting that this degree of overlap is unlikely to have arisen by chance .", "Conversely , a similar comparison of the genes induced by unfolded DD with the erUPR genes induced by tunicamycin suggests that there is not significant overlap between the two cellular responses ( Figure 4A ) . 10 . 7554/eLife . 07687 . 008Figure 4 . Comparison of unfolded DD stress with known stress responses .", "( A ) Venn diagram showing pairwise comparisons of the genes induced by unfolded DD ( gray ) with the genes induced by the HS response ( HSR ) ( red ) , the erUPR ( green ) , and the p53-mediated response to DNA damage ( blue ) .", "The list of p53-induced genes is from Kenzelmann Broz et al . ( 2013 ) .", "The p-values are calculated by hypergeometric test .", "( B–E )", "Heatmap representations of genes responding to unfolded DD or HS .", "The 50 most significantly induced genes are shown for each condition to facilitate comparison with the other perturbations and timepoints .", "The genes are sorted by statistical significance for the unfolded DD at 135 min ( panel B ) and 405 min ( panel C ) and for HS at 45 min ( panel D ) and 135 min ( panel E ) .", "( F ) Heatmap showing how 39 genes encoding proteasome subunits respond to either unfolded DD or HS .", "For panels B–F , black indicates no significant changes in transcript levels . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 00810 . 7554/eLife . 07687 . 009Figure 4—figure supplement 1 . Comparison of the unfolded DD stress with proteasome inhibition . Venn diagram showing pairwise comparisons of the genes induced by unfolded DD ( gray ) with the genes induced by proteasome inhibition using bortezomib ( brown ) The p-values are calculated by hypergeometric test . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 009 The appearance of the ‘p53 pathway’ in our gene expression analysis led us to perform a comparison between the list of 790 genes induced by the unfolded DD and the 508 genes significantly up-regulated by p53 in response to DNA damage ( Figure 4A ) ( Kenzelmann Broz et al . , 2013 ) .", "This analysis revealed 90 genes in common ( p-value = 1 . 07e-13 ) .", "Considered together , these results suggest that the acute appearance of unfolded DD elicits a co-ordinated transcriptional response in NIH3T3 cells .", "Gene expression analysis highlights biological processes and biological pathways associated with protein homeostasis and stress responses as over-represented in the lists of responsive genes .", "Furthermore , the cellular response to unfolded DD is distinct from both the known HSR and the erUPR in the secretory pathway .", "Similarly , since the ‘ubiquitin-proteasome pathway’ appears in our gene expression analysis , we also treated cells expressing DD-GFP with the proteasome inhibitor , bortezomib , followed by mRNA-seq analysis at the same times as the other perturbations .", "Treatment with bortezomib induced the expression of 583 genes , with 65 in common with the unfolded DD perturbation .", "The overlap between these two sets of induced genes is not statistically significant ( p-value = 2 . 42e-1 ) , suggesting that the unfolded DD creates a cellular stress that is distinct from simple proteasome inhibition ( Figure 4—figure supplement 1 , Supplementary file 5 ) .", "We were concerned that the inclusion of statistically less significant genes induced by both perturbations made the cellular response to unfolded DD and the HSR appear to be dissimilar .", "To explore this possibility , we plotted the 50 genes that were most significantly induced by either the unfolded DD or HS at both timepoints ( Figure 4B–E ) .", "Looking at the 50 most significant genes induced by unfolded DD at the early timepoint , we find 28 of these genes are also significantly changed at the later timepoint , and all of these are induced ( Figure 4B ) .", "However , comparison of the top 50 early genes induced by the unfolded DD with the HSR reveals only two significantly induced genes in common at the early HSR timepoint and 15 genes at the late HSR timepoint .", "A similar comparison of the 50 most significant genes induced by the unfolded DD at the late timepoint ( Figure 4C ) reveals only three in common with the early HSR timepoint and 15 in common with the late timepoint ( 13 induced and 2 reduced ) .", "To make the comparison in the reverse sense , comparison of the 50 most significantly induced genes at the early HSR timepoint ( Figure 4D ) reveals 48 in common with the late timepoint ( all of them induced ) .", "However , comparison of the top 50 early HSR responders with the early unfolded-DD timepoint finds only 8 genes in common ( 3 induced and 5 repressed ) , and comparison with the late unfolded-DD timepoint reveals 13 in common ( 2 induced and 11 repressed ) .", "Likewise , comparison of the 50 most significantly induced HSR genes at the late timepoint ( 135 min ) with the genes induced by the unfolded DD finds 8 in common ( 7 induced and 1 repressed ) with the earlier timepoint and 13 in common ( 10 induced and 3 repressed ) with the later timepoint ( Figure 4E ) .", "This lack of concordance between the genes induced by the unfolded DD and the genes induced by HS is suggestive , but certainly not conclusive , that the cellular-signaling pathways used to respond to these two perturbations are not identical .", "If the HSR shared common signaling mechanisms with the cellular response to unfolded DD , then one would expect to see similar changes in gene-expression profiles ( Brannon et al . , 2010 ) .", "When analyzing the responsive genes in an unbiased fashion , relying solely on the statistical significance of the observed changes , we see no such concordance ( Figure 4B–E ) .", "Another way to approach the issue is to look at groups of genes that are known to share a common function .", "For example , cells experiencing proteotoxic stress might respond by producing more proteasomes to rid the cell of unfolded protein ( Vilchez et al . , 2013 ) .", "When we looked at 39 genes that encode proteasome subunits , we found that the appearance of the unfolded DD causes changes in 16 of these genes , all of them up-regulated ( Figure 4F ) .", "The HSR induces changes in only 3 genes , all of them down-regulated .", "The limited degree of overlap between these two sets of induced genes leads us to conclude that these two cellular stress responses are mechanistically distinct , though some degree of overlap cannot be excluded .", "We quantified cellular levels of p53 protein following withdrawal of S1 by immunoblotting .", "The levels of p53 rose at 45 and 135 min , but p53 levels were elevated but falling by 405 min ( Figure 5A ) .", "This result is consistent with the transcript analysis from both mRNA-seq and RT-qPCR in which the mRNA levels of p53 target genes such as Mdm2 and Cdkn1a were strongly induced at 135 min but were lower by 405 min following the appearance of the unfolded DD ( Figure 2 ) . 10 . 7554/eLife . 07687 . 010Figure 5 . p53 protein accumulates upon appearance of the unfolded DD .", "( A ) S1 was withdrawn from NIH3T3 cells expressing the DD-GFP fusion protein for the indicated times , and equal amounts of total protein were immunoblotted with antibodies against p53 or GAPDH .", "( B ) S1 was withdrawn from cells expressing DD-GFP , and the cells were fixed at the indicated times .", "The levels of p53 and GFP were quantified using indirect immunofluorescence , and Hoechst 33 , 342 was used to image nuclei .", "( C ) Quantification of the p53 signal observed in whole cells , cytoplasm , or nuclei from immunofluorescence data .", "The Welch test was used to calculate p-values .", "( D ) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against p53 , cultured for 2 days , then RNA was harvested for RT-qPCR analysis 135 min following withdrawal of S1 .", "Transcript levels for the indicated genes were normalized to GAPDH and expressed as fold-change relative to unperturbed cells .", "siNT = non-targeting control . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 01010 . 7554/eLife . 07687 . 011Figure 5—figure supplement 1 . Investigating potential sources of p53 activation .", "( A ) S1 was withdrawn from cultures of NIH3T3 cells stably transduced with the DD-GFP fusion protein , and the rate of DD-GFP degradation was quantified using analytical flow cytometry .", "( B ) Unfolded DD was induced in cells expressing DD-GFP by S1 withdrawal , and cells were harvested at the indicated times .", "Lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies .", "Tubulin is the loading control .", "Methyl methanesulfonate ( MMS ) was used as the positive control for DNA damage .", "( C ) Unfolded DD was induced in NIH3T3 cells that were treated with vehicle or with inhibitors of the ATM or ATR kinases .", "RNA was harvested at 135 min and analyzed using RT-qPCR .", "ATM inhibitor: 3 μM KU-55933 and the ATR inhibitor: 2 μM 4-{4-[ ( 3R ) -3-methylmorpholin-4-yl]-6-[4- ( methylsulfonyl ) piperidin-4-yl]-pyrimidin-2-yl}-1H-indole .", "( D ) Unfolded DD was induced in NIH3T3 cells stably expressing DD-mCherry that were treated with vehicle or CM-H2DCFDA .", "2 hr of treatment with 10 μM MG132 was the positive control . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 01110 . 7554/eLife . 07687 . 012Figure 5—figure supplement 2 . Involvement of other transcription factors .", "( A ) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against HSF1 , cultured for 2 days , then RNA was harvested for RT-qPCR analysis 135 min following withdrawal of S1 .", "Transcript levels for the indicated genes were normalized to GAPDH and expressed as fold-change relative to unperturbed cells .", "siNT = non-targeting control .", "( B ) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against NRF2 , cultured for 2 days , then RNA was harvested for RT-qPCR analysis 135 min following withdrawal of S1 .", "Transcript levels for the indicated genes were normalized to GAPDH and expressed as fold-change relative to unperturbed cells .", "siNT = non-targeting control .", "( C ) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against p53 , cultured for 2 days , then RNA was harvested for RT-qPCR analysis to quantify the RNA level of p53 .", "( D ) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against HSF1 , cultured for 2 days , then RNA was harvested for RT-qPCR analysis to quantify the RNA level of HSF1 .", "( E ) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against NRF2 , cultured for 2 days , then RNA was harvested for RT-qPCR analysis to quantify the RNA level of NRF2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 012 To gain further insight into the role of p53 in response to unfolded DD , we used immunofluorescence analysis to quantify p53 levels and subcellular localization in the DD-GFP cells .", "Before withdrawal of S1 , p53 levels were low and DD-GFP levels were high ( Figure 5B ) .", "Levels of p53 rose by 45 min following the appearance of the unfolded DD and remained elevated through 405 min .", "Consistent with FACS analysis ( Figure 5—figure supplement 1A ) , levels of DD-GFP remained relatively high through 135 min but significantly lower by 405 min .", "This experiment also shows that p53 is enriched in nuclei upon appearance of the unfolded DD , and statistical analysis of the immunofluorescence images supports these conclusions that p53 levels significantly rise and that p53 localizes to the nucleus ( Figure 5C ) .", "It is possible that p53 levels rise as a result of an indirect secondary effect caused by the unfolded DD .", "DNA damage is well known to activate p53 ( Kenzelmann Broz et al . , 2013 ) , so we triggered the appearance of the unfolded DD and then immunoblotted cell lysates for various markers of DNA damage including phospho-KAP1 , phospho-RNA32 , phospho-Chk1 , and γ-H2AX ( Figure 5—figure supplement 1B ) .", "None of these hallmarks of DNA damage were significantly induced , suggesting that the DD-GFP-expressing cells were not experiencing DNA damage .", "ATM and ATR are kinases that are essential for cells to respond to DNA damage ( Cimprich and Cortez , 2008 ) , so we used chemical inhibitors of ATM and ATR to assess the potential role of these kinases in the cellular response to the appearance of unfolded DD ( Figure 5—figure supplement 1C ) ( Hickson et al . , 2004; Couch et al . , 2013 ) .", "Neither inhibitor affected the cellular response to the unfolded DD , suggesting that DNA damage signaling mediated by ATM or ATR is not responsible for the increased levels of p53 .", "Oxidative stress can also induce p53 , so we used CM-H2DCFDA as a sensor for reactive oxygen species in DD-mCherry-expressing cells upon withdrawal of S1 .", "We observed no significant signal by analytical flow cytometry upon creation of the unfolded DD ( Figure 5—figure supplement 1D ) .", "We conclude that cellular changes in p53 levels are a response to the unfolded DD and not to indirect sequelae such as DNA damage or reactive oxygen species .", "To further validate the role of p53 in the cellular response to unfolded DD , we used RNA interference to knockdown p53 in DD-GFP-expressing cells .", "We then performed RT-qPCR to quantify changes in mRNA levels for various target genes following withdrawal of S1 .", "Knockdown of p53 diminished the DD-dependent induction of p53 targets such as Mdm2 , Cdkn1a , and Eda2r ( Figure 5D ) .", "However , increased mRNA levels were not affected for some of the responsive genes including Ubc , Zfand2a , and Gclm ( Figure 5D ) .", "These results suggest the involvement of other factors during the response to unfolded DD .", "Since we see induction of HS proteins and oxidative stress genes , we performed used RNAi to knockdown Hsf1 and Nrf2 , two transcription factors that mediate cellular responses to HS and oxidative stress .", "Cells were treated with siRNA reagents to knockdown these two genes and RT-qPCR was used to quantify mRNA levels of two dozen genes that are induced by the unfolded DD .", "Although most of the responsive genes were unaffected by knockdown of HSF1 and NRF2 , loss of these factors did affect the induction of some responsive genes .", "Loss of Hsf1 attenuated the induction of several HS proteins ( Hspa1b , Hsph1 , Dnajb1 ) and loss of Nrf2 affected the induction of oxidative stress genes ( Hmox , Osgin , Sqstm1/p62 ) , respectively .", "These studies are far from comprehensive , but we can conclude that the cellular response to unfolded DD involves several stress-responsive transcription factors ( Figure 5—figure supplement 2 ) .", "Cells have evolved protein QC surveillance mechanisms to monitor discrete cellular compartments such as the secretory pathway ( erUPR ) and mitochondria ( mitoUPR ) , so we considered the possibility that the unfolded DD may elicit different transcriptional responses if unfolded protein were to appear in the nucleus or in the cytosol .", "We tagged the DD-GFP fusion protein with a nuclear localization signal and created an NIH3T3 cell line that expressed the DD-GFP fusion protein primarily in the nucleus .", "A complementary cell line was created , expressing a DD-GFP-mCherry fusion protein that was tagged with a nuclear export sequence .", "FACS-based cell sorting was used to select cells that express both the nucleus-localized and the nucleus-excluded DD-GFP at similar levels ( Figure 6—figure supplement 1A ) .", "Fluorescence micrographs of both cell lines treated with S1 revealed that the targeted DD-GFP fusion proteins are present primarily in their expected compartments ( Figure 6A ) .", "Analytical flow cytometry was used to quantify the rates of DD-GFP degradation upon withdrawal of S1 , revealing that the nuclear DD-GFP was degraded with faster kinetics than the cytosolic DD-GFP ( Figure 6—figure supplement 1B ) . 10 . 7554/eLife . 07687 . 013Figure 6 . Nuclear or cytosolic unfolded proteins elicit distinct responses .", "( A ) Images of live NIH3T3 cells stably expressing nuclear DD-GFP or cytosolic DD-GFP .", "Hoechst 33 , 342 was used to image nuclei .", "( B , C )", "Unfolded DD was induced by withdrawal of S1 , and transcript levels of specific genes were quantified using RT-qPCR at the indicated times .", "Genes that respond more strongly to nuclear unfolded DD are shown in panel B , and genes that are induced by cytosolic unfolded DD are shown in panel C . Transcript levels were normalized to GAPDH and expressed relative to unperturbed samples . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 01310 . 7554/eLife . 07687 . 014Figure 6—figure supplement 1 . Additional comparisons of nuclear vs cytosolic unfolded protein stress .", "( A ) Comparison of fluorescent level between nuclear DD cells and cytosolic DD cells by analytical flow cytometry .", "( B ) Flow cytometry data of the kinetics of nuclear DD-GFP and cytosolic DD-GFP degradation .", "NIH3T3 cells stably transduced with the DD-GFP fusions were cultured with S1 1 μM .", "The S1 was withdrawn and the decrease in fluorescence was monitored .", "( C ) S1 was withdrawn from cultures of NIH3T3 cells stably expressing the nuclear or the cytosolic DD-GFP fusion protein , and transcript levels of the designated genes were quantified using RT-qPCR .", "Transcript levels were normalized to GAPDH and expressed relative to unperturbed samples . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 014 Unfolded DD was created in each cell line , and cells were harvested at 45 , 135 , and 405 min for transcript analysis using RT-qPCR .", "The mRNA levels of several genes , predominantly known p53 targets such as Cdkn1a , Gadd45a , and Mdm2 respond to the appearance of either the nuclear or cytosolic unfolded DD ( Figure 6B , Figure 6—figure supplement 1C ) .", "Other genes including DNA polymerase kappa and Trp53inp1 are more sensitive to the unfolded DD in the nucleus .", "Conversely , we observed that transcript levels of many genes are relatively insensitive to the appearance of unfolded DD in the nucleus but are strongly induced by cytosolic unfolded DD ( Figure 6C , Figure 6—figure supplement 1C ) .", "Genes falling into this group include chaperones ( Hsp90aa1 , Hspa1b , Hspa8 , Dnajb1 ) , proteasome subunits , known protein QC surveillance proteins ( Bag3 , Zfand2a , Gclm , p62/Sqstm1 ) , and two of the four mammalian ubiquitin genes ( Ubb and Ubc ) .", "In order to assess how cellular fitness might be affected by the appearance of the unfolded DD , we co-cultured the DD-GFP-expressing cells with unmodified NIH3T3 cells ( Sancho et al . , 2013 ) .", "We created unfolded DD by withdrawing S1 for 45 , 135 , or 405 min .", "Cells were co-cultured for an additional 17 hr in S1-containing media and then analyzed by flow cytometry to quantify the ratio of DD-GFP-expressing cells to control cells ( Figure 7A ) .", "We observed that the amount of time cells were exposed to the unfolded DD inversely correlated with replicative fitness .", "Creating unfolded DD for a period as brief as 45 min caused a significant and reproducible reduction in the population of DD-GFP cells , and 405 min of exposure to unfolded DD caused the original ratio of 61:39 ( DD-GFP:control cells ) to fall to 51:49 .", "Cells that express threefold lower amounts of the DD do not experience this replicative fitness defect ( Figure 7A ) .", "In attempt to identify the source of this replicative fitness defect , we used RNAi to knockdown p53 , Hsf1 , and Nrf2 in separate populations of cells .", "Loss of p53 reduces the replicative phenotype exhibited by cells challenged with the unfolded DD , whereas loss of Hsf1 or Nrf2 did not reveal any detectable effect in cycling cells ( Figure 7—figure supplement 1A–C ) .", "Thus , there is a replicative cost for cells challenged with the unfolded DD , and this response appears to be mediated , at least in part , by p53 . 10 . 7554/eLife . 07687 . 015Figure 7 . The cUPR provides protection against subsequent proteotoxic stress .", "( A ) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1 .", "The stabilizing S1 was withdrawn for 45 , 135 , or 405 min and then re-administered to the cells for an additional 17 hr .", "Analytical flow cytometry was used to quantify the relative populations of each cell type .", "The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP that induce the unfolded DD stress response ( ufDD , black ) as well as the cell line expressing threefold lower levels of DD-GFP ( low ufDD , gray ) as shown in Figure 2B .", "( B ) Cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in a 24-well plate .", "One group of cells was not exposed to stress from either the unfolded DD or 30 μM aqueous sodium arsenite ( Ctl ) .", "The second group was exposed to arsenite only ( ARS ) .", "The third group experienced the unfolded DD by withdrawal of S1 ( shown as ufDD ) , and the fourth group was exposed to the unfolded DD followed by arsenite stress ( ufDD + ARS ) .", "The relative populations of each cell type were quantified using analytical flow cytometry , with the control sample as the reference point for cells expressing DD-GFP .", "Expected = predicted fraction of DD-GFP cells assuming the unfolded DD and arsenite are additive .", "*** p-value < 0 . 001 by Welch's test , **** p-value < 0 . 0001 by Welch's test . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 01510 . 7554/eLife . 07687 . 016Figure 7—figure supplement 1 . The effect of transcription factors on cell growth .", "( A ) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1 .", "The stabilizing S1 was withdrawn for 45 , 135 , or 405 min and then re-administered to the cells for an additional 17 hr .", "Analytical flow cytometry was used to quantify the relative populations of each cell type .", "The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of p53 ( blue ) as well as the cell line experiencing non-target siRNA ( siNT , black ) .", "( B ) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1 .", "The stabilizing S1 was withdrawn for 45 , 135 , or 405 min and then re-administered to the cells for an additional 17 hr .", "Analytical flow cytometry was used to quantify the relative populations of each cell type .", "The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of HSF1 ( red ) as well as the cell line experiencing non-target siRNA ( siNT , black ) .", "( C ) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1 .", "The stabilizing S1 was withdrawn for 45 , 135 , or 405 min and then re-administered to the cells for an additional 17 hr .", "Analytical flow cytometry was used to quantify the relative populations of each cell type .", "The experiment was performed in triplicate for populations of cells expressing high levels of DD-GFP experiencing siRNA of NRF2 ( green ) as well as the cell line experiencing non-target siRNA ( siNT , black ) .", "( D ) Cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in a 24-well plate .", "One group of cells was not exposed to stress from either the unfolded DD or 10 μM MG132 ( Ctl ) .", "The second group was exposed to MG132 only ( MG132 ) .", "The third group experienced the unfolded DD by withdrawal of S1 ( shown as ufDD ) , and the fourth group was exposed to the unfolded DD stress followed by proteotoxic stress ( ufDD + MG132 ) .", "The relative populations of each cell type were quantified using analytical flow cytometry , with the control sample as the reference point for cells expressing DD-GFP .", "Expected = predicted fraction of DD-GFP cells assuming unfolded DD and MG132 are additive .", "** p-value < 0 . 05 by Welch's test , ** p-value < 0 . 01 by Welch's test , *** p-value < 0 . 001 by Welch's test . DOI: http://dx . doi . org/10 . 7554/eLife . 07687 . 016 We used a similar co-culture experiment to examine whether the cellular response to the appearance of unfolded DD protects cells from the adverse effects of unfolded protein .", "Cells expressing DD-GFP were mixed with NIH3T3 cells and co-cultured in the presence of S1 .", "The S1 was then either withdrawn for 5 hr or S1 was maintained in the media .", "At the 5-hr timepoint , S1 was re-administered , and the cells were co-cultured for an additional hour to allow recovery from the unfolded DD .", "At this point , the co-cultured cells were then treated with either vehicle or with 30 μM sodium arsenite ( Yun et al . , 2008 ) , cultured overnight , and analyzed at 24 hr ( post appearance of unfolded DD ) by flow cytometry .", "Both proteotoxic stresses affect the replicative potential of cells expressing the DD-GFP fusion protein .", "If the effects of the two stresses were additive , one could estimate an expected ratio of the DD-GFP:control cells ( Figure 7B ) .", "Cells that responded to the appearance of the unfolded DD were strongly cross-protected from arsenite toxicity .", "A similar effect was seen when a different proteotoxic stress , the proteasome inhibitor MG132 , was used to challenge the co-cultured cells ( Figure 7B , Figure 7—figure supplement 1D ) .", "These results suggest that the transcriptional response induced by the appearance of the unfolded DD provides a fitness benefit by protecting cells against other forms of proteotoxic stress ( Calabrese et al . , 2007; Morimoto , 2011 ) ." ], [ "Our understanding of the cellular responses to protein-folding stress has been achieved through the use of relatively blunt experimental perturbations .", "Culturing eukaryotic cells at elevated temperatures or in the presence of hydrogen peroxide affects not only proteins but virtually every molecule in the cell ( Zunino et al . , 2001 ) .", "Nevertheless , these perturbations have enabled many insights into the HSR and other protein-folding stress responses .", "Studies of the erUPR in the secretory pathway rely heavily upon the use of small molecules such as tunicamycin , thapsigargin , or dithiothreitol , and ethidium bromide is used to rapidly stress the protein-folding environment within mitochondria .", "Genetic methods to introduce stress are more specific but not as acute .", "Transgenes encoding folding-defective mutants of cellular proteins ( e . g . , CPY* ) have been valuable for studies of protein folding and trafficking ( Knop et al . , 1996 ) .", "However , there are few examples of mutant proteins being used to activate the HSR , despite the conventional wisdom that the HSR responds to unfolded proteins ( Wolff et al . , 2014 ) .", "Mutant proteins that are prone to misfold affect cellular fitness when constitutively expressed ( Geiler-Samerotte et al . , 2011 ) .", "However , the chronic nature of these genetically encoded stressors allows cells to adapt to their presence , rendering these perturbations unsuitable for studies of cellular responses that are induced by acute stress .", "Given the success achieved using the acute , small molecule perturbants described above , we were optimistic that the ability to rapidly create a specific unfolded protein would illuminate cellular surveillance mechanisms that otherwise would be difficult to observe ( Chu et al . , 2013 ) .", "The DDs we employed possess many attributes that make them attractive as conditional unfolded proteins .", "DDs are genetically encoded , providing opportunities to control subcellular localization .", "Additionally , DDs are maintained in a metabolically stable , folded conformation when bound to their stabilizing ligands , but withdrawal of the stabilizing ligand initiates the rapid unfolding of the DD ( Egeler et al . , 2011 ) .", "Considered together , these features endow the DDs with the specificity inherent in genetic approaches combined with the temporal advantages derived from the use of small molecule pharmacology ( Rakhit et al . , 2014 ) .", "The cellular response to unfolded DD shares some functional similarities with the HSR and the erUPR .", "The transcriptional response is dose-dependent in the sense that lower levels of unfolded DD trigger a weak response or no response relative to higher levels of unfolded protein .", "Presumably , cells do not need to respond to the unfolded DD if they can degrade it rapidly , similar to the erUPR is not activated until endoplasmic reticulum-associated degradation is either overwhelmed orincapacitated ( Travers et al . , 2000 ) .", "Additionally , as is observed with other stress responses , cells that mount the response to the unfolded DD pay a replicative fitness cost relative to cells that do not .", "However , a benefit accrues to cells that initiate this transcriptional response in the form of cross-protection from other forms of proteotoxic stress .", "Despite these similarities , the cellular response to the unfolded DD and the HSR appears to be mechanistically distinct , although it is important to recall the challenges inherent in such a comparative analysis .", "First , the perturbations are fundamentally different .", "The discrete unfolded DD is qualitatively and quantitatively different from the variety of changes wrought by HS .", "HS will cause many proteins to unfold , and the overall amount of unfolded protein will likely be higher than can be attained using the unfolded DD .", "Additionally , HS causes hundreds or thousands of different proteins to unfold .", "This variety of stressors may present unique challenges for cellular QC surveillance pathways .", "Finally , HS affects all cellular molecules including nucleic acids and membranes .", "Temporal differences present a second potential challenge for such a comparison .", "Given that the perturbations are fundamentally different in nature and intensity , the cellular response to two different stresses might begin at different times relative to the experimental initiation of the perturbations .", "Taken to its logical limit , even two identical cellular responses that are compared out-of-phase with each other ( i . e . , initiated at different times ) may appear unrelated by gene expression analysis .", "So , lists of responsive genes should include any gene that responds to a stimulus in a statistically significant manner at any of the measured timepoints .", "Even using such inclusive definitions for all three perturbations we employed in this study , the response to unfolded DD has little in common with the erUPR ( Figure 4A ) .", "Genes that facilitate protein homeostasis in the secretory pathway may not necessarily be beneficial for protein folding in the cytosol or nucleus , so one might reasonably expect these two responses to be unrelated .", "Conversely , given the shared biological goals of the cellular response to unfolded DD and the HSR , one might expect to find overlap in the lists of responsive genes .", "Gene expression analysis suggests that is the case: the number of induced genes found in common for both perturbations , though modest , is greater than would be expected by chance ( Figure 4A ) .", "Not surprisingly , several chaperone genes are induced by both the unfolded DD and the HSR including Hsp90ab1 , Hspa1b , Hsph1 , Hspa8 , Bag3 , Dnajb1 , Dnajb2 , St13/Hip , and Hspb8 .", "However , many stress response genes are uniquely induced by the unfolded DD ( Supplementary file 2 ) .", "For example , the appearance of unfolded DD , but not the HSR , induces the expression of genes involved in aggresome formation such as Hdac6 , Vcp/p97 , several dynenin genes ( Dync1h1 , Dync1li1 , Dynll2 , Dctn2 , Dctn3 , and Dctn4 ) , and the minus end-directed kinesins Kif3c and Kifc1 .", "The unfolded DD uniquely induces genes involved in autophagy including Ulk1 , Tsc2 , Wdr45 , Atg2a and Atg2b , Vps11 , Gabarapl1 and Gabarapl2 , and cathepsins A and D . Many of the genes encoding proteasome subunits are induced by the unfolded DD but not the HSR ( Figure 4F ) , and several other UPS genes are uniquely up-regulated by the unfolded DD including ubiquitin ligases ( Keap1 , Ube4a , Ube4b , Huwe1 , Klhl21 , Ube2o , Ubr2 , Ubr3 , Ubr4 , Herc2 , Mdm2 ) and deubiquitinating enzymes ( Usp14 , Usp19 , Usp20 , Usp27x , Usp3 , Usp5 ) .", "Finally , many apoptosis-related genes are uniquely induced by the appearance of unfolded DD including Fas , Wrap53 , Apaf1 , Tnfrsf10b/DR5 , Eda2r , Parp2 , Parp4 , and Bak1 .", "One key difference between these two stress responses is the presence of a p53-dependent arm in the cellular response to unfolded DD .", "The involvement of p53 may explain many genes uniquely induced by unfolded DD , for example , the selective induction of the genes encoding proteasome subunits .", "However , mRNA-seq and ChIP-seq analyses of p53 target genes implicate only three of these genes ( Psmb3 , Psmc3 , Psmd13 ) as direct p53 targets , out of the 16 that are uniquely induced by the appearance of unfolded DD ( Kenzelmann Broz et al . , 2013 ) .", "Indeed , RNAi-mediated knockdown of p53 demonstrated that this transcription factor is involved in the induction of some , but not all , of the genes induced by unfolded DD ( Figure 5D ) .", "Similar findings were observed when Hsf1 or Nrf2 were knocked down .", "This clear difference between the two cellular stress responses , not explained by these three stress-response pathways , suggests the involvement of at least one additional ( not yet identified ) arm of the cellular response to unfolded DD that is not shared by the HSR .", "The biophysical properties of p53 may explain its absence from the HSR .", "At 37°C , the p53 core domain unfolds in vitro with a half-time of only 9 min , so it is possible that p53 is unable to maintain its folded , functional conformation when cells are exposed to the higher temperatures used to initiate the HSR ( Friedler et al . , 2003 ) .", "If one were able to generate large amounts of unfolded protein at 37°C ( i . e . , simulate the HSR under standard culture conditions ) , p53 might be involved in the response .", "Indeed , treatment of mammalian cells with proteasome inhibitors , which causes misfolded proteins to accumulate , leads to increased p53 levels and the induction of p53-dependent proteins such as Mdm2 and Cdkn1/p21 ( Lopes et al . , 1997; Pandit and Gartel , 2011 ) .", "Targeting the folded DD to either the nucleus or the cytosol allowed us to create unfolded protein in a specific cellular compartment .", "This unique experimental feature revealed that a subset of the genes induced by unfolded DD respond more strongly to nucleus-localized unfolded DD , whereas other responsive genes are more sensitive to unfolded cytosolic protein .", "The creation of unfolded protein in the nucleus induces primarily p53-dependent genes , implying that p53 ( either directly or indirectly ) may be responsible for protein QC surveillance in the nucleus .", "Gardner has characterized a nuclear E3 ubiquitin ligase in yeast called San1p that appears to recognize and ubiquitylate unfolded or misfolded proteins ( Rosenbaum et al . , 2011 ) .", "Although SAN1 orthologs have not been found in higher eukaryotes , metazoans may likewise have evolved mechanisms to perform protein QC surveillance in the nucleus .", "The fact that p53 responds to the appearance of unfolded protein , especially its sensitivity to unfolded protein in the nucleus , suggests that p53 may play a central role in nuclear protein QC surveillance in addition to its role as the guardian of the genome .", "In support of this hypothesis , Sawa and co-workers have reported that mutant huntingtin protein binds directly to p53 , leading to higher p53 levels and the induction of p53-responsive genes ( Bae et al . , 2005 ) .", "Although p53 appears to respond to unfolded protein that is created in either the nucleus of the cytosol , many of the genes that are induced by unfolded DD are strongly induced by cytosolic unfolded protein but respond modestly or not at all when the unfolded protein is created in the nucleus ( Figure 6C ) .", "Several chaperones , proteasome subunits , ubiquitin genes , and redox-responsive genes fall into this category .", "This result suggests that there are one or more protein QC surveillance pathways that monitor unfolded protein in a compartment-specific manner .", "The fact that cells respond so differently to the appearance of nuclear or cytosolic unfolded DD may have mechanistic implications for the cellular response to unfolded DD .", "We know that newly unfolded DD is rapidly ubiquitylated , which might deplete , at least temporarily , cellular levels of ubiquitin .", "One potential mechanism to initiate the cellular response to unfolded DD might include a futile cycle involving the production and rapid degradation of a transcription factor ( Barton and Sontag , 2013 ) .", "When conditions favor homeostasis , the surveillance transcription factor would be constitutively ubiquitylated and degraded .", "However , under conditions of proteotoxic stress , when ubiquitin becomes unavailable , the surveillance transcription factor would be stabilized and become competent to regulate the expression of genes involved in restoring homeostasis to the cell .", "The mitoUPR operates in this fashion with ATFS-1 in the role of the surveillance factor ( Nargund et al . , 2012 ) .", "Similarly , the Nrf2 transcription factor is degraded by the E3 ligase Keap1 until oxidative stress disables Keap1 , allowing Nrf2 levels to rise and regulate redox-responsive stress genes ( Kobayashi et al . , 2004 ) .", "A similar mechanism might explain the cellular response to unfolded DD , with p53 and other as yet unknown transcription factors in the roles of surveillance factors .", "However , one would expect the pool of cellular ubiquitin to rapidly partition between the nucleus and cytosol .", "Assuming rapid ubiquitin equilibration , the subcellular location of the unfolded protein should not affect the cellular response .", "The cellular response should be initiated by either cytosolic or nuclear unfolded protein , and the ensuing transcriptional programs should be identical .", "This is not what we observe , so it seems unlikely that ubiquitin depletion alone triggers the transcriptional response to unfolded DD .", "The health of an organism depends , at least in part , on the ability of its protein QC surveillance mechanisms to ensure that misfolded proteins are degraded ( Chiti and Dobson , 2006 ) .", "There is evidence in humans that these surveillance systems become less effective with age , leading to higher levels of aberrantly folded proteins , particularly in post-mitotic cells such as neurons ( Douglas and Dillin , 2010 ) .", "Protein misfolding and aggregation is correlated with , and in some cases likely causative of , a variety of neurodegenerative diseases including Alzheimer's , Parkinson's , and Huntington's diseases ( Morimoto , 2008; Knowles et al . , 2014 ) .", "It is well known that cells try to rescue aberrantly folded proteins by refolding them using chaperones or destroy them via the proteasome or autophagy ( Hartl et al . , 2011 ) .", "However , we are only beginning to understand the basic mechanisms by which cells target cytosolic and nuclear proteins for degradation when the proper folded state cannot be achieved .", "One of the barriers to progress in this field is the lack of good tools and methods that would allow us to move beyond simple correlations to mechanistically probe the specific relationships between protein folding and the etiology of diseases where protein misfolding has been implicated .", "The use of the DDs to illuminate a new cellular response used to maintain protein homeostasis highlights how useful these molecular tools can be .", "An increased understanding of the cellular response to the appearance of unfolded DD may also point to new strategies for diagnosing and ultimately treating unmet medical needs such as neurodegenerative diseases and cancer .", "Many features of this stress response remain to be discovered , including the molecular mechanisms that initiate the transcriptional response as well as the identities of the genes that contribute most strongly toward maintaining a healthy protein-folding environment ( Gibney et al . , 2013 ) .", "Connections between protein QC surveillance mechanisms and disease are of particular interest , although hints are readily apparent .", "Mutations in proteins involved in the UPS have been implicated in neurological disease ( e . g . , Ube3a in Angelman syndrome and Ube3c in autism spectrum disorders ) ( Greer et al . , 2010; O'Roak et al . , 2012 ) .", "The key sensor in the HSR , Hsf1 , has recently been shown to regulate in malignant cancer cells a transcriptional program that is distinct from the program induced by HS ( Mendillo et al . , 2012 ) .", "Finally , although a firm mechanistic picture is not yet clear , there is convincing evidence of an inverse correlation between Parkinson's disease and cancer , with a similar pattern becoming clear for Alzheimer's disease and cancer ( Driver et al . , 2012 ) .", "The previously unappreciated role of p53 in protein QC surveillance might provide a mechanistic basis for this anticorrelation ." ], [ "Constructs were subcloned using standard molecular biology techniques into the PiggyBac Transposon System vector pB ( System Biosciences , Mountain View , CA ) , which the Super PB transposase recognizes transposon-specific inverted terminal repeat sequences located on both ends of the transposon vector and moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites .", "We made plasmids with FKBP-derived DD-sfGFP , FKBP-derived DD-mCherry , FKBP-derived Nuclear DD-sfGFP , ecDHFR-derived DD-sfGFP , and FKBP-derived Cytosolic DD-sfGFP .", "For Nuclear DD-sfGFP , we placed the PPKKKRKV sequence , a nuclear localization signal from SV40 Large T-antigen , N-terminus of DD-GFP .", "Cytosolic DD-sfGFP was made by integrating nuclear exporting sequence from REV protein ( LQLPPLERLYLD ) in N-terminus and mCherry in C-terminus .", "Also , low-expressing DD-sfGFP and sfGFP constructs were cloned by the retroviral expression vector pBMN , which features long terminal repeats that contain all necessary elements for gene expression once integrated into the mammalian genome .", "The NIH3T3 cell line ( ATCC CRL-1658 ) was cultured in DMEM ( Dulbecco's Modified Eagle Medium ) supplemented with 10% heat-inactivated donor bovine serum ( Invitrogen ) , 2 mM glutamine , 100 U/ml penicillin , and 100 μg/ml streptomycin .", "All other cell lines were cultured with 10% heat-inactivated fetal bovine serum ( Invitrogen ) , 2 mM glutamine , 100 U/ml penicillin , and 100 μg/ml streptomycin .", "Retrovirus was produced by transfecting ΦNX ecotropic cells with pBMN plasmids using standard TransIT-LT1 ( Mirus ) protocols .", "Viral supernatants were harvested 48 hr post-transfection and filtered ( 0 . 45 µm , Fisher ) .", "NIH3T3 cells were incubated with the retroviral supernatants supplemented with 4 µg/ml polybrene for overnight at 37°C .", "Cells were cultured in growth media for 48–72 hr to allow for viral integration .", "Infected cells were sorted by FACS for high-GFP level cells .", "Cells were plated at 10 × 104 cells per well of a 6-well plate 12 hr prior to transfection .", "Cells were co-transfected with plasmid of pB vector and PiggyBac Transposase vector following standard protocols using TransIT-LT1 ( Mirus ) .", "Cells were cultured in growth media for about a week for stable integration , and transfected cells were sorted by FACS for high-GFP level cells .", "To remove S1 from culture media for DD-GFP or GFP cells , we directly added purified 500 µM FKBP ( F36V ) protein in cultured media to make final concentration of 5 µM FKBP ( F36V ) protein .", "To withdraw trimethoprim from DHFRDD-GFP cells , cells were washed twice with culture media then incubated with the fresh media .", "BL21 cells expressing His6-FKBP ( F36V ) protein ( Egeler et al . , 2011 ) or His6-FKBP ( F36V ) -sfGFP protein were cultured in Luria–Bertani media at 37°C until optical density at 600 nm reached 0 . 5–0 . 7 .", "Protein expression was induced with 0 . 5 mM isopropyl β-D-1- thiogalactopyranoside at 20°C overnight , then cells were harvested by centrifugation at 4500 RPM for 20 min at 4°C .", "Cells were lysed in phosphate-buffered saline ( PBS ) buffer supplemented with 1 mg/ml lysozyme ( Invitrogen ) and left on ice for 30 min .", "After sonication , Triton X-100 ( Sigma ) was added to 1% , and after 30 min incubation on ice , the solutions were centrifuged at 19 , 000 RPM for 30 min at 4°C .", "The soluble fraction was bound to Ni-NTA resin at 4°C for 1 hr .", "The resin was washed several times with wash buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 20 mM imidazole at pH 8 ) until eluent was free of protein by absorbance at 280 nm , then protein was eluted with elution buffer ( 50 mM NaH2PO4 , 300 mM NaCl , 250 mM imidazole at pH 8 ) .", "Eluted solutions were combined and dialyzed into dialysis buffer ( PBS with 4% glycerol ) and stored at −80°C .", "Cells were trypsinized and resuspended in culture media before flow cytometry experiments .", "Sorting was performed at the Stanford Shared FACS Facility using BD FACSAria sorter .", "For co-culture growth experiments , cells were trypsinized and resuspended in culture media before flow cytometry experiments .", "Then , we analyzed cells by FACSCalibur with no less than 25 , 000 events represented .", "Collected data were analyzed using FlowJo software .", "Total RNA was harvested using QIAShredder columns ( Qiagen ) and RNAEasy kit ( Qiagen ) .", "Poly ( A ) RNAs were purified from total RNA using Dyna oligo ( dT ) beads ( Invitrogen ) , and fragmented using RNA Fragmentation reagents ( Life Technologies ) .", "For first-strand cDNA synthesis , we used fragmentated RNA , random hexamer primers ( Life Technologies ) , 10xRT buffer ( Life Technologies ) , 25 mM or 50 mM MgCl2 ( Life Technologies ) , 0 . 1 M DTT , RNaseOUT ( Life Technologies ) , 10 mM dNTP mix ( Life Technologies ) , and SuperScript III Reverse Transcriptase ( Life Technologies ) in 20 µl reaction volume .", "Samples were subjected to second-strand cDNA synthesis with DNA polymerase I ( Invitrogen ) , 500 mM Tris-HCl pH 7 . 8 buffer , 50 mM MgCl2 ( Life Technologies ) , 0 . 1 M DTT , RNaseH ( Life Technologies ) , 10 mM dNTP mix ( Life Technologies ) .", "Samples were then subjected to end repair using End-It DNA End-Repair Kit ( epicentre ) .", "We followed Illumina's genomic DNA library preparation protocol with some modifications .", "We used Ligafast ( Promega ) and a 1:40 dilution of the ligation adaptor mix for the adaptor ligation step .", "In addition , we used Encore SP+ Complete RNA seq kit ( NuGEN ) and Mondrian SP+ system ( NuGEN ) for preparing cDNA library for samples .", "The quality and amount of cDNA samples were checked by BioAnalyzer ( Agilent ) and Qubit ( Life Technologies ) .", "Deep sequencing was performed with Illumina Hiseq 2000 in Stanford Sequencing Service Center .", "Sequencing data have been submitted to GEO ( accession numbers GSE65504 , GSE65636 , and GSE71145 ) .", "Illumina reads were aligned to the mm9 mouse genome assembly and analyzed against RefSeq using the DNAnexus platform ( https://dnanexus . com/ ) .", "Any genes with read count of zero were removed from the sets .", "Then , differential gene expression analyses were performed by using edgeR , ( http://www . bioconductor . org/ ) .", "The lists of up- or down-regulated genes were obtained using a FDR of 0 . 05 as a cutoff .", "Gene enrichment analyses were performed using DAVID ( http://david . abcc . ncifcrf . gov/ ) .", "All statistical analyses were performed in R ( www . rproject . org ) or Prism 6 ( http://www . graphpad . com/scientific-software/prism/ ) .", "Hierarchical clustering with centered correlation and centroid linkage was implemented using Cluster 3 . 0 ( http://bonsai . hgc . jp/∼mdehoon/software/cluster/software . htm#ctv ) .", "Java TreeView software ( jtreeview . sourceforge . net/ ) was used to create heatmaps .", "Total RNA was harvested using QIAShredder columns ( Qiagen ) and RNAEasy kit ( Qiagen ) .", "We used 300 ng–1000 ng of total RNA , and treated samples with RQ1 RNase-Free DNase ( Promega ) .", "For reverse transcription of RNAs , we used DNase-treated total RNA , a mixture of random hexamer primers ( Life Technologies ) and oligo dT primers ( Life Technologies ) , 10xRT buffer ( Life Technologies ) , 25 mM or 50 mM MgCl2 ( Life Technologies ) , 0 . 1 M DTT , RNaseOUT ( Life Technologies ) , 10 mM dNTP mix ( Life Technologies ) , and SuperScript III Reverse Transcriptase ( Life Technologies ) in 10 µl reaction volume .", "RT-qPCR analyses were carried out using Power SYBR Green PCR Master Mix ( Life Technologies ) on 7900HT Fast Real-Time PCR System ( Applied Biosystems ) .", "Primer sequences are included in Supplementary file 6 .", "Cells were washed with PBS and lysed with the buffer .", "Lysates were resolved by SDS-PAGE ( Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis ) and transferred to PVDF ( Polyvinylidene Fluoride ) membrane ( Millipore ) as previously described ( Egeler et al . , 2011 ) .", "Detection was achieved using ( Horseradish Peroxidase ) HRP-conjugated secondary antibodies and Millipore Immobilon .", "Western blot quantification was performed using ImageJ ( http://imagej . nih . gov/ij/ ) .", "Antibodies were anti-GAPDH ( mouse , 6C5 , Abcam ) , anti-GFP ( mouse , JL-8 , Clontech ) , anti-HA ( rat , 3F10 , Roche ) , anti-tubulin ( mouse , DM1A , Sigma ) , anti-p53 ( rabbit , CM5 , Vector Lab ) , anti-phospho-Ser824-Kap1 ( rabbit , Bethyl Laboratory ) , anti-phospho-Ser345-Chk1 ( rabbit , 133D3 , Cell Signaling ) , anti-phospho-S4/S8-RPA32 ( rabbit , Bethyl Laboratory ) , anti- γH2AX ( rabbit , 20E3 , Cell Signaling ) , anti-phospho-Ser51-eIF2α ( rabbit , Cell Signaling ) , and anti-Ub ( mouse , VU-1 , LifeSensors ) .", "Cells were washed with PBS and fixed in 4% paraformaldehyde , permeabilized with 100 µg/ml digitonin ( Sigma ) in PBS and blocked with 2% BSA ( Bovine Serum Albumin ) , 2% FBS ( Fetal Bovine Serum ) in PBS .", "Cells were incubated with 1:500 p53 CM5 antibody ( Vector Lab ) in PBS with 2% BSA , 2% FBS .", "After PBS washes , samples were incubated with 1:1000 Alexa 594-labeled secondary antibody ( Invitrogen ) and 2 µg/ml Hoechst 33 , 342 in PBS with 2% BSA , 2% FBS .", "After the second round of PBS washes , samples were mounted with ProLong Gold Antifade Reagent ( Invitrogen ) .", "Images were captured on a Zeiss Axioskop 2 epifluorescence microscope equipped with a QICAM FAST 1394 digital CCD camera .", "Quantification of images was performed using CellProfiler ( www . cellprofiler . org ) .", "We used these siRNA reagents: SMARTpool siGENOME Mouse Trp53 ( M-040642-02-0005 ) , SMARTpool ON-TARGET plus Mouse Hsf1 ( L-040660-01-0005 ) , SMARTpool ON-TARGET plus Mouse Nrf2 ( L-040766-00-0005 ) .", "siRNAs were transfected using Dharmafect1 and as described previously ( Chu et al . , 2013 ) .", "Cells stably expressing DD-GFP and unmodified NIH3T3 cells were mixed and plated in 24-well plates .", "S1 ligand was withdrawn for various times .", "Cells were cultured for an additional 17 hr in S1-containing media and then analyzed by flow cytometry to quantify the ratio of DD-GFP-expressing cells to control cells .", "To assess protective benefits , we used a similar co-culture experiment to examine the potential for the appearance of the unfolded DD to provide a protective benefit to cells exposed to a second source of proteotoxic stress .", "Cells expressing DD-GFP were mixed with NIH3T3 cells and co-cultured in the presence of S1 .", "The S1 was either withdrawn for 5 hr to trigger the cellular response or S1 was maintained in the media .", "At the 5-hr timepoint , a media change was used to re-administer S1 and the cells were cultured for an additional hour .", "At this point , cells were treated with either vehicle or with a second stress ( 30 µM sodium arsenite or 10 µM MG132 ) , cultured overnight , and analyzed at 24 hr by flow cytometry .", "Cells that did not experience either stress were defined as the control and the ratio of GFP+:GFP− cells at the end of the experiment was set to 100 .", "The effects of the various treatments ( unfolded DD only , arsenite or MG132 only , or both unfolded DD and arsenite or MG132 ) were then quantified using by flow cytometry .", "Expected effect of both stresses was calculated by multiplying the two mean values of either single-stress condition , and the error was computed by error propagation ." ] ]
[ "Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins .", "Chief among these is the heat shock response ( HSR ) , which is assumed to respond to unfolded proteins in the cytosol and nucleus alike .", "In this study , we probe this axiom further using engineered proteins called ‘destabilizing domains’ , whose folding state we control with a small molecule .", "The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response , which is distinct from the HSR and other known pathways that respond to unfolded proteins .", "The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol , although unfolded protein in either compartment engages the p53 network .", "This response provides cross-protection during subsequent proteotoxic stress , suggesting that it is a central component of protein quality control networks , and like the HSR , is likely to influence the initiation and progression of human pathologies ." ]
[ "The majority of cellular proteins must be folded into a precise shape to work correctly .", "But when cells experience stressful conditions such as high temperatures and harmful chemicals , proteins start misfolding or even completely unfold .", "Misfolded proteins can be highly toxic .", "As a result , cells use several different groups of molecules that work together in pathways to both detect and then refold incorrectly folded proteins .", "Protein misfolding has mainly been studied under extreme experimental conditions that cause hundreds of different proteins to misfold at the same time .", "In 2006 , researchers developed a technique that can be used to switch a protein between a folded and an unfolded state .", "Now , Miyazaki et al . —including several of the researchers involved in the 2006 work—have used this technique to investigate how a cell responds when a single protein unfolds .", "The sudden appearance of the unfolded protein increased the expression of several genes .", "Some of these genes are part of known pathways , but the majority were not known to respond to protein-folding stress .", "The cell also reacts differently depending on whether the unfolded protein is made in the cell nucleus , or the cytosol ( the fluid that surrounds the nucleus and other cellular components ) .", "Miyazaki et al . discovered that a network of molecules controlled , in part , by a protein called p53 activates the response to unfolded proteins in either the nucleus or the cytosol .", "Cells that activated this network were also better able to protect themselves from further stress .", "This newly discovered pathway is likely to play an important role in monitoring the quality of the proteins made in the cell .", "The next step involves identifying the cellular sensor ( s ) that recognize the unfolded protein as well as the transcription factors that are responsible for increasing gene expression .", "With a more complete picture of this new response , it will be possible to knock-out specific pathways to determine their roles ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology" ]
Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes
elife-06156-v2
[ [ "Cells respond to various signals by activating different types of RTKs and committing to specific cell-fate decisions ( Katz et al . , 2007 ) .", "A remarkable property of this system is that different RTKs can elicit distinct cellular responses through the same signal transduction machinery ( Marshall , 1995; Kholodenko et al . , 2006 ) .", "In several cases , signalling specificity results from differences in amplitude and duration of the intracellular signalling cascades ( Marshall , 1995; Maroun et al . , 2000; Nagashima et al . , 2007 ) .", "For example , in PC12 cells , EGF stimulation of EGFR leads to transient Erk phosphorylation and cell proliferation , whereas NGF binding to TrkA leads to sustained Erk phosphorylation and cell differentiation ( Marshall , 1995 ) .", "Differences in signalling amplitude and duration can arise from positive or negative feedback loops within the same signalling pathway ( Santos et al . , 2007 ) or activation of additional signalling components ( York et al . , 1998 ) .", "To explain such differences , it has been proposed that both EGF and NGF stimulation induce a specific ‘molecular context’ that determines the topology of the signal transduction network ( Santos et al . , 2007 ) .", "How such a topology is determined for different RTKs and whether it is the sole determinant of signal specificity is unclear ( Kholodenko , 2007 ) .", "Insights into this problem may be provided by the spatio-temporal distribution of RTKs along the endosomal system .", "The detection of phosphorylated receptors and signalling adaptors in endosomes ( Di Guglielmo et al . , 1994; Vieira et al . , 1996; Sorkin , 2001; Teis et al . , 2002; Lampugnani et al . , 2006; Galperin and Sorkin , 2008; Schenck et al . , 2008; Coumailleau et al . , 2009 ) led to the concept that signalling is initiated at the plasma membrane but continues in endosomes ( Di Guglielmo et al . , 1994 ) .", "Indeed , inhibition of endocytosis by blocking Dynamin function causes significant alterations in signalling specificity ( Vieira et al . , 1996 ) .", "However , recent studies challenged this concept arguing that EGFR signalling occurs primarily at the plasma membrane ( Damke et al . , 1994; Brankatschk et al . , 2012; Sousa et al . , 2012 ) .", "Interestingly , a recent systems survey of endocytosis ( Collinet et al . , 2010 ) revealed an unexpected tight control in the number , size , and cargo content for EGF-positive endosomes , raising the question of why is EGF packaging in endosomes so accurately controlled ?", "Here , we hypothesized that the tight control of the endosomal distribution of EGF could serve to regulate signal transmission .", "We tested this hypothesis by quantitatively analysing the endosomal distribution of EGFR as an RTK model system in endosomes and evaluating its impact on cell-fate decisions ." ], [ "To measure the content of p-EGFR in individual endosomes , we used two independent assays ( for a detailed description see ‘Materials and methods’ and Figure 1—figure supplement 1 ) .", "First , we modified a FRET-FLIM microscopy assay previously used to measure the spatial distribution of p-EGFR at the plasma membrane ( Wouters and Bastiaens , 1999; Verveer et al . , 2000 ) .", "The assay measured the FRET signal between EGFR-GFP and an anti-phospho-tyrosine antibody ( p-Tyr-ab ) labelled with AlexaFluor 555 .", "Since FLIM microscopy lacks the spatial resolution to analyse the receptor activation at a sub-cellular level , we modified the assay into a high-resolution FRET microscopy assay .", "However , instead of the total cell signal , we measured the distribution of EGFR and p-EGFR at the level of individual endosomes resolved by high-resolution confocal microscopy and quantitative automated image analysis ( Rink et al . , 2005; Collinet et al . , 2010 ) ( Figure 1—figure supplement 2 ) .", "To avoid artefacts of overexpression , we used HeLa cells transfected with a bacterial artificial chromosome ( BAC ) transgene stably expressing EGFR-GFP under its endogenous promoter ( Poser et al . , 2008 ) .", "In these cells ( Figure 1—figure supplement 3A ) , the uptake of EGF was only ∼twofold higher compared to endogenous ( Figure 1—figure supplement 3B ) .", "However , the transport kinetics were similar ( Figure 1—figure supplement 3C ) .", "Second , we measured p-EGFR with an antibody against a specific phospho-tyrosine residue ( Tyr1068 ) .", "Both assays gave very similar results ( Figure 1—figure supplement 4 ) .", "As the FRET assay is not restricted to a single phosphorylation site that can change over time ( Morandell et al . , 2008 ) , we used it as a primary assay in further experiments .", "Under the fixation conditions used , we observed no significant difference in the morphology ( Figure 1—figure supplement 5A ) or area ( Figure 1—figure supplement 5B ) of EGFR-positive endosomes ( Video 1 ) .", "For every time point , ∼15 , 000 endosomes from over 200 cells were analysed . 10 . 7554/eLife . 06156 . 003Video 1 . Live-cell imaging of EGFR endocytosis . HeLa EGFR-GFP BAC cells were imaged with a spinning disk microscope after 1 minute of EGF stimulation with 10 ng/ml EGF .", "Movie shows maximal projection of 3 z-slices of 0 . 8 mm thickness . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 003 Continuous stimulation with EGF triggered the internalization of EGFR into endosomes ( Figure 1 and Figure 1—figure supplement 2 ) .", "The total amount of endosomal EGFR peaked after 15 min and decreased , reflecting ( 1 ) down-regulation of surface receptors ( Wiley et al . , 1991 ) and ( 2 ) their degradation ( Dunn and Hubbard , 1984 ) over time ( Figure 1A , green curve ) .", "On the other hand , the total p-EGFR levels reached a maximum already at 10 min , followed by a phase of decay ( Figure 1A , red curve ) .", "Comparison of decay kinetics for both curves after 15 min showed that de-phosphorylation of p-EGFR occurred faster than degradation ( τdecay EGFR = 88 . 13 ± 14 . 49 , τdecay p-EGFR = 30 . 97 ± 1 . 69 , for details see ‘Materials and methods’ ) .", "Our FRET measurements are thus consistent with previously reported EGFR transport and phosphorylation kinetics determined by biochemical and microscopic methods ( Di Guglielmo et al . , 1994; Burke et al . , 2001 ) . 10 . 7554/eLife . 06156 . 004Figure 1 . Cells keep a constant amount of p-EGFR in endosomes .", "( A ) Time course of total integral intensity of EGFR ( green ) and p-EGFR ( red ) in endosomes measured by a FRET microscopy assay in HeLa EGFR BAC cells after continuous stimulation with 10 ng/ml EGF .", "The total integral intensity is defined as the sum of integral intensities of all endosomes in an image normalized by the area covered by the cells ( for details see ‘Materials and methods’ and Supplementary information ) .", "( B ) Time course of mean integral intensity per endosome for total EGFR ( green curve ) and p-EGFR ( red curve ) as in ( A ) .", "Intensity curves ( A–B ) were normalized to the intensity value at 10 min .", "Crosses show the corresponding values after 1 min of EGF stimulation and incubation in ligand-free medium for 10 or 30 min ( pulse-chase ) .", "( C ) Time course of histogram distributions of the total EGFR integral intensity per endosome upon EGF stimulation as in ( A ) .", "( D ) Time course of histogram distributions of the p-EGFR integral intensity per endosome upon EGF stimulation as in ( A ) .", "In both graphs , receptors in CCVs are responsible for the width of the distribution at 3 min ( red curves in C and D ) .", "For comparison , histogram amplitude in B and C were normalized by each curve integral .", "In each graph , the integral intensity values were scaled by the mode of the histogram at 10 min .", "The experimental points from all histograms were fitted with a log-normal distribution .", "( E–F )", "Distribution of p-EGFR in endosomes as a function of EGF concentration after continuous stimulation for 30 min .", "Mean number of endosomes with EGFR ( green curve ) and p-EGFR ( red curve ) per 1000 μm2 of the area covered by cells ( E ) and mean integral intensity of EGFR ( green curve ) and p-EGFR ( red curve ) per endosome ( F ) .", "On panel ( F ) curves were normalized to the intensity value at 10 ng/ml EGF .", "Lines are hyperbolic fits ( E ) or least square fits ( F ) to the experimental points .", "In both cases insets show the same graphs in linear scale .", "The different magnitude of the error bars in ( E ) and ( F ) is due to the averaging by the total number of images ( E ) or the total number of endosomes ( F ) .", "In all cases , points show mean ± SEM .", "All measurements were done in three independent experiments with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 00410 . 7554/eLife . 06156 . 005Figure 1—figure supplement 1 . Bleed-through correction for p-EGFR detection by FRET microscopy .", "( A ) Representative image of HeLa cells with no EGFR-GFP expression stained with an anti-p-Tyr antibody directly labelled with AlexaFluor 555 used to quantify the amount of fluorescence bleed-through .", "Scale bars , 10 μm .", "( B ) Distribution of the ratio of FRET-p-Tyr maximum intensities of individual colocalized objects in the FRET and p-Tyr channel .", "Since there is no GFP fluorescence , these objects give an estimation of fluorescence bleed-through ( filled circles ) .", "The continuous black line is the fit of three Gaussian components ( shown in coloured dashed lines ) .", "The mean and variance of the red and blue curves were used for image corrections ( see ‘Materials and methods’ for details ) .", "( C ) Mean FRET-p-Tyr intensity distribution before ( black curve ) and after correction ( red curve ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 00510 . 7554/eLife . 06156 . 006Figure 1—figure supplement 2 . EGFR and p-EGFR measurements by FRET microscopy .", "( A ) Representative images of HeLa EGFR-GFP BAC cells after continuous stimulation with 10 ng/ml EGF for the indicated time points .", "EGFR-GFP fluorescence is shown in green , the corrected p-EGFR intensity is shown in red , and DAPI-stained nuclei are shown in blue .", "Measurements from each individual endosome were used for all quantifications .", "Scale bars , 10 μm .", "( B–C )", "Time course of histogram distributions of the total p-EGFR ( B ) or EGFR ( C ) integral intensity per endosome upon EGF stimulation as in Figure", "1 . The histogram shows the number of vesicles per 1000 μm2 of the area covered by cells .", "Intensity values were scaled by the mode of the histogram at 10 min .", "In all graphs , experimental points were fitted with a log-normal distribution .", "Points show the mean from three independent experiments with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 00610 . 7554/eLife . 06156 . 007Figure 1—figure supplement 3 . BAC expression of EGFR-GFP does not change EGF transport kinetics .", "( A ) Representative Western blot of comparing the expression of EGFR and EGFR-GFP in HeLa Kyoto and HeLa EGFR-GFP BAC cells .", "The lower band corresponds to the untagged receptor , whereas the upper band corresponds to EGFR-GFP , which is absent in HeLa Kyoto cells .", "( B ) Time course of EGF integral intensity in endosomes in HeLa Kyoto ( black curve ) and HeLa EGFR-GFP BAC cells ( red curve ) .", "Intensity curves were normalized to the intensity value at 10 min for HeLa Kyoto cells .", "( C ) Comparison of both time courses after dividing the HeLa EGFR-GFP BAC cells by", "2 . Squares show the difference between both curves .", "Experimental points show mean ± SEM from one representative experiment with a total of ∼150 cells per time point and condition .", "Time courses were fitted as in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 00710 . 7554/eLife . 06156 . 008Figure 1—figure supplement 4 . Validation of FRET measurements with a specific anti-Tyr1068 antibody . Representative images of p-EGFR staining by an antibody against a single phospho-tyrosine residue of EGFR after 0 , 10 , and 30 min of continuous stimulation with 10 ng/ml EGF .", "Scale bars , 10 μm .", "Time course of the p-EGFR mean integral intensity per endosome measured by standard immunofluorescence ( blue ) and by FRET assay ( red ) .", "For comparison , both curves were normalized to the value at 10 min .", "Experimental points were fitted as in Figure 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 00810 . 7554/eLife . 06156 . 009Figure 1—figure supplement 5 . PFA fixation does not significantly change endosome EGFR endosome morphology .", "( A ) Representative images of HeLa BAC cells expressing EGFR-GFP after 20 min of stimulation with 10 ng/ml EGF before ( left panel ) or after fixation ( right panel ) .", "Scale bars , 10 μm .", "( B ) Histogram distribution of EGFR endosome area before ( blue ) and after fixation ( red ) .", "The histogram shows the number of EGFR endosomes per 1000 μm2 of the area covered by cells .", "Measurements were taken from ∼500 cells from one representative experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 00910 . 7554/eLife . 06156 . 010Figure 1—figure supplement 6 . The total amount of p-EGFR in endosomes decays with the same kinetics as the number of endosomes with p-EGFR . Time course of total integral p-EGFR intensity in endosomes ( red ) and endosomes with p-EGFR ( black ) per 1000 μm2 of the area covered by cells ( black ) after stimulation with 10 ng/ml EGF as in Figure", "1 . Points show mean ± SEM .", "All measurements were done in three independent experiments with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01010 . 7554/eLife . 06156 . 011Figure 1—figure supplement 7 . p-EGFR has a narrower integral intensity per endosome distribution than the total EGFR at late time points .", "( A–E )", "Histogram distributions for the p-EGFR ( red ) or total EGFR ( green ) integral intensity per endosome at 5 ( A ) , 10 ( B ) , 15 ( C ) , 30 ( D ) , and 60 ( E ) min of continuous stimulation with 10 ng/ml EGF .", "For comparison , the amplitude of all histograms was normalized by the curve integral and the integral intensity was scaled by the mode of each histogram .", "In all graphs , experimental points were fitted with a log-normal distribution .", "Points show the mean from three independent experiments with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01110 . 7554/eLife . 06156 . 012Figure 1—figure supplement 8 . The mean amount of p-EGFR per endosome increases at high concentrations of EGF .", "( A ) Mean integral intensity of EGFR ( green curve ) and p-EGFR ( red curve ) per endosome upon stimulation with different EGF concentrations for 30 min ( B ) Time course of mean integral intensity of p-EGFR per endosome after continuous stimulation with 10 ng/ml ( black curve ) or 100 ng/ml ( red curve ) EGF .", "Curves were normalized by the intensity value at 10 min for 10 ng/ml EGF .", "Experimental points were fitted as in Figure 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01210 . 7554/eLife . 06156 . 013Figure 1—figure supplement 9 . The mean p-EGFR amount per endosome does not correlate with endosome area at late time points after EGF stimulation .", "( A ) Mean p-EGFR integral intensity per endosome as a function of endosome area upon 10 ( black curve ) or 30 min ( red curve ) of EGF stimulation .", "Both curves were normalized to the intensity value at 1 μm2 for 10 min stimulation .", "( B ) Histogram distribution of endosome area upon 10 ( black curve ) or 30 min ( red curve ) of EGF stimulation .", "Each histogram was normalized by its respective curve integral .", "Points show mean ± SEM .", "All measurements were done in three independent experiments with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 013 We next determined the distribution of EGFR and p-EGFR in individual endosomes .", "The number of endosomes with p-EGFR decayed with similar kinetics as the total p-EGFR signal ( τdecay N-p-EGFR = 45 . 24 ± 11 . 39 vs τdecay p-EGFR = 30 . 97 ± 1 . 69; compare red with black curve in Figure 1—figure supplement 6 ) .", "The mean content of total EGFR per endosome increased over time and then rapidly decayed reaching steady state , due to the balance of continuous EGF uptake and degradation ( Figure 1B , green curve ) .", "After a rapid increase , the mean content of p-EGFR in each endosome stabilized to a fairly constant level after ∼20 min ( Figure 1B , red curve ) .", "Similar results were obtained when EGF was pulsed for 1 min and chased for different periods of time ( Figure 1B , blue and black points ) .", "To determine how the endosomal content of p-EGFR originates over time , we compared the distributions of EGFR and p-EGFR content per endosome .", "The width of distribution of total EGFR increased with time ( Figure 1C ) , due to the fact that , as EGF continues to flow in , it first enters small early endosomes and progressively accumulates in larger ones ( Rink et al . , 2005 ) .", "In contrast , the p-EGFR distribution first widened like that of total EGFR but then became almost twofold narrower than that of EGFR ( Figure 1—figure supplement 7A–E compare the red and green curves ) and stabilized after 30 min ( Figure 1D ) .", "These results suggest an unexpected behaviour of p-EGFR , which over time stabilizes at a constant mean level per endosome .", "Surprisingly , the mean amount of p-EGFR in endosomes was not ligand dependent .", "We stimulated cells with different concentrations of EGF for 30 min , when the amount of p-EGFR per endosome reached its steady state ( Figure 1B , D ) .", "Once again , we found that EGFR and p-EGFR behaved very differently .", "The number of endosomes containing EGFR saturated already at low concentrations of EGF ( 0 . 5–1 . 0 ng EGF; Figure 1E , green curve ) whereas the amount of total EGFR per endosome increased almost linearly ( Figure 1F , green curve ) .", "This is expected because the higher the concentration of EGF , the higher the internalization of EGFR , whereas the number of receiving endosomes does not change significantly .", "In contrast , the number of endosomes with p-EGFR augmented with increasing EGF concentrations ( Figure 1E , see red curve in semi-logarithmic scale , inset in linear scale ) .", "Strikingly , the mean amount of p-EGFR per endosome remained fairly constant , despite the EGF concentration varying almost over three orders of magnitude ( Figure 1F , red curve ) .", "Therefore , increasing concentrations of EGF resulted in an increase in the number of endosomes with the same mean package of p-EGFR .", "Importantly , such a packaging is saturable because at high EGF concentrations the mean p-EGFR content per endosome was no longer constant with time ( Figure 1—figure supplement 8 ) .", "The finding that endosomes contain a constant mean level of p-EGFR is striking .", "We performed several control experiments to verify that this is not an artefact caused by the FRET method or the assay .", "First , the mean amount of p-EGFR per endosome did increase at EGF concentrations higher than 10 ng/ml ( Figure 1—figure supplement 8 ) , indicating that the value measured is not artificially fixed , for example , by limited antigen accessibility .", "Second , a similar constant mean value of p-EGFR per endosomes was estimated with an independent method using the Tyr1068 antibody ( Figure 1—figure supplement 4 ) .", "Third , the narrow distribution of p-EGFR per endosome may simply reflect the sorting into endosomes of regular size .", "Whereas at 10 min the p-EGFR amount per endosome increased with the endosome area ( Figure 1—figure supplement 9A , black curve ) , at 30 min ( steady state , Figure 1B ) , the same mean amount was present in small and large endosomes alike ( Figure 1—figure supplement 9A , red curve ) .", "Therefore , the amount of activated receptors per endosome is independent of endosome area .", "Finally , we verified that it is not a phenomenon peculiar to HeLa cells but also occurring in non-immortalized , non-cancer cell lines .", "Using the anti-phosphoTyr1068 antibody , we found that in primary mouse hepatocytes upon EGF stimulation the mean amount of p-EGFR per endosome saturated at ∼20 min whereas the mean amount of EGF continued to grow ( data not shown ) , indicating that the packaging of p-EGFR in endosomes is not peculiar to a signalling-aberrant cancerous cell line .", "In which endocytic compartment was p-EGFR packaged so uniformly ?", "Nearly 80% of p-EGFR colocalized with the early endosomal marker EEA1 throughout the time course ( Figure 2—figure supplement 1A ) .", "Less than 10% of p-EGFR colocalized with APPL1 after 15 min showing that it passed this endosomal compartment ( Miaczynska et al . , 2004 ) ( Miaczynska et al . , submitted ) .", "Very little p-EGFR colocalized with LAMP-1 , a marker of late endosomes and lysosomes ( Figure 2—figure supplement 1C ) .", "One possibility is that the packages of p-EGFR may reflect incorporation into intra-luminal vesicles ( ILV ) of multi-vesicular bodies ( MVB ) .", "This possibility was ruled out using a previously described differential detergent solubilisation method ( Malerød et al . , 2007 ) .", "We could determine that a large fraction of EGFR was not accessible to antibodies upon digitonin permeabilization , reflecting sequestration into ILVs ( Malerød et al . , 2007; Piper and Katzmann , 2007 ) ( see Suppl . information and Figure 2A , B ) .", "In contrast , p-EGFR was always detectable suggesting that it was not within ILVs . 10 . 7554/eLife . 06156 . 014Figure 2 . The constant mean amount of p-EGFR per endosome corresponds to receptor clusters that are regulated by Hrs and PTPN11 . ( A ) Representative images of total EGFR and p-EGFR after staining with saponin or digitonin permeabilization methods .", "Immunofluorescence staining of LBPA is shown as a control marker for ILVs in MVBs .", "Scale bars , 10 μm .", "( B ) Integral intensity of EGFR , p-EGFR , and LBPA ( mean ± SEM ) after permeabilization with digitonin or saponin .", "**p < 0 . 005 by a two-tailed t-test .", "Measurements were done in three independent experiments with a total of ∼150 cells per condition .", "( C ) Time course of mean integral intensity per endosome for ub-EGFR ( blue curve ) upon EGF stimulation as in Figure 1A .", "p-EGFR is included for comparison .", "( D ) Representative STORM images of p-EGFR ( red ) stained using a rabbit monoclonal anti-p-EGFR ( Tyr 1068 ) antibody overlaid on top of a high magnification confocal image of EGFR ( green ) .", "Left panels show clusters of p-EGFR upon stimulation with EGF for 10 or 30 min .", "Right panels show clusters of p-EGFR upon stimulation with EGF for 30 min in Hrs down-regulation or mock treatment .", "( E ) Time course of the mean p-EGFR integral intensity per endosome in Hrs ( red ) , Snf8 ( blue ) , Vps24 ( green ) , or mock-treated cells ( black ) ( using three different siRNA oligonucleotides per gene ) .", "All curves were normalized by the intensity value at 10 min for the mock sample .", "Points show mean ± SEM from three different siRNAs per gene .", "Scale bar , 1 μm .", "( F ) Integral intensity distribution of p-EGFR per endosome after down-regulation for 72 hr of PTPN11 ( red ) or in mock treatment ( black ) after continuous stimulation with 10 ng/ml EGF for 30 min .", "Red points show the average distribution of three different siRNAs .", "Experimental points were fitted as in Figure 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01410 . 7554/eLife . 06156 . 015Figure 2—figure supplement 1 . The majority of the p-EGFR is located in EEA1-positive endosomes .", "( A ) Time course of integral intensity of total EGFR ( green curve ) and p-EGFR ( red curve ) colocalizing with EEA1 after continuous stimulation with 10 ng/ml EGF for different time points .", "Intensity curves were normalized to the intensity value at 10 min ( B–C ) Fraction of the total integral intensity of EGFR ( green curve ) or p-EGFR ( red curve ) colocalized with EEA1 ( B ) or LAMP-1 ( C ) .", "In all cases , points show mean ± SEM .", "Measurements were done in three independent replicates with a total of ∼150 cells per time point or condition .", "Time courses were fitted to obtain the decay half-time τ ( for details on the fitting procedure see ‘Materials and methods’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01510 . 7554/eLife . 06156 . 016Figure 2—figure supplement 2 . ub-EGFR measurements by FRET microscopy .", "( A ) Representative images of HeLa EGFR-GFP BAC cells after continuous stimulation with 10 ng/ml EGF for the indicated time points .", "EGFR-GFP fluorescence is shown in green , the corrected ub-EGFR intensity is shown in red .", "Measurements from each individual endosome were used for all quantifications .", "Scale bars , 10 μm .", "( B–C )", "Heat map of the 2D co-distribution of ub-EGFR and EGFR ( B ) or ub-EGFR and p-EGFR ( C ) integral intensity per endosome upon EGF stimulation as in Figure 1 after 10 , 30 , or 60 min . ub-EGFR and EGFR are well correlated , whereas the distribution of p-EGFR is significantly narrower than ub-EGFR at 30 and 60 min .", "Heat maps show the result of one representative experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01610 . 7554/eLife . 06156 . 017Figure 2—figure supplement 3 . Quantification of number of EGFR and pEGFR molecules per endosome .", "( A ) Distribution histogram of the differences in intensity between individual endosomes in consecutive frames during sequential photo-bleaching of EGFR-GFP ( see ‘Materials and methods’ for details ) .", "( B ) Difference between the number of positive and negative events in ( A ) for each ΔIntensity value .", "( C ) Distribution histogram of the differences in intensity between individual endosomes in consecutive frames during sequential photo-bleaching of p-EGFR ( see ‘Materials and methods’ for details ) .", "( D ) Difference between the number of positive and negative events in ( C ) for eachΔIntensity value .", "The local amplitude maxima of the periodic function in ( B ) and ( D ) give an estimate of the change in intensity values when 1 , 2 , 3 , … , n number of molecules are bleached .", "( E ) Distribution of the number of molecules of EGFR-GFP and p-EGFR in individual endosomes after stimulation with 10 ng/ml EGF for 10 min . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01710 . 7554/eLife . 06156 . 018Figure 2—figure supplement 4 . Hrs , but not ESCRT-II or ESCRT-III components , increases the mean p-EGFR amount per endosome .", "( A–B )", "Histogram distributions of the total EGFR ( green ) or p-EGFR ( red ) integral intensity per endosome after 30 min of EGF stimulation as in Figure 5 for Hrs ( A ) or mock-treated cells ( B ) .", "( C ) Representative images of HeLa EGFR BAC cells after continuous stimulation with 10 ng/ml EGF for 30 min and Hrs , Snf8 , or Vps24 knock-down .", "Scale bars , 10 μm .", "( D ) Changes in EGFR at the endosomal surface after knock-down of different ESCRT components measured by the differential permeabilization assay shown in Figure 2A Bar graphs show mean ± SEM .", "Measurements were done in three independent experiments using three different siRNA oligonucleotides with a total of ∼150 cells .", "In all graphs , experimental points were fitted with a log-normal distribution .", "Points show the mean from three independent experiments with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01810 . 7554/eLife . 06156 . 019Figure 2—figure supplement 5 . Kinetics of Shc1 recruitment to endosomes .", "( A ) Representative images of HeLa EGFR-GFP BAC cells before ( left panel ) and after 10 min stimulation with 10 ng/ml EGF ( right panel ) .", "EGFR fluorescence is shown in green and Shc1 is shown in red .", "( B ) Time course of mean integral intensity of Shc1 per endosome ( black curve ) .", "The mean intensity of p-EGFR per endosome ( red curve ) is included for comparison .", "( C ) Mean integral intensity of Shc1 per endosome as a function of EGF concentration .", "The curve was normalized to the intensity value at 10 ng/ml EGF .", "The solid line is a least square fits to the experimental points .", "In all cases , points show mean ± SEM .", "Measurements were done in three independent replicates with a total of ∼150 cells per time point or condition .", "Time courses were fitted as in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 01910 . 7554/eLife . 06156 . 020Figure 2—figure supplement 6 . Pharmacological inhibition of EGFR kinase rapidly decreases the total p-EGFR in endosomes only at high but not low EGF concentrations . Time course of the total p-EGFR integral intensity upon stimulation with 10 ( black and green curves ) or 100 ng/ml ( red and blue curves ) of EGF .", "AG1478 ( green and blue curves ) was added 10 min after stimulation with EGF and remained in the medium throughout the time course .", "All curves were normalized by the intensity value at 10 min for the DMSO—10 ng/ml sample .", "Experimental points were fitted as in Figure", "1 . Points show mean ± SEM of ∼150 cells per time point and condition from one representative experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 02010 . 7554/eLife . 06156 . 021Figure 2—figure supplement 7 . Pharmacological inhibition of EGFR kinase activity increases the mean p-EGFR amount per endosomes .", "( A ) Representative images of HeLa EGFR BAC cells after continuous stimulation with 10 ngl/ml EGF for 30 min .", "Inhibitors were added 10 min after stimulation with EGF and remained in the medium throughout the time course .", "Scale bars , 10 μm .", "( B ) Time course of the mean p-EGFR integral intensity per endosome in AG1478 ( red ) , Gefitinib ( blue ) , Lapatinib ( green ) , or DMSO-treated cells ( black ) .", "All curves were normalized by the intensity value at 10 min for the mock sample .", "Experimental points were fitted as in Figure", "1 . Measurements for AG1478 were done in three independent experiments; measurements from Gefitinib and Lapatinib show a representative experiment with a total of ∼150 cells per time point and condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 02110 . 7554/eLife . 06156 . 022Figure 2—figure supplement 8 . Phosphatases can control the p-EGFR packaging in endosomes .", "( A ) Total p-EGFR integral intensity after continuous stimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells and down-regulation of the indicated phosphatase or treatment with transfection reagent only .", "( B ) Mean p-EGFR integral intensity per endosome after continuous stimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells and down-regulation of the indicated phosphatase or treatment with transfection reagent only .", "In both cases , bars are colour coded according to their respective phosphatase .", "All phosphatases were down-regulated with at least three oligos .", "Bars show mean ± SEM of all images for each oligo . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 022 How do the kinetics of p-EGFR endosomal packaging compare with the kinetics of receptor dephosphorylation and ubiquitylation ?", "After 10 min of EGF stimulation , the pool of p-EGFR in EEA1-positive endosomes ( Figure 2—figure supplement 1A , red curve ) decayed faster than total EGFR ( Figure 2—figure supplement 1A , green curve; τdecay EGFR = 56 . 03 ± 5 . 72 , τdecay p-EGFR = 37 . 08 ± 4 . 19 ) , possibly due to de-phosphorylation or preferential removal of p-EGFR from early endosomes .", "The latter can be excluded since the fraction of p-EGFR in EEA1-positive endosomes remained almost constant throughout the time course ( Figure 2—figure supplement 1B ) .", "EGFR ubiquitylation is required for its internalization into endosomes , and this is dependent on EGFR phosphorylation and the recruitment of the c-Cbl E3 ligase ( Sigismund et al . , 2013 ) .", "To compare the levels of ubiquitylated EGFR ( ub-EGFR ) with those of p-EGFR within the endosomal system , we modified the FRET assay using an anti-ubiquitin antibody ( Figure 2—figure supplement 2A ) .", "The kinetics of ub-EGFR were significantly different from those of p-EGFR .", "Whereas the levels of p-EGFR peaked at 15 min , ub-EGFR reached its maximum at 30 min after stimulation ( Figure 2C , compare red with blue curves ) and decreased more slowly than p-EGFR at later times , probably reflecting deubiquitylation prior to receptor sequestration into ILVs ( Piper and Katzmann , 2007 ) .", "These results are consistent with the fact that the appearance of p-EGFR precedes that of ub-EGFR ( Umebayashi et al . , 2008 ) .", "Moreover , ub-EGFR had a similar distribution to that of EGFR ( Figure 2—figure supplement 2B ) but significantly wider than p-EGFR ( Figure 2—figure supplement 2C ) , suggesting that the mechanisms responsible for stabilizing the mean levels of p-EGFR per endosome are not correlated with receptor ubiquitylation .", "Our data suggest the existence of a saturable mechanism adjusting the amount of p-EGFR in each individual endosome .", "Such a constant mean amount may be due to the formation of small clusters within early endosomes .", "To test this possibility , we imaged the spatial distribution of p-EGFR in endosomes using the anti-EGFR phosphoTyr1068 antibody by super-resolution microscopy .", "Using direct Stochastic Optical Reconstruction Microscopy ( dSTORM ) ( Lampe et al . , 2012 ) , we could indeed visualize clusters of p-EGFR ( Figure 2D , left panel ) that decreased in size between 10 and 30 min of EGF internalization , in agreement with the narrowing of p-EGFR distribution over time ( Figure 1D ) .", "To determine the number of molecules in the clusters , we used two methods .", "First , we developed a new method to estimate the number of fluorescent molecules in light microscopy images by measuring the intensity fluctuations during photo-bleaching over time ( for details see ‘Materials and methods’ and Figure 2—figure supplement 3 ) .", "Based on the fluorescence signal from the anti-phosphoTyr1068 antibody and EGFR-GFP , we estimated an average of 102 ± 38 and 76 ± 29 ( Mean ± SEM ) molecules of EGFR and p-EGFR per endosome 30 min after EGF ( 10 ng/ml ) internalization ( Figure 2—figure supplement 3 ) , corresponding to 707 ± 265 and 527 ± 202 molecules per μm3 of endosomal volume ( apparent , assessed by light microscopy ) , respectively .", "A hundred EGFR molecules would require ∼12 clathrin-coated vesicles for delivery to endosomes ( see ‘Materials and methods’ ) .", "We also estimated the total number of GFP-EGFR per cell and found values ( 29 , 000 ) well in agreement with previous estimates for HeLa cells ( see ‘Materials and methods’ and Figure 2—figure supplement 1A , B ) .", "Second , based on the size of receptor from the PDB database ( structure ID: 3NJP ) , we calculated that 83 ± 25 ( Mean ± SEM , N = 1456 ) receptors could fit in the apparent area of p-EGFR visualized by dSTORM , a value which is remarkably in agreement with the fluorescence intensities estimates .", "To further validate that the constant mean amount of p-EGFR per endosome corresponds to receptor clusters , we performed a focused RNAi screen on established components of the endosomal receptor sorting machinery ( CHLCb , CHLCa , Hip1 , Hip1R , Htt , Tom1 , Tollip , Tom1L1 , Tom1L2 , Hrs , Snf8 , Vps24 ) .", "We found that only Hrs depletion resulted in a continuous accumulation of p-EGFR in endosomes with time ( Figure 2E ) .", "At the same time , the p-EGFR intensity distribution widened similar to that of total EGFR ( Figure 2—figure supplement 4A , B ) .", "The silencing of Hrs also caused an increase in the size of p-EGFR clusters within each endosome as revealed by dSTORM ( Figure 2D , right panel ) .", "Interestingly , this is not due to the inhibition of ILV formation , as down-regulation of Snf8 and Vps24 , members of the ESCRT-II and ESCRT-III complexes , respectively ( Piper and Katzmann , 2007 ) , reduced the sequestration of EGFR into the endosomal lumen ( Figure 2—figure supplement 4D ) but did not have significant effects on the amount of p-EGFR per endosome ( Figure 2E , blue and green curves ) .", "Thus , the constant mean amount of p-EGFR in endosomes likely corresponds to the receptor clusters observed by super-resolution microscopy .", "This raises the question of whether p-EGFR can be accessible to downstream signalling components .", "Therefore , we measured the recruitment of a direct downstream effector of p-EGFR , Shc1 .", "The kinetics of Shc1 recruitment to endosomes precisely mimic the kinetics of p-EGFR ( Figure 2—figure supplement 5 ) arguing that the p-EGFR clusters are signalling competent .", "Upon internalization , EGF enters the early endosomal network and , similar to LDL ( Rink et al . , 2005 ) , following endosome homotypic fusion and fission reactions , accumulates in few large endosomes prior to transfer to late endosomes .", "A mechanism must exist that prevents the continuous accretion of p-EGFR upon endosome fusion .", "A simple mechanism could be that the de-phosphorylation rate increases with the increase in p-EGFR per endosome .", "When two endosomes fuse , the resulting endosome should contain the sum of EGFR and p-EGFR of the original endosomes .", "However , given such de-phosphorylation rate dependency , the amount of p-EGFR would return to the level prior to fusion , thus stabilizing the mean amount of p-EGFR per endosome .", "A prediction of this hypothesis is that the kinase activity of EGFR in endosomes controls its own dephosphorylation .", "To test this , we inhibited the EGFR kinase activity pharmacologically with AG1478 , lapatinib or gefitinib 10 min after EGF stimulation ( to prevent alterations on receptor internalization ) and determined the effects on the receptors already internalized and phosphorylated .", "We compared low with high concentrations of EGF , that is , under conditions of saturation of p-EGFR packaging in endosomes ( Figure 1—figure supplement 8 ) .", "At low EGF concentrations , when the packaging mechanism is not saturated , the total amount of p-EGFR was not significantly reduced by the inhibitors ( Figure 2—figure supplement 6 compare black and green curves ) .", "This behaviour argues that the packages of p-EGFR in endosomes are protected from the phosphatases .", "In addition , the inhibitors caused a continuous accumulation of p-EGFR in fewer and larger endosomes over time ( Figure 2—figure supplement 7 ) .", "In contrast , adding the inhibitor after stimulation with high concentrations of EGF caused a sharp reduction in the total amount of p-EGFR ( Figure 2—figure supplement 6 compare red and blue curves ) , as observed previously ( Kleiman et al . , 2011 ) .", "This means that the kinase activity of EGFR is necessary to maintain the levels of p-EGFR in individual endosomes .", "These results support the idea that the dephosphorylation of p-EGFR in endosomes indeed depends on the EGFR activity within the endosomal packages .", "Which phosphatases are responsible for controlling p-EGFR packaging in endosomes ?", "To identify them , we performed a focused RNAi screen against 21 protein tyrosine phosphatases ( PTP ) expressed in HeLa cells ( Tarcic et al . , 2009 ) .", "Hits were defined if silencing satisfied three conditions: ( 1 ) it increased the total amount of p-EGFR in endosomes and ( 2 ) increased the mean amount of p-EGFR per endosome , and ( 3 ) the phenotype was observed with at least two siRNAs per gene .", "Five phosphatases , PTP4A1 , PTPN11 , PTPN9 , PTPN18 , and PTPRK , increased the amount of p-EGFR in individual endosomes ( Figure 2F and Figure 2—figure supplement 8 ) .", "Interestingly , PTPN11 is an EGFR interactor ( Deribe et al . , 2009 ) whose activity is enhanced upon tyrosine phosphorylation ( Agazie and Hayman , 2003 ) , suggesting a molecular mechanism whereby p-EGFR could regulate its own de-phosphorylation in endosomes .", "What are the consequences of such mechanism for signal transduction ?", "To address these questions and generate testable predictions , we developed a mathematical model that describes the amount of total intracellular p-EGFR over time .", "Previously , excellent models have been developed that quantitatively describe EGFR endocytosis and signalling ( Felder et al . , 1992; French et al . , 1994; Kholodenko et al . , 1999; Kholodenko , 2002; Resat et al . , 2003 ) .", "However , although all these models described in detail the dynamics of ligand binding , dimer formation and endocytosis , recycling and degradation of the receptor , they did not consider the trafficking dynamics of the phosphorylated receptors with respect to the dynamics of the endosomal network because these data were not available .", "Our new experimental data brought two new concepts .", "First , dephosphorylation and degradation of p-EGFR occur sequentially but are uncoupled .", "Second , the amount of p-EGFR is controlled at the level of individual endosomes .", "These new concepts require further development of the existing EGFR mathematical models .", "Our model was formulated as a set of ordinary differential equations ( ODE , see ‘Materials and methods’ and Figure 3 ) describing ( 1 ) the total amount of EGFR and p-EGFR at the plasma membrane as a function of ligand binding , ( 2 ) endocytosis of p-EGFR and its indirect effects on EGFR endocytosis , and ( 3 ) distribution of cargo between early endosomes at different stages of maturation ( e . g . , formation of MVB ) .", "For this , we considered the processes of receptor internalization , dephosphorylation , degradation , recycling , endosome fusion and fission .", "As in previous models ( French et al . , 1994; Resat et al . , 2003 ) , we described time course kinetics of total cellular p-EGFR , surface and endosomal EGFR and p-EGFR .", "Importantly , our model also describes the total number of p-EGFR-positive endosomes and mean amount of p-EGFR per endosome ( see ‘Materials and methods’ for details ) .", "To account for the observed stabilization of the mean amount of p-EGFR per endosome over time ( Figure 1 ) , the dependency of p-EGFR dephosphorylation on EGFR kinase activity ( Figure 2—figure supplement 6 , 7 ) and the fact that the mechanism is saturable ( Figure 1—figure supplement 8 ) , we included a sigmoidal dependency of the p-EGFR dephosphorylation rate on the amount of p-EGFR per endosome .", "The model was then fitted to the experimental data from the p-EGFR time course ( Figure 3A , B ) .", "Figure 3C shows that this simple theoretical model can reproduce our observations of a constant mean amount of p-EGFR per endosome in a wide range of EGF concentrations when fitted to the experimental data .", "Importantly , a model without this non-linear dephosphorylation dependency could correctly describe the total amount of EGFR and p-EGFR in endosomes ( Figure 3—figure supplement 1A , B ) but did not agree with the measurements for the mean amount of p-EGFR per endosome ( Figure 3—figure supplement 1C ) , thus supporting the sigmoidal dependency of the p-EGFR de-phosphorylation rate on the amount of p-EGFR per endosome ( Figure 3 ) .", "Previous models did not include this non-linear term because data on the distribution of p-EGFR in individual endosomes was not available . 10 . 7554/eLife . 06156 . 023Figure 3 . Mathematical model of p-EGFR predicts signalling amplitude and duration depends on early endosome fusion/fission rate . Parameters of the mathematical model were fitted to the experimentally measured number of p-EGFR endosomes , total integral intensity of p-EGFR , mean integral intensities of p-EGFR per endosome and total vesicular EGFR .", "The experimental data were obtained in a time course of EGF stimulation at four concentrations ( 0 . 5 , 1 . 0 , 5 . 0 , and 10 ng/ml , colour coded as indicated ) .", "The fit results are presented on panels ( A–C ) .", "The experimental data and model predictions are drawn as filled circles and solid curves , respectively .", "( A ) Number of p-EGFR endosomes per 1000 μm2 of cell area .", "( B ) Total integral intensity of p-EGFR measured by FRET .", "The scaling factors that convert arbitrary numbers of the model to the experimental data were found by the least square procedure ( see ‘Materials and methods’ ) .", "( C ) Comparison of mean integral intensity of p-EGFR per endosome measured experimentally ( filled circles ) and mathematical model ( solid curves ) of the time course of p-EGFR upon EGF stimulation as in Figure 1A .", "The concentration of EGF is colour coded as presented .", "( D ) Model predictions of the total amount of p-EGFR in endosomes as a function of EGF concentration and in the presence of different homotypic early endosome fusion rates ( colour coded as indicated ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 02310 . 7554/eLife . 06156 . 024Figure 3—figure supplement 1 . A mathematical model without the non-linear phosphorylation dependency cannot describe the mean amount of p-EGFR per endosome . Parameters of a mathematical model with a first-order dephosphorylation rate were fitted to the experimental data as in Figure 3 .", "( A ) Total integral intensity of EGFR .", "( B ) Total integral intensity of p-EGFR measured by FRET .", "( C ) Mean integral intensity of p-EGFR per endosome .", "The experimental data and model predictions are drawn as filled circles and solid curves , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 024 An unexpected prediction of our model is that the total de-phosphorylation rate , and thus the total amount of p-EGFR , is dependent on the fusion/fission rate of the endosomes ( Figure 3D ) .", "If so , could this have an effect on signal transduction ?", "To test these hypotheses , we reduced early endosome homotypic fusion by lowering the intracellular concentration of established components of the endosome tethering and fusion machinery , EEA1 , Rabenosyn5 , Vps45 ( Christoforidis et al . , 1999; Ohya et al . , 2010 ) , Syntaxin-6 and Syntaxin-13 ( Brandhorst et al . , 2006 ) that play no direct role in signalling .", "These genes were down-regulated by RNAi in combinations and only partially ( ∼50–70% depletion for each protein , Figure 4A ) to achieve a significant inhibition of endosome fusion and yet prevent or reduce cell toxicity .", "This procedure caused a mild redistribution of EGFR to endosomes of smaller size ( <0 . 5 μm2 cross-section area , for details see ‘Materials and methods’ and Figure 6—figure supplement 2 ) ( Figure 4C , D ) .", "Similar results were obtained upon depletion of a second combination of genes ( EEA1 , Stx13 , Stx6 , not shown , see below ) .", "Note that these treatments generated a pattern of endosomes similar to that observed in different cell types and under different culture conditions ( see below , Figure 6 ) and neither altered the surface levels of EGFR ( Figure 4—figure supplement 1 ) nor its kinetics of uptake ( Figure 4B ) and exit from endosomes , that is , recycling and degradation ( Figure 4—figure supplement 2 ) .", "We also excluded potential effects on endosome acidification , because blocking it with bafilomycin did increase both p-EGFR and total EGFR ( Figure 4—figure supplement 3 ) .", "Remarkably , under our experimental conditions of mild down-regulation of the early endosomal fusion machinery the packaging of active receptors was unaffected as shown by both the time course and the steady-state mean ( constant ) amount of p-EGFR per endosome ( Figure 4E; see above , Figure 1B ) .", "In contrast , the total number of endosomes with p-EGFR and their life-time augmented ( Figure 4F ) , resulting in a net increase in the total amount and life-time of p-EGFR ( Figure 4G ) .", "Notably , reduction of the endosome fusion rate in the mathematical model ( ∼40% , in line with the depletion of tethering proteins , Figure 4A ) is sufficient to reproduce fairly well the experimental increase in p-EGFR endosomes observed ( Figure 4F ) .", "These results support the hypothesis that EGFR activation can be modulated by the endosomal system .", "Since p-EGFR de-phosphorylation precedes EGFR degradation ( see above , Figure 1A , Figure 2—figure supplement 2 ) and EGFR degradation is unaffected ( Figure 4B , Figure 4—figure supplement 2 ) , we deduce that the effect on the life-time of p-EGFR caused by reduced endosomal fusion is primarily due to reduced de-phosphorylation . 10 . 7554/eLife . 06156 . 025Figure 4 . Increasing the number and life-time of p-EGFR endosomes results in prolonged EGFR activation .", "( A ) Protein down-regulation of EEA1 and Rabenosyn5 72 hr after siRNA transfection .", "RT-PCR showed an 80% reduction in Vps45 mRNA levels ( data not shown ) .", "( B ) Time course of EGFR integral intensity in endosomes after partial protein depletion of EEA1 , Rabenosyn5 , and Vps45 ( red curve ) or mock treatment ( black curve ) .", "Cells were given a 1-min pulse of 10 ng/ml EGF , washed and chased for the indicated time points before fixation .", "( C ) Representative images of HeLa EGFR BAC cells after EEA1 , Rabenosyn5 , and Vps45 knock-down or treatment with transfection reagent only ( mock ) .", "Scale bars , 10 μm .", "( D ) Shift in the EGFR-endosome area distribution toward smaller endosomes after EEA1 , Rabenosyn5 , and Vps45 knock-down .", "The values of the histograms of endosome area distribution for the control and knock-down conditions were normalized and subtracted .", "The curve shows the relative increase ( above zero ) or reduction ( below zero ) in the number of endosomes for each area bin ( in logarithmic scale ) ( for details see ‘Materials and methods’ and Figure 6—figure supplement 2 ) .", "Experimental points were fitted with two log-normal distributions .", "( E–G )", "Changes in p-EGFR endosomes in EEA1 , Rabenosyn5 , and Vps45 knock-down ( red curve ) or mock-treated ( black curve ) cells after continuous stimulation with 10 ng/ml EGF .", "Time courses of the mean integral intensity of p-EGFR per endosome ( E ) , mean number of p-EGFR endosomes determined experimentally ( squares ) or predicted by the mathematical model ( solid curves ) for a 37% endosomes fusion rate ( red curve ) compared to control ( black curve ) ( F ) , and total p-EGFR integral intensity in endosomes ( G ) measured as in Figure", "1 . Intensity curves were normalized to the intensity value at 10 min for mock-treated cells .", "Experimental points show mean ± SEM .", "All measurements were done in three independent experiments with a total of ∼150 cells per time point or condition .", "Time courses were fitted as in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 02510 . 7554/eLife . 06156 . 026Figure 4—figure supplement 1 . Knock-down of fusion machinery does not change EGFR distribution at the plasma membrane in HeLa cells . Cells were stimulated with 100 ng/ml EGF-AlexaFluor 488 for 10 min on ice to prevent receptor endocytosis .", "The AlexaFluor 488 signal was enhanced by detection with a specific antibody to detect the amount of EGFR at the plasma membrane .", "Scale bar , 10 μm .", "( A ) Representative images of HeLa EGFR BAC cells after EEA1 , Rabenosyn5 , and Vps45 knock-down or treatment with transfection reagent only ( mock ) .", "( B ) Total intensity of EGF-AlexaFluor 488 ( Mean ± SEM ) in knock-down or control cells .", "The total intensity was normalized to the fraction of the area covered by cells .", "Measurements were done in three independent replicates with a total of ∼150 cells per time point or condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 02610 . 7554/eLife . 06156 . 027Figure 4—figure supplement 2 . Knock-down of fusion machinery does not change EGFR degradation in HeLa cells .", "( A–B )", "Time course of EGFR degradation after partial protein depletion of the three endosomal fusion components EEA1 , Rabenosyn5 , and Vps45 or mock treatment and continuous stimulation with 10 ng/ml EGF for the indicated times in the presence of 10 μg/ml cyclohexamide in HeLa EGFR BAC cells .", "( A ) Representative EGFR and γ-Tubulin Western blots and ( B ) their quantification for EEA1 , Rabenosyn5 , and Vps45 knock-down ( red curve ) or mock-treated ( black curve ) samples .", "Points show mean ± SEM from three independent experiments .", "Lines are linear fits to the experimental points . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 02710 . 7554/eLife . 06156 . 028Figure 4—figure supplement 3 . Blocking endosome acidification with Bafilomycin increases both total EGFR and p-EGFR , but not the mean amount of p-EGFR per endosome .", "( A ) Time course of p-EGFR integral intensity in endosomes after incubation with 50 nM BafilomycinA1 ( red curve ) or 1% DMSO ( blue curve ) for 30 min and during the remaining of the time course .", "( B ) Time course of EGFR integral intensity in endosomes after incubation with 50 nM BafilomycinA1 ( red curve ) or 1% DMSO ( blue curve ) for 30 min and during the remaining of the time course .", "( C ) Time course of the mean p-EGFR integral intensity per endosome after incubation with 50 nM BafilomycinA1 ( red curve ) or 1% DMSO ( blue curve ) for 30 min and during the remaining of the time course .", "Experimental points show mean ± SEM .", "All measurements were done in three independent experiments with a total of ∼150 cells per time point and condition .", "Time courses were fitted as in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 028 Increased EGFR phosphorylation results in sustained Erk signalling ( Sasagawa et al . , 2005; Nakakuki et al . , 2010 ) and this leads to the phosphorylation and stabilization of the immediate early gene product c-Fos ( Nakakuki et al . , 2010 ) .", "We asked whether the redistribution of endosomal EGFR could be sufficient to induce sustained Erk activation and c-Fos phosphorylation .", "Indeed , upon EGF stimulation , both the amplitude and duration of Erk1/2 phosphorylation were increased in the depleted cells compared to control ( Figure 5A , B ) .", "Consistently , c-Fos phosphorylation was also higher after 30 min of EGF stimulation ( Figure 5C , D ) .", "The fact that the amount and life-time of total EGFR in endosomes remained unvaried in these experiments ( Figure 4B ) eliminates the trivial possibility that the observed changes are due to modulation of receptor degradation . 10 . 7554/eLife . 06156 . 029Figure 5 . Redistribution of endosomal EGFR increases the amplitude and duration of MAPK signalling .", "( A–B )", "Time course of Erk1/2 phosphorylation after partial protein depletion of the three endosomal fusion components EEA1 , Rabenosyn5 , and Vps45 or mock treatment and continuous stimulation with 10 ng/ml EGF for the indicated times in HeLa EGFR BAC cells .", "( A ) Representative phospho-Erk1/2 and Erk1/2 Western blots and ( B ) their quantification for EEA1 , Rabenosyn5 , and Vps45 knock-down ( red curve ) or mock-treated ( black curve ) samples .", "Points show mean ± SEM from three independent experiments .", "The time course was fitted as in Figure", "1 . ( C–D ) Nuclear c-Fos phosphorylation in EEA1 , Rabenosyn5 , and Vps45 knock-down or mock-treated cells as in ( A ) after 30 min of EGF stimulation .", "( C ) Representative images of EEA1 and phospho-c-Fos immunostaining in EEA1 , Rabenosyn5 , and Vps45 knock-down or mock-treated cells .", "Scale bars , 20 μm .", "( D ) Total intensity of nuclear phospho-c-Fos in EEA1 , Rabenosyn5 , and Vps45 knock-down or mock-treated cells .", "Bar graph shows mean ± SEM .", "Measurements were done in three independent experiments from a total of ∼1000 cells per condition .", "*p < 0 . 05 by a 2-tailed t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 029 The experiments on HeLa cells and the theoretical analysis raise the question of whether modulation of early endosome homotypic fusion is a general mechanism to regulate signal amplitude and duration .", "If this were the case , we would predict that growth factors with different signalling outputs ( amplitude and duration ) differentially modulate the endosomal distribution ( i . e . , endosome number , size , and cargo content ) .", "To test this prediction , we examined different growth factors and cellular systems .", "First , we used primary mouse hepatoblasts where HGF promotes their proliferation ( Tanimizu et al . , 2003 ) .", "In these cells , HGF but not EGF elicits a sustained Erk response ( Figure 6—figure supplement 1 ) .", "Indeed as predicted , stimulation of hepatoblasts with HGF caused a strong shift in the distribution of early endosomes toward smaller sizes ( Figure 6A , red curve Figure 6B ) , whereas EGF had the opposite effect ( Figure 6A , green curve , Figure 6B ) .", "Second , we turned to an in vitro model of reference for cell-fate decisions , PC12 cells .", "In PC12 cells , EGF stimulation leads to transient Erk phosphorylation and cell proliferation , whereas NGF leads to sustained Erk phosphorylation and cell differentiation ( Marshall , 1995 ) .", "Consistent with our results in primary mouse hepatoblasts , NGF stimulation in PC12 cells caused a significant shift in the distribution of early endosomes toward smaller sizes compared with EGF ( Figure 6C , D ) .", "Moreover , NGF itself was distributed to a larger number of smaller endosomes in comparison with EGF ( Figure 6E , F ) .", "Altogether , these data argue that the modulation of endosome fusion , reflected by the changes in endosome number and size , is a general property of growth factors .", "These data further suggest that signalling amplitude and duration can be regulated by changes in the fusion rate of endosomes ( see Table 1 ) . 10 . 7554/eLife . 06156 . 030Figure 6 . Growth factors differentially shift the distribution of the number and size of endosomes .", "( A ) Representative images of primary mouse hepatoblasts after stimulation with 10 ng/ml EGF or HGF for 30 min ( B ) Shift in the EEA1-positive endosome area distribution after stimulation with HGF ( red curve ) or EGF ( green curve ) .", "The values of the histograms of endosome area distribution for growth factor stimulated and non-stimulated cells were normalized and subtracted .", "The curve shows the relative increase ( above zero ) or reduction ( below zero ) in the number of endosomes for each area bin ( in logarithmic scale ) .", "HGF stimulation increased while EGF decreased the proportion of endosomes smaller than 0 . 2 μm2 .", "( C–F )", "PC12 cells after stimulation for 30 min with 100 ng/ml EGF or 50 ng/ml NGF .", "( C ) Representative images of EEA1-positive endosomes .", "( D ) Shift in the EEA1-positive endosome area distribution after stimulation with NGF ( red curve ) or EGF ( green curve ) measured as in ( B ) .", "NGF stimulation increased while EGF slightly decreased the proportion of endosomes smaller than 0 . 2 μm2 .", "( E ) Representative images of EGF or NGF .", "( F ) Differences in the area distribution of endosomal NGF and EGF measured as in ( B ) .", "NGF is enriched in endosomes smaller than 0 . 2 μm2 relative to EGF .", "For all graphs points show the mean ± SEM of experimental distributions .", "Measurements were done in three independent experiments with n ∼150 cells per condition .", "In all graphs , experimental points were fitted with two log-normal distributions .", "Image scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 03010 . 7554/eLife . 06156 . 031Figure 6—figure supplement 1 . HGF triggers sustained Erk1/2 activation in primary mouse hepatoblasts .", "( A–B )", "Time course of Erk1/2 phosphorylation after continuous stimulation with 10 ng/ml HGF or EGF for the indicated times in mouse primary hepatoblasts .", "( A ) Representative phospho-Erk1/2 and Erk1/2 Western blots and ( B ) its quantification for HGF ( red curve ) or EGF ( black curve ) stimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 03110 . 7554/eLife . 06156 . 032Figure 6—figure supplement 2 . Quantification of the difference between two area distributions . The differences between two endosome area distributions are measured as follows: ( 1 ) The binned histograms of the endosome area are built from the measurements of individual vesicles with bins linear in a logarithmic scale .", "( 2 ) The histograms are normalized on their integrals , i . e . , histograms are scaled to have the sum of values in all bins equal to one .", "( 3 ) The histogram from the control condition is subtracted from the respective histograms of interest .", "The relative enrichment ( red lines ) or depletion ( black lines ) in the population of vesicles is calculated by the integral over a particular area interval . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 03210 . 7554/eLife . 06156 . 033Table 1 . Changes in endosome number and areaDOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 033Cell typeEndosome marker or cargoGrowth factorEndosome number*Endosome area ( μm2 ) Increase in number of smaller vesiclesHeLa#EGFREGF22 ± 90 . 518 ± 0 . 023 ( control = 0 . 629 ± 0 . 029 ) 9 . 53% ± 0 . 014 ( <0 . 4 μm2 ) E14 . 5 hepatoblastEEA1EGF−6 ± 180 . 286 ± 0 . 02 ( control = 0 . 294 ± 0 . 02 ) −0 . 91% ± 0 . 003 ( <0 . 3 μm2 ) E14 . 5 hepatoblastEEA1HGF18 ± 120 . 276 ± 0 . 02 ( control = 0 . 294 ± 0 . 02 ) 2 . 05% ± 0 . 002 ( <0 . 3 μm2 ) PC12EEA1EGF3 ± 100 . 471 ± 0 . 05 ( control = 0 . 461 ± 0 . 05 ) −1 . 03% ± 0 . 01 ( <0 . 3 μm2 ) PC12EEA1NGF23 ± 160 . 454 ± 0 . 05 ( control = 0 . 461 ± 0 . 05 ) 2 . 2% ± 0 . 01 ( <0 . 3 μm2 ) PC12EGFEGF316 ± 460 . 276 ± 0 . 003–PC12NGFNGF341 ± 50 . 245 ± 0 . 0075 . 15% ± 0 . 01 ( <0 . 3 μm2 , difference from EGF-endosomes ) *Endosome number is expressed as the difference from the control or non-stimulated cells .", "The value shows the number of endosomes per 1000 μm2 of area covered by cells .", "#HeLa cells after knock-down of EEA1 , Rabenosyn5 , and Vps45 .", "All values show mean ± SEM .", "Finally , we tested whether the differences in endosomal distribution can be , at least in part , causative of the different cell-fates triggered by EGF and NGF in PC12 cells .", "If so , we would predict that redistributing EGF to a larger number of small endosomes as seen in PC12 stimulated with NGF would be sufficient to switch signalling specificity and induce differentiation of PC12 cells .", "Therefore , we applied the same protocol of partial protein depletion previously used for HeLa cells ( Figure 4 ) in PC12 cells and consistently observed a mild redistribution of EGF into smaller endosomes ( Figure 7—figure supplement 1A , C , D ) .", "Also in this case , the partial depletion did not result in major changes in EGF transport kinetics in PC12 cells ( Figure 7—figure supplement 1B ) , but increased the phosphorylation of both Erk ( Figure 7—figure supplement 2A , B ) and c-Fos ( Figure 7—figure supplement 2C , D ) upon stimulation with EGF .", "Next , we stimulated PC12 cells with EGF or NGF for 24 hr and analysed for neurite formation and β-III tubulin expression as markers of differentiation ( Ohuchi et al . , 2002 ) and for EdU incorporation as a measure of proliferation ( Figure 7A ) .", "Stimulation with NGF increased the number of cells with neurites ( Figure 7B , quantification in Figure 7C ) and positive for β-III tubulin ( Figure 7B , quantification in Figure 7D ) , and reduced cell proliferation ( Figure 7A , quantification in Figure 7E ) , the opposite of the stimulation with EGF .", "Remarkably , upon redistribution of endosomes , EGF increased process formation ( Figure 7B , quantification in Figure 7C ) , β-III tubulin expression ( Figure 7B , quantification in Figure 7D ) , and reduced cell proliferation ( Figure 7A , quantification in Figure 7E ) .", "The type of response was therefore similar to that of NGF , although the efficacy was lower .", "Nevertheless , these results show that a mild reduction of homotypic early endosome fusion was sufficient to modify cell fate and induce neuronal differentiation of PC12 cells . 10 . 7554/eLife . 06156 . 034Figure 7 . Redistribution of endosomal EGF is sufficient to trigger neuronal differentiation in PC12 cells .", "( A–B )", "Representative images of PC12 cells after partial protein depletion of either EEA1 , Rabenosyn5 , and Vps45 or EEA1 , Syntaxin-6 , and Syntaxin-13 , or mock treatment and stimulation with 100 ng/ml EGF or 50 ng/ml NGF for 24 hr .", "Scale bars , 50 μm .", "( B ) A high-resolution image of single cells to highlight the changes in β-III tubulin expression and neurite formation .", "β-III tubulin is shown in green , nuclei are shown in blue , and EdU-positive nuclei are shown in pink .", "Scale bars , 10 μm .", "Note that in Figure 6C , E , the short incubation times did not permit neurite outgrowth .", "( C ) Increase in the number of cells with β-III tubulin-positive processes longer than 1 μm compared to mock-treated cells after EGF stimulation .", "( D ) Increase in β-III tubulin expression measured by the total intensity of the cytoplasmic β-III tubulin immunostaining .", "The total intensity per image was normalized by the image area covered by cells .", "( E ) Number of proliferating cells measured by EdU incorporation .", "The number of EdU-positive nuclei was divided by the total number of nuclei .", "In all cases , data show mean ± SEM .", "For each parameter , pair-wise comparisons were done against EGF-stimulated mock-treated cells .", "*p < 0 . 05 , **p < 0 . 005 by Fisher's LSD test .", "All measurements were done in three independent experiments with a total of ∼15000 cells per condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 03410 . 7554/eLife . 06156 . 035Figure 7—figure supplement 1 . Knock-down of fusion machinery redistributes endosomal EGF in PC12 cells .", "( A ) Partial protein depletion of Syntaxin-6 and Syntaxin-13 72 hr after electroporation .", "Protein reduction of EEA1 and Rabenosyn5 was similar to that in HeLa cells ( not shown ) .", "( B ) Time course of EGF integral intensity in endosomes after EEA1 , Rabenosyn5 , and Vps45 ( red curve ) or EEA1 , Syntaxin-6 , and Syntaxin-13 knock-down ( blue curve ) or mock treatment ( black curve ) .", "Cells were given a 1-min pulse of 100 ng/ml of EGF-AlexaFluor 555 , washed and chased for the indicated time points before fixation .", "Curves were normalized to the intensity value at 10 min for mock-treated cells .", "Points show mean ± SEM .", "All measurements were done in three independent replicates with a total of ∼150 cells per time point or condition .", "Time courses were fitted as in Figure 1 ( C–D ) Shift in the EGF-endosome area distribution after EEA1 , Rabenosyn5 , and Vps45 ( C ) or EEA1 , Syntaxin-6 , and Syntaxin-13 ( D ) knock-down measured as in Figure 3 .", "Endosomes smaller than 0 . 2 μm2 ( cross-sectional area ) are increased after EEA1 , Rabenosyn5 , and Vps45 ( C ) or EEA1 , Syntaxin-6 , and Syntaxin-13 knock-down ( D ) .", "Points show the mean ± SEM .", "All measurements were done in four independent replicates with a total of ∼200 cells per time point or condition .", "Experimental points were fitted with two lognormal distributions . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 03510 . 7554/eLife . 06156 . 036Figure 7—figure supplement 2 . Redistribution of endosomal EGF is sufficient to increase MAPK activation in PC12 cells .", "( A–D )", "Analysis of MAPK activation in PC12 cells after partial protein depletion of either EEA1 , Rabenosyn5 , and Vps45 or EEA1 , Syntaxin-6 , and Syntaxin-13 , or mock treatment and stimulation with 100 ng/ml EGF or 50 ng/ml NGF for 30 min ( A ) Representative images of Erk1/2 activation by immunofluorescence in PC12 cells .", "phospho-Erk1/2 is shown in green and nuclei are shown in blue .", "Scale bars , 10 μm .", "( B ) Increase in phospho-Erk1/2 intensity compared to EGF-treated control cells .", "The total intensity was normalized by the fraction of the area covered by cells .", "( C ) Representative images of c-Fos phosphorylation by immunofluorescence in PC12 cells .", "phospho-c-Fos is shown in green .", "Scale bar , 25 μm .", "( D ) Increase in nuclear phospho-c-Fos intensity compared to EGF-treated control cells .", "In all cases , data show mean ± SEM .", "For each parameter , pair-wise comparisons were done against EGF-stimulated mock-treated cells .", "*p < 0 . 05 , **p < 0 . 005 by Fisher's LSD test .", "All measurements were done in three independent experiments with a total of ∼500 cells per condition . DOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 036" ], [ "Genomic studies have revealed that signalling pathways exert a profound effect on the endosomal system ( Pelkmans et al . , 2005; Stasyk et al . , 2007; Collinet et al . , 2010 ) .", "Parameters such as number of endosomes and size are tightly controlled in the case of EGF endocytosis ( Collinet et al . , 2010 ) .", "Our results provide a rationale for such modulation and a novel framework for interpreting and predicting the signalling response of phosphorylated RTKs .", "In homogeneous assays ( e . g . , by Western blot ) , the total levels of active RTKs can be observed to rapidly decay with time in most signalling systems ( Dunn and Hubbard , 1984; Burke et al . , 2001; Sousa et al . , 2012 ) .", "These methods , however , measure the average steady state of an entire cell population and lack the spatial information .", "Here , we employed quantitative high- and super-resolution microscopy to resolve details of this process with sub-cellular resolution and high sensitivity .", "We discovered that the mean amount of p-EGFR per endosome was fairly constant over time and p-EGFR was found in small clusters in early endosomes .", "The endosomal network is shaped by the balance of endosome fusion and fission ( Foret et al . , 2012 ) and this balance is also necessary for the formation of the endosomal clusters of p-EGFR .", "Modulation of the endosomal fusion/fission machinery manifests itself as a change in the size of endosomes ( Sigismund et al . , 2008 ) ( Figure 6 ) .", "Shifting the balance toward smaller endosomes through inhibition of fusion increased the number and reduced the size of endosomes , consequently expanding the number and life-time of p-EGFR clusters .", "Although the inhibition of endosome fusion was very mild , we cannot exclude the possibility that it may alter the recruitment and/or activity of signalling components by yet unknown mechanisms .", "On the other hand , for the interpretation of phenotypes upon perturbations on signalling , it is also important to consider the impact they have on the endosomal network ( Collinet et al . , 2010 ) .", "By analogy with synaptic transmission ( Edwards , 2007 ) , the packages of p-EGFR in early endosomes could be considered as quanta of signalling molecules .", "The concept of phosphorylated RTK quanta is reminiscent of analogue-to-digital communication systems , where a continuous variable ( e . g . , extracellular growth factor concentration ) is transformed into a sequence of binary levels ( e . g . , phosphorylated RTK quanta in endosomes ) .", "An analogue-to-digital switch was described for Ras nanoclusters at the plasma membrane ( Tian et al . , 2007 ) .", "In the case of endosomal digital signalling , our mathematical model predicts that it could serve two functions .", "First , it provides a mechanism to regulate signal amplitude and duration following RTK internalization .", "As a consequence , the total de-phosphorylation rate becomes dependent on the fusion/fission rate of the endosomes .", "This is interesting in view of the specific modulation of the endosome fusion/fission rates by growth factors ( Figure 6 , see below ) .", "Second , it acts as a noise dampening system ( Ladbury and Arold , 2012 ) , suppressing the noise due to , for example , fluctuations of EGF in the extracellular medium , expression levels of EGFR on the cell surface , etc .", "An increase in the amount of p-EGFR would result in faster de-phosphorylation rates .", "In contrast , low concentrations of EGF or EGFR would result in low de-phosphorylation rates .", "The middle point between the two extremes is the hallmark of signalling resilience .", "In addition , such a digital system may facilitate the integration of signalling information from different RTKs into a single , correct cell-fate decision .", "Our results highlight the importance of measuring the spatio-temporal distribution of signalling molecules using quantitative image analysis approaches to gain a deeper understanding of signal transduction regulation .", "What is the molecular machinery responsible for the formation of the clusters and how is the number of p-EGFR molecules regulated ?", "Clearly , the clustering mechanism is saturable ( Figure 2A , B ) , as very high concentrations of EGF above some threshold suppress the correct endosomal packaging in addition to changes in the entry routes and signal output ( Sigismund et al . , 2008 ) .", "We found that both Hrs and a few phosphatases , notably PTPN11 ( SHP2 ) , specifically regulate the amount of receptors within the p-EGFR clusters and their size .", "Hrs is known to interact with EGFR and regulate its degradation together with other components of the ESCRT machinery ( Umebayashi et al . , 2008 ) .", "However , the effect of Hrs on the size of the p-EGFR clusters appears to be independent of the formation of ILVs , as suggested by the fact that Snf8 and Vps24 down-regulation does not produce the same effect .", "Our mathematical model revealed that a correlation between p-EGFR dephosphorylation rate and p-EGFR amount per endosome can explain the mean constant size of p-EGFR quanta .", "We can envisage various non-exclusive mechanisms that can account for this correlation .", "One possible mechanism is a scaffold with a characteristic size that binds to p-EGFR and protects it from phosphatases .", "This hypothesis correlates higher total EGFR kinase activity to higher p-EGFR dephosphorylation , but only indirectly .", "Increasing the concentration of EGF in the medium would lead to a higher rate of delivery of p-EGFR to endosomes through vesicles which have no scaffold .", "If scaffold formation were rate limiting , the increased flux of p-EGFR into endosomes would reduce the fraction of protected p-EGFR thus exposing it to dephosphorylation .", "A caveat of this model is that , as the fusion of endosomes proceeds over time , multiple quanta would be expected to be brought together , increasing the mean amount of p-EGFR per endosome .", "This expectation is in contradiction with our experimental data ( Figure 1B , D ) .", "With this model , additional factors must thus be taken into account to explain why multiple quanta cannot co-exist on the same endosomes .", "The finding that Hrs knock-down increases the levels of p-EGFR suggests a different scaffold-based model .", "Instead of acting as a p-EGFR protective scaffold ( or part of a scaffold ) , Hrs could exert the opposite function and stabilize the unphosphorylated EGFR , preventing its re-phosphorylation ( Kleiman et al . , 2011 ) .", "Since the activity of Hrs is negatively regulated by p-EGFR ( Row et al . , 2005; Bache et al . , 2002 ) , this model is compatible with the data showing loss of quanta and increase in endosomal p-EGFR levels upon Hrs knock-down ( Figure 2D , E ) .", "However , this hypothesis alone can neither explain the formation of quanta nor the finding that blocking p-EGFR kinase activity does not change the total levels of p-EGFR over time ( Figure 2—Figure supplement 6 ) .", "Another mechanism is based on Turing Instability ( Turing , 1952 ) ( a reaction-diffusion mechanism ) .", "This mechanism is perhaps less intuitive but widely spread in biological processes , such as symmetry breaking and pattern formation in morphogenesis ( Kondo and Miura , 2010 ) .", "It is based on the observation that p-EGFR recruits and phosphorylates PTPN11 ( SHP2 ) in a phosphor-tyrosine dependent manner ( Deribe et al . , 2009 ) , thus enhancing its phosphatase activity ( Agazie and Hayman , 2003 ) .", "Briefly , p-EGFR would recruit and activate the phosphatase SHP2 , forming a negative feedback loop .", "The phosphatase would diffuse on the surface of endosomes , dephosphorylating p-EGFR molecules before being itself inactivated in the absence of further interactions with p-EGFR .", "Such reaction-diffusion mechanism within a specific parameter range is known to form spatially restricted clusters of active molecular species ( Turing Instability ) ( Turing , 1952 ) , in this particular case quanta of p-EGFR on endosomes .", "A transient increase in p-EGFR after an endosome fusion event would increase the recruitment and/or activity of SHP2 , re-establishing the p-EGFR quanta through dephosphorylation .", "If the characteristic length of Turing Instability is larger than endosome size , then multiple quanta cannot co-exist within a single endosome .", "The Turing Instability hypothesis explains the observed increase in p-EGFR quanta size after EGFR kinase inhibition , keeping the total p-EGFR levels unchanged ( Figure 2—figure supplement 6 , 7 ) , as well as the increase in total endosomal p-EGFR upon inhibition of endosome fusion ( Figure 4G ) .", "However , it does not explain the effect of Hrs knock-down .", "A combination of the Turing instability and Hrs-mediated ( negative ) scaffold mechanisms is more consistent with our observations .", "The regulation of endosomal packing reported in our study is likely not restricted to EGFR alone but is a general property , as different growth factors affect the endosomal network according to their specific signal output and cellular context ( Figure 6 ) .", "Hrs and SHP2 are also recruited by other RTKs ( Agazie and Hayman , 2003; Row et al . , 2005 ) .", "The relative affinity of SHP2 to different receptors could lead to larger or smaller quanta , thus tuning the specificity of the signalling response .", "RTK quanta with different sizes could also result from differential phosphorylation of Hrs by RTKs ( Row et al . , 2005 ) , given that the relative amount of Hrs on endosomes depends on its phosphorylation state ( Urbé et al . , 2000 ) .", "By which mechanisms can RTKs regulate the endosomal network ?", "It has been shown that RTKs can modulate the activity of the transport machinery .", "For example , activation of p38 MAP kinase causes phosphorylation of the Rab5 effectors EEA1 and Rabenosyn-5 , enhancing their recruitment to endosomes and consequently stimulating early endosome fusion ( Macé et al . , 2005; Cavalli et al . , 2001 ) .", "RTK stimulation also modulates the nucleotide cycle of Rab5 via activation of the Rab5 GEF RIN1 ( Tall et al . , 2001 ) or inactivation of the Rab5 GAP RN-tre ( Lanzetti et al . , 2000 ) .", "Therefore , we predict that in general RTK ligands that stimulate the endosomal fusion machinery ( such as EGF ) will have a short phosphorylation half-life , whereas ligands that change the fusion/fission balance in favour of smaller endosomes ( such as NGF ) will have a long phosphorylation half-life .", "The combined effects of quanta size regulation through Hrs and SHP2 and modulation of fusion/fission will give a specific signalling amplitude and duration in different cell types stimulated with different ligands .", "We propose that the shape of distribution of the endosomal network can serve as a predictive parameter of the signalling status of the cell .", "Our results support the concept of endosomes as signalling platforms ( Di Guglielmo et al . , 1994 ) , a view recently shared for the β2-adrenoceptor ( Irannejad et al . , 2013 ) but opposed by other studies ( Brankatschk et al . , 2012; Sousa et al . , 2012 ) .", "This apparent contradiction can be explained by the fact that , under normal conditions of endocytosis , only the small fraction of p-RTK in endosomes are protected from inactivation and degradation , and can thus contribute to signal propagation .", "A feature of the p-EGFR clusters is that , with the increase in the local concentration , the stability of the active EGFR dimer ( Chung et al . , 2010 ) and signalling properties ( Verveer et al . , 2000 ) would also be increased .", "By blocking endocytosis , the levels of active receptors are artificially increased at the cell surface ( Sousa et al . , 2012 ) , bypassing the normal requirement for endosomal regulation .", "Our observations raise many more questions concerning the molecular mechanisms of quanta formation and their impact on cell fate decision .", "Clearly , the variety of models on quanta formation requires future experimental tests to determine the correct mechanism and reveal its molecular details .", "In addition , it will be important to validate our observations in an in vivo animal model to demonstrate that the dynamics of the endosomal network reflect the signalling activity by RTK under physiological conditions ." ], [ "To reduce the consequences of EGFR overexpression , we used HeLa Kyoto cells transfected with a bacterial artificial chromosome ( BAC ) transgene stably expressing EGFR-GFP under its endogenous promoter ( Poser et al . , 2008 ) .", "Cells were incubated for different times in serum-free medium with 10 ng/ml EGF ( Invitrogen , California , USA ) or for 30 min with 0 . 05 , 0 . 1 , 0 . 25 , 0 . 5 , 1 , 2 . 5 , 5 , 7 . 5 , or 10 ng/ml EGF .", "Cells were then fixed and processed for immunofluorescence as previously described ( Collinet et al . , 2010 ) using a mouse monoclonal anti-phospho-tyrosine 4G10 antibody ( Millipore , California , USA ) directly labelled with AlexaFluor 555 ( Molecular Probes , Invitrogen ) .", "For colocalization measurements , samples were also incubated with rabbit polyclonal anti-EEA1 ( Rink et al . , 2005 ) or mouse monoclonal anti-LAMP-1 ( BD Biosciencies , California , USA ) antibodies .", "Images were acquired using a laser-scanning confocal microscope ( Duoscan , Zeiss ) with a 63×/1 . 4 oil objective .", "Multicolour images were acquired in three sequential scans: GFP fluorescence and AlexaFluor 555 fluorescence were detected simultaneously with two different detectors using 488 and 561 nm laser light and a 505/530 band-pass filter or a 593 nm long-pass spectral range in a META detector ( Zeiss ) ; FRET signal was detected with 458 nm excitation and a 593 nm long-pass spectral range in a META detector ( Zeiss ) .", "10 images per time point were collected , and each image was the maximum projection of four confocal sections of ∼1 μm thickness with 0 . 5 μm step .", "For the comparison between live and fixed cells , images were acquired with an automated spinning-disk confocal microscope ( OPERA , Evotec Technologies-PerkinElmer ) with a 40×/0 . 9 NA water immersion objective .", "EGFR-GFP was excited with a 488 nm laser and detected with a 520/35 nm filter .", "DAPI for nuclei identification was excited in a separate exposure with a 405 nm laser and detected a 450/50 nm filter .", "Eighty images per condition were acquired .", "Every image contained on average 20 cells .", "Image analysis was performed using custom designed image analysis software ( MotionTracking ) as previously described ( Rink et al . , 2005; Collinet et al . , 2010 ) .", "The ‘integral intensity’ corresponds to the integral of fluorescent marker intensity per endosome .", "The ‘total integral intensity’ is defined as the sum of integral intensities of all endosomes in an image normalized by the area covered by the cells .", "The ‘endosome cross-sectional area’ was measured as the apparent fluorescent area ( in µm2 ) of an endosome ( above the half-maximum value of fluorescence intensity of each structure ) .", "Since MotionTracking approximates real image intensity by a sum of analytical functions ( Rink et al . , 2005 ) , the resulting area and intensity have no pixel granularity .", "p-EGFR or ub-EGFR was first identified on the basis of triple colocalization between objects detected by the EGFR ( 488 nm laser excitation and 505/530 nm bandpath emission filter ) , anti-p-Tyr antibody p-Tyr-ab for p-EGFR or anti-mono and polyubiquitynilated conjugates ( FK2 ) ( Enzo Biosciences , New York , USA ) ( 561 nm laser excitation and a 593 nm long-pass filter ) , and FRET ( 458 nm laser excitation and a 593 nm long-pass filter ) channels ( Figure 1—figure supplement 1A ) .", "Colocalization was scored by cross-sectional overlap >30% .", "The FRET signal was corrected for spectral bleed-through ( SBT ) .", "Two major processes contribute to the SBT: ( 1 ) the GFP fluorescence bleed-through in the FRET channel and ( 2 ) direct excitation of AlexaFluor 555 by the 458 nm laser .", "We performed control experiments to estimate SBT for subsequent correction .", "To estimate GFP fluorescence bleed-through , we imaged EGFR-GFP BAC HeLa cells in the FRET channel ( excitation 458 nm ) without p-Tyr-ab staining .", "The signal in the FRET channel was below our detection limit and , therefore , we omitted correction in the subsequent analysis .", "The SBT by direct excitation of AlexaFluor 555 was estimated by quantification of FRET vesicles that colocalized with p-Tyr-ab or ub-ab , but not with EGFR ( bleed-through control ) .", "Following the approach of Gordon et al . , ( 1998 ) , the correction in this case will be , ( 1 ) F=I−k·T , where F is the corrected intensity in the FRET channel , I is the raw intensity in the FRET channel , T is the intensity in the p-Tyr-ab channel , k=<Icontrol><Tcontrol> is the bleed-through coefficient ( ratio of means ) calculated from control vesicles .", "Unfortunately , this correction method provided a good estimation of the average FRET signal , but when applied to individual endosomes it gave negative intensities for a substantial ( 30–40% ) number of cases , thus precluding the estimation of mean intensity per endosome .", "In order to identify the source of negative intensities , we calculated the distribution of ratios of intensities in the FRET channel to the intensities in the p-Tyr-ab channel per endosome ( Figure 1—figure supplement 1B ) .", "This distribution is broad and one can conclude that correction by Equation 1 will inevitably produce negative values in some cases .", "We fitted the distribution by the sum of three Gaussian components ( Figure 1—figure supplement 1B , red , green , and blue dashed lines ) .", "By using control cells that did not express EGFR-GFP , we tested that the first two components correspond to SBT ( direct excitation of AlexaFluor 555 by the 458 nm laser ) .", "Next , we developed a probabilistic model to find the expected FRET signal , given the p-Tyr-ab signal and the constants ( µ , σ ) of Gaussian distribution of the intensities ratios .", "The distribution of ratios is P ( m ) dm=12πσe− ( m−μ ) 22σ2dm .", "We denoted m=I−FT the ratio of bleed-through signal in the FRET channel to the signal in the p-Tyr-ab channel for individual endosomes .", "After this substitution , the probability to obtain a FRET signal F is P ( F ) dF=P ( m ( F ) ) dmdFdF=12πσTexp ( − ( F− ( I−μT ) ) 22σ2T2 ) dF .", "Since the bleed-through cannot be higher than the measured signal I , we can calculate the expectation of the FRET signal as: 〈F〉=∫0IF·P ( F ) dF∫0IP ( F ) dF .", "After substitution and integration , we get: ( 2 ) 〈F〉=I−μT+2πσT ( 1−e−12 ( μσ ) 2 ) −sgn ( I−μT ) · ( 1−e−12 ( I−μTσT ) 2 ) erf ( μ2σ ) +erf ( I−μT2σT ) .", "One can see that", "( a ) if I − µT > 0 ( i . e . , SBT is small relative to the true FRET signal ) , then the last term in the formula is very small and 〈F〉≈I−μT in agreement with Gordons' formula;", "( b ) even if I − µT < 0 ( i . e . , SBT is large relative to the true FRET signal or the FRET signal is absent ) , the Equation 2 always gives small , but positive values .", "As such , Equation 2 provides a good estimation of the expected FRET intensity given the measured intensities in the FRET and p-Tyr-ab channels .", "Next , we developed this approach further by taking into account that the real bleed-through distribution is the sum of two Gaussians with mean values µ1 , µ2 , standard deviations σ1 , σ2 and their contribution in the total distribution a1 , a2 .", "Following the same approach as above we get: ( 3 ) 〈F〉=I− ( a1μ1+a2μ2 ) T+2π ( a12σ12+a22σ22 ) T ( 1−e−M ) −sgn ( N ) · ( 1−e−N2 ) erf ( M ) +erf ( N ) , where M= ( μ12σ1 ) 2+ ( μ22σ2 ) 2 and N=I− ( a1μ1+a2μ2 ) T2 ( a12σ12+a22σ22 ) T .", "The example of FRET correction by Equation 3 is presented on Figure 1C .", "To validate the FRET measurements , cells were treated with EGF , stained using a rabbit monoclonal anti-p-EGFR ( Tyr 1068 ) antibody , and imaged using a laser-scanning confocal microscope using the same protocols as described above .", "EGFR-GFP BAC cells were incubated with 10 ng/ml EGF for 30 min , fixed and stained with the rabbit monoclonal anti-p-EGFR ( Tyr 1068 ) antibody as described above .", "One field of view was sequentially acquired to record bleaching of GFP and fluorescently labelled secondary antibodies .", "The resulting time series was segmented with MotionTracking as described above , individual objects were tracked for consecutive frames , and the fluorescence intensity of every endosome between two consecutive frames was subtracted to build the ΔIntensity distribution ( Figure 2—figure supplement 3A , C ) .", "The width of distribution is mostly determined by the fluctuations of intensities .", "However , due to bleaching , the distribution is slightly skewed toward negative values .", "First , the ΔIntensity was binned .", "Then the difference between frequencies of negative and positive ΔIntensity of equal absolute values was plotted as function of ΔIntensity ( Figure 2—figure supplement 3B , D ) .", "We named it neg-double-difference function .", "Since every bin of neg-double-difference function in the vicinity of the first peaks contained ∼2500 events , random fluctuations were strongly suppressed and the averaging revealed the discrete structure of bleaching , that is , bleaching of individual molecules .", "The local amplitude positive maxima correspond to discrete intensity changes when 1 , 2 , 3 , … , n molecules are bleached ( see e . g . , arrows on peaks at 280 , 560 , and 800 integral intensity units in Figure 2—figure supplement 3B ) .", "We estimated that one molecule of GFP and alexa555-antibody corresponds to the integral intensity units at the first peak of the neg-double-difference function ( 280 and 190 integral intensity units for EGFR-GFP and alexa555 , respectively ) .", "This method estimated directly the number of EGFR-GFP molecules .", "The total number of EGFR molecules per endosome was corrected for the ratio of endogenous and BAC EGFR-GFP expressions ( 1 . 29 ± 0 . 07 , based on WB quantification ) .", "Since the method estimated the number of fluorophores , in the case of antibodies with unknown labelling stoichiometry and epitope accessibility , the result has to be corrected for a scaling factor .", "The scaling factor of antibody labelling was estimated as 1 . 9 by comparison of EGFR and p-EGFR distributions at 3 and 5 min of EGF stimulation ( 10 ng/ml ) , when most internalized EGFR are still phosphorylated ( Sorkin and Goh , 2009 ) , with distributions at 30 min .", "We used this fluorescence intensity-based method also to estimate the number of EGFR molecules in both clathrin-dependent and independent vesicles .", "From geometrical calculations , assuming that an uncoated CCV has a diameter of 90 nm and an EGFR dimer has a diameter of ∼15 nm and luminal domain of ∼10 nm , we estimate that a CCV can contain up to 70 EGFR molecules .", "However , this calculation does not take into account that vesicles contain multiple types of transmembrane proteins and thus , the value can only be an upper limit .", "Therefore , we estimated the number of EGFR molecules per diffraction-limited , EEA1-negative vesicle that can be observed following 5 min of 10 ng/ml EGF stimulation .", "Using this method , we estimated 8 . 5 ± 3 . 5 molecules/vesicle .", "Based on this value , we calculated that ∼12 vesicles are required to deliver the 102 EGFR molecules/EEA1-positive endosome .", "Time courses were fitted with the sum of two exponential terms: one for growth and one for decline .", "( 4 ) Ae−tτ1+Be−tτ2 The constant of the decline exponent τ2 was used as an estimation of the decay time of the corresponding process .", "The fitting of the experimental data was done according to an optimization scheme previously described ( Press et al . , 1992 ) .", "To discriminate p-EGFR exposed on the surface of endosomes from p-EGFR sequestered into ILVs , we used a differential detergent solubilisation method as previously described for protease protection assays ( Malerød et al . , 2007 ) .", "Cells were fixed and permeabilized with saponin 0 . 1% for 10 min or digitonin 0 . 001% for 1 min .", "After permeabilization , cells were washed with PBS and stained with a mouse monoclonal anti-phospho-tyrosine-AlexaFluor 555 antibody ( Millipore ) , a mouse monoclonal anti-LBPA ( a gift by J Gruenberg , University of Geneva ) antibody , or a mouse monoclonal anti-GFP ( Roche , Switzerland ) antibody together with a goat anti-mouse-AlexaFluor 555 antibody ( Molecular Probes , Invitrogen ) to reveal the antigen signal .", "Membrane permeabilization with saponin allows access of antibodies both to the cytosol and the luminal content of endosomes , whereas digitonin only to the cytosol .", "Upon digitonin permeabilization , the staining of LBPA , a marker of ILVs and EGF or EGFR was strongly reduced in comparison with saponin permeabilization ( Figure 2A ) , consistent with their localization predominantly within the endosomal lumen .", "After 30 min of EGF stimulation , the endosomal , but not the plasma membrane EGFR staining was strongly reduced in cells permeabilized with digitonin compared with saponin , probably reflecting the internalization of receptors into ILVs ( Figure 2A , B ) .", "In contrast , the p-EGFR levels were only moderately reduced upon permeabilization with digitonin compared with saponin extraction ( Figure 2A , B ) , suggesting that the majority of p-EGFR faced the cytosolic surface of endosomes and was not within ILVs .", "To measure Shc1 recruitment to endosomes , cells were permeabilized with saponin and stained with a rabbit polyclonal anti-Shc1 antibody ( BD Biosciences ) .", "Image acquisition , correction , and analysis proceeded as described above .", "Cells were stimulated for different times with 10 ng/ml EGF and fixed as described above .", "To detect p-EGFR in endosomes , cells were stained using a rabbit monoclonal anti-p-EGFR ( Tyr 1068 ) antibody .", "For dSTORM microscopy , the samples were mounted on medium optimized for enhanced switching between fluorescent and non-fluorescent states as previously described ( van de Linde et al . , 2011; Lampe et al . , 2012 ) .", "Imaging was performed using a H3 Andor spinning disk microscope with a 100× objective as previously described ( van de Linde et al . , 2011; Lampe et al . , 2012 ) .", "First , the binned histograms of endosome area were built with bin widths linear in a logarithmic scale .", "Then , the histograms were normalized on their integrals , that is , histograms were scaled to have the sum of values in all bins equal to one .", "Finally , the histogram from the control condition was subtracted from the respective histograms of the different conditions ( Figure 6—figure supplement 2 ) .", "To describe the time course of the formation of a mean constant amount of p-EGFR per endosome during endocytosis , we postulated a sigmoidal dependency of the dephosphorylation rate on the amount of p-EGFR per endosome .", "The rationale for this is that if the amount of p-EGFR per endosome is above a critical value , dephosphorylation is significantly increased , whereas if the amount is lower , dephosphorylation is decreased .", "The delay between EGF stimulation and onset of internalization of p-EGFR into early endosomes is well documented ( Burke et al . , 2001; Wiley , 2003 ) .", "This delay includes EGF binding to receptor ( ∼3 min ) , CCV formation ( ∼1–2 min ) and delivery of p-EGFR to early endosomes .", "In order to keep the model as simple as possible , we described these mechanisms in a coarse grained model by an exponential delay with constant δτ .", "Since the dephosphorylation rate depends on the amount of p-EGFR per endosome , we expanded the mass flux equation usually applied in these cases with an equation that describes the number of endosomes carrying p-EGFR .", "Our experimental data suggest a significant redistribution of EGFR from the plasma membrane into endosomes even at very low doses of EGF ( see Figure 1E , green curve . Compare 0 . 5 with 10 ng/ml ) .", "A simple mechanism to explain this is the internalization of ligand-unoccupied EGFR upon EGF stimulation , for example by formation of EGFR oligomers at the plasma membrane ( Ariotti et al . , 2010; Hofman et al . , 2010 ) .", "Another possible mechanism includes transient activation of p38 ( Faust et al . , 2012 ) by EGFR signalling that leads to acceleration of unoccupied receptor internalization ( Zwang and Yarden , 2006; Faust et al . , 2012 ) .", "Therefore , we modelled the rate of EGFR-positive vesicle formation as Kv=kv0+kv1SpqQvq+Spq , where Sp is p-EGFR on plasma membrane , q is Hill coefficient , Qv is a characteristic constant .", "We considered that the ratio of EGF loaded/unloaded EGFR in the vesicles is equal to the weighted ratio p-EGFR/EGFR on plasma membrane with weight factor w .", "Importantly , the use of this term in the model gave the best description of the time course of total EGFR , but was not essential to explain the p-EGFR dynamics in individual endosomes ( data not shown ) .", "( 5 ) dSmdt=−kin·Sm·cEGF ( 1−e− ( tδτ ) 2 ) +kout·Smp−Kv·SmSm+w·Smp·Sm+kre_out·Sre ( 6 ) dSmpdt=kin·Sm·cEGF ( 1−e− ( tδτ ) 2 ) −kout·Smp−Kv·w·SmpSm+w·Smp·Smp ( 7 ) dSpedt=Kv·w·SmpSm+w·Smp·Smp− ( β1+Sper ( Q·Npe ) r+Sper ( β2−β1 ) ) ·Spe ( 8 ) dSedt=Kv·SmSm+w·Smp·Sm+ ( β1+Sper ( Q·Npe ) r+Sper ( β2−β1 ) ) ·Spe−kreSe−kleSe ( 9 ) dSredt=kreSe−kre_out·Sre ( 10 ) dNpedt=Kvsvw·SmpSm+w·Smp·w·Smp−ρ·Npe2+f·Npe , where , Sm is the amount of non-phosphorylated EGFR on the plasma membrane , cEGF is the amount of EGF in the extracellular medium , Smp is the amount of p-EGFR on plasma membrane , Se is the non-phosphorylated EGFR on early endosomes , Spe is the amount of non-phosphorylated EGFR on early endosomes , Sre is the amount of EGFR on recycling endosomes , Kv is the rate of EGF-stimulated EGFR-positive vesicle formation ( see above ) , Npe is the number early endosomes with p-EGFR , kin is the rate of EGF binding to EGFR , kout is the rate of EGF release from EGFR , kre is the rate of sorting of EGFR from early to recycling endosomes , kle is the rate of sorting of EGFR from early to late endosomes , kre_out is the rate of delivery of EGFR from recycling endosomes to plasma membrane , sv is the mean amount of p-EGFR per endocytic vesicle , β1 , β2 are minimum and maximum dephosphorylation rates , r is a Hill coefficient of dephosphorylation rate , Q is the characteristic amount of p-EGFR at which the dephosphorylation has ½ maximal rate , ρ is the early endosome homotypic fusion rate ( measured as number of events/minute/endosome ) , f is the early endosome homotypic fission rate .", "Equation 5 describes the total amount of non-phosphorylated EGFR on the plasma membrane ( Sm ) .", "The first term describes the loss of non-phosphorylated EGFR that becomes phosphorylated upon EGF binding .", "The second term describes the increase in non-phosphorylated EGFR upon release of EGF from p-EGFR and concomitant dephosphorylation .", "The third term describes the amount of non-phosphorylated EGFR which is internalized upon EGF-stimulated endocytosis ( see above ) .", "The last term describes the recycling of EGFR to the plasma membrane .", "Equation 6 describes the total amount of p-EGFR on the plasma membrane ( Smp ) .", "The first term describes the phosphorylation of EGFR upon EGF binding , the second the dephosphorylation following EGF release , and the third its endocytosis .", "Equation 7 describes the amount of p-EGFR in EEA1-positive early endosomes ( Spe ) .", "The first term describes the endocytosis of p-EGFR and the second its dephosphorylation .", "Note that the equation includes a sigmoidal function β1+Sper ( Q·Npe ) r+Sper ( β2−β1 ) for the dephosphorylation rate .", "Equation 8 describes the amount of non-phosphorylated EGFR on early endosomes ( Se ) .", "The first term describes EGF-stimulated endocytosis of ligand-free EGFR .", "The second term describes the increase in the amount of non-phosphorylated EGFR through dephosphorylation of p-EGFR .", "The third and fourth terms describe the sorting of EGFR to recycling and late endosomes , respectively .", "Equation 9 describes the amount of EGFR on recycling endosomes ( Sre ) .", "The first term describes the delivery of EGFR from early endosomes and the second its recycling to the plasma membrane .", "Equation 10 describes the number of EEA1-positive early endosomes containing p-EGFR ( Npe ) .", "For simplicity , we considered that the p-EGFR is evenly distributed between endosomes .", "The first term describes the endocytosis of p-EGFR , the second the homotypic fusion of early endosomes , and the third their homotypic fission .", "The model was fitted to the experimental data which included time courses of p-EGFR and EGFR colocalization to EEA1 ( Kalaidzidis et al . , 2015 ) upon stimulation with four different concentrations ( 0 . 5 , 1 . 0 , 5 . 0 , and 10 . 0 ng/ml ) of EGF ( Figure 3A , B ) .", "Fitting was performed with FitModel software ( Zeigerer , 2012 ) ( http://pluk . mpi-cbg . de/projects/fitmodel ) .", "Since the amount p-EGFR was measured experimentally in arbitrary FRET intensity units , the modelled amount of p-EGFR was scaled before a comparison with the experimental data .", "The scaling factor was found by the least square formula scale=∑i=1Ndi·siσi2∑i=1Nsi2σi2 , where di , σi; i = 1…N are experimental values and their SEMs; si are model predictions for the respective time points .", "The model prediction of p-EGFR modulation by reduction of the early endosome homotypic fusion rate is presented on Figure 3C .", "The model and fit parameters are provided in the text format and in the format of FitModel software in the Source code 1 ( Model . zip ) .", "HeLa EGFR BAC cells were reverse transfected with 5 nM siRNA oligonucleotides per gene using the oligonucleotides given in Table 2 . 10 . 7554/eLife . 06156 . 037Table 2 . List of siRNAs used for down-regulation of endosomal proteinsDOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 037Gene namesiRNA librarysiRNA IDEEA1Ambion Silencer139147Rabenosyn5Ambion Silencer292470Vps45Ambion Silencer136363HrsQiagenSI00067305HrsQiagenSI00288239HrsQiagenSI02659650Vps24Invitrogen148627Vps24Invitrogen148628Vps24QiagenSI00760515Snf8Invitrogen140086Snf8QiagenSI00375641Snf8QiagenSI00375648 Transfection was carried out using Interferin ( Polyplus transfection ) together with the selected oligonucleotides following the manufacturer's instructions or treated only with Interferin ( mock ) .", "72 hr after transfection total protein extracts were prepared to measure down-regulation of the targeted proteins by western blotting using antibodies previously described for EEA1 and Rabenosyn5 ( Collinet et al . , 2010 ) .", "To measure the redistribution of EGFR in endosomes , cells were incubated with 10 ng/ml EGF ( Invitrogen ) , fixed , and processed for quantitative microscopy .", "Image acquisition and analysis were done as described earlier .", "Measurement of p-EGFR was done using the FRET assay described above .", "To measure EGFR transport kinetics , cells were incubated in serum-free medium for 1 min with 10 ng/ml EGF ( Invitrogen ) , washed with serum-free medium , and chased for different time points .", "Cells were then fixed and samples were processed for quantitative microscopy analysis as explained above .", "To measure degradation of EGFR , cells were incubated for 1 hr with 10 μg/ml Cyclohexamide before stimulation with 10 ng/ml EGF for different time points .", "Total protein extracts were prepared and analysed by western blotting using rabbit monoclonal anti-EGFR ( Cell Signaling , New England BioLabs , Massachusetts , USA ) and mouse anti γ-tubulin ( Antibody Facility , MPI-CBG , Germany ) antibodies .", "To measure activated EGFR at the plasma membrane , cells were incubated with 100 ng/ml EGF-AlexaFluor 488 for 10 min on ice to prevent endocytosis , fixed with PFA , stained with a rabbit anti-AlexaFluor 488 antibody fraction ( Invitrogen ) to enhance the fluorescent signal and imaged as described above .", "HeLa EGFR BAC cells were reverse transfected with the protocol described above with 5 nM of the oligonucleotides in Table 3 .", "After 72 hr , cells were stimulated with 10 ng/ml EGF for 30 min , fixed with PFA , and stained using a rabbit monoclonal anti-p-EGFR ( Tyr 1068 ) antibody as described above .", "Images were acquired with an automated spinning-disk confocal microscope ( OPERA , Evotec Technologies-PerkinElmer ) with a 40×/0 . 9 NA water immersion objective .", "Settings were adjusted to minimize pixel-intensity saturation and maximize the dynamic range .", "Around 30 images for each siRNA oligonucleotide were collected .", "Images with less than three cells were excluded from analysis .", "Image analysis was performed with MotionTracking as described above . 10 . 7554/eLife . 06156 . 038Table 3 . List of genes for PTP siRNA screenDOI: http://dx . doi . org/10 . 7554/eLife . 06156 . 038Gene symbolGene IDsiRNa IDSequence 5′–3′PTPN1357835783-HSS108838UCACAUUUCUGAACCAACUAGACAAPTPN1357835783-HSS184076CAUCAGACUCUAAGCAACAUGGUAUPTPN1357835783-HSS184077CCAUUGAGGGUAAUCUCCAGCUAUUPTPN1357835783-NM_080683 . 1_1459GAAACACCCUUUGAAGGCAACUUAAPTPRK57965796-HSS108869CCCAUCCAAGUGGAAUGUAUGUCUUPTPRK57965796-HSS108870GGUCAUUCUUGAAACUGAUACUUCAPTPRK57965796-HSS184093CCGCGCAAAGGAUACAACAUCUAUUPTPRK57965796-NM_002844 . 2_975CCGCUUCCUUCAGAUUGCAAGAAGUPTPRA57865786-HSS108844CCAGUUCACGGAUGCCAGAACAGAAPTPRA57865786-HSS108845GCAUUCUCAGAUUAUGCCAACUUCAPTPRA57865786-HSS108846GGCACCAACAUUCAGCCCAAAUAUAPTPRA57865786-NM_080841 . 2_1383CGCCUCAUCACUCAGUUCCACUUUAPTPRR58015801-HSS108880AGUUGAGGUUCUGGUUAUCAGUGUAPTPN957805780-HSS108830CCCUCAUUGACUUCUUGAGAGUGGUPTPRR58015801-HSS108882GGUACACCUCAUGGCCUGAUCACAAPTPN957805780-HSS108831ACCUCAUGAGGAACCUCUUCGUUCUPTPRR58015801-HSS184097CAAGAGAGAAGAGGGUCCAACGUAUPTPN957805780-HSS184065CGCUGUCUUGGAAUGUGGCUGUCAAPTPRR58015801-NM_130846 . 1_1022CAGUGGCAAGGAGAAAGCCUUCAUUPTPN957805780-NM_002833 . 2_1369CAUCCAAGAGUUGGUGGACUAUGUUPTPN257715771-HSS108817GGAAGACUUAUCUCCUGCCUUUGAUPTPN257715771-HSS108818GAGCGGGAGUUCGAAGAGUUGGAUAPTPN257715771-HSS184039GAGAUUCUCAUACAUGGCUAUAAUAPTPN257715771-NM_002828 . 2_1178CCGAUGUACAGGACUUUCCUCUAAADUSP218441844-HSS140936GCUCUGCCACCAUCUGUCUGGCAUAPTPN357745774-HSS108820GGCGUGGUACAGACCUUUAAAGUUAPTPRE57915791-HSS108853UCUGGGAAUGGAAAUCCCACACUAUDUSP218441844-HSS140937GCUGCUGUCCCGAUCUGUGCUCUGAPTPN357745774-HSS108821GAGCUGUCCGCUCAUUUGCUGACUUPTPRE57915791-HSS108854ACGAGACUUUCUGGUCACUCUCAAUDUSP218441844-HSS140938GGCAUCACAGCCGUCCUCAACGUGUPTPN357745774-HSS108822CCACCCGGGUAUUAUUGCAGGGAAAPTPRE57915791-HSS108855GGAACAGUAUGAAUUCUGCUACAAADUSP218441844-NM_004418 . 3_925UGGACGAGGCCUUUGACUUCGUUAAPTPN357745774-NM_002829 . 2_621CAAUCAGAAGCAGAAUCCUGCUAUAPTPRE57915791-NM_130435 . 2_1499GAGCAGGAUAAAUGCUACCAGUAUUPTPRF57925792-HSS108856CCCAUCAUCCAAGACGUCAUGCUAGPTPRF57925792-HSS108858GGACAGCAGUUCACGUGGGAGAAUUPTPRF57925792-HSS184088CAGCUGUGCCCUUUAAGAUUCUGUAPTPRF57925792-NM_130440 . 2_6013CAGCUUUGACCACUAUGCAACGUAAPTP4A280738073-HSS140957GAUAACUCACAACCCUACCAAUGCUDUSP618481848-HSS176270GAGAGCAGCAGCGACUGGAACGAGAPTP4A280738073-HSS140958GCGUUCAAUUCCAAACAGCUGCUUUDUSP618481848-HSS176271UGGCAUUAGCCGCUCAGUCACUGUGPTP4A280738073-HSS188476GGUUCGAGUUUGUGAUGCUACAUAUDUSP618481848-HSS176272UGGCUUACCUUAUGCAGAAGCUCAAPTP4A280738073-NM_080392 . 2_1123UCGAGUUUGUGAUGCUACAUAUGAUDUSP618481848-NM_022652 . 2_1097CAUGUGACAACAGGGUUCCAGCACAPTPRM57975797-HSS108871CCGAGUGAGGCUGCAGACAAUAGAAPTP4A31115611156-NM_007079 . 2_423UCAGCACCUUCAUUGAGGACCUGAAPTPN182646926469-HSS120076GCUGCCUUAUGAUCAGACGCGAGUAPTPN1457845784-HSS108841UCAUGGGAAUGAAGAAGCCUUGUAUPTPRM57975797-HSS108872CAGGCUCUGGUUACAGGGCAUUGAUPTP4A31115611156-NM_007079 . 2_460UACCACUGUGGUGCGUGUGUGUGAAPTPN182646926469-HSS120077UCGAGAGAUAGAGAAUGGGCGGAAAPTPN1457845784-HSS108843GCCGCUGAUGUUGGCAGCAUUGAAUPTPRM57975797-HSS108873CCCGACGCUUCAUUGCUUCAUUUAAPTP4A31115611156-NM_007079 . 2_473CGUGUGUGUGAAGUGACCUAUGACAPTPN182646926469-HSS120078CCCACCUGACUUCAGUCUCUUUGAUPTPN1457845784-HSS184078GAUAUCAGUAUUACCUGCAAGUCAAPTPRM57975797-NM_002845 . 3_1217CCGACGCUUCAUUGCUUCAUUUAAUPTP4A31115611156-NM_007079 . 2_678CCAUCAACAGCAAGCAGCUCACCUAPTPN182646926469-NM_014369 . 2_835UCAGUCUCUUUGAUGUGGUCCUUAAPTPN1457845784-NM_005401 . 3_3394CACGAAGUUUCGAACGGAUUCUGUUPTPN157705770-HSS108816GAGUGAUGGAGAAAGGUUCGUUAAAPTPN157705770-HSS184025CAUGAAGCCAGUGACUUCCCAUGUAPTPN157705770-HSS184026CGAGAGAUCUUACAUUUCCACUAUAPTPN157705770-NM_002827 . 2_507CAGAGUGAUGGAGAAAGGUUCGUUAPTPRJ57955795-HSS108867GCGACUUCAUAUGUAUUCUCCAUCAPTPRJ57955795-HSS184091CGGGUUCUUCUUGAAAGCAUUGGAAPTPRJ57955795-HSS184092GAGCAGCCAUGAUGCAGAAUCAUUUPTPRJ57955795-NM_002843 . 3_1838CGGGUAGAAAUAACCACCAACCAAAPTPN1257825782-HSS108835GCCACAGGAAUUAAGUUCAGAUCUAPTP4A178037803-HSS111748GCAACUUCUGUAUUUGGAGAAGUAUPTPN1257825782-HSS108836GCCUCUUGAUGAGAAAGGACAUGUAPTP4A178037803-HSS111749UCAAAGAUUCCAACGGUCAUAGAAAPTPN1257825782-HSS108837UCUGAUGGUGCUGUGACCCAGAAUAPTP4A178037803-HSS111750CCAACCAAUGCGACCUUAAACAAAUPTPN1257825782-NM_002835 . 2_554CAGGACACUCUUACUUGAAUUUCAAPTP4A178037803-NM_003463 . 3_1382AACCAGAUUGUUGAUGACUGGUUAAPTPN1157815781-HSS108834ACAUGGAACAUCACGGGCAAUUAAAPTPN1157815781-HSS184068CAGACAGAAGCACAGUACCGAUUUAPTPN1157815781-HSS184069GAAAGGGCACGAAUAUACAAAUAUUPTPN1157815781-NM_002834 . 3_5519CAGGAUGCCUUUGUUAGGAUCUGUA 72 hr after transfection , HeLa EGFR BAC cells were stimulated with 10 ng/ml EGF for different time points .", "Then , total protein extracts were prepared and analysed by western blotting using rabbit monoclonal anti-phospho-Erk1/2 ( Thr202/Tyr204 ) ( Cell Signaling , New England BioLabs ) and mouse monoclonal anti-Erk1/2 ( Cell Signaling , New England BioLabs ) antibodies .", "For quantification , phospho-Erk1/2 intensity values were first normalized by the total Erk1/2 signal to control for differences in lane loading .", "For every blot , these values were normalized by the mean intensity amplitude per blot and then scaled by the mean difference between knock-down and mock-treated samples per experiment to account for experimental variability .", "To measure c-Fos activation , cells were stimulated with 10 ng/ml EGF for 30 min , fixed with PFA , and permeabilized with 0 . 5% Triton in PBS and 5% BSA as a blocking reagent .", "Cells were stained with a rabbit monoclonal anti-phospho-c-Fos ( Ser32 ) antibody ( Cell Signaling , New England BioLabs ) and processed for image analysis .", "To measure Erk1/2 activation in PC12 cells , cells were stimulated with 100 ng/ml EGF or 50 ng/ml NGF and stained with a rabbit monoclonal anti-phospho-Erk1/2 ( Thr202/Tyr204 ) ( Cell Signaling , New England BioLabs ) using the same protocol as above .", "10 images per condition were acquired using a laser-scanning confocal microscope ( Duoscan , Zeiss ) with a 40×/1 . 3 oil objective .", "Image analysis was carried out as described above .", "All animal studies were conducted in accordance with German animal welfare legislation and in strict pathogen-free conditions in the animal facility of the Max Planck Institute of Molecular Cell Biology and Genetics , Dresden , Germany .", "Protocols were approved by the Institutional Animal Welfare Officer ( Tierschutzbeauftragter ) , and necessary licenses were obtained from the regional Ethical Commission for Animal Experimentation of Dresden , Germany ( Tierversuchskommission , Landesdirektion Dresden ) .", "Foetal hepatic cells were isolated from C57BL/6JOlaHsd mice , maintained in the animal facility of the MPI-CBG .", "Pregnancies were dated by the presence of a vaginal plug ( embryonic day ( E ) 0 . 5 ) .", "Hepatoblasts were prepared from E14 . 5 liver as described previously ( Kamiya et al . , 1999 ) .", "Delta-like 1 ( Dlk1 ) + hepatoblasts were isolated from the E14 . 5 hepatic cells as described previously with minor modifications ( Tanimizu et al . , 2003 ) .", "Briefly , cells were blocked with an anti-mouse CD16/32 ( BD Biosciences ) and stained with a FITC-conjugated anti-Dlk1 antibody ( MBL International , Massachusetts , USA ) followed by anti-FITC Microbeads ( Miltenyi Biotec GmbH , Germany ) .", "The labelled cells were separated using a MACS Cell Separation Column ( Miltenyi Biotec ) .", "Dlk1+ cells were resuspended in DMEM ( PAA Laboratories GmbH , Austria ) containing 5% FBS , 2 mM L-glutamine ( PAA Laboratories GmbH ) , 100 μM MEM Non-Essential Amino Acids ( PAA Laboratories GmbH ) , 0 . 1 μM dexamethasone ( Sigma–Aldrich ) , 100 Units/ml penicillin ( PAA Laboratories GmbH ) , 100 μg/ml streptomycin ( PAA Laboratories GmbH ) , and 4% BD Matrigel Basement Membrane Matrix ( BD Biosciences ) , and seeded on a μ-slide 8-well ( Ibidi GmbH , Germany ) coated with fibronectin ( Sigma–Aldrich , Germany ) .", "To measure Erk1/2 activation , cells were starved for 24 hr before stimulation with either 10 ng/ml EGF or HGF ( R&D systems , Minnesota , USA ) .", "Total cell lysates were prepared and analysed using the same protocol and antibodies described above .", "We used a clone of PC12 cells , PC12 Nsc-1 ( Cellomics Inc . , Maryland , USA ) cells , due to their increased growth rate and decreased cell clumping , which facilitate imaging experiments ( Hahn et al . , 2009 ) .", "Cells were grown following the manufacturer's instructions .", "PC12 cells were starved for 36 hr before stimulation either with 100 ng/ml EGF ( Invitrogen ) or 50 ng/ml NGF ( R&D Systems ) for 30 min .", "E14 . 5 Dlk1+ hepatoblasts were starved for 24 hr before stimulation with either 10 ng/ml EGF or HGF ( R&D systems ) .", "Then , cells were fixed with 3% para-formaldehyde and stained with a mouse monoclonal anti-EEA1 ( BD Biosciences Pharmingen ) .", "A fluorescently conjugated goat anti-mouse-AlexaFluor 555 secondary antibody ( Molecular Probes , Invitrogen ) revealed the antigen signal .", "Image acquisition and image analysis were performed as described above .", "PC12 Nsc-1 cells were electroporated with 100 nM Stealth Select siRNA oligonucleotides ( Invitrogen ) ( EEA1: 5′—GAA AGC AGC UCA ACU UGC UAC UGA A—3′ , 3′—UUC AGU AGC AAG UUG AGC UGC UUU C—5′; Rabenosyn5: 5′—GGG CCU CAC ACU GAU CUU GCC UAU U—3′ , 3′—AAU AGG CAA GAU CAG UGU GAG GCC C—5′; Vps45: 5′—GAC CCG GCA UGA AGG UAC UUC UCA U—3′ , 3′—AUG AGA AGU ACC UUC AUG CCG GGU C—5′; Syntaxin-13: 5′—CCA AGG UGA UCU GAU UGA UAG CAU A—3′ , 3′—UAU GCU AUC AAU CAG AUC ACC UUG G—5′; Syntaxin-6: 5′—GGA UGC UGG AGU GAC GGA UCG AUA U—3′ , 3′—AUA UCG AUC CGU CAC UCC AGC AUC C—5′ ) or electroporated without siRNAs ( Mock ) using the Amaxa Cell line Nucleofector Kit V ( Lonza , Switzerland ) following the manufacturer's instructions .", "36 hr after electroporation , cells were placed in serum-free medium .", "72 hr after electroporation total protein extracts were prepared to measure down-regulation of the targeted proteins by western blotting with a mouse monoclonal anti-Syntaxin-13 ( Synaptic Sytems , Germany ) or a mouse monoclonal anti-Syntaxin-6 ( Transduction Laboratories , BD Biosciences ) antibody .", "To measure EGF transport , cells were stimulated with 100 ng/ml EGF-Alexafluor 555 ( Molecular Probes , Invitrogen ) for 1 min , washed with serum-free medium , and chased for different times .", "Then , cells were fixed and processed for microscopy as described above .", "Cells were starved for 36 hr and then stimulated in serum-free medium with 100 ng/ml EGF ( Invitrogen ) or 50 ng/ml NGF ( R&D Systems ) for 24 hr at 37°C and 5% CO2 .", "During the last 3 hr , 5-ethynyl-2′ –deoxyuridine ( EdU ) was added at a final concentration of 10 μM .", "Then , cells were fixed and stained with Click-iT AlexaFluor 647 Azide ( Molecular Probes , Invitrogen ) following the manufacturer's instructions .", "Afterwards , cells were stained with a mouse monoclonal anti-β-III tubulin antibody ( Chemicon International , Millipore ) and a fluorescently conjugated goat anti-mouse-AlexaFluor 555 ( Molecular Probes , Invitrogen ) to reveal the antigen signal .", "Nuclei were stained with DAPI .", "20 images per condition were acquired using a laser-scanning confocal microscope ( Duoscan , Zeiss ) with a 20×/0 . 8 objective .", "Image processing was carried out as described above .", "Images were inspected manually for process formation; cells with processes were defined as those having thin β-III tubulin-positive processes longer than 1 μm .", "The β-III tubulin expression was measured by total immunofluorescence intensity normalized by the frame area covered by cells to account for frame-to-frame variability in cell number ." ] ]
[ "An outstanding question is how receptor tyrosine kinases ( RTKs ) determine different cell-fate decisions despite sharing the same signalling cascades .", "Here , we uncovered an unexpected mechanism of RTK trafficking in this process .", "By quantitative high-resolution FRET microscopy , we found that phosphorylated epidermal growth factor receptor ( p-EGFR ) is not randomly distributed but packaged at constant mean amounts in endosomes .", "Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant .", "By mathematical modelling , we found that this mechanism confers both robustness and regulation to signalling output .", "Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation .", "We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response ." ]
[ "Molecules called growth factors can stimulate cells to grow , divide , or differentiate into more specialised cell types .", "Cells detect these molecules via proteins called receptor tyrosine kinases that span their surface membrane .", "The growth factor binds to the portion of the receptor outside the cell , which makes the receptor send signals to the cell's nucleus that change how the cell grows , divides , or specialises .", "Different growth factors and receptor tyrosine kinases affect cell development in different ways .", "However , it was unclear how this occurred , as the receptors all send signals via the same signalling pathways .", "Some researchers proposed that specific responses could be triggered if some receptor tyrosine kinases activated these pathways more strongly than other receptors , or if they activated the pathways for different lengths of time .", "Now , Villaseñor et al . have looked at a receptor tyrosine kinase for a growth factor called EGF .", "Activated EGF receptors are marked with a phosphate group ( or ‘phosphorylated’ ) and are then removed from the surface membrane and packaged into structures within the cell called endosomes .", "Villaseñor et al . found that different endosomes contain the same mean amount of phosphorylated EGF receptor .", "When exposed to higher EGF concentrations , the cells respond by increasing the number of endosomes , and so the average number of phosphorylated EGF receptors in each endosome remains almost constant .", "Villaseñor et al . used mathematical modelling to show that this mechanism , which they refer to as an ‘analogue-to-digital conversion’ , ensures a robust signal , and can regulate the signalling of the activated receptors in both space and time .", "Different growth factors either increase or decrease the number and size of endosomes in various cell types .", "Moreover , Villaseñor et al . found that changing how the phosphorylated EGF receptor is distributed between endosomes alters how the cells interpret the signal and differentiate when they are exposed to EGF .", "These findings mean that the signalling activity of a cell could be predicted from the number and size of its endosomes .", "Moreover , the findings also suggest that interfering with this mechanism could change cell behaviour , for example , it could stop cancer cells proliferating and force them to differentiate instead ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "computational and systems biology" ]
Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length
elife-26947-v1
[ [ "Animal cells within a tissue often display a striking uniformity in cell size ( Ginzberg et al . , 2015; Lloyd , 2013 ) .", "What are the mechanisms that ensure a single common target size for the numerous individual cells within a tissue ?", "In other words , how is size variability suppressed ?", "To date , most attempts to address this question have focused on the so-called cell size checkpoint – a mode of regulation that inhibits cell cycle progression for cells that are smaller than a target size ( Wood and Nurse , 2015; Neufeld and Edgar , 1998 ) .", "In 1965 , Killander and Zetterberg showed that populations of cultured mouse fibroblasts that are born small compensate with longer periods of growth in the G1 phase of cell cycle ( Killander and Zetterberg , 1965 ) .", "This suggests a model whereby size checkpoints selectively delay G1 progression of small cells , resulting in a negative correlation of birth size and G1 length .", "Following Zetterberg’s early studies , additional evidence for size checkpoints in animal cells have been published by several other groups ( Shields et al . , 1978; Darzynkiewicz et al . , 1979; Gao and Raff , 1997; Dolznig et al . , 2004; Tzur et al . , 2009; Kafri et al . , 2013 ) .", "Nevertheless , there remained skepticism regarding the existence of a coordination between cell size and cell cycle in animal cells ( Lloyd , 2013 ) .", "This skepticism was largely , but not exclusively , owed to studies on Schwann cells which showed that growth and division can be influenced separately by different types of extracellular factors ( Conlon and Raff , 2003; Rathmell et al . , 2000 ) .", "For a more comprehensive discussion of these disputes , see Lloyd ( 2013 ) .", "A more substantial body of research on cell size checkpoints is found in studies on yeast ( Rupes , 2002 ) .", "In 1977 , evidence supporting the existence of size checkpoints were demonstrated in both budding and fission yeast ( Fantes and Nurse , 1977; Johnston et al . , 1977 ) .", "One point of difference between the two yeast species is that , while budding yeast compensates for smaller birth size by extending the duration of G1 , fission yeast predominately ( but not exclusively ) employs a compensatory lengthening of G2 .", "Nevertheless , the control mechanisms are similar: size uniformity is promoted by a mechanism that establishes a negative correlation between cell size and the duration of cell cycle .", "In fact , similar size-dependent regulation of cell cycle duration has also been discovered in bacteria ( Willis and Huang , 2017 ) and plants ( Serrano-Mislata et al . , 2015 ) .", "How is cell size sensed and communicated to the cell cycle machinery ?", "Two general class of models have been proposed: a geometric model and a titration model ( Wood and Nurse , 2015 ) .", "The geometric mechanism is , perhaps , best exemplified by a series of studies relating size of fission yeast to the localized activity of two proteins , Pom1 and Cdr2 ( Martin and Berthelot-Grosjean , 2009; Moseley et al . , 2009 ) .", "In fission yeast S . pombe , Pom1 forms polar cortical gradients along the cell , peaking at the cell tips , while Cdr2 is localized to a medial band of cortical nodes .", "While still controversial ( Wood and Nurse , 2013 ) , the suggestion is that inhibitory signals from the pole-localizing Pom1 declines as cell grow longer , allowing activation of Cdr2 and entry into mitosis .", "Several alternate geometric models of size sensing have been proposed and are reviewed in Wood and Nurse ( 2015 ) .", "In addition to the geometric models , several titration models have been proposed in budding yeast S . cerevisiae .", "These models have focused on the G1 cyclin Cln3 and cell cycle inhibitor Whi5 as genetic perturbations of either genes significantly affects cell size ( de Bruin et al . , 2004; Costanzo et al . , 2004 ) .", "During G1 , it was suggested that level of G1/S promoters such as Cln3 increase , while concentration of cell cycle inhibitors such as Whi5 decease due to dilution by growth of cell volume ( Wang et al . , 2009; Schmoller and Skotheim , 2015 ) .", "These effects jointly determine the timing of S phase entry and cell size .", "Despite the various models of mechanisms that correlate cell size and G1 length , a perturbation that breaks this correlation has not yet been reported .", "In the present study , we relied on a chemical screen which identified that in animal cells , inhibition of the p38 MAPK pathway results in loss of the coordination between size and G1 length .", "The mammalian p38 MAPK pathway participates in numerous biological processes , including the regulation of cell cycle checkpoints .", "In response to DNA damage or oxidative stress , p38 is activated and induces a cell cycle arrest ( Thornton and Rincon , 2009; Ambrosino and Nebreda , 2001 ) .", "Hyperosmotic conditions that shrink cell volume also strongly activate p38 ( Han et al . , 1994; Moriguchi et al . , 1996; New and Han , 1998 ) .", "Furthermore , upregulation of the p38 MAPK pathway can lead to increased cell size ( Clerk et al . , 1998; Kudoh et al . , 1998; Molnár et al . , 1997; López-Avilés et al . , 2005; Cully et al . , 2010 ) .", "These observations raise the possibility that p38 may function to regulate cell size checkpoints in animal cells ." ], [ "To identify the molecular pathways linking cell size and cell cycle progression , we searched for perturbations that disrupt this link .", "We screened two compound libraries , known as the Novartis MOA ( Mechanism-of-Action ) box and Kinome box , which together included over 3000 compounds .", "The MOA Box contains an annotated list of compounds that are dynamically managed and curated to maximize coverage of targets , pathways , and bioactivity space ( Figure 1—figure supplement 1 ) .", "The MoA library was specifically designed to facilitate biological discovery by screening and profiling experiments .", "The Kinome Box is a library containing a wide range of kinase inhibitors that were selected based on their efficiency and specificity ( primary targets IC50 <1 μM , <25 total targets ) .", "Uniquely , all compounds included in our screen are ones that are thoroughly annotated not only for their primary targets but also the lower affinity targets ( off-targets ) .", "The advantage of this is that phenotypes associated with compound treatments can be statistically associated with particular signaling pathways despite the off-target effects that are inevitably associated with screened chemicals ( see Materials and methods - Analysis of the compound screen ) .", "To initiate the screen , unsynchronized HeLa cells were treated with compounds for 24 hr .", "After the compound treatments , we used a four-color labeling strategy to measure both cell size and cell cycle stage of each individual cell in each of the screened conditions .", "A combination of three labels ( DAPI and the two FUCCI cell cycle reporters , mAG-hGem and mKO2-hCdt1 ) were used to report cell cycle stage .", "While mAG-hGem and mKO2-hCdt1 report progression through G1 ( Sakaue-Sawano et al . , 2008 ) , DAPI labels progression through S phase .", "To measure cell size , we stained the cells with Alexa Fluor conjugated succinimidyl ester ( SE ) as a fourth color label .", "SE is a protein dye that accurately quantifies the total macromolecular protein mass in single cells and correlates with a cell’s dry mass ( Kafri et al . , 2013 ) .", "Following compound treatments and the four-color labeling procedure , samples were imaged with fluorescence microscopy and images were processed with an automated image-processing pipeline that we developed to quantify fluorescence intensity per single cell ( Figure 1A ) .", "With this screen assay , each drug treatment yielded a multi-dimensional joint distribution of cell size and cell cycle stage ( Figure 1B ) .", "To simplify the analysis , we used this data to classify cells from each of the screened conditions into discrete cell cycle stages ( Figure 1—figure supplement 2 ) .", "In the end , this procedure resulted with detailed data on how each of the screened compounds influences cell count , average cell size , and size variability in each of the separate cell cycle stages .", "In an unsynchronized population , the proportion of cells in a particular cell cycle stage reflects the duration of that stage relative to the entire cell cycle ( Kafri et al . , 2013 ) .", "Plotting the proportion of cells in early G1 , from each of the screened conditions , against the average size of early G1 cells , revealed a negative correlation between the two quantities ( Figure 1C and D ) .", "Chemical treatments that reduced the size of early G1 cells typically resulted in larger proportions of cells in G1 , suggesting that the duration of G1 depends on a cell’s initial size .", "To confirm this possibility , we used time-lapse microscopy to track nuclear size of live cells , which reaffirmed that cells born with smaller nucleus spend longer times in G1 ( Figure 3G ) .", "In an accompanying manuscript authored by Ginzberg et al . ( Ginzberg et al . , 2017 ) , we demonstrate that nuclear size is a faithful proxy of cell size .", "Altogether , these results support the hypothesis that G1 length is regulated in a size-dependent manner to promote uniformity among cells .", "To investigate the pathways that link cell size and G1 length , we assembled the list of compounds that altered average cell size , the fraction of cells in G1 , or both ( i . e . outliers on the plot in Figure 1C and Figure 1—figure supplement 3 , see Materials and methods - Analysis of the compound screen ) .", "We then sorted these ‘hits’ into two different categories .", "The first , which we call on-axis compounds , consisted of compounds that perturbed both cell size and the proportion of cells in G1 , but did so in a manner that maintained the coordination between the two .", "In other words , on-axis compounds produced reciprocally correlated influences on cell size and the fraction of cells in G1 .", "The second category of hits , which we call off-axis compounds , are compounds that disproportionally affected either size or cell cycle progression .", "Off-axis compounds resulted in paired values of cell size and G1 fraction that lie off-axis to the trend defined by the majority of screened conditions in Figure 1C .", "We reasoned that off-axis compounds may function by perturbing mechanisms that coordinate cell size with G1 length .", "To identify the pathways associated with either the off-axis or on-axis phenotypes , we used the compound annotations to perform target-enrichment analysis with Fisher’s exact test ( see Materials and methods -Analysis of the compound screen ) .", "Specifically , we sought proteins and pathways that are significantly overrepresented in the combined target list of either the on-axis or off-axis compounds .", "This analysis avoids the risk of formulating a hypothesis based on a single compound .", "Interestingly , we found that the proteins most frequently targeted by the on-axis compounds are PI3K , Akt and mTOR ( Figure 1E ) .", "This suggests that while the PI3K/Akt/mTOR pathway regulates cell size , this pathway is not involved in the coordination of cell size and the length of G1 .", "By contrast , the MAP Kinases are highly enriched among the targets of off-axis compounds ( Figure 1F ) .", "Notably , multiple components of the p38 MAPK pathway , including p38α ( MAPK14 ) , MK2 ( MAPKAPK2 , a direct substrate of p38 ) and TAK1 ( MAP3K7 , an essential upstream activator of p38 ) are ranked in the top 1% of significant targets ( p-value<0 . 01 ) .", "Over 35 compounds that target p38 pathway components , highlighted in Figure 1F , consistently displayed an off-axis phenotype .", "This suggests that the p38 pathway is involved in the coordination of G1 length with cell size .", "As an additional test for pathways that control cell size , we ranked compounds based on their influence on cell size variability ( Figure 1—figure supplements 4 and 5 ) .", "Using a similar target-enrichment approach , we generated a list of candidate proteins whose inhibition is associated with increased variation of cell size .", "This analysis showed that compounds that target p38 pathway components are associated with increased cell size variability .", "Notably , MK2 , a downstream substrate of p38 was the highest-ranking target in this list .", "These results hint that p38 pathway may coordinate cell size with progression through G1 in a manner that promotes cell size uniformity .", "To test whether the p38 MAPK pathway coordinates G1 length with cell size , we developed a robust assay to quantify this coordination in non-cancerous RPE1 cells ( Figure 2A ) .", "The mTOR inhibitor , rapamycin , is an on-axis compound that perturbs cell size while maintaining the coordination of size with G1 length .", "As demonstrated by live-cell imaging , treatment with 5 nM rapamycin slows cellular growth rates by almost half ( Figure 3—figure supplement 1 ) .", "However , as seen in Figure 3C and D , this reduced growth causes only a slight decrease in cell size as its effect is largely buffered by an increased cell cycle duration .", "This phenomenon is further characterized in Ginzberg et al . , 2017 .", "To quantify the strength of coordination between cell size and G1 length , we treated unsynchronized populations with a series of concentrations of rapamycin ( see Materials and methods –Compound treatment ) .", "As shown in Figure 2A , higher concentrations of rapamycin result in smaller G1 cells and longer durations of G1 .", "This experimental procedure reveals a robust and reproducible negative correlation between cell size and G1 length ( Figure 2A ) .", "We then used this assay to test if p38 is required for the negative correlation of cell size with G1 length .", "We examined a panel of specific inhibitors against MAPK pathways ( see Materials and methods –Compound treatment ) and tested whether any of these inhibitors break the negative correlation induced by the rapamycin concentration series .", "Our results show that , consistently , inhibitors of p38 disrupt the negative correlation of size and the proportion of cells in G1 ( Figure 2B–E , Figure 2—figure supplements 1 , 2 and 3 ) .", "This result was observed with multiple chemical inhibitors of p38 that are distinct in chemical structure ( Gaestel et al . , 2007 ) , making it less likely that the observed phenotype is owed to off-target effects .", "These results strongly implicate the p38 pathway in the coordination of G1 length with cell size .", "By contrast , inhibitors of JNK or ERK/MEK , do not significantly weaken the correlation of size and G1 length .", "Instead , these inhibitors shift the correlation to larger or smaller values of cell size ( Figure 2D and E ) .", "To further examine the involvement of p38 in the coordination between size and G1 length , we used time-lapse microscopy to track live cells and directly correlate their G1 length and birth size .", "Consistent with the conclusions of the bulk measurements , inhibition of p38 results in a weakened correlation of nucleus size and G1 length on a single-cell level ( Figure 3H and J , Figure 3—figure supplement 2 ) .", "Also , as expected from perturbations of size checkpoints , inhibition of p38 increases the variability in cell size ( Figure 3—figure supplement 3A ) .", "In direct contrast to the effects of p38 inhibition , treating cells with the mTOR inhibitor , rapamycin , seems to strengthen the correlation of size and G1 length ( Figure 3I ) .", "One possible reason for this is that , by shrinking cells , rapamycin subjects a greater proportion of cells to the control of a size checkpoint .", "In addition to weakening the correlation of G1 length and cell size , p38 inhibitors also cause a reduction in average cell size ( Figure 2B and D ) .", "To understand this cell shrinkage , we performed two sets of complementary measurements , both aimed at examining the influence of p38 inhibition on cell growth and cell cycle progression .", "In the first set of measurements , we used time-lapse microscopy to collect proliferation curves and growth curves of single cells throughout their entire cell cycle .", "Imaged cells expressed mAG-hGem , a cell cycle reporter ( Sakaue-Sawano et al . , 2008 ) , enabling accurate detection of the G1/S transition ( see Materials and methods - Automated lineage tracking ) .", "In the second set of measurements , we performed time-course experiments to periodically collect samples of unsynchronized proliferating cells .", "For each collected sample , we measured the total bulk protein mass and total cell count , which were then used to estimate the average rate of cell growth and the average rate of cell division ( see Materials and methods - Estimation of cell cycle durations and growth rate from bulk measurements ) .", "As shown in Figure 3 , p38 inhibition results in shorter cell cycles ( Figure 3B and D , Figure 3—figure supplement 3B ) , with a consequential increase in rates of cell division ( Figure 3A ) .", "In addition , the results show that the shortened cell cycle length caused by p38 inhibitors is owed to a decrease in G1 length ( Figure 3E and Figure 3—figure supplement 3C ) with no significant changes in the lengths of S or G2 phases ( Figure 3F , Figure 3—figure supplement 3D E ) .", "At G1/S transition , cells subject to p38 inhibition have smaller nucleus size as compared to control ( Figure 3C ) .", "This decrease in cell size is caused by a shorter growth duration ( in G1 ) rather than a slower growth rate .", "This conclusion is independently demonstrated by each of the two experiments described above , both showing similar growth rates for p38-inhibited cells and control cells ( Figure 3—figure supplement 3F , Figure 3—figure supplement 1 ) .", "Altogether , this suggests that the p38 pathway mediates a cell size checkpoint at the G1 transition , selectively allowing large but not small cells to progress into S phase .", "Potentially , this model also explains why p38 inhibitors reduce cell size .", "By disrupting the cell size checkpoint , p38 inhibitors cause cells to progress through the G1/S transition with prematurely small size , causing an overall reduction in cell size .", "Mammalians have four isoforms of p38 MAPK ( p38α , p38β , p38γ and p38δ ) .", "Using the same assay described in Figure 2A , we examined how knockdown of the p38 MAPKs affects the coordination of cell size and G1 length .", "Interestingly , using siRNA to knockdown p38γ or p38δ , significantly weakens or eliminates the negative correlation between cell size and proportion of cells in G1 , while knockdown of p38α and p38β only partially weakens this correlation ( Figure 4A and B , Figure 4—figure supplements 1 and 2 ) .", "Faced with this result , one may wonder why chemical inhibitors of p38α had such a dramatic influence on a coordination that is mediated primarily by p38γ and p38δ ?", "As shown in two comprehensive studies ( Davis et al . , 2011; Karaman et al . , 2008 ) , two of tested p38 inhibitors ( SB203580 and BIRB-796 ) also target p38γ and/or p38δ ( Figure 4—source data 1 ) at their working concentrations ( Figure 4—figure supplement 2 ) .", "Combined with the siRNA knockdown of the p38 isoforms , it suggests that the coordination between cell size and G1 progression is mainly mediated through p38γ and p38δ .", "An additional difference between the chemical and genetic inhibition of p38 is a greater reduction in cell size that results from the chemical inhibition .", "A possible explanation for this difference is the lower specificity of chemical inhibitors as compared to genetic perturbations .", "Functional redundancy of the different p38 isoforms , for example , can partially compensate siRNA treatments but not pharmacological perturbations .", "Alternatively , it may be that transfection with siRNA activates nonspecific cellular responses , such as upregulation of innate immune pathways , that lead to an increase in size ( Snijder et al . , 2012; Marques and Williams , 2005; Grumont et al . , 2004 ) .", "Nonetheless , these results collectively demonstrate that both chemical and genetic inhibition of p38 activity result in loss of coordination between cell size and G1 length .", "To further test the relationship of p38 and the control of cell size , we used siRNA to perturb the activity of MKK3/6/4 , which are upstream activators of the p38 pathway .", "As a control , we also knocked down MKK7 , an upstream regulator of JNK .", "Consistent with the results of the chemical inhibitors , knocking down either MKK3 , MKK6 or MKK4 disturbs the negative correlation between cell size and proportion of cells in G1 ( Figure 4C and D , Figure 4—figure supplement 1 ) .", "By contrast , knocking down MKK7 leaves the coordination of G1 and cell size intact .", "This result further supports a model whereby the coordination of cell size and cell cycle is specific to p38 and not the other MAPK pathways .", "The p38 MAPK pathway mediates cell cycle checkpoints by modulating multiple cell cycle regulators including cyclin D , cyclin E , p53 and CDC25 ( Thornton and Rincon , 2009; Lavoie et al . , 1996; Mikule et al . , 2007; Yee et al . , 2004 ) .", "To test the mechanisms of the p38-mediated cell size checkpoint , we treated cells with inhibitors of mTOR , rapamycin and Torin-2 , to induce a size-dependent lengthening of G1 .", "Several cell cycle regulators were assayed including positive indicators of G1/S transition ( Cyclin D , Cyclin E , p-Rb , Cdc25A ) and negative regulators of G1 progression ( p27Kip1 ) ( Figure 2C ) .", "Surprisingly , inhibition of p38 reversed the influence of mTOR inhibition on p27 ( Figure 2C , Figure 2—figure supplement 4 ) .", "Cells subject to mTOR inhibition display increased levels of p27 , a CDK inhibitor that functions to prolong G1 .", "By contrast , when these mTOR-inhibited cells are co-treated with inhibitors of p38 , p27 levels are reduced .", "These results raise the intriguing possibility that the p38-dependent lengthening of G1 in small cells is mediated by the cell cycle inhibitor , p27 .", "However , it is worth noting that , since p38 has long been implicated with the G1 arrest that follows environmental stress conditions , several different G1 regulators in addition to p27 have been previously identified as p38 effectors ( Thornton and Rincon , 2009; Lavoie et al . , 1996; Mikule et al . , 2007; Yee et al . , 2004 ) .", "It is , therefore , not unlikely that the p38 mediated G1 lengthening is redundantly mediated by multiple effectors , making this connection an intriguing subject for future investiations .", "The model suggesting that p38 regulates a cell size checkpoint is contingent on the hypothesis that the activity of the p38 MAPK pathway is somehow dependent on cell size .", "Figure 2C shows that p38 is , indeed , activated when cell size is decreased by inhibition of mTOR .", "While this result is consistent with size-dependent p38 activation , it could also have an alternative explanation .", "Namely , it may be that p38 is directly activated by molecular signals resulting from mTOR inhibition , in a manner that is independent of the influence of mTOR on cell size .", "To test this possibility , we relied on the difference in the time scales associated with cell growth and the time scales associated with mTOR activity .", "As shown in Figure 5A , we designed a ‘size-recovery experiment’ in which cells were released from a 20 hr Torin-2-mediated inhibition of mTOR and allowed to recover in size .", "The prolonged mTOR inhibition reduced average cell size by over 15% .", "Note that cells subject to Torin-2 treatment were still cycling ( Figure 5—figure supplement 1 ) .", "When Torin-2 is washed out , mTOR activity is restored within 1 hr ( Figure 5B ) , whereas recovery of cell size proceeds slowly , over a period of 14 hr .", "This experimental design provides an opportunity to observe cells that are smaller than their target size , but have long been relived from mTOR inhibition ( Figure 5B ) .", "Western-blots of lysates from cells during the recovery phase show that the p38 MAPK remains upregulated long after mTOR has resumed normal levels of activity ( Figure 5B ) and that the dynamics of p38 activity mirror the dynamics of the recovery of cell size rather than the recovery of mTOR activity .", "To further test whether p38 is selectively upregulated in small G1 cells , we employed a p38 Kinase Translocation Reporter ( KTR ) ( Regot et al . , 2014 ) that quantitatively reports p38 activation in live cells .", "This assay involves a fluorescent reporter that , once phosphorylated by p38 MAPK , shuttles from the nucleus to the cytoplasm .", "The ratio of cytoplasmic to nuclear ( C/N ) fluorescence represents a quantitative measure of p38 activation ( see Materials and methods -Image processing and cell segmentation ) .", "In addition to the KTR , cells were also stained with DAPI to discriminate the G1 , S and G2 subpopulations .", "As expected , cells treated with a p38 inhibitor display a lower C/N ratio as compared to untreated cells ( Figure 5C , Figure 5—figure supplement 2 ) .", "Using a similar assay as described in Figure 2A , cells expressing the p38 KTR were treated with increasing concentrations of rapamycin for a period of 22 hr , resulting in a dose-dependent decrease in cell size .", "Consistent with our hypothesis , decreases in cell size lead to upregulated p38 activity ( Figure 5C , Figure 5—figure supplements 2 , 3 ) .", "To exclude the possibility that differences in KTR localization are a direct result of mTOR inhibition rather than the influence of mTOR inhibition on cell size , we also performed measurements on cells six hours after the mTOR inhibitor was washed out .", "Confirming our hypothesis , the negative correlation of cell size and p38 activity persists for at least 6 hr after cells are released from mTOR inhibition , long after normal mTOR activity is resumed .", "Interestingly , in cells released from mTOR inhibition , p38 activity correlates with cell size only in G1 , but not in S or G2 ( Figure 5—figure supplement 4 ) .", "This result is consistent with a model whereby size-dependent p38 activity regulates the G1/S transition .", "As an additional control , we expressed dual color KTRs in the cells , reporting the activity of both p38 and JNK .", "These measurements showed that while p38 activity is negatively correlated with cell size , JNK activity is not ( Figure 5C ) .", "The sustained p38 activity , which proceeds hours after mTOR activity has resumed normal levels of activity , supports the hypothesis that it is the reduction in cell size and not inhibition of mTOR that activates p38 .", "However , an alternative possibility is that the sustained p38 activity is due to intrinsically slow inactivation kinetics of the p38 pathway , in a manner that is independent of cell size .", "To test this possibility , we exposed cells to short treatment of anisomycin or hyperosmotic shock , both of which strongly activate p38 ( Figure 5E ) without interfering with cell mass .", "Note although hyperosmotic shock does reduce cell volume , it does not reduce cell mass .", "Cell mass is the product of anabolic processes , largely regulated by mTOR , that changes over time scales of hours .", "By contrast , cell volume is a labile phenotype that changes over short time scales and is regulated by various ion channels and transporters ( Lang et al . , 1998 ) .", "Further , as a complimentary approach , we exposed cells to cycloheximide , a protein-translation inhibitor that significantly reduces cell mass but does not inhibit mTOR ( Figure 5D ) .", "In fact , as previously reported ( Bar-Peled and Sabatini , 2014; Sancak et al . , 2008 ) , cycloheximide activates mTOR due to increased intracellular pools of amino acids .", "Figure 5D and E show that sustained p38 activity , which persists after perturbations are removed , is observed only in conditions that decreases cell size .", "Specifically , 6 hr after cells had been released from the chemical treatments , increased p-p38 level was observed in cells that had been treated with Torin-2 or cycloheximide ( Figure 5D , Figure 5—figure supplement 5 ) , while p-p38 level of cells that had been treated with anisomycin or osmotic shock was indistinguishable from control ( Figure 5E ) .", "These results suggest that the sustained activity of p38 accompanying the recovery of cell size is a result of reduction in cell size rather than mTOR inhibition or slow inactivation kinetics of p38 signaling .", "As a final test for the hypothesis that p38 plays a role in the maintenance of cell size , we asked if p38 activity is required for cells that are recovering from perturbations of cell size .", "To test this , we repeated the size recovery experiment shown in Figure 5A , in the presence or absence of several different p38 inhibitors ( Figure 6A ) .", "Figure 5A shows that it takes approximately 25 hr for cells to fully recover their size following release from Torin-2 inhibition .", "We suspected that p38 inhibitors may either delay this process or inhibit recovery altogether .", "To discriminate these two options , we performed cell size measurements not only at times that fall within the normal time scales of recovery ( 6 hr , 24 hr ) , but also at time points that significantly exceed this range ( 30 hr , 48 hr ) .", "Indeed , our results show that even at the late time point of 48 hr after Torin washout , cell size is significantly reduced .", "Anecdotally , while p38 inhibition prevents the recovery in cell size at the late time points , a partial recovery is observed at 6 hr post Torin washout .", "This partial recovery , at early time points , is not unexpected .", "When cells are subject to mTOR inhibition ( Torin-2 ) , their growth rate is significantly reduced .", "Once mTOR inhibition has ceased , growth rate leaps back to its normal high levels , causing an immediate increase in cell size that is apparent in the early time points ( 6 hr post Torin-2 washout ) .", "However , to fully recover their size , cells must also lengthen the duration of growth by invoking the cell size checkpoint in G1 .", "As our results suggest , it is this process that becomes defective when p38 is inhibited , which manifests its influence on size only at the later time points .", "As additional controls , we also used inhibitors against two other MAPKs , ERK and JNK .", "Consistent with our expectation , inhibition of JNK or ERK do not repress recovery in cell size ( Figure 6B ) .", "Further , inhibition of p38 increased proliferation rates as compared to control ( Figure 6C ) , supporting the hypothesis that p38 activation inhibits cell cycle progression of cells that are smaller than their appropriate size .", "Interestingly , the influence of p38 inhibition on the recovery in cell size persists long after the inhibitors are washed out ( Figure 6—figure supplement 1 ) .", "While Torin-2-treated cells begin to recover their size immediately after mTOR inhibition is relieved , cells that are co-treated with both Torin-2 and a p38 inhibitor display a marked delay in recovery ( Figure 6—figure supplement 1 ) .", "This delay may hint that the cell growth cycle depends on a commitment point that is cell-cycle-stage-dependent , analogous to the restriction point that regulates the cell division cycle ( Blagosklonny and Pardee , 2002 ) ." ], [ "It has been over five decades since the first discovery of the connection between cell size and cell cycle in mammalian cells ( Killander and Zetterberg , 1965 ) .", "However , the molecular mechanism underlying this coordination between size and cell cycle remained elusive .", "In this study , we relied on a small molecule screen to identify that the p38 MAPK pathway is involved in the coordination cell cycle progression with cell size .", "At the heart of our methodological approach was an analysis that identified an axis of coordination relating cell size and cell cycle progression ( Figure 1C ) .", "Namely , we showed that almost all screened conditions that decrease cell size also bring about an increase in the proportion of G1 cells .", "Based on this , we classified the screen hits into two categories: on-axis compounds and off-axis compounds .", "Compounds that perturb cell size or cell cycle progression but maintain a coordination between the two were called on-axis compounds .", "By contrast , compounds that disrupt the coordination of cell cycle and cell size were called off-axis compounds .", "p38 MAPK pathway is highly enriched for the off-axis phenotype , implicating its role in the coordination of cell size with G1 length .", "Follow-up experiments with specific p38 inhibitors or genetic knockdown of the pathway confirm that inhibition of p38 disturbs the coordination between size and G1 length ( Figure 2 , Figure 3 and Figure 4 ) .", "We further show that reduction in size results in upregulation of p38 activity ( Figure 5 ) , and that p38 activity is required for cells to recover their original size ( Figure 6 ) by extending the duration of G1 .", "By probing for the major regulators of G1-S transition , our results suggest that the cell cycle inhibitor p27 may play a role in how p38 affects cell cycle to maintain cell size uniformity ( Figure 2 ) .", "However , it is worth noting that , in addition to p27 , previous literature has implicated several other G1 regulators as p38 effectors .", "It is , therefore , not unlikely that the influence of p38 on G1 length is redundantly mediated by multiple branches of signaling .", "In future investigations , it will be interesting to examine the specific mechanisms that explain how p38 promotes cell size uniformity and , crucially , identify how p38 ‘senses’ cell size .", "Our results reveal that the p38 MAPK pathway may be involved in a ‘cell-size checkpoint’ that regulates G1 progression in a cell-size dependent manner .", "Together , this study presents the p38 MAPK pathway as a key new component of cell size control in animal cells .", "Interestingly , while inhibitors of JNK or ERK/MEK also induce changes in cell size , they do not eliminate the correlation of cell size and G1 length ( Figure 2D ) .", "Instead , these inhibitors shift this correlation to larger or smaller values of cell size .", "This may suggest that instead of disrupting a size checkpoint ( as p38 inhibitors do ) , ERK/MEK or JNK inhibitors changed the target size with which that checkpoint is associated .", "While our study focused on the correlation of cell size and G1 length , we also observed a weak negative correlation between G1 length and cellular growth rates , as measured by time-lapse microscopy , using nuclear size as a proxy of cell size ( Figure 3—figure supplement 1 ) .", "Such connection between cellular growth rate and G1 length has been previously reported in cultured cells with more accurate methods ( Son et al . , 2012 ) as well as in the context of tissues ( Datar et al . , 2000 ) .", "It remains to be seen whether and how the correlation of G1 length with cell size and the correlation of G1 length with growth rate are mechanistically related .", "Mechanisms by which the p38 MAPK pathway regulates cell cycle progression are well-established in literature ( Thornton and Rincon , 2009; Lavoie et al . , 1996; Mikule et al . , 2007; Yee et al . , 2004 ) .", "Here , we have shown that such p38-dependent cell cycle control is regulated by cell size .", "Previously , it has been shown that p38 is activated by hyperosmotic conditions that shrink cell volume ( Han et al . , 1994; Moriguchi et al . , 1996; New and Han , 1998 ) .", "The results reported here show that the p38 pathway is activated in cells with reduced cell mass ( Figure 5 ) , suggesting that a common pathway responds to changes in both cell volume and cell mass .", "This finding is not trivial , since , unlike cell mass , cell volume is a labile phenotype that changes and recovers over rapid time scales ( minutes ) and is modulated by ion channels and transporters ( Lang et al . , 1998 ) .", "In contrast , cell mass changes more slowly ( over hours ) and is modulated by protein translation and central carbon metabolism .", "Given the conclusions of our study , the influence of p38 signaling on cell size in animal models is a definite point of curiosity .", "While literature on cell size in animal tissues is abundant ( Ginzberg et al . , 2015; Hall et al . , 2004; Lloyd , 2013 ) , focus on size uniformity or size variability is quite sparse .", "For example , growth in cell size has been primarily associated with activity of the mTOR pathway ( Fingar et al . , 2002 ) .", "mTOR regulates cell size by promoting protein translation and metabolism ( Saxton and Sabatini , 2017 ) , both of which translate into cellular growth rate .", "In this study , we observed a decrease in size following p38 inhibition .", "However , this reduction in size is not due to inhibition of mTOR , which would slow down cellular growth rates .", "Instead , p38 inhibition reduces cell size by shortening the duration of growth in G1 ( Figure 3 ) .", "Defects in cell size and growth were previously implicated with genetic perturbations of p38 in mammalian tissues such as liver , bone and heart ( Tormos et al . , 2013; Thouverey and Caverzasio , 2015; González-Terán et al . , 2016 ) .", "Since there are four isoforms of p38 in mammals , it is interesting to ask whether these isoforms are functionally diverged with respect to their influence on cell size ?", "Our results suggest that the coordination of G1 length with cell size depend more on p38γ and p38δ and less on p38α or p38β .", "Interestingly , a recent study by González-Terán et al . ( 2016 ) reported that mice lacking p38γ and p38δ have smaller cardiomyocytes and lower heart mass , whereas p38α knockout mice display no growth defect in heart ( Nishida et al . , 2004 ) .", "The authors found knockout of p38γ and/or p38δ lead to higher level of mTOR inhibitor DEPTOR , resulting in lower mTOR activity .", "This may suggest that , in addition to extending cell cycle duration of small cells , p38 may also regulate growth in size by activating mTOR during development .", "Last , while the present study focuses on p38 and its involvement in the regulation of cell size , we believe that the multi-parametric analysis developed in the context of the study have merit on its own .", "In the small molecule screen , every screened condition was assayed by single-cell measurements on thousands of unsynchronized cells , distributed over the entire cell cycle ( Figure 1B and Figure 1—figure supplement 2 ) .", "This single-cell data enabled detection of compounds which affect the phenotype of interest in a cell-cycle-dependent manner .", "Conventionally , pharmacological screens are performed to identify compounds that inhibit a particular process or activity .", "By contrast , the screen strategy that we developed herein sought not to inhibit a given pathway or activity but to inhibit the coordination between two biological processes ( cell size and G1 length ) .", "We envision , that such an approach may have general utility .", "Cancer , for example , is caused not only by the mitogenic activity of an oncogene , but also by defects in cross-talk and/or feedback linking different branches of signaling .", "Disrupting the coordination between such disparate branches of signaling may prove important .", "Finally , our phenotypic screen with a biologically-annotated set of compounds ( the MOA box ) demonstrates the power of applying a systematic ‘forward genetics’ approach to chemical biology .", "To conclude , in this study , we show that p38 pathway is involved in the coordination of cell size and G1 length .", "Aberrantly small cells display higher levels of p38 activity and grow in G1 for longer periods of time .", "Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells , resulting in faster proliferation , smaller cell size and increased heterogeneity in size .", "These results suggest a model whereby the p38 MAPK pathway functions downstream of a cell-size-sensing process and feeds information about cell size to regulators of cell cycle ." ], [ "Succinimidyl ester conjugated to Alexa Fluor 647 and DAPI were purchased from Life Technologies ( Burlington , ON ) .", "The following small molecule inhibitors were purchased from Selleckchem ( Houston , TX ) : Rapamycin , Torin2 , SB203580 , VX-702 , SB202190 ( FHPI ) , BIRB 796 ( Doramapimod ) , FR 180204 , PD184352 ( CI-1040 ) , PD98059 , JNK Inhibitor II , JNK Inhibitor IX .", "Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters ( KTR ) were a kind gift from Markus Covert ( Addgene plasmids No . 59151 and 59155 ) .", "ON-TARGETplus SMARTpool siRNAs for the genes of interest as well non-targeting negative control siRNAs were obtained from Dharmacon ( Lafayette , CO ) .", "Phospho-p38 MAPK ( Thr180/Tyr182 ) antibody ( RRID:AB_2139682 ) for immunofluorescence was purchased from Cell Signaling ( Beverly , MA ) .", "HeLa ( ATCC , RRID:CVCL_0030 ) and the retinal pigmented epithelial ( RPE1 , ATCC , RRID:CVCL_4388 ) cell lines stably expressing the degron of Geminin fused to Azami Green were cultured in DMEM medium ( Life Technologies ) supplemented with 10% Fetal Bovine Serum ( FBS , Wisent , Montreal , QC ) at 37°C in a humidified atmosphere with 5% CO2 .", "RPE1 cells stably co-expressing H2B conjugated to mTurquoise and Geminin conjugated mVenus were cultured in DMEM/F12 medium ( Life Technologies ) supplemented with 10% FBS .", "RPE1 cells stably expressing the JNK or p38 MAPK KTRs were generated as described below .", "Briefly , Human embryonic kidney HEK293T ( ATCC , RRID:CVCL_0063 ) cells were maintained in DMEM ( Wisent ) supplemented with 10% FBS at 37°C and 5% CO2 .", "The lentiviral transfer plasmids encoding the KTRs were co-transfected with plasmids encoding the packaging genes ( viral Gag-pol ) and the envelope gene , VSV-G , into the packaging HEK293T cells using jetPRIME transfection reagent ( Polyplus Transfection New York , NY ) .", "The medium was changed 24 hr post-transfection and the viral supernatants were harvested 48 hr later , passed through a 0 . 45-μm syringe filter and frozen at −80°C .", "The retrovirus was thawed at room temperature and then used to transduce RPE1 cells with the respective lentiviral transduction particles .", "Resistant clones were selected in 4 μg/mL puromycin ( InvivoGen , San Diego , CA ) for 3 days , isolated using cloning cylinders , and subsequently expanded and maintained in puromycin-containing DMEM medium .", "In all experiments , cell density was monitored to avoid over-confluence and contact inhibition .", "All cell lines used in this study have been tested for and confirmed to be negative for mycoplasma contamination .", "Two internal Novartis sets of tool compounds were screened , the publicly known subset of compounds in the Mechanism-of-Action ( MOA ) Box ( 1609 compounds ) and a Kinome Box ( 1637 compounds ) .", "The MOA Box is a dynamically managed annotated list of compounds that cover target , pathway , and bioactivity space as comprehensively as possible to facilitate biological discovery by screening and profiling experiments .", "Bioactivity annotations were derived from integrated in-house assays and external assay data sources containing a mixture of qualitative target assignments ( Thomson Reuters Integrity , DrugBank , Novartis-nominated MOA Box members ) or quantitative dose response-type experimental assay data ( chEMBL , GVK GOSTAR , Novartis assay data ) .", "Compounds were prioritized per target based on availability , amount of target evidence and , if available , clinical phase , while limiting the number of compounds selected from any one assay , publication , or patent .", "At the time of screening , chemical modulators of 903 unique human primary targets were represented based on hand-annotation , typically by 1–5 compounds/target where a compound-target association was made .", "In total , 42% of MOA Box compounds have only one assigned primary target , and a mean of 2 primary targets .", "However , additional target coverage for MOA Box compounds could be inferred from , the integrated sources listed above , comprising 1964 total unique targets , such that 11% of members have only one inferable target and a median of 6 inferable targets .", "Target enrichment analysis , described below , was carried out using all possible targets ( assigned and inferred from data ) .", "The target class coverage by percent of the MOA Box is as follows ( Figure 1—figure supplement 1 ) : Non-kinase Enzymes ( 28% ) , Kinases ( 16% ) , GPCR ( 15% ) , Proteases ( 12% ) , Ion Channel ( 11% ) , Unclassified ( 4% ) , Transporters ( 3% ) , Other Receptors ( 3% ) , Nuclear Hormone Receptors ( 3% ) , Cytokines ( 3% ) , and <1% each of Transcriptional Regulators , Signal Transducers and Structural Molecules .", "To assemble the Kinome Box , all physically-available inhibitors with publically-known structures having IC50 values < 1 μM for a human kinase were filtered for only those inhibiting <25 total kinases in integrated internal and external data .", "A total of 473 human kinases were covered by 1637 compounds ( mean and median number of targets per compound equal to 5 . 4 and 2 , respectively ) .", "The screen was performed in 384-well μclear microplates ( Greiner Bio-one , Monroe , NC ) .", "On day 1 , 2000 cells per well were seeded in 30 μl medium .", "On day 2 , compound was added to a final concentration of 1 μM , 3 μM or 10 μM ( or 2 μM , 6 μM or 20 μM for Kinome Box ) from a 2 mM or 10 mM stock solution using a Biomek FX with 384-well head ( Beckman Coulter , Indianapolis , IN ) .", "The DMSO concentration was kept below or at 0 . 5% v/v .", "To reduce stochastic noise and promote overall screen accuracy , each compound was screened with three concentrations and duplicates .", "The cells were seeded into 96-well μclear microplates ( Greiner Bio-one , Monroe , NC ) 16 hr prior to drug treatment .", "To perturb the p38 , JNK and Erk pathways , the following specific inhibitors were used at the concentrations indicated in the figure legends .", "All compounds were dissolved in DMSO and diluted in DMEM when used .", "The concentrations have been selected to avoid severely interfering with cell proliferation or cause noticeable cell death .", "Twenty-four hours post compound treatment ( control wells were treated with DMSO ) , the cells were treated and incubated with Rapamycin ( 30 , 3 , 0 . 3 , 0 . 1 and 0 . 03 nM ) for an additional 24 hr before fixation and staining , or for whole cell lysis .", "DMSO only ( 0 . 01–0 . 5% v/v in DMEM ) was used as a negative control for all the compound treatment experiments .", "To search for an appropriate working concentration of the MAPK inhibitors , we tested a series of different concentrations starting with the compounds IC50 , as published by the vendors .", "However , it is worth noting that the IC50 of a given compound may vary from one cell line to another .", "Also , many reported IC50 are based on cell-free assays which highly depend on assay conditions and are usually much lower than the working concentrations that are relevant for cell culture .", "As an example , the reported IC50 ( cell-free assay ) of SB203580 on p38 ranges from 0 . 19 nM ( Gaestel et al . , 2007 ) to 7 μM ( Wei et al . , 2007 ) .", "Because of these inherent inaccuracies in published IC50s , a range of concentrations for each of the chemical inhibitors were tested .", "To start , we performed four serial dilutions of each compound , where the lowest tested concentration was the compounds published IC50 , as advertised by the vendor website .", "Compound concentrations that resulted in a significant decrease in cell number ( e . g . half the cell number of the control wells ) were deemed toxic and excluded from following analyses .", "For example , the compound PD184382 resulted in decreased cell count when used at concentrations higher than 300 nM .", "Conversely , if a given concentration ( of a compound ) resulted in a phenotype that is indistinguishable from wild-type , the entire concentration series for that specific compound was recalibrated to start at a higher concentration value .", "Examples of the latter include the compounds , TAK-715 and JNK inhibitor II .", "Using this procedure of calibration , we selected a specific working concentration for each of the compounds used in the study .", "To examine the process of recovery in cell size , cells were treated with Torin2 ( 50 and 25 nM ) for 24 hr , washed with PBS and re-incubated with fresh media for the times indicated .", "Cells were resuspended using trypsin and cell size and cell density was measured using the Multisizer 4 Coulter Counter ( Beckman-Coulter , Mississauga , ON ) or collected for whole cell lysis at time points indicated in the figures .", "In the experiment using p38 or JNK KTRs , cells were seeded in 96 well plates and treated with rapamycin ( 30 , 3 , 0 . 3 , 0 . 1 , 0 . 03 nM and DMSO controls ) for 24 hr .", "The cells were either fixed and stained or washed with PBS and replaced with fresh DMEM media for 6 hr before fixation and staining .", "RPE1-Geminin cells were seeded in 6-well plates at a density of 2 × 105 cells/well .", "Twenty four hours post-seeding , the cells were transfected with SMARTpool siRNA ( 25 nM ) using DharmaFECT one transfection reagent according to the manufacturer's instructions .", "Twenty hours post transfection , the cells were re-suspended using 0 . 05% trypsin-EDTA ( Wisent ) and re-seeded into 96-well µclear microplates ( Greiner Bio-one , Monroe , NC ) at a density of 5 , 000 cells/well .", "Six hours after re-seeding , cells were treated with rapamycin ( 30 , 3 , 0 . 3 , 0 . 1 and 0 . 03 nM ) or DMSO control for 24 hr before fixation and staining .", "The experimental procedures were optimized so there is no observable cell cycle arrest or cell death .", "Specifically , the siRNA-transfected cells were washed-out of transfection reagent and cultured in regular media when used for the assay with rapamycin concentration series .", "Cells were observed to continuously increase in number during the assay and proliferate with a similar rate as non-transfected cells .", "The procedures followed the Cell Signaling protocol .", "Briefly , cells were first fixed with 4% PFA for 15 min , blocked in blocking buffer for 1 hr .", "Afterwards , samples were incubated in primary antibody at 4°C overnight , and subsequently secondary antibody for 1 hr before imaging .", "Cells were fixed in 4% paraformaldehyde ( Electron Microscopy Sciences , Hatfield , PA ) for 10 min , followed by permeabilization in cold methanol at −20°C for 5 min .", "Cells were stained for cell size with 0 . 4 μg/mL Alexa Fluor 647 conjugated succinimidyl ester ( SE ) for 2 hr to nonspecifically label total protein content .", "The cells were then labeled for DNA with 1 μg/mL DAPI for 10 min .", "The cells were either imaged using an IN Cell Analyzer 2000 HCA system ( GE Healthcare Life Sciences , Pittsburgh , PA ) microscope at 10X magnification ( compound screen ) or using the Operetta High-Content Imaging System ( Perkin Elmer , Woodbridge , ON ) at 20X magnification ( compound treatment and time-lapse experiments ) .", "To prepare whole cell lysates , cells were rinsed with ice-cold PBS and solubilized with RIPA Lysis Buffer ( Boston Bio-Products , Boston MA ) [50 mM Tris-HCl , 150 mM NaCl , 5 mM EDTA , 1 mM EGTA , 1% NP-40 , 0 . 1% SDS and 0 . 5% sodium deoxycholate , pH 7 . 4] supplemented with protease and phosphatase inhibitor Cocktail ( Thermo Scientific , Burlington , ON ) .", "Protein concentration was determined using the BCA protein assay ( Thermo Scientific , Burlington , ON ) and suspended with 4X Bolt LDS Sample Buffer and 10X Bolt Reducing Agent and heated for 10 min at 70°C .", "Samples of equal protein were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting for proteins as indicated .", "The antibodies used for immunoblotting were all purchased from Cell Signaling Technology ( Beverly , MA ) .", "All western-blot results in the figures have been reproduced in replicate experiments with cell lysates samples prepared in independent experiments .", "Automated image-processing pipelines have been developed using Matlab ( RRID:SCR_001622 ) .", "The general scheme includes three steps: correction for background fluorescence and/or uneven illumination , cell segmentation , and feature quantification .", "Each step has been optimized according to microscopes , experimental design ( e . g . fixed or live cell ) and the features needed .", "The same image processing pipeline and parameters were applied identically to all images from the same experiment .", "To correct for background fluorescence , a background image was constructed and applied per channel .", "There are two scenarios to construct a background image: either by averaging images containing few or no cells ( in the compound screen ) ; or by averaging the background region across all images collected ( experiments with KTR cells , and in the time-lapse imaging ) .", "Uneven illumination was corrected for images from the compound screen in which the problem is obvious .", "A flat-field image has been constructed and applied for all channels .", "The flat-field image was constructed based on the fact that 2N peak in DNA distribution is invariant to cell positions in the image .", "In the step of cell segmentation , nucleuses were first spotted through a nuclear channel ( DNA or H2B ) , and segmented by seed-based watershed .", "When cell channel exists , the segmented nucleuses were further used as seeds to segment cells ( SE channel ) by watershed .", "The nuclear and cell border were detected with thresholds in corresponding channels , which were either automatically detected or manually selected based on need .", "Following segmentation , each individual cell was processed to collect for its quantitative features , including total/average fluorescence per channel , nuclear size , etc .", "To calculate the activity of the KTR , the fluorescent intensity of the KTR channel was collected and integrated within either the cytoplasmic region ( identified by SE channel ) or the nuclear region ( identified by DAPI channel ) to calculate the ratio of cytoplasmic to nuclear intensity .", "Note the integrated intensity correlates with the amount of fluorescent protein while intensity/brightness is usually used as an estimate for protein concentration .", "Further , measurements with integrated intensity is more robust to morphological changes , while brightness measured with epiflourescence could change dramatically depending on the thickness of the cell .", "As the experiment used in this study undergo extended treatment ( over 20 hr ) and the cells could change their morphology under treatment , it is more reasonable to use integrated intensity to estimate shuttling of proteins .", "Cells were first partitioned , according to the nuclear DNA level , into G1 ( 2N ) , S ( 2 N-4N ) and G2 ( 4N ) phase .", "Progression in G1 phase was further divided , based on fluorescence of nuclear Geminin ( and nuclear Cdt1 if available ) into early G1 ( low Geminin ) , late G1 ( medium Geminin , high Cdt1 ) , and G1/S transition ( higher Geminin , medium to high Cdt1 ) .", "The thresholds were automatically detected based on distribution of DNA , log ( Geminin ) and log ( Cdt1 ) .", "Cells were treated with inhibitors and fixed every 20 hr over a period of 3 days .", "The rate of cell proliferation and protein accumulation were calculated by fitting a log model ( assuming that cells proliferate and increase protein content exponentially ) .", "To calculate the duration for each cell cycle stage , the cells were first portioned into G1 , S and G2 to calculate the proportion of cells in each cell cycle stage .", "The duration of a cell cycle stage is then calculated with the following formula: t=T⋅PDF2−CDF , where t is the duration of the specific cell cycle stage , PDF is the proportion of the cells in that stage , and CDF is the proportion of cells in or earlier than that stage .", "This formula is a simplification of the ERA method ( Kafri et al . , 2013 ) .", "The analysis was performed per plate separately , due to observed variability among plates .", "After image-processing of the compound screen datasets , every well captures approximately 4000 cells .", "For each well , both cell size at early G1 stage ( S ) and proportions of cells in early G1 stage ( P ) were collected .", "The minimum volume ellipsoid ( MVE ) estimator ( Rousseeuw , 1985; Rousseeuw and van Zomeren , 1990 ) was applied to pick a smallest volume ellipsoid that consists of around 50% of the total sample .", "The MVE estimator was a reliable tool to identify robust trend and detect outliers in multivariate data .", "The subsample of minimum volume ellipsoid was used to calculate the robust covariance matrix ( rCov ) , from which correlation coefficient between S and P was calculated per plate .", "Further , we calculated the Mahalanobis distance from each data point ( a well ) to the center of the ellipsoid , and performed thresholding ( 5% significance ) to the Mahalanobis distance to detect the outlier wells .", "Intuitively , the outlier wells display drastic distinctive phenotype in early G1 cell size and/or its G1-length compared with the control wells ( and most drug treatments ) .", "Based on rCov , the principle vector of [S , P] was calculated and the angle of each outlier well with reference to the major principle vector was computed .", "The outlier angles were used to classify on/off-axis outliers: outliers with an angle smaller than 45 degree were classified as on-axis outliers , and otherwise off-axis outliers .", "The on/off-axis outliers were further filtered separately to lower false-positive rate .", "In the compound screen , each compound has been tested for at least six times ( three concentrations with duplicates ) ; and compounds that have only been identified as outlier once among all treatments were excluded from further analysis .", "Next with the robust outliers , we performed the target enrichment analysis using hypergeometric test ( Fisher’s exact test ) for each known target of the outliers to identify the target proteins that are significantly highly represented in the outlier compounds .", "To identify compound treatments that increase cell size variability , median absolute deviation ( MAD ) , a robust measure of variability , of cell size was calculated per well .", "We observed that cell size MAD is linearly correlated with median cell size ( r = 0 . 964 ) .", "Accordingly , we normalize the MAD by median cell size to obtain a robust measure of cell-to-cell variability within cell populations .", "For each well , its normalized MAD is compared to that of the control wells in the same plate to calculate a MAD score .", "To select for compound treatments with perturbed size variability , a threshold in MAD score was calculated based on the distribution of MAD scores of all control wells ( 1% significance ) .", "The outliers with higher MAD scores compared with the threshold were filtered for repeatability and enriched for target proteins the same way as described above .", "RPE1 cells with stable expression of H2B-mTurquoise and Geminin-mVenus were seeded in 96-well μclear microplates ( Greiner Bio-one , Monroe , NC ) and grown in the incubator for at least 6 hr prior to imaging .", "The cells were imaged with the Operetta High-Content Imaging System .", "During the imaging , the plate was incubated in the live cell chamber ( 37°C , 5% CO2 ) and grown in the FluoroBrite DMEM supplemented with FBS , L-glutamine and Sodium Pyruvate .", "As the cells were previously cultured in regular DMEM and displayed suboptimal cell proliferation after switching to FluoroBrite DMEM , the cells were grown in FluoroBrite medium for a period of 2 weeks to adapt to the new medium before the time-lapse experiments .", "Widefield fluorescent images of H2B-mTurquoise and Geminin-mVenus were collected every 15 min at 20X magnification for 50 hr .", "Under this experimental setting , the microscope could support imaging of up to four wells every time .", "The live-cell images were first processed with the cell-segmentation pipeline to obtain single-cell features including cell position , fluorescent intensity and nuclear size .", "Subsequently , cells from each time point T were compared with the ones from T + 1 to track for cell motion and division by searching for the globally optimal matches between neighboring time points ( Kanade et al . , 2011 ) .", "Parameters used for tracking were automatically calculated based on distribution of cell features and confirmed in subsamples by eye .", "As output from tracking , each track starts with either appearance ( first time point , or cell move into image field ) or cell birth ( with known mother and sister cell ) , and end with disappearance ( last time point , or cell move out of image field ) or cell division ( with known daughter cells ) .", "After tracking , the individual cell tracks were further filtered to select for accurate tracks with the full cell cycle captured .", "Specifically , the algorithm selects for cell tracks that start with typical features of cell birth , end with typical features of mitosis and have relatively smooth fluorescence dynamics .", "Further , for each full cell cycle track , swift rise in Geminin level was detected to quantify early G1 duration .", "Initial nuclear size is estimated by averaging the first nine data points ( ~2 . 5 hr ) after cell birth to decrease noise .", "Similarly , nuclear size at G1/S transition is estimated by averaging the ±4 data points ( ~2 . 5 hr ) at time point of G1/S transition .", "Nuclear size measurements collected during mitosis were excluded from analysis , as H2B-mTurquoise does not accurately depict nuclear size during mitosis when nuclear envelope breaks down and chromosome condenses .", "To calculate growth rate in nuclear size , dynamics of individual nuclear size over entire cell cycle was first fitted with a smoothing spline model .", "The dynamics of the growth rate is then calculated with the first derivative of the fitting result ." ] ]
[ "Animal cells within a tissue typically display a striking regularity in their size .", "To date , the molecular mechanisms that control this uniformity are still unknown .", "We have previously shown that size uniformity in animal cells is promoted , in part , by size-dependent regulation of G1 length .", "To identify the molecular mechanisms underlying this process , we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression .", "Small cells display higher p38 activity and spend more time in G1 than larger cells .", "Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells , resulting in faster proliferation , smaller cell size and increased size heterogeneity .", "We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly , to increase cell size uniformity ." ]
[ "Animal cells come in many different sizes .", "In humans , for example , egg cells are thousands of times larger than sperm cells .", "Yet cells of any given type are often strikingly similar in size .", "The cells that line the surface of organs including the skin and kidneys are especially uniform; in fact a loss of size uniformity in certain tumors is a sign of malignancy .", "What kind of regulation could enable separate cells within a tissue to have the same size ?", "One possibility is that each type of cell is programmed with a specific target size , and that a cell can sense if it strays from its target and compensate with longer or shorter periods of growth .", "Animal cells sensing their own size was first reported in the 1960s , and since then much research in this area has focused on “cell size checkpoints” .", "These mechanisms stop cells that are too small from progressing through the series of events that allow one cell to divide in two , which is known as the cell cycle .", "Supporting the existence of size checkpoints , studies in yeast have repeatedly shown that cells that start off smaller tend to grow for longer during stages of cell cycle named G1 and G2 .", "Several researchers have proposed different mechanisms to explain how information about a cell’s starting size influences the length of its cell cycle to result in the negative correlation between these two factors .", "However , as yet no one had managed to find a way to break this negative correlation , which would greatly help scientists to confirm the actual mechanisms that cells use to sense their size .", "To address this , Liu , Ginzberg et al . first looked for chemicals that , when added to human cells , stopped cell size from being correlated with the time taken to complete a cell cycle .", "This search revealed that information about cell size is communicated to regulators of the cell cycle via a signaling pathway that involves an enzyme known as the p38 MAPK .", "Liu , Ginzberg et al . then showed that this specific pathway is activated in small but not large cells , where it slows the small cells’ progress through the cell cycle .", "As expected , inhibiting the p38 enzyme also broke down the relationship between time spent in G1 and cell size , and led to the human cells growing to a range of different sizes .", "These findings now pave the road to answering a fundamental question in cell biology: what is the elusive cell size sensor ?", "Understanding how cells sense their size will open a window onto how quantitative information is programmed , sensed and communicated within living cells .", "These findings will shed also new light onto how cells specialize into cell types of different sizes , and what happens when cells lose the ability to sense or regulate their size in diseases like cancers ." ]
2018
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "cancer biology" ]
The CUL5 ubiquitin ligase complex mediates resistance to CDK9 and MCL1 inhibitors in lung cancer cells
elife-44288-v3
[ [ "Cancer cells frequently manipulate the intrinsic apoptotic pathway to evade cell death and expand their proliferative capacity .", "Aberrant increases in levels of anti-apoptotic proteins in the BCL-2 family , such as amplification of MCL1 , have been widely implicated in the transformation of cancer cells and the development of resistance to current therapies ( Kelly and Strasser , 2011 ) .", "BCL-2 family members are classified based on the conservation of their BCL-2 homology ( BH ) domains: multi-domain proteins BAK , BAX and BOK serve as apoptosis executors in the mitochondria; proteins containing only the BH3 domain ( BH3-only ) promote BAK/BAX activation .", "Anti-apoptotic proteins such as MCL1 , Bcl-xL and BCL-2 inhibit apoptosis by antagonistic binding to pro-apoptotic BH3-only proteins as well as BAK and BAX ( Czabotar et al . , 2014 ) .", "High-resolution investigation of somatic copy number alterations has revealed that gene amplification of MCL1 and BCL2L1 ( Bcl-xL ) are key determinants of survival in many cancers , including breast cancer , non-small cell lung cancer ( NSCLC ) , multiple myeloma , acute myeloid leukemia , and B-cell acute lymphoblatic leukemia ( Goodwin et al . , 2015; Koss et al . , 2013; Xiao et al . , 2015; Zhang et al . , 2011 ) .", "Amplification of MCL1 is a prognostic indicator for disease severity and progression , making it an attractive therapeutic target ( Campbell et al . , 2018; Yin et al . , 2016 ) .", "In an effort to restrict the action of anti-apoptotic proteins , numerous compounds have been developed that mimic BH3-only proteins ( BH3-mimetics ) .", "Unfortunately , the first BH3-mimetics that specifically antagonized Bcl-xL were associated with significant thrombocytopenia , thus complicating their therapeutic use ( Lessene et al . , 2013; Leverson et al . , 2015a; Tao et al . , 2014 ) .", "Small-molecule inhibition of MCL1 has recently gained significant attention ( Figure 1A ) , and compounds that selectively target MCL1 are currently in clinical trials ( Abulwerdi et al . , 2014; Burke et al . , 2015; Caenepeel et al . , 2018; Kotschy et al . , 2016; Leverson et al . , 2015b; Tron et al . , 2018; Phase I Study of S64315 Administred Intravenously in Patients With Acute Myeloid Leukaemia or Myelodysplastic Syndrome ) .", "Promising reports of direct BH3-mimetic MCL1 inhibitors in preclinical hematological malignancies show potent efficacy with low cytotoxicity ( Kotschy et al . , 2016; Leverson et al . , 2015b ) .", "However , assessment of MCL1 inhibitors in solid breast tumors showed little single agent activity unless combined with a chemotherapeutic agent ( Merino et al . , 2017 ) .", "Co-dosing Bcl-xL and MCL1 inhibitors to achieve effective treatment may be complicated by severe accompanying side effects .", "Beyond direct inhibitors of the BCL2 family of proteins , inhibitors of cyclin-dependent kinase 9 ( CDK9 ) can indirectly target MCL1 .", "CDK9 inhibition restricts transcription elongation thus exploiting all mRNAs and proteins that have short-lived half-lives .", "Due to its short half-life , MCL1 is one of several targets that is particularly susceptible to acute CDK9i treatment , and other ( proto- ) oncogenes such as MYC are also CDK9i targets ( Figure 1A ) ( Akgul et al . , 2000; Gregory et al . , 2015; Huang et al . , 2014a; Lemke et al . , 2014 ) .", "Although CDK9 inhibition suppresses MCL1 expression , it does not affect levels of other anti-apoptotic proteins such as Bcl-xL , exposing a potential vulnerability in CDK9i treatment such that cancers may already have or develop a mode of resistance .", "Selective CDK9 inhibitors have shown promising results in preclinical murine models; however , they have a limited therapeutic window and must be acutely dosed for clinical applications due to their global effects on transcription ( Garcia-Cuellar et al . , 2014; Hellvard et al . , 2016 ) .", "In order to truly harness the power of CDK9 inhibitors ( CDK9i ) or MCL1 inhibitors ( MCL1i ) , it is imperative to uncover additional targets that may sensitize cells to these treatments .", "Since CDK9i shares at least one of its targets with MCL1i , we reasoned that a genome wide search could uncover shared factors that modulate the therapeutic activity of these compounds and suggest approaches to resensitize otherwise resistant tumor cells .", "As lung cancer is the leading cause of cancer mortality and most NSCLC patients develop resistance to first-line treatment , we performed genome-wide CRISPR inhibition ( CRISPRi ) screens in a NSCLC line resistant to both CDK9 and MCL1 inhibition ( Siegel et al . , 2016 ) .", "We discovered that disruption of multiple members of the cullin 5-RING ubiquitin ligase ( CRL5 ) complex markedly resensitized cells to both CDK9i and MCL1i .", "The CRL5 complex targets pro-apoptotic BH3-only proteins Bim and Noxa for proteasomal degradation .", "Epistatic knockdown of Bim or Noxa in a CUL5 knockout background rendered cells once again resistant to CDK9i , but did not affect increased sensitivity to MCL1i .", "Our data indicate that members of the CRL5 complex modulate the apoptotic threshold and could be attractive targets for future combination therapy to treat otherwise resistant NSCLC ." ], [ "We assessed a panel of NSCLC lines for their sensitivity to CDK9i ( AZD5576 ) or MCL1i ( AZD5991 ) ( Figure 1B and Table 1 ) .", "Several cell lines with amplified MCL1 ( copy number ≥3 ) were highly sensitive to CDK9i and MCL1i , consistent with overexpression of MCL1 to escape apoptosis .", "We also found several NSCLC lines resistant to CDK9i and MCL1i despite being MCL1-amplified .", "Increased Bcl-xL expression correlated with the MCL1-amplified lines that were resistant to CDK9i and MCL1i .", "Conversely , MCL1-amplified cell lines with low levels of Bcl-xL were sensitive to CDK9i and MCL1i .", "Both MCL1i and CDK9i induce apoptosis and cell death in a sensitive cell line ( H23 ) after just 6 hr , with saturated cell death and caspase activation at 1 μM .", "Conversely , a resistant line ( LK2 ) was about 100-fold more resistant to CDK9i and MCL1i ( Figure 1C ) .", "After extended treatment ( 24 hr ) , CDK9i induced a significant decrease in cell viability in both the resistant and sensitive cell lines , highlighting off-target toxicity presumably stemming from global transcriptional arrest independent of targeting short half-life transcripts such as MCL1 and MYC ( Figure 1D ) .", "This emphasizes the need for a tightly regulated treatment window for CDK9i to avoid non-specific cell death .", "While Bcl-xL amplification correlates with resistance to MCL1-targeting drugs , preclinical studies suggest dual inhibition of MCL1 and Bcl-xL may not be a clinically viable option due to toxic side effects .", "We sought to identify alternate pathways that could resensitize resistant LK2 lung cancer cells to CDK9i or MCL1i .", "Prolonged CDK9i treatment induces non-specific cell death , possibly due to its polypharmacology , and so a growth-based screen measuring cell abundance after extended compound exposure was inappropriate .", "Instead , we developed a positive selection FACS-based screen for apoptosis during acute exposure to maximize on-target cell death and minimize non-specific cell death ( Figure 2A ) .", "We exposed a clonal LK2 CRISPRi cell line transduced with a genome-wide CRISPRi-v2 guide RNA library ( Materials and methods and Figure 2A ) to either 3 μM CDK9i for 6 hr or 1 μM MCL1i for 12 hr .", "Per-cell apoptosis was assayed with 0 . 5 μM Cell Event , which fluoresces upon cellular caspase activation , and apoptosing cells were separated by genome-scale fluorescence activated cell sorting ( FACS ) .", "To identify vehicle effects , we also performed duplicate genome-wide sorts with DMSO .", "Bulk untreated cells were harvested on the same day as the FACS sort to provide an accurate ‘background’ sampling of sgRNAs and eliminate confounding effects from essential genes that had dropped out of the population .", "We measured quantitative differences in sgRNA frequency by deep sequencing and integrated enriched or depleted sgRNAs into gene-level hits by comparing sorted samples to the untreated control using ScreenProcessing ( Figure 2—source datas 1–3 ) and MAGeCK ( Figure 2—source datas 4–7 ) ( Horlbeck et al . , 2016; Li et al . , 2014 ) .", "No significant gene-level hits were detected in the DMSO control ( Figure 2—figure supplement 1B ) , indicating that gene calls in CDK9i- or MCL1i-treated samples were derived from drug action .", "We found multiple sgRNAs targeting Bcl-xL led to re-sensitization of LK2 cells to CDK9i and MCL1i ( Figure 2B–C ) , confirming reports that depletion of both MCL1 and Bcl-xL initiates apoptosis ( Goodwin et al . , 2015; Xiao et al . , 2015; Zhang et al . , 2011 ) .", "Downregulation of the mitochondrial porin VDAC2 also promoted apoptosis in drug-treated cells ( Figure 2B–C ) , consistent with its proposed role in BAK/BAX sequestration at the mitochondrial membrane ( Cheng et al . , 2003; Chin et al . , 2018; Lauterwasser et al . , 2016 ) .", "EIF4G2 ( DAP5 ) was identified as a hit in the CDK9i screen ( Figure 2B ) , and this gene has been implicated in stimulating cap-independent translation of anti-apoptotic protein BCL-2 ( Marash et al . , 2008 ) .", "Comparing the most significantly enriched genes from both screens showed an overlap between MCL1i and CDK9i .", "The CDK9i screen had many more hits , consistent with its polypharmacology of transcriptional inhibition ( Figure 2—figure supplement 1C ) .", "Despite separate mechanisms of drug action and potentially distinct targets , significantly more of the top resensitization hits were shared between CDK9i and MCL1i than are expected by chance ( exact hypergeometric test p<6 . 4×10−9 ) .", "These shared hits are part of two physical complexes , one potentially involved in specialized translation and the other involved in protein degradation .", "First , knockdown of multiple components of the eukaryotic translation initiation factor 3 ( eIF-3 ) complex ( EIF3H and EIF3M ) resensitize LK2 cells to CDK9i and MCL1i ( Figure 2B–C and Figure 2—figure supplement 1D–E ) .", "eIF-3 is reported to bind a highly specific program of messenger RNAs involved in cell proliferation and apoptosis ( Lee et al . , 2015 ) .", "We speculate that this complex could be involved in mediating translational activation of certain anti-apoptotic proteins or repression of pro-apoptotic proteins .", "Regulation of apoptosis by eIF-3 warrants further investigation , but we opted to focus on the second , better-studied complex .", "Knockdown of multiple members of a cullin-RING ubiquitin ligase complex ( CRL ) including CUL5 , UBE2F and RNF7 resensitize LK2 cells to CDK9i and MCL1i ( Figure 2B–C and Figure 2—figure supplement 1D–E ) .", "The cullin 5 ( CUL5 ) scaffold was identified as a top resensitizing hit in both screens .", "Repression of UBE2F was a sensitizing hit in the MCL1i screen ( Figure 2C and Figure 2—figure supplement 1E ) .", "Knockdown of Ring Finger Protein 7 ( RNF7 ) , a catalytic subunit that binds to the CUL5 scaffold , also sensitizes cells to both CDK9i and MCL1i ( Figure 2—figure supplement 1D–E ) .", "Cullin-RING ligases are emerging as attractive cancer targets and a novel class of small molecule neddylation inhibitors have recently been developed ( Soucy et al . , 2009 ) .", "While the cullin 3 complex ( CRL3 ) is more typically associated with cancer phenotypes , there is little data on the function of CRL5 in tumorigenesis or resistance .", "We therefore sought to further examine the role of the CRL5 complex in resensitizing LK2 cells to CDK9 and MCL1 inhibition .", "CUL5 serves as a protein scaffold in the CRL5 complex , forming a platform for RNF7 , UBE2F , Elongin B/C , and a substrate adaptor to recruit and ubiquitinate a target substrate ( Figure 3A ) ( Kamura et al . , 2004 ) ( Huang et al . , 2009 ) ( Mahrour et al . , 2008; Okumura et al . , 2016 ) .", "We made stable dCas9-KRAB LK2 cell lines that expressed an individual CRISPRi sgRNA to knockdown CUL5 , RNF7 , UBE2F , or a non-targeting ( NT ) control ( Figure 3B ) .", "CUL5 and RNF7 appeared to be dependent on each other for stability such that when either protein was knocked down , levels of the corresponding protein were also diminished ( Figure 3C ) .", "Depletion of UBE2F resulted in the disappearance of a higher molecular weight band corresponding to neddylated CUL5 ( Figure 3B–C ) .", "Using two independent stable LK2 CRISPRi lines targeting CUL5 , RNF7 , UBE2F or Bcl-xL , we validated that individual knockdowns of each gene resensitized the cell to both MCL1i and CDK9i ( Figure 3D and Figure 3—figure supplement 1A ) .", "Bcl-xL served as a positive control based on previous reports that dual inhibition of Bcl-xL and MCL1 induces apoptosis ( Goodwin et al . , 2015; Xiao et al . , 2015; Zhang et al . , 2011 ) .", "Assessing cleaved PARP ( c-PARP ) levels , we found that depletion of CUL5 , RNF7 , UBE2F or Bcl-xL significantly induced PARP cleavage when combined with CDK9i or MCL1i ( Figure 3E ) .", "In the absence of drug , knockdown of Bcl-xL alone led to substantial apoptosis but this was not the case for knockdown of CUL5 , RNF7 or UBE2F ( Figure 3E ) .", "This suggests that inactivation of CRL5 could be less toxic than inhibition of Bcl-xL and that inhibition of CRL5 may be better suited to co-administration with CDK9 and MCL1 inhibitors .", "To further validate the synergy between inhibition of CRL5 and CDK9i or MCL1i , we made isogenic CRISPR-Cas9 knockouts of CUL5 and UBE2F in the LK2 background ( Materials and methods and Figure 3—figure supplement 1B–C ) .", "Cells did not tolerate extended culturing of CRISPRi-mediated stable knockdowns of RNF7 , but knockouts of CUL5 and UBE2F were viable .", "Challenging CUL5 or UBE2F knockout cells with CDK9i or MCL1i induced high levels of apoptosis at 1 μM of either compound , whereas wild type cells showed no response even at 10 μM ( Figure 3F , Figure 3—figure supplement 1D ) .", "To assess effects on long-term survival and proliferation , we performed clonogenic assays where the CUL5 and UBE2F knockout clones were exposed to an acute treatment of CDK9i or MCL1i ( Figure 3G–H ) .", "Drug treatment greatly reduced colony-forming ability in the CUL5 and UBE2F knockout cells , indicating efficient and persistent resensitization of these cells .", "Taken together , multiple lines of evidence indicate that the resistance of LK2 cells to both CDK9 and MCL1 inhibition can be overcome by depletion of the CRL5 complex .", "To further investigate how CRL5 protects LK2 cells from CDK9i and MCL1i , we asked whether depletion of Elongin B and Elongin C also synergized with these drugs .", "Individual knockdown of Elongin B by CRISPRi-induced apoptosis at levels similar to that of CUL5 knockdown , but knockdown of Elongin C had minimal effect ( Figure 3—figure supplement 2A–B ) .", "Elongin B and C have often been described as functioning in a complex , but our results suggest a potential separation of function ( Okumura et al . , 2012 ) .", "In an attempt to find the CRL5 substrate adaptor responsible for resensitization to MCL1 and CDK9 inhibitors , we used CRISPRi to individually knock down a large number of CUL5 substrate adaptors annotated in literature and tested them for synergy with MCL1i and CDK9i ( Okumura et al . , 2016 ) .", "None of the literature-proposed substrate adaptors analyzed induced apoptosis following CDK9i or MCL1i treatment ( Figure 3—figure supplement 2C and Figure 3—source data 1 ) , but we cannot rule out redundancy between substrate adaptors .", "The CRL5 substrate adaptors responsible for resensitization to MCL1 inhibition remain to be identified .", "We searched for the substrate of CRL5 that potentiates resensitization to CDK9 or MCL1 inhibition .", "p53 , a master regulator of apoptosis , can be a target of CRL5 during viral infection ( Cai et al . , 2006; Querido et al . , 2001; Sato et al . , 2009 ) .", "However , loss of CUL5 did not lead to accumulation of p53 ( Figure 3—figure supplement 2D ) , nor affected MCL1 and Bcl-xL levels ( Figure 3—figure supplement 2E ) .", "Pro-apoptotic proteins are required for efficient induction of cell death , and CRL5 could target pro-apoptotic proteins for proteasomal degradation .", "Indeed , Noxa was recently proposed to be a substrate of CRL5 ( Zhou et al . , 2017 ) .", "We interrogated the levels of all eight BH3-only proteins using individual CRISPRi knockdowns of CUL5 , RNF7 , UBE2F and a NT control .", "Knockdown of CRL5 components increased protein levels of two BH3-only proteins , Noxa and Bim ( Figure 4A ) .", "After treatment with CDK9i or MCL1i , we also found that CUL5 knockdown had increased levels of both proapoptotic proteins Noxa and Bim while all other BH3-only proteins remained unchanged ( Figure 4B–C ) .", "Interestingly , CDK9i treatment alone reduced levels of Noxa and Bim through transcriptional downregulation , presumably related to the half-lives of these mRNAs ( Figure 4D ) .", "Knockdown of CUL5 together with CDK9i rescued protein levels of Noxa and Bim but not mRNA levels , consistent CRL5-mediated post-translational degradation of Noxa and Bim .", "We used epoxomicin ( proteasomal inhibitor ) and folimycin ( lysosomal inhibitor ) to confirm that CRL5 targets Noxa and Bim for proteasomal degradation .", "Folimycin treatment did not affect the abundance of Noxa or Bim ( Figure 4E ) .", "However , epoxomicin treatment causes accumulation of Bim in LK2 parental cells .", "Epoxomicin treatment had no additional effect on Bim in CUL5 knockout cells .", "Hence , under these conditions CRL5 appears to be the primary ligase for Bim .", "Noxa accumulates in both parental and CUL5 knockout cells after epoxomicin treatment ( Figure 4E ) .", "These data indicate that Noxa is proteasomally regulated , but suggest that Noxa can be targeted by additional ubiquitin ligases .", "We were unable to demonstrate direct ubiquitination of Noxa and Bim by CRL5 via ubiquitin co-immunoprecipitation ( Figure 4—figure supplement 1 ) , leaving open the possibility that these proteins are indirect substrates .", "We examined whether Noxa and/or Bim were required to resensitize CUL5-CRISPRi cells to CDK9i or MCL1i using RNAi .", "If induction of apoptosis in CUL5-deficient cells requires Noxa and/or Bim , then removal of either or both factors should make the cells re-resistant to CDK9i and MCL1i .", "Knocking down BAK , the apoptosis effector required for cell death , prevented apoptosis induced by CDK9i or MCL1i in CUL5-CRISPRi cells ( Figure 5A–B and Figure 5—figure supplement 1 ) .", "Knocking down Noxa in CUL5-CRISPRi cells almost completely prevented CDK9i-induced apoptosis ( Figure 5A and Figure 5—figure supplement 1A ) .", "Knocking down Bim in CUL5-CRISPRi cells did not prevent CDK9i-induced apoptosis , and simultaneously knocking down both Noxa and Bim in CUL5-CRISPRi cells did not further inhibit apoptosis beyond what was observed with knockdown of Noxa alone .", "These data indicate that in CDK9i-treated cells lacking CUL5 , initiation of apoptosis is primarily dependent on Noxa .", "Surprisingly , knocking down Noxa and/or Bim in CUL5-CRISPRi cells exposed to MCL1i still led to high levels of apoptosis ( Figure 5B and Figure 5—figure supplement 1B ) .", "The difference in apoptotic dependence between MCL1i and CDK9i could be due to transcriptional downregulation of Noxa and Bim during CDK9i ( Figure 4D ) or from downregulation of other pro-apoptotic factors , whose loss in conjunction with Noxa depletion leads to abrogation of apoptosis .", "The mechanism underlying CRL5-mediated resensitization to MCL1i is still unclear , and our data highlight differences between direct inhibition of MCL1 and indirect downregulation via CDK9 that can also affect other factors .", "In conclusion , we find that depletion of key components of the CRL5 family of ubiquitin ligases sensitizes otherwise resistant cells to CDK9 and MCL1 inhibition .", "We show that the CRL5 complex targets pro-apoptotic proteins Noxa and Bim for degradation , and increased Noxa abundance upon CRL5 knockdown/out contributes to CDK9i sensitivity .", "However , more work is needed to elucidate the mechanistic distinctions by which CRL5 depletion re-sensitizes cells to both CDK9i and MCL1i ( Figure 5C ) ." ], [ "Cullin-RING complexes comprise the largest class of ubiquitin ligases and regulate a diverse array of biological processes ( Petroski and Deshaies , 2005 ) .", "While CUL3 is implicated in a wide array of cancer biologies , relatively little is known about the physiological functions of many CUL5-containing ubiquitin ligases .", "A few reports have implicated genetic alterations in CUL5 in cancer progression and our unbiased screens reveal that the CUL5-RNF7-UBE2F ubiquitin ligase can modulate the apoptotic threshold of LK2 cells in response to multiple emerging cancer therapeutics ( Burnatowska-Hledin et al . , 2004; Fay et al . , 2003; Lubbers et al . , 2011; Xu et al . , 2012 ) .", "The CUL5 ligase complex regulates the stability of Noxa and Bim , while increased Noxa abundance through disruption of CUL5 underlies re-sensitization to a CDK9 inhibitor .", "The discovery that inactivation of the CRL5 ligase can resensitize LK2 cells to both CDK9 and MCL1 inhibition suggests a possible co-treatment strategy , as well as a potential biomarker for patient stratification .", "Based on our data , patients with lung cancers that exhibit loss of function mutations in either CUL5 , RNF7 , UBE2F or Elongin B that coincide with high expression of MCL1/Bcl-xL could be good candidates for treatment with CDK9i or MCL1i .", "These patients may be unlikely to have pre-existing CRL5-mediated resistance to treatment .", "There are currently no small-molecule inhibitors that target specific components of the CRL5 complex .", "However , several potent inhibitors that broadly inhibit the ubiquitin-proteasome system have been developed .", "MLN4924 inhibits the NEDD8 activating enzyme ( NAE ) , thereby preventing conjugation of NEDD8 onto all cullin scaffolds , and shows significant antitumor activity in clinical trials ( Soucy et al . , 2009 ) .", "Similiarly , treatment with MLN7243 , a small molecule inhibitor of the ubiquitin activating enzyme ( UAE ) currently in clinical trials , depletes cellular ubiquitin conjugates and also demonstrates antitumor activity in xenografts ( Hyer et al . , 2018 ) .", "Several clinically approved proteasome inhibitors such as bortezomib and carfilzomib are currently used in the treatment of multiple myeloma and lymphomas ( Kuhn et al . , 2007; Richardson et al . , 2005 ) .", "Although a therapeutic window exists for these compounds , patients also experience adverse side effects due to the widespread effects of proteasome inhibition , complicating the possibility of co-dosing with CDK9i or MCL1i .", "Compounds have also been developed that directly manipulate protein-protein interactions within specific ligases .", "The small molecule CC0651 selectively inhibits Cdc34A , an E2 for SCF-type CRLs , and has been shown to impede proliferation of cancer cells ( Ceccarelli et al . , 2011; Huang et al . , 2014b ) .", "Another example is the synthesis of a potent inhibitor ( ligand 7 ) targeting the von Hippel-Landau ( VHL ) substrate adaptor , thereby preventing the degradation of HIF-1α , a potential treatment for chronic anemia ( Galdeano et al . , 2014 ) .", "While ubiquitin ligases are challenging targets for small molecule development , their importance in myriad disease states have spurred efforts to manipulate their activity .", "Our results in LK2 cells suggest that targeting CRL5 could be used to combat innate and acquired resistance to therapeutics that directly or indirectly affect MCL1 .", "Additional work will be necessary to establish the mechanistic distinctions between CRL5’s ability to set the apoptotic response to distinct small molecules , as well as the clinical applicability of targeting CRL5 in cancer ." ], [ "The LK2 cell line was obtained from AstraZeneca and maintained in RPMI supplemented with 10% ( v/v ) fetal bovine serum , 100 units/mL penicillin and streptomycin , GlutaMAX , sodium pyruvate and non-essential amino acids .", "All cells tested negative for Mycoplasma contamination using the MycoAlert Plus Mycoplasma detection kit from Lonza .", "AstraZeneca licensed the CDK9 inhibitor ( AZD5576 ) originally published as PC585 ( Garcia-Cuellar et al . , 2014 ) and supplied it to us for this study .", "The MCL1 inhibitor ( AZD5991 ) is synthesized by AstraZeneca ( Tron et al . , 2018 ) .", "Purified Cas9 ribonucleoproteins ( RNPs ) complexed with sgRNA pairs flanking coding exon one for CUL5 or UBE2F were nucleofected into LK2s to completely disrupt all coding potential as previously described ( Lingeman et al . , 2017 ) .", "Nucleofected pools were seeded at single cell densities by FACS sorting .", "Clones were initially screened by western blot for loss of protein and confirmed by Sanger sequencing .", "Individual analysis of sgRNA phenotypes sgRNA protospacers targeting CUL5 , RNF7 , UBE2F , Elongin B , Elongin C , Bcl-xL , Noxa and a negative control non-targeting protospacer were individually cloned into BstXI/BlpI-digested pCRISPRia-v2 ( Addgene #8432 ) by ligating annealed complementary synthetic oligonucleotide pairs .", "The sgRNA expression vectors were packaged into lentivirus as previously described and successful transductants were selected with puromycin at a final concentration of 2 . 5 μg/mL .", "Protospacer sequences are in Supplementary file", "1 . Lentivirus was produced by transfecting HEK293T with standard packaging vectors using the TransIT-LT1 Transfection Reagent ( MIR 2306; Mirus Bio LLC ) .", "Viral supernatant was collected at 48 hr and 72 hr after transfection , filtered through a 0 . 45 μm polyethersulfone syringe filter , snap-frozen and stored at −80°C for future use .", "For screens , viral titrations were performed by transducing LK2 cells at serial dilutions and assessing BFP ( present on the sgRNA-encoding plasmid ) percentages 48 hr following transduction .", "The viral dilution resulting in ~15% BFP-positive cells was used to transduce cells for the screens .", "The human CRISPRi v2 library contains 5sgRNAs/annotated TSS of each gene comprising a total of 104 , 535 sgRNAs .", "In order to maintain 500X coverage ( representation ) of each guide at all times , ~50×106 cells need to be transduced with one sgRNA .", "To ensure delivery of one sgRNA/cell a low MOI of ~0 . 3 is used , which results in not every cell getting transduced , thus 5–6 fold more cells need to be transduced in order to maintain 500X coverage .", "Replicate cultures of 3 × 108 cells were plated in twenty 15 cm dishes and transduced at an MOI of ~0 . 3 in the presence of 4 μg/mL polybrene .", "Cells were split into 2 . 5 μg/mL puromycin and selected for 4 days .", "Cells were passaged into regular media and recovered for 2 days .", "60 × 106 were collected as the ‘background’ sample .", "60 × 106 cells were treated with 3 μM CDK9i and 0 . 5 μM Cell Event for 6 hr . 60 × 106 cells were treated with 1 μM MCL1i and 0 . 5 μM Cell Event for 12 hr . 60 × 106 cells were treated with DMSO ( 1:10 , 000X dilution ) and 0 . 5 μM Cell Event for 12 hr .", "After treatment , cells were trypsinized , washed and fixed in 1% formaldehyde/PBS .", "The entirety of the cell population was then FACS-sorted where GFP-positive ( apoptosing ) cells were isolated .", "As previously mentioned , this was done in duplicate .", "Genomic DNA was purified from each cell population with blood purification kits ( Machery-Nagel , Nucleospin blood L or XL , depending on cell number ) and the sgRNA-encoding region was enriched , amplified , and processed for sequencing on the Illumina Hiseq 2500 .", "TruSeq index sequences unique to each cell population were used to multiplex samples .", "Data analysis was performed as described ( Horlbeck et al . , 2016; Li et al . , 2014 ) .", "Briefly , sequence reads from the Illumina HiSeq 2500 were trimmed , aligned to CRISPRi v2 sgRNA library , counted and normalized .", "For the Python-based ScreenProcessing pipeline , sgRNA phenotypes and negative control gene phenotypes were determined along with Mann-Whitney P values .", "The top three guide-level phenotypes were collapsed to produce the gene-level phenotype score .", "For genes with multiple annotated transcription start sites ( TSSs ) , phenotypes were calculated for each TSS and the TSS with the lowest Mann Whitney p-value was used to represent the gene .", "For MAGeCK , all sgRNAs/gene ( including both TSSs if there are two ) are ranked based on p-values from mean variance modeling .", "A robust ranking aggregation ( RRA ) algorithm is then used to call genes that are significantly enriched or depleted based on p-value and false discovery rate ( FDR ) .", "For qPCR , RNA was extracted with the RNeasy Mini Kits ( Qiagen ) .", "cDNA was produced from 1 μg of purified RNA using the iScript Reverse Transcription Supermix for RT-qPCR ( Bio-Rad Laboratories ) .", "qPCR reactions were performed with the SsoAdvanced Universal SYBR Green Supermix ( Bio-Rad Laboratories ) in a total volume of 10 μl with primers at final concentrations of 500 nM .", "Primer sequences are included in Supplementary file", "2 . The thermocycler was set for 1 cycle of 95°C for 30 s , and 40 cycles of 95°C for 5 s and 55°C for 15 s , respectively .", "Fold enrichment of the assayed genes over the control HPRT and/or GAPDH loci were calculated using the 2−ΔΔCT method as previously described ( Livak and Schmittgen , 2001 ) .", "Cell Event reagent was added to cells for the duration of the drug or vehicle treatment , at a final concentration of 0 . 5 μM .", "Cells were harvested by trypsinization and analyzed on a flow cytometer , either the BD FACSARIA for the screen or an Attune Nxt cytometer ( ThermoFisher ) for the follow-up experiments .", "Cells were plated at 5000 cells/well of a 384-well white opaque plates in corresponding cell growth media .", "Cells were treated with compounds at indicated concentrations for 6 hr ( 37°C , 5% CO2 ) with a final DMSO concentration of 0 . 3% .", "Caspase-3/7 activation was subsequently determined using a Caspase-Glo 3/7 Reagent ( Promega Corporation ) as described in manufacturer's instructions .", "Dose-response curves were plotted and analyzed ( including EC50 determination ) using GraphPad Prism .", "Percentage of caspase activation was calculated against the maximum caspase activation value ( 100% ) obtained with a combination of MCL1 , BCL2 , and Bcl-xL inhibition .", "Cells were plated at 5 , 000 cells/well of a 384-well white opaque plates in corresponding cell growth media .", "Cells were treated with compounds at indicated concentrations for 24 hr ( 37°C , 5% CO2 ) with a final DMSO concentration of 0 . 3% and assayed for viability using the CellTiter-Glo Reagent ( Promega Corporation ) as described in manufacturer's instructions .", "Results were normalized to the samples without treatment at time 0 .", "GI50 values were calculated using nonlinear regression algorithms in Prism .", "Primary antibodies against the following proteins were used: CUL5 ( ab184177; Abcam ) ; RNF7 ( ab181986; Abcam ) ; UBE2F ( ab185234; Abcam ) ; Noxa ( OP180; Millipore ) ; Bim ( ab32158; Abcam ) ; Bid ( ab32060; Abcam ) ; Puma ( ab9643; Abcam ) ; Bad ( ab62465; Abcam ) ; Bik ( ab52182; Abcam ) ; Beclin ( ab207612; Abcam ) ; Bmf ( ab9655; Abcam ) ; MCL1 ( D35A5 #5453; CST ) ; Bcl-xL ( 54H6; CST ) ; c-PARP ( 19F4 #9546; CST ) Ubiquitin ( P4D1 #3936; CST ) ; HA ( C29F4; CST ) ; GAPDH ( 14C10 #2118; CST ) ; p53 ( DO-1 sc-126; Santa Cruz Biotechnology ) ; HSP90 ( sc-69703; Santa Cruz Biotechnology ) .", "For each protein antibody , manufacturer’s recommended dilutions were used .", "Mouse or rabbit immunoglobulin G was visualized at a 1:10 , 000 dilution: donkey anti-mouse 680 ( 925–68022; LI-COR ) ; donkey anti-rabbit 680 ( 925–68023; LI-COR ) ; donkey anti-mouse 800 ( 925–32212; LI-COR ) ; donkey anti-rabbit 800 ( 925–32213; LI-COR ) .", "Blots were imaged on an Odyssey CLx Imaging System ( LI-COR ) .", "LK2 wild type and CUL5-KO cells were transfected with equal amounts of His-Ubiquitin and either HA-Noxa or HA-Bim .", "48 hr after transfection , cells were treated with 100 μM epoxomicin for 8 hr .", "Cells were harvested and lysed in either denaturing buffer ( 8M Urea , 300 mM NaCl , 0 . 5% NP-40 , 50 mM Na2HPO4 , 50 mM Tris pH8 . 0 ) or lysis buffer from ThermoFisher ( Pierce HA-Tag Magnetic IP/CoIP Kit #88838 ) .", "Denatured extracts were bound to magnetic Ni-NTA beads ( Qiagen #36111 ) , while other lysed extracts were bound to magnetic anti-HA beads .", "Beads were washed five times and eluted in 1X Laemmli buffer by boiling for 5 min .", "Samples were loaded on a gel and processed for immunoblotting .", "Cells were reverse-transfected in six-well plates using RNAiMAX ( Thermo Fisher Scientific ) with 50 nM siRNA .", "48 hr following siRNA treatment , cells were treated for the Cell Event apoptosis assay as indicated and also harvested to verify knockdown by qRT-PCR .", "BCL2L11 ( Bim ) siRNA SMARTpool was from Dharmacon ( L-004383-00-0005 ) and BAK siRNA was obtained from Ambion ( Life Technologies 4457298 ) .", "Six-well plates were seeded at 500 cells/well and allowed to attach for 12 hr .", "Cells were treated with DMSO or 1 µM CDK9i or 1 µM MCL1i for 8 hr .", "Drug treatments were washed out and plates were replenished with fresh media .", "2 weeks later colonies were stained with 0 . 5% crystal violet and 6% glutaraldehyde in water .", "Plates were scanned using a flatbed scanner and colonies were scored on ImageJ ." ] ]
[ "Overexpression of anti-apoptotic proteins MCL1 and Bcl-xL are frequently observed in many cancers .", "Inhibitors targeting MCL1 are in clinical development , however numerous cancer models are intrinsically resistant to this approach .", "To discover mechanisms underlying resistance to MCL1 inhibition , we performed multiple flow-cytometry based genome-wide CRISPR screens interrogating two drugs that directly ( MCL1i ) or indirectly ( CDK9i ) target MCL1 .", "Remarkably , both screens identified three components ( CUL5 , RNF7 and UBE2F ) of a cullin-RING ubiquitin ligase complex ( CRL5 ) that resensitized cells to MCL1 inhibition .", "We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex .", "Accumulation of Noxa caused by depletion of CRL5 components was responsible for re-sensitization to CDK9 inhibitor , but not MCL1 inhibitor .", "Discovery of a novel role of CRL5 in apoptosis and resistance to multiple types of anticancer agents suggests the potential to improve combination treatments ." ]
[ "Organisms keep their tissues healthy by instructing damaged or unwanted cells to kill themselves via a controlled process known as apoptosis .", "Cancer cells , however , are able to evade death by increasing the level of proteins that block apoptosis , such as MCL1 .", "Researchers have recently developed new drugs that can inhibit the action of the MCL1 protein .", "But a number of cancers have become resistant to these inhibitors .", "So , one important question is whether other proteins in cancer cells could be drugged , together with MCL1 , to overcome or even avoid this resistance .", "Now , Kabir et al . have addressed this question by searching the genome of human lung cancer cells , which were resistant to treatment , for targets that could improve the performance of two MCL1 inhibitors .", "This involved reducing the level of every protein in these cells one by one using a genetic technique known as CRISPR-Cas9 , and looking for cells that lost their resistance to the MCL1 inhibitor .", "From these genetic screens , Kabir et al . identified three proteins that are part of complex called CRL5 .", "Inactivating this protein complex caused cancer cells to become more sensitive to the MCL1 inhibitor .", "Further biochemical experiments showed that CRL5 may contribute to drug resistance by reducing the levels of two proteins that promote apoptosis .", "These findings suggest that inhibiting CRL5 in combination with MCL1 could combat drug resistance .", "Although there are currently no drugs against CRL5 , future experiments determining how CRL5 and MCL1 are linked could identify new drug targets and improve existing cancer treatments ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "plant biology", "microbiology and infectious disease" ]
Biosynthesis of a sulfated exopolysaccharide, synechan, and bloom formation in the model cyanobacterium Synechocystis sp. strain PCC 6803
elife-66538-v1
[ [ "Bacterial extracellular polysaccharides establish biofilms for nutrient supply and stress avoidance , and they sometimes support cellular activities such as motility and infectivity ( Woodward and Naismith , 2016 ) .", "Generally , the polysaccharide chains consist of a few types of sugars ( with or without chemical modifications ) and are anchored on cells ( capsular polysaccharides , CPS ) or exist as nonanchored exopolysaccharides ( EPS , also called released polysaccharides ) .", "Nonetheless , their molecular structures vary greatly , for example , branching schemes , sugar constituents , and modifications , and thus their physical properties also vary .", "Bacterial extracellular polysaccharides and lipopolysaccharides are produced and exported via three distinct pathways: the Wzx/Wzy-dependent pathway , ABC-dependent pathway , and synthase-dependent pathway ( Schmid et al . , 2015 ) .", "Many bacteria can produce several extracellular polysaccharides , and production often depends on environmental conditions .", "Some extracellular polysaccharides have been appropriated for use as biopolymers for food ( i . e . cellulose as nata de coco ) , food additive ( i . e . xanthan as a thickener or an emulsifier ) , cosmetics ( i . e . hyaluronic acid for viscosupplementation ) , and medicine ( i . e . alginate for drug delivery ) .", "Cyanobacteria , the oxygenic photoautotrophic bacteria that inhabit almost every ecosystem on Earth , contribute to the global photosynthetic production ( Flombaum et al . , 2013; Mangan et al . , 2016 ) .", "Cyanobacteria produce various extracellular polysaccharides to form colonies , which are planktonic or attached on solid surfaces , likely to stay in a phototrophic niche in nature ( De Philippis , 1998 ) .", "A notable example is the water bloom , a dense population of cyanobacterial cells that floats on the water surface and often produces cyanotoxins and extracellular polysaccharides ( Huisman et al . , 2018 ) .", "The extracellular polysaccharides are also important for photosynthetic production of cyanobacteria and their application ( Kehr and Dittmann , 2015; Kumar et al . , 2018 ) .", "However , very little is known about their biosynthesis except for extracellular cellulose .", "A thermophilic cyanobacterium ( Thermosynechococcus vulcanus ) accumulates cellulose to form tight cell aggregation ( Kawano et al . , 2011 ) .", "This cellulose is produced by cellulose synthase tripartite system unique to cyanobacteria , and its biosynthesis is regulated by temperature and light ( Enomoto et al . , 2015; Maeda et al . , 2018 ) .", "In the cyanobacterial genomes , there are still many putative genes for extracellular polysaccharide biosynthesis .", "Interestingly , many cyanobacterial extracellular polysaccharides are sulfated , that is , as a sugar modification ( Pereira et al . , 2009 ) .", "Sulfated polysaccharides are also produced by animals ( as glycosaminoglycan in the extracellular matrix such as heparan sulfate ) and algae ( as cell wall components such as carrageenan ) but are scarcely known in other bacteria or plants ( Ghosh et al . , 2009 ) .", "Major examples of cyanobacterial sulfated polysaccharides are spirulan from Arthrospira platensis ( vernacular name , ‘Spirulina’ ) , sacran from Aphanothece sacrum ( vernacular name , ‘Suizenji-Nori’ ) , and cyanoflan from Cyanothece sp .", "CCY 0110 ( Mota et al . , 2020; Mouhim et al . , 1993; Okajima et al . , 2008 ) .", "These sulfated polysaccharides were found in their biofilms and may be relevant to some ecological functions in cyanobacteria ( Fujishiro et al . , 2004 ) .", "In addition , the bioactivities ( antiviral , antitumor , and anti-inflammatory ) of sulfated polysaccharides from cyanobacteria were reported , too ( Flores et al . , 2019a; Hayashi et al . , 1996; Ngatu et al . , 2012 ) .", "However , very little is known about their biosynthesis machinery and physiological functions .", "On the other hand , biosynthesis and modification of animal sulfated polysaccharides have been extensively studied because of their importance to tissue protection , tissue development , and immunity ( Karamanos et al . , 2018; Sasisekharan et al . , 2006 ) and potential applications in healthy foods , biomaterials , and medicines ( Jiao et al . , 2011; Wardrop and Keeling , 2008 ) .", "The poor understanding about cyanobacterial sulfated polysaccharide biosynthesis is probably due to low or no accumulation of sulfated polysaccharides in typical model species ( Pereira et al . , 2009 ) .", "More than three decades ago , Panoff et al . reported sulfated polysaccharides in two related model cyanobacteria , Synechocystis sp .", "PCC 6803 and Synechocystis sp .", "PCC 6714 ( hereafter Synechocystis 6803 and Synechocystis 6714 ) ( Panoff et al . , 1988 ) .", "Recently , Flores et al . confirmed sulfated polysaccharides and reported their enhanced accumulation in a sigma factor sigF mutant for global cell surface regulation in Synechocystis 6803 ( Flores et al . , 2019b ) .", "In parallel , several papers have studied genes that could be involved in extracellular polysaccharides biosynthesis in Synechocystis 6803 , but no clear results were obtained about the sulfated polysaccharide ( Fisher et al . , 2013; Foster et al . , 2009; Jittawuttipoka et al . , 2013; Pereira et al . , 2019 ) .", "Here , we found that a motile substrain of Synechocystis 6803 showed bloom-like cell aggregation and sulfated EPS production , but a non-motile substrain ( a standard substrain for photosynthesis study ) did not .", "By gene disruption and overexpression , we first identified a whole set of genes responsible for sulfated EPS biosynthesis and its regulatory system , opening the way to engineering of their production ." ], [ "We fortuitously found that a motile substrain of Synechocystis 6803 produces EPS and forms floating cell aggregates resembling a typical cyanobacterial bloom .", "We established a two-step culture regime ( 2-day bubbling culture and subsequent standing culture without bubbling under continuous light ) for reproducible formation of bloom-like aggregates ( Figure 1A , B ) .", "The first ( bubbling ) step allows for cell propagation and EPS production , whereas the second ( standing ) step allows for massive cell aggregation and flotation , even though Synechocystis 6803 does not possess genes for intracellular gas vesicles ( Harke et al . , 2016 ) .", "In Synechocystis 6803 , cell flotation accompanying the generation of extracellular gas bubbles was suppressed by inactivation of photosynthesis ( Figure 1C ) , suggesting that gas derived from photosynthesis drives the upward movement of cells embedded in viscous EPS .", "The non-motile , glucose-tolerant substrain—commonly used for photosynthesis research—did not aggregate or float .", "We first isolated crude viscous EPS from the bloomed culture by membrane filtration ( Figure 1—figure supplement 1A ) .", "The crude EPS consisted of polysaccharide but very little protein or nucleic acid and its abundance remained unchanged during the second culture step ( Figure 1—figure supplement 1B ) .", "As a common feature of diverse EPS biosynthesis systems in bacteria , membrane-bound glycosyltransferases are particularly important ( Schmid et al . , 2015 ) .", "In the Synechocystis 6803 genome , 59 genes were annotated as glycosyltransferases .", "Twelve of them were uncharacterized and predicted to encode transmembrane helices .", "So , we disrupted five of them initially and found that slr5054 is essential for bloom formation ( Figure 1D , E and Figure 1—figure supplement 2 ) .", "The viscous EPS preparation was improved by removing cells before filtration to avoid cell-associated polysaccharides such as CPS ( Figure 1F ) .", "∆slr5054 lacked most of the EPS present in the wild type ( WT ) , whereas the CPS and free polysaccharide fractions were similar in the WT and ∆slr5054 ( Figure 1G ) .", "Then , we performed Alcian blue staining to examine the acidity of the EPS ( Figure 1—figure supplement 3 ) .", "Generally , sulfated polysaccharides are stained at pH 0 . 5 condition , while acidic polysaccharides , which contain sulfate groups and/or carboxylate groups ( such as uronic acids and carboxylate modification ) , are stained at pH 2 . 5 condition ( Bellezza et al . , 2006 ) .", "The EPS from WT was clearly stained under both pH conditions , strongly suggestive of the sulfate modification .", "Gene cluster for the biosynthesis of viscous EPS slr5054 resides on a megaplasmid , pSYSM , in a large gene cluster ( sll5042–60 ) , which we named xss ( extracellular sulfated polysaccharide biosynthesis ) ( Figure 2A , xssA–xssS ) .", "This cluster includes two genes for sulfotransferases ( xssA , xssE ) , eight genes for glycosyltransferases ( xssB , xssC , xssG , xssI , xssM , xssN , xssO , xssP ) , three genes for the polysaccharide polymerization system ( Wzx/flippase; xssH , Wzy/polymerase; xssF , and polysaccharide co-polymerase [PCP]; xssK ) , one gene for a putative transcriptional regulator ( xssQ ) , a pair of genes for the bacterial two-component phosphorelay system ( xssR , xssS ) , and genes encoding several small proteins of unknown function ( Supplementary file 1 , Figure 2A ) .", "All genes except those of unknown function were disrupted individually with a read-through cassette , and segregation was confirmed by colony PCR ( Figure 2—figure supplement 1 ) .", "Bloom formation and sugar content of the EPS fraction were reduced in many mutants ( Figure 2B , C ) .", "In particular , bloom formation was completely abolished in ∆xssA , ∆xssB , ∆xssF , ∆xssH , ∆xssK , ∆xssM , ∆xssN , and ∆xssP , in which EPS accumulation was also suppressed .", "Certain glycosyltransferase mutants ( ∆xssC , ∆xssG , ∆xssI , ∆xssO ) formed blooms but accumulated little EPS , and neither bloom formation nor EPS accumulation was substantially altered in one sulfotransferase mutant ( ∆xssE ) .", "In general , the Wzx/Wzy system in bacteria produces various EPS , lipopolysaccharides , and CPS through four steps:", "( i ) biosynthesis of a heterooligosaccharide repeat unit on a lipid linker at the cytoplasmic side of the plasma membrane by a series of glycosyltransferases and modification enzymes ,", "( ii ) flip-out of the unit to the periplasmic side by Wzx ,", "( iii ) polymerization by transfer of the nascent polysaccharide chain to the repeat unit by Wzy , and", "( iv ) export of the EPS chain through the periplasm and outer membrane via PCP and the outer-membrane polysaccharide export protein ( OPX ) ( Islam and Lam , 2014; Schmid et al . , 2015 ) .", "It is very likely that the xss cluster harbors a whole set of genes for the Wzx/Wzy-dependent pathway except a gene for OPX .", "The sensory histidine kinase mutant ∆xssS accumulated a much larger amount of EPS than WT , whereas mutants of the cognate response regulator xssR and transcriptional regulator xssQ had a null phenotype with regard to both bloom formation and EPS accumulation ( Figure 2D , E , Supplementary file 2 ) .", "The double mutant ∆xssS/∆xssR had a phenotype similar to that of ∆xssR .", "Overexpression of xssR or xssQ ( OX-xssR , OX-xssQ ) resulted in strong bloom formation as well as marked accumulation of viscous EPS , similar to that seen for ΔxssS .", "The combination of xssQ disruption and xssR overexpression ( ΔxssQ + OX xssR ) abrogated bloom formation and EPS accumulation , whereas the combination of xssQ overexpression and xssR disruption ( ΔxssR + OX xssQ ) resulted in a pronounced phenotype of bloom formation and EPS accumulation .", "These results suggested that the sensor histidine kinase XssS suppresses the response regulator XssR , leading to activation of the transcriptional activator XssQ .", "Notably , the OX-xssR and OX-xssQ strains formed sticky , non-motile , biofilm-like colonies on agar plates that could be picked by tweezers ( Figure 2F ) .", "XssQ is a new type of the signal transduction ATPase with numerous domains ( STAND ) protein , because it harbors an N-terminal helix-turn-helix transcriptional DNA-binding domain ( Figure 2—figure supplement 2 ) .", "Typical STAND proteins possess a three-domain module with ATPase activity and are involved in processes such as apoptosis and immunity in animals , plants , and some bacteria ( Danot et al . , 2009 ) .", "Using real-time quantitative PCR ( qPCR ) , we compared gene expression in the xss cluster for WT , ΔxssS , and ΔxssQ ( Figure 3A ) .", "Expression of five genes ( xssA , xssB , xssE , xssN , xssP ) was very low in ∆xssQ compared with WT , whereas that of xssF , xssH , and xssK was not substantially affected .", "These results suggested that XssQ transcriptionally activates genes encoding sulfotransferases and certain glycosyltransferases but not genes for polymerization and export via the Wzx/Wzy system .", "qPCR analysis of gene expression in ∆xssS revealed a tendency for slight up-regulation of xssB , xssE , xssN , and xssP .", "We next performed RNA-seq of WT , ΔxssS , and ΔxssQ to analyze the transcriptome ( Figure 3—figure supplement 1 ) .", "The genes down-regulated in ΔxssQ and up-regulated in ΔxssS were mostly xss genes .", "In detail , the regulated genes were xssA-E and xssL-P , which were roughly consistent with the qPCR analysis ( Figure 3A ) .", "We conclude that xssA-E and xssL-P were specifically regulated by XssS/XssR/XssQ .", "In a previous report , xssA–xssE and xssL–xssP were up-regulated at low temperature in another substrain of Synechocystis 6803 ( Kopf et al . , 2014b ) .", "To test this in our substrain , we measured the sulfated EPS accumulation of WT culture at 20°C , and it was 3 . 1-fold greater than that at normal growth temperature ( 31°C; Figure 2D , E and Supplementary file 2 ) .", "This result suggests that XssS/XssR/XssQ is a temperature sensor for xss gene expression .", "We aligned nucleotide sequences near the transcription start sites of the regulated genes ( xssA , xssE , xssL , xssN , and xssP ) to find the consensus sequences for XssQ binding ( Figure 3B ) , according to the differential RNA-seq-type transcriptomic analysis of Synechocystis 6803 ( Kopf et al . , 2014b ) .", "There are single or tandem consensus sequence , AAGTTXXAC .", "To confirm the binding of XssQ to this region , we performed electrophoretic mobility shift assay ( EMSA ) using purified recombinant XssQ protein and a PCR-amplified DNA fragment of xssE upstream ( Figure 3C ) .", "The band position of the radiolabeled probe DNA shifted reflecting the concentration of XssQ .", "This shift was largely eliminated by excess addition of the unlabeled native competitor , but not by addition of the mutant competitor with mutations in the consensus region .", "These results suggest that XssQ recognizes the consensus sequence of xssE and other target genes for their transcriptional activation .", "The EPS fractions of WT and the overproduction mutant ( ∆xssS ) were subjected to chemical composition analysis ( Table 1 , Figure 3—figure supplement 2 ) .", "EPS from ∆xssS consisted of only four types of monosaccharides and sulfate with the near stoichiometric molar ratio of rhamnose:mannose:galactose:glucose:sulfate of 1:1:1:5:2 .", "This finding roughly fits with the gene number , that is , eight glycosyltransferase genes and two sulfotransferase genes .", "We assumed that the sulfated EPS of ∆xssS is produced by the xss cluster in Synechocystis 6803 and designated ‘synechan’ .", "On the other hand , the EPS fraction from WT likely contained unknown polysaccharides consisting of mannose , fucose , and xylose in addition to synechan of the ∆xssS .", "It should be mentioned that the membrane filtration is effective to collect synechan selectively from the ‘free’ polysaccharides , which have been mixed together with viscous molecules in literature .", "There is no candidate gene in the xss-carrying plasmid for the OPX protein of the Wzx/Wzy system , whereas sll1581 , an OPX homolog , was found on the main chromosome .", "Disruption of sll1581 ( Δsll1581 ) abolished bloom formation and EPS accumulation ( Figure 2D , E ) .", "Thus , the chromosomal OPX protein Sll1581 ( XssT ) appears to serve as the outer-membrane exporter for synechan .", "Interestingly , Synechocystis 6803 possesses xssT ( OPX gene ) and sll0923 ( a second PCP-2a gene ) on the main chromosome and xssK ( PCP-2a gene ) on the plasmid pSYSM , whereas its close relative Synechocystis 6714 harbors only homologs of sll0923 and xssT but lacks the entire plasmid carrying the xss cluster .", "This suggested that XssT serves as an OPX for dual function for both XssK and Sll0923 .", "It is likely that Synechocystis 6803 acquired pSYSM and borrowed the chromosomal OPX gene xssT to produce synechan or , alternatively , Synechocystis 6714 may have lost the plasmid ." ], [ "Summarizing these data , we propose models for synechan biosynthesis apparatus including OPX and temperature-responsive regulation ( Figure 4A , B and Figure 2—figure supplement 2 ) .", "The model of the Xss apparatus fits well with the known Wzx/Wzy-dependent apparatus represented by xanthan biosynthesis in Xanthomonas campestris ( Katzen et al . , 1998 ) .", "The eight glycosyltransferases including XssP ( the priming glycosyltransferase ) produce oligosaccharide repeat unit of eight sugars , which is consistent with the sugar composition of synechan .", "These findings suggest that the xss cluster on the pSYSM plasmid harbors a whole set of genes for synechan biosynthesis except the OPX gene ( xssT on the main chromosome ) .", "Notably , the cluster harbors two sulfotransferase genes , which have not been found to our knowledge in other bacterial gene clusters for extracellular polysaccharide biosynthesis .", "Sulfotransferases , XssA and XssE , belong to distinct subfamilies of bacterial sulfotransferases .", "We found many sulfotransferase genes in various cyanobacterial genomes by Pfam search ( PF00685 , PF03567 , PF13469 ) .", "They are mostly found in gene clusters for putative extracellular polysaccharide biosynthesis ( Wzx/Wzy-type and ABC-type ) ( Figure 4—figure supplement 1 ) .", "It should be noted that they are more or less partial as a cluster for extracellular polysaccharide biosynthesis system , whereas the xss cluster appears to be complete except the OPX gene in Synechocystis 6803 .", "It is well established that the polysaccharide moiety of membrane-anchored lipopolysaccharides and CPS of bacteria are produced and exported by the Wzx/Wzy-dependent or ABC transporter–dependent pathways , whereas free EPS , that is , xanthan and cellulose , are produced by the Wzx/Wzy-dependent and synthase-dependent pathways but not by the ABC transporter–dependent pathway ( Schmid et al . , 2015; Willis and Whitfield , 2013 ) .", "In the literature , a sulfated CPS was reported in A . platensis ( formerly Spirulina platensis ) ( Mouhim et al . , 1993 ) .", "This sulfated CPS may be produced by an ABC transporter-type gene cluster in Figure 4—figure supplement 1 .", "Gene disruption will confirm such predictions deduced from the gene cluster analyses , although targeted disruption is not so easy in many cyanobacteria due to poor transformation efficiency except Synechocystis 6803 .", "In contrast , no sulfated polysaccharide has been reported in the other bacteria , though many sulfotransferases are also registered in Pfam database .", "Some of them are known to transfer a sulfuryl group to lipo-oligosaccharides in rhizobia ( Nod factor ) and mycobacteria ( Mougous et al . , 2002 ) .", "XssQ , a STAND protein with a DNA binding domain is indeed the transcriptional activator for xssE and other induced genes .", "XssQ homologs are found widely throughout the cyanobacteria but the set of XssS/XssR/XssQ is found near the gene cluster for sulfated EPS biosynthesis with sulfotransferases in many cyanobacteria ( Figure 4—figure supplement 2 , Supplementary file 3 ) .", "Consensus sequences are also found in upstream of some genes in the cluster , suggesting that the XssS/XssR/XssQ system may operate universally for induction of sulfated EPS production under certain environmental conditions such as cold temperature .", "Acidic polysaccharides containing uronic acids and other carboxylic groups are common in bacteria , but sulfated polysaccharides are produced exclusively by cyanobacteria ( Pereira et al . , 2009 ) .", "To speculate on the physiological significance of sulfated polysaccharides , we summarized the distribution of sulfotransferase genes in cyanobacteria of various habitats ( Supplementary file 3 ) .", "Species living in high salinity environments such as a salt lake or seawater mostly produce sulfated polysaccharides .", "Abundant sulfotransferases are found in the marine Trichodesmium that forms bloom-like colonies to collect dust-bound iron for nutrition ( Rubin et al . , 2011 ) .", "Sulfated polysaccharides may play a role for adsorbing metal ions such as iron in saline environments .", "Consistently , the xss genes are up-regulated in iron deficient conditions in Synechocystis 6803 ( Kopf et al . , 2014b ) .", "The freshwater species Synechocystis sp .", "PCC 6714 lacks the xss genes including sulfotransferases and salt-resistance genes that are present in the more salt-resistant Synechocystis 6803 ( Kopf et al . , 2014a ) .", "A cyanobacterial sulfated polysaccharide , sacran , shows much higher capacity for saline absorption than does uronic acid-containing polysaccharides , whereas both absorb pure water efficiently ( Okajima et al . , 2008 ) .", "This fact may also support the role of sulfated polysaccharides for water retention in aquatic or terrestrial habitats .", "There are many putative genes that may be involved in biosynthesis and export of extracellular polysaccharides and lipopolysaccharides in the genome of Synechocystis 6803 ( Fisher et al . , 2013; Pereira et al . , 2015 ) .", "Many of them have been disrupted for characterization .", "With regard to the Wzx/Wzy-dependent system , sll5052 ( xssK ) disruption mutant showed no clear phenotypes for bloom formation or EPS production ( Jittawuttipoka et al . , 2013 ) , probably because the parent strain did not produce discernable amount of EPS like our non-motile strain or large EPS molecules were removed by membrane filtration from their EPS preparation .", "Similarly , the deletion mutant of sll5049 ( xssH ) did not show any defect in EPS or CPS accumulation , though related mutants ( ∆sll0923 for a second PCP-2a ) were shown to be depleted slightly of both CPS and EPS ( Pereira et al . , 2019 ) .", "These results contrast with our null phenotype of ∆sll5052 ( xssK ) and ∆sll5049 ( xssH ) , probably because of the difference in the parent strains .", "In addition , we found that our ∆sll0923 did not show any defect in the bloom formation .", "On the other hand , disruption of sigF ( slr1564 ) for a sigma factor of global cell surface regulation increased three- to fourfold accumulation of sulfated EPS preparation ( Flores et al . , 2019a; Flores et al . , 2019b ) .", "The proteome analysis of ∆sigF revealed many ( more than 160 ) proteins except for any Xss proteins were up-regulated , leaving the sulfated EPS biosynthesis pathway elusive .", "The sugar composition of the EPS of their WT is somehow similar to our WT , likely reflecting varied mixture of synechan and unknown polysaccharides .", "Moreover , the sugar composition of the EPS fraction of ∆sigF was different for WT or synechan from our ∆xssS .", "The global cell surface regulator sigF may affect accumulation of various polysaccharides .", "To get insights into the difference in bloom formation between the motile and non-motile substrains , we compared transcriptome data ( Supplementary file 4 ) .", "It is evident that many xss genes on the plasmid are expressed several times higher in the motile substrain than the non-motile one except for xssT on the main chromosome , despite that the nucleotide sequence of the xss gene cluster was completely conserved between them .", "This fact suggests a possibility that another signaling via XssS/XssR/XssQ may contribute to the difference in bloom formation and synechan production between the substrains .", "Moreover , cell aggregation of another motile substrain of Synechocystis 6803 requires type IV pili apparatus , which drives cell motility ( Conradi et al . , 2019 ) .", "Cell aggregation of another non-motile substrain also requires the type IV pili but this aggregation was enhanced by disruption of the OPX gene ( xssT ) ( Allen et al . , 2019 ) .", "Thus , cell aggregation and bloom formation may be complex phenomena of extracellular polysaccharides and type IV pili in Synechocystis 6803 .", "The cyanobacterial bloom rapidly accumulates in populations of cyanobacterial cells floating on the water surface , which often produce potent cyanotoxins ( hepatotoxins , neurotoxins , etc . ) ( Merel et al . , 2013 ) .", "Blooms are thought to be supported mainly by cellular buoyancy due to intracellular proteinaceous gas vesicles constructed by gas vesicle proteins ( Beard et al . , 2002; Walsby , 1994 ) .", "Moreover , recent studies suggested that extracellular polysaccharides are also important for the bloom formation ( Chen et al . , 2019 ) .", "Some papers reported that the cells without gas vesicle can form blooms by EPS-dependent manner after artificial addition of divalent cations ( Ca2+ or Mg2+ ) ( Dervaux et al . , 2015; Wei et al . , 2019 ) .", "On the other hand , our study demonstrated that the gas-trapped EPS is sufficient for bloom formation of Synechocystis 6803 , which does not produce gas vesicles , without addition of any divalent cations ( Figure 4B ) .", "This result is consistent with reports that cyanobacteria without gas vesicles form booms in natural environments , including freshwater lakes ( Casero et al . , 2019; du Plooy et al . , 2015; Steffen et al . , 2012 ) .", "Finally , sulfated polysaccharides are expected to be healthy foods , industrial materials and medicines ( Jiao et al . , 2011; Wardrop and Keeling , 2008 ) .", "Some sulfated EPS from Synechocystis 6803 showed antitumor activity ( Flores et al . , 2019a ) , though this EPS may not be identical to synechan .", "The Xss-dependent biosynthesis of synechan in Synechocystis 6803 should be a good model for studies of other cyanobacterial sulfated polysaccharides .", "Combinatorial expression of sulfotransferases and glycosyltransferases from other cyanobacteria in Synechocystis cells will provide clues to their functions .", "Heterologous expression of Synechocystis xss genes in other organisms will also open a possibility of large-scale production of modified synechan species .", "Further molecular studies of xss genes and related genes from the database should accelerate screening and potential applications of cyanobacterial sulfated polysaccharides ." ], [ "The motile substrain PCC-P of the unicellular cyanobacterium Synechocystis sp .", "PCC 6803 , which exhibits phototaxis ( Yoshihara et al . , 2000 ) and forms bloom-like aggregates , was used as the WT in this work .", "A non-motile glucose-tolerant substrain , which has been widely used for studies of photosynthesis , was used for comparison ( Chin et al . , 2018 ) .", "Cells were maintained in 50 mL of BG11 liquid medium ( Stanier et al . , 1971 ) under continuous illumination of fluorescent lamps from outside ( 30 µmol photons/m2/s ) with bubbling of 1% CO2 in air at 31°C , or on 1 . 5% agar plates .", "Cell density was monitored at 730 nm .", "Primers used are listed in Supplementary file", "5 . Plasmids and mutants were constructed as described ( Chin et al . , 2018 ) .", "In brief , the DNA fragments , antibiotic-resistance cassettes , the trc promoter , and plasmid vectors were amplified by PCR using PrimeSTAR MAX DNA polymerase ( Takara , Shiga , Japan ) and combined using the In-Fusion System ( Takara ) .", "The resulting plasmid constructs were confirmed by DNA sequencing .", "Gene disruption was performed in two different ways .", "One method was replacement of a large portion of a targeted gene ( s ) with an antibiotic-resistance cassette .", "The other method was replacement of the translation initiation codon with a stop codon .", "In both cases , the screening cassette without the terminator was inserted in the direction of the targeted gene ( s ) to allow transcriptional read-through of the downstream gene ( s ) .", "For overexpression , gene expression was constitutively driven by the strong trc promoter in two ways: integration of a target gene with the strong trc promoter into a neutral site near slr0846 or IS203c , or replacement of the target-gene promoter with the trc promoter .", "Natural transformation and subsequent homologous recombination were performed as described ( Chin et al . , 2018 ) .", "The antibiotic concentration for the selection of transformants was 20 μg/mL chloramphenicol , 20 μg/mL kanamycin , and/or 20 μg/mL spectinomycin .", "Complete segregation of the transformed DNA in the multicopy genome was confirmed by PCR using primers listed in Supplementary file 5 , and the transformants are listed in Supplementary file", "6 . The bloom was reproducibly formed using the two-step culture regime we developed in this work .", "Before the bloom formation experiment , cells were precultured once in liquid after transfer from plates .", "In the first step , cells inoculated at OD730 = 0 . 2 were grown with vigorous aeration under continuous light at 31°C or 20°C for 48 hr .", "Typically the cell density reached OD730 ~ 2 .", "In the second step , the culture was shifted to the standing condition without bubbling under the same continuous light for another 48 hr ( or longer ) for cells to rise to the surface .", "Regarding the mutants of transmembrane glycosyltransferases , bloom formation was examined after 168 hr of the second-step culture .", "The final concentration of the photosynthesis inhibitor DCMU ( 3- ( 3 , 4-dichlorophenyl ) -1 , 1-dimethylurea ) was 100 μM .", "The fractionation method to isolate the crude EPS is shown in Figure 1—figure supplement 1A .", "The viscous materials including cells after the second step of culture were collected by filtration using a 1 . 0 μm pore PTFE membrane ( Millipore ) .", "The trapped materials were gently and carefully recovered from the membrane using MilliQ water with the aid of flat-tip tweezers .", "The collected sample was vortexed and then centrifuged at 20 , 000 × g for 10 min to remove cells .", "The supernatant constituted the crude EPS that contained viscous EPS and possibly CPS .", "The refined fractionation method to isolate EPS is shown in Figure 1F .", "The entire culture at the end of the first step , which did not contain gas bubbles , was first centrifuged at 10 , 000 × g for 10 min to remove cells and CPS and then filtered through a 1 . 0 μm pore PTFE membrane .", "The trapped EPS was carefully recovered as described above .", "The flowthrough of the filtration was regarded as free polysaccharides , which were recovered by ethanol precipitation .", "CPS was released from the cell pellet by vigorous vortexing with MilliQ water and recovered by centrifugation to remove cells ( 20 , 000 × g for 10 min ) .", "Total sugar was quantified using the phenol-sulfate method ( DuBois et al . , 1956 ) .", "A 100 μL aliquot of 5% ( w/w ) phenol was added to 100 μL of a sample in a glass tube and vortexed three times for 10 s .", "Then , 500 μL of concentrated sulfuric acid was added , and the tube was immediately vortexed three times for 10 s and then kept at 30°C for 30 min in a water bath .", "Sugar content was measured by absorption at 487 nm using a UV-2600PC spectrophotometer ( Shimadzu , Japan , Tokyo ) .", "Any contamination of the BG11 medium was evident by slight background coloration .", "This background was subtracted on the basis of the extrapolation of absorption at 430 nm , where the coloration due to sugars was minimal .", "Glucose was used as the standard .", "Some EPS samples were highly viscous , so we vortexed and sonicated them before measurement .", "Statistical significance was determined using Welch’s t test .", "The collected EPS samples were dialyzed with MilliQ water and then freeze-dried for 3 days .", "Sugar composition was analyzed by Toray Research Center , Inc ( Tokyo , Japan ) .", "A part of the fluffy sample ( WT , 0 . 298 mg; ΔxssS , 0 . 203 mg ) was dissolved in 200 μL of 2 M trifluoroacetic acid and hydrolyzed at 100°C for 6 hr .", "The treated sample was vacuum-dried with a centrifugal evaporator , redissolved in 400 μL MilliQ water , and filtered through a 0 . 22 μm pore filter .", "This sample was used for the analysis .", "Monosaccharide composition was determined by HPLC with the LC-20A system ( Shimadzu ) .", "For neutral sugars , the column was TSK-gel Sugar AXG ( TOSOH , Japan ) and the temperature was 70°C .", "The mobile phase was 0 . 5 M potassium borate ( pH 8 . 7 ) at 0 . 4 mL/min .", "Post-column labeling was performed using 1% ( w/v ) arginine and 3% ( w/v ) boric acid at 0 . 5 mL/min , 150°C .", "For uronic acids , the column was a Shimpack ISA-07 ( Shimadzu ) and the temperature was 70°C .", "The mobile phase was 1 . 0 M potassium borate ( pH 8 . 7 ) at 0 . 8 mL/min .", "Post-column labeling was performed using 1% ( w/v ) arginine and 3% ( w/v ) boric acid at 0 . 8 mL/min , 150°C .", "The detector was an RF-10AXL ( Shimadzu ) , with excitation at 320 nm and emission at 430 nm .", "The standard curves were prepared for each monosaccharide with standard samples .", "The SO42− content was determined by anion exchange column chromatography using the ISC-2100 system ( Thermo Fisher Scientific , Waltham , MA ) .", "The column was eluted via a gradient of 0–1 . 0 M KOH .", "The separation column was IonPac ASI l-HC-4 μm ( Thermo Fisher Scientific ) .", "Electric conductivity was used for detection .", "The polysaccharides were stained with 1% Alcian blue 8GX ( Merck ) for 10 min in 3% acetic acid ( pH 2 . 5 ) or 0 . 5 N HCl ( pH 0 . 5 ) as previously described ( Di Pippo et al . , 2013 ) .", "The qPCR was performed as described in our previous work ( Maeda et al . , 2018 ) .", "Cells were harvested by centrifugation at 5000 × g for 10 min at 4°C .", "Cell disruption and RNA extraction were done using an RNeasy Mini kit for bacteria ( Qiagen , Venlo , The Netherlands ) .", "In addition , cells were disrupted five times by mechanical homogenization with zirconia beads ( 0 . 1 mm diameter ) in a microhomogenizing system ( Micro Smash MS-100 , TOMY SEIKO , Tokyo , Japan ) at 5000 rpm for 40 s .", "For cDNA preparation , RNA was reverse-transcribed using random primers ( PrimeScript RT reagent kit with gDNA eraser , Takara ) .", "Real-time PCR was performed using the THUNDERBIRD SYBR qPCR Mix ( Toyobo ) and the Thermal Cycler Dice Real Time System II ( Takara ) .", "The transcript level in each strain was normalized to the internal control ( rnpB ) .", "The primers used are listed in Supplementary file", "5 . The expression and purification of recombinant His-tagged proteins and EMSA were performed as described in our previous works ( Hirose et al . , 2010; Maeda et al . , 2014 ) .", "In brief , His-tagged XssQ was expressed using pET28a vector system and Escherichia coli C41 ( DE3 ) strain .", "The protein was purified by Histrap HP column ( Cytiva , Tokyo , Japan ) and AKTA prime system ( Cytiva ) .", "For probe and native competitor , the upstream region of xssE was amplified with the primer set xssEup-1F/2R ( total 251 bp ) .", "As a mutant competitor , the same region of the chemically synthesized DNA fragment containing mutations in the two consensus sequences was used for amplification with the same primer set as mentioned above .", "Labeling of the DNA probe , electrophoresis , and autoradiography were performed as described ( Midorikawa et al . , 2009 ) .", "We incubated the aliquots of the XssQ protein ( 0 , 500 , 1000 , or 3000 ng/lane ) with the radiolabeled probe for 30 min at room temperature .", "For competition , 3000 ng of XssQ was incubated with the probe and 20 pmol of unlabeled competitors ( native or mutant ) .", "RNAs for RNA-seq analysis were extracted as described above .", "Library construction for RNA sequencing analysis was conducted as described previously ( Ohbayashi et al . , 2016 ) .", "The average number of raw read pairs per sample was 2 . 84 million .", "The reads were trimmed using CLC Genomics Workbench ver . 12 . 0 ( QIAGEN ) with the following parameters; Phred quality score >30; ambiguous nucleotides allowed: 1; automatic read-through adaptor trimming: yes; removing the terminal 15 nucleotides from the 5’ end; and removing truncated reads of less than 20 nucleotides in length .", "Trimmed reads were mapped to the all genes in Synechocystis sp .", "PCC 6803 using CLC Genomics Workbench ver . 12 . 0 with the following parameters; mismatch cost: 2; indel cost: 3; length fraction: 0 . 8; similarity fraction: 0 . 9; and maximum number of hits for a read: 10 .", "In the comparison between WT and mutants ΔxssS , and ΔxssQ ( Figure 3—figure supplement 1 ) , the genome information ( accession numbers , chromosome: CP003265 , pSYSM: CP003266 , pSYSX: CP003269 , pSYSA: CP003267 , pSYSG: CP003268 , pCA2 . 4: CP003270 , pCB2 . 4: CP003271 , and pCC5 . 2: CP003272 ) was used as a reference , and in the comparison between the motile WT ( PCC-P ) and non-motile ( NM ) substrains ( Supplementary file 4 ) , the genome information ( accession numbers , chromosome: AP012276 , pSYSM: AP004310 , pSYSX: AP006585 , pSYSA: AP004311 , pSYSG: AP004312 , pCA2 . 4: CP003270 , pCB2 . 4: CP003271 , and pCC5 . 2: CP003272 ) was used as a reference .", "Reads per kilobase per million mapped reads ( RPKM ) were calculated using CLC Genomics Workbench ver . 20 . 0 .", "Original sequence reads were deposited in the DRA/SRA database with the following accession numbers , DRA011755 .", "The accession number of BioProject was PRJDB11449 .", "The sequences of the proteins were obtained from NCBI ( http://www . ncbi . nlm . nih . gov/ ) and Pfam ( http://pfam . xfam . org/ ) ( Finn et al . , 2016 ) .", "The domain architecture was searched using the Simple Modular Architecture Research Tool , SMART ( Letunic et al . , 2015 ) .", "Glycosyltransferase classifications were based on the CAZy database ( http://www . cazy . org/ ) ( Henrissat , 1991; Lombard et al . , 2014 ) .", "Amino acid sequence similarity was evaluated by NCBI BLAST search ." ] ]
[ "Extracellularpolysaccharides of bacteria contribute to biofilm formation , stress tolerance , and infectivity .", "Cyanobacteria , the oxygenic photoautotrophic bacteria , uniquely produce sulfated extracellular polysaccharides among bacteria to support phototrophic biofilms .", "In addition , sulfated polysaccharides of cyanobacteria and other organisms have been focused as beneficial biomaterial .", "However , very little is known about their biosynthesis machinery and function in cyanobacteria .", "Here , we found that the model cyanobacterium , Synechocystis sp .", "strain PCC 6803 , formed bloom-like cell aggregates embedded in sulfated extracellular polysaccharides ( designated as synechan ) and identified whole set of genes responsible for synechan biosynthesis and its transcriptional regulation , thereby suggesting a model for the synechan biosynthesis apparatus .", "Because similar genes are found in many cyanobacterial genomes with wide variation , our findings may lead elucidation of various sulfated polysaccharides , their functions , and their potential application in biotechnology ." ]
[ "Bacteria are single-cell microorganisms that can form communities called biofilms , which stick to surfaces such as rocks , plants or animals .", "Biofilms confer protection to bacteria and allow them to colonize new environments .", "The physical scaffold of biofilms is a viscous matrix made of several molecules , the main one being polysaccharides , complex carbohydrates formed by many monosaccharides ( single sugar molecules ) joined together .", "Cyanobacteria , also known as blue-green algae , are a type of bacteria that produce oxygen and use sunlight as an energy source , just as plants and algae do .", "Cyanobacteria produce extracellular polysaccharides that contain sulfate groups .", "These sulfated polysaccharides are also produced by animals and algae but are not common in other bacteria or plants .", "One possible role of sulfated , extracellular polysaccharides in cyanobacteria is keeping cells together in the floating aggregates found in cyanobacterial blooms .", "These are visible discolorations of the water caused by an overgrowth of cyanobacteria that occur in lakes , estuaries and coastal waters .", "However , little is known about how these polysaccharides are synthesized in cyanobacteria and what their natural role is .", "Maeda et al . found a strain of cyanobacteria that formed bloom-like aggregates that were embedded in sulfated extracellular polysaccharides .", "Using genetic engineering techniques , the researchers identified a set of genes responsible for producing a sulfated extracellular polysaccharide and regulating its levels .", "They also found that cell aggregates of cyanobacteria can float without having intracellular gas vesicles , which was previously thought to enable blooms to float .", "The results of the present study could have applications for human health , since many sulfated polysaccharides have antiviral , antitumor or anti-inflammatory properties , and similar genes are found in many cyanobacteria .", "In addition , these findings could be useful for controlling toxic cyanobacterial blooms , which are becoming increasingly problematic for society ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "microbiology and infectious disease", "genetics and genomics" ]
Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes
elife-60083-v1
[ [ "The acquisition of genomic data continues to exceed significantly the pace at which corresponding functional information can be obtained ( Kemble et al . , 2019 ) .", "This aphorism applies to Mycobacterium tuberculosis , etiological agent of tuberculosis ( TB ) and the leading cause of death globally from an infectious disease ( WHO , 2019 ) .", "Despite considerable activity ( Satta et al . , 2018 ) in the two decades subsequent to the pioneering release of the first complete mycobacterial whole-genome sequence ( Cole et al . , 1998 ) , the M . tuberculosis genome still contains a large number of genes of unknown or hypothetical function ( Mazandu and Mulder , 2012; Satta et al . , 2018 ) .", "Moreover , functional validation is lacking even for many annotated genes , undermining fundamental understanding of mycobacterial metabolic and cellular functions which impact pathogenicity and drug susceptibility .", "There is consequently a pressing need for tractable , high-throughput approaches that can inform mycobacterial gene function rapidly and at scale .", "The most commonly applied functional genomics methods in bacteria – transposon-sequencing ( Tn-Seq ) ( van Opijnen and Camilli , 2013 ) and , increasingly , CRISPR-interference ( CRISPRi ) -Seq ( de Wet et al . , 2018; Wang et al . , 2018; Lee et al . , 2019 ) – combine pooled mutagenesis with next-generation sequencing , returning quantitative estimates of fitness ( via relative abundance ) of mutants in a particular growth condition .", "Common to these approaches is that they report on the capacity of the cells to produce biomass , thereby excluding other – potentially subtler – measures of the physiological consequences of target gene disruption .", "To overcome this limitation , some studies have utilized novel combinations of pooled approaches with cellular stains and sorting ( Rego et al . , 2017; Baranowski et al . , 2018 ) , or with imaging and in situ sequencing ( Camsund et al . , 2020 ) to provide alternative readouts of genetic function .", "However , these methods are restricted in their resolution and throughput .", "As for many bacterial pathogens , mycobacterial functional genomics has focused primarily on the identification of essential genes , employing Tn-based investigations under a variety of conditions ( Sassetti et al . , 2003; DeJesus et al . , 2017 ) , in different strain ( Carey et al . , 2018 ) and mutant ( Kieser et al . , 2015 ) backgrounds , and in models of infection ( Sassetti and Rubin , 2003; Rengarajan et al . , 2005 ) .", "The development of genetic tools for conditional knockdown mutagenesis via targeted gene silencing ( utilizing engineered hypomorphic strains carrying promoter replacement and/or targeted protein-degradation mutations ) has subsequently enabled gene-by-gene explorations of essential gene function ( Kim et al . , 2011; Kim et al . , 2013; Schnappinger and Ehrt , 2014 ) .", "However , while these approaches have ensured increasingly refined ( conditional ) essentiality predictions ( DeJesus et al . , 2017 ) , there is still an absence of whole-cell functional data for the majority of essential genes .", "Here , we couple key recent advances in mycobacterial CRISPRi ( Rock et al . , 2017 ) with quantitative microscopic imaging ( Ducret et al . , 2016 ) in developing a platform enabling whole-cell characterization of essential gene knockdown in the non-pathogenic model mycobacterium , M . smegmatis .", "Leveraging available gene essentiality and CRISPRi guide efficacy data ( de Wet et al . , 2018 ) , we describe the construction of an arrayed collection of 276 validated CRISPRi mutants targeting essential M . smegmatis homologs of M . tuberculosis genes .", "Applying high-throughput quantitative imaging to the arrayed library , we implement a bespoke analytic pipeline to probe essential gene function at scale .", "From the ‘phenoprints’ generated via this approach , we demonstrate the potential for preliminary assignment of gene function and illustrate the capacity to analyze metabolic and macromolecular synthesis pathways using clustering analyses .", "Notably , these observations provide evidence supporting a mycobacterial restriction-modification system , and identify filamentation as a defining mycobacterial response to histidine starvation but not to other amino acid auxotrophies .", "Finally , we demonstrate the potential utility of this approach in elucidating antimicrobial mechanism-of-action ( MOA ) , supporting its potential incorporation as a complementary tool in current TB drug discovery pipelines .", "Consistent with our intention to generate a community-accessible resource , all data from the screen are made available via an interactive database ( https://timdewet . shinyapps . io/MorphotypicLanscape/ ) ." ], [ "We aimed to construct an arrayed library of inducible CRISPRi mutants ( Figure 1A ) targeting essential M . smegmatis genes ( de Wet et al . , 2018 ) .", "To this end , we leveraged an optimized mycobacterial CRISPRi system ( Rock et al . , 2017 ) and previously generated genome-wide CRISPRi-Seq data ( de Wet et al . , 2018 ) from which we were able to identify the set of highest efficiency single-guide ( sg ) RNAs targeting 294 essential M . smegmatis genes with direct M . tuberculosis homologs ( Supplementary file 1 ) .", "CRISPRi produces polar effects on downstream genes in polycistronic operons ( Rock et al . , 2017 ) .", "To ascertain operonic structure directly , we utilized published data on the transcriptional landscape of wild-type M . smegmatis mc2155 during exponential growth in standard laboratory media ( Martini et al . , 2019 ) .", "Of our targeted set of 294 genes , 229 genes ( ~78% ) were not located in identified operons ( Supplementary file 2 ) .", "For these genes , it was expected that any knockdown phenotype should solely reflect the impact of silencing the targeted essential gene .", "Of the remaining 65 genes within operons , 20 possessed downstream transcriptional start sites which would be expected to abrogate polar effects .", "This left 34 genes whose genomic context was likely to complicate the interpretation of any knockdown phenotype .", "We nevertheless considered this group of 34 genes worth pursuing given our ability to evaluate the results in the context of the corresponding essentiality calls from Tn-Seq ( Dragset et al . , 2019 ) and in comparison to phenotypes observed for related ( or ‘shared pathway’ ) genes .", "Following large-scale cloning , all 294 CRISPRi constructs were electroporated into an M . smegmatis ParB-mCherry reporter mutant ( Santi and McKinney , 2015 ) .", "This strain was chosen as background to enable visualization of the oriC proximate region of the chromosome during imaging assays , thereby providing information about chromosome location dynamics and copy number as a function of essential gene knockdown .", "To validate the identity and essentiality of each M . smegmatis mutant strain , we performed Sanger sequencing of the inserted sgRNA and whole-cell spotting assays of ATc sensitivity .", "Cloning was repeated for mutants which failed the validation screen .", "This process yielded 276 validated CRISPRi mutants ( Figure 1B , Figure 1—figure supplement 1 ) , 93% of the initial target set .", "According to a review of the literature ( Supplementary file 3 ) , approximately 90% of the strains in our library lacked whole-cell morphological characterization in either M . tuberculosis or M . smegmatis .", "Furthermore , almost 40% of the targeted genes and their protein products had no biochemical or structural information ( Supplementary file 3 ) .", "High-throughput , quantitative imaging has been productively utilized in a number of bacterial systems to rapidly characterize the impact of genetic alterations on cellular function ( Peters et al . , 2016; Liu et al . , 2017; Campos et al . , 2018 ) or to determine antimicrobial MOA ( Nonejuie et al . , 2013 ) .", "Until very recently ( Smith et al . , 2020 ) , an equivalent approach had not been described for mycobacteria .", "Therefore , on initiating this study , we chose to investigate the use of imaging to phenotype the CRISPRi mutant library following ATc-dependent transcriptional silencing .", "This required the development of tools for the extraction of large-scale quantitative data describing mycobacterial cell morphology .", "To determine the optimal duration of ATc exposure , we performed time-lapse microscopy of knockdown mutants of three well-characterized essential genes ( Videos 1 and 2 ) – the cell-division mediator , ftsZ ( Dziadek et al . , 2003 ) , the elongasome anchor , wag31 ( Kang et al . , 2008 ) , and the inosine monophosphate dehydrogenase , guaB2 ( Singh et al . , 2017; Park et al . , 2017 ) .", "Time-lapse microscopy established that fully penetrant phenotypes were manifest at 18 hr post ATc exposure for all three genes .", "This represents approximately 6–7 doubling times of the wild-type M . smegmatis mc2155 parental strain ( Logsdon et al . , 2017 ) , or a dilution factor of pre-existing protein of approximately 64 to 128 ( 26 - 27 ) -fold .", "For both wag31 and ftsZ mutants , lytic cell death was observable after the 18 hr time-point .", "While guaB2 knockdown did not produce as distinctive a visual phenotype as the other two genes , the formation of minicells and a decline in growth rate was evident from 18 hr .", "Although this test set comprised only three mutants , we were encouraged that , for these essential genes with distinct cellular functions , the 18 hr timepoint appeared suitable to ensure sufficient knockdown and dilution of pre-existing protein to yield a detectable phenotype while maintaining suitable cell numbers for imaging .", "We therefore proceeded to apply the same experimental conditions in phenotyping the full library , recognizing that the pragmatic utilization of a single , ‘terminal’ endpoint might not be optimal for every mutant in the collection .", "Adopting the 18 hr CRISPRi duration , we derived a protocol for medium-throughput imaging of batches of 24 strains ( Figure 2A ) .", "Exponential-phase cultures were treated with ATc overnight for 18 hr prior to spotting onto 2% agarose pads for semi-automated imaging .", "Phase contrast and fluorescence images of ParB-mCherry localizations were captured for 263 of the 276 mutant strains .", "Additionally , an empty vector control strain was treated and repeatedly imaged in the same manner .", "To confirm the reproducibility of data obtained from the single-timepoint , single-replicate imaging workflow , we selected 137 strains for re-imaging on separate days .", "Image analysis tools such as MicrobeJ ( Ducret et al . , 2016 ) or Oufti ( Paintdakhi et al . , 2016 ) extract detailed quantitative descriptions of bacterial morphology and protein localization ( Figure 2—figure supplement 1 ) .", "However , automated image analysis is complicated by out-of-focus objects , or cells that are too close together to separate adequately – a particular concern for mycobacteria with their proclivity to clump ( Cheng et al . , 2014 ) .", "To overcome these complications , machine learning-based classifiers have been utilized in E . coli for post-processing clean-up ( Campos et al . , 2018 ) .", "To extract quantitative descriptions of mutant morphologies and ParB-mCherry localizations across our imaging dataset , we utilized the ImageJ package , MicrobeJ ( Ducret et al . , 2016 ) , with a machine learning-based post-processing clean-up ( Figure 2B ) .", "For this purpose , we trained an Averaged Neural Network with 22 , 936 manually classified objects , sampled from across our imaging dataset .", "Receiver Operator Characteristic ( ROC ) analysis of our classifier produced an Area Under the Curve ( AUC ) of 0 . 952 when applied to a reserved test set ( Figure 2C ) .", "Our curated dataset contains morphological descriptions for 163559 cells across all 263 imaged mutant and empty vector control strains , with a mean of 568 cells per mutant ( ranging from a minimum of 24 to a maximum of 3861 cells ) .", "Reassuringly , when comparing the mean cell lengths of the 137 replica-imaged knockdown mutants following ATc induction , the imaging and analytic pipeline showed high reproducibility ( Figure 2D , r = 0 . 88 , Pearson’s ) .", "There are relatively few published descriptions of the morphological impacts of essential gene silencing ( Supplementary file 3 ) .", "We aimed to use our extracted imaging data to generate a comprehensive repository of morphological changes following CRISPRi-mediated essential gene knockdown in M . smegmatis .", "For each gene , a nearest-neighbor approach was applied to identify the representative morphotype which was in turn incorporated into an atlas of morphological changes consequent on essential gene silencing ( Figure 3 ) .", "Visual inspection of the atlas revealed distinctive alterations in morphology in a large number of knockdown mutants .", "Moreover , it appeared that silencing certain cellular functions or pathways produced consistent morphological changes .", "In general , the responses could be broadly categorized into either elongation – occasionally with bulging and/or branching – or shortening , with a variety of alterations in cell width and roundness .", "For example , components of DNA metabolism ( dna- prefix genes ) , the divisome ( fts- genes ) , and protein translation ( rpl- and rps- genes ) produced cellular elongation .", "To our surprise , a filamentation response was also observed for components of the histidine biosynthesis pathway ( his- genes ) , but not for other amino acid biosynthesis pathways .", "In contrast , components of cell-wall homeostasis – including peptidoglycan ( ponA1 ) , arabinogalactan ( aftA ) and mycolic acid synthesis ( inhA and mmpL3 ) – were associated with distinctive shortening of cells , often in combination with bulging .", "To quantitate these observations , we adapted an approach developed for E . coli ( Campos et al . , 2018 ) .", "Single-cell data were reduced to normalized , gene-level descriptors of morphology .", "For each mutant , the mean and coefficient of variation ( CV ) of each morphological feature were extracted and normalized to the distribution of empty vector control samples with a Z-score transformation ( Figure 4—figure supplement 1A ) .", "For a particular mutant and feature , the Z-score reflects how many standard deviations the mutant feature is away from the mean of the control strains .", "That is , it provides a measure of the extent to which transcriptional inhibition of the targeted essential gene impacts a specific morphological characteristic .", "In total , 206 ( 78% ) strains returned at least one Z-score >3 , or <-3 .", "Certain gross changes in morphology were more frequently observed than others ( Figure 4—figure supplement 1B ) .", "For example , many mutants exhibited marked increases in mean curvature , roundness and feret ( the calliper diameter ) .", "Conversely , few mutants exhibited major alterations in the number of ParB foci ( maxima ) per cell , suggesting that the number of replication origins ( oriCs ) per cell – and , by implication , ploidy – was maintained even under essential gene knockdown .", "To test for associations between morphological changes and functional classes of genes , we extracted clusters of orthologous groups ( COG ) annotations for all 276 essential genes before testing for statistical enrichment with changes in morphology .", "For each morphological feature , we tested for COG enrichment of the mutants with Z-scores > 3 or<-3 .", "This analysis revealed that statistical enrichments were largely consistent with visual inspection of the cell atlas ( Figure 4—figure supplement 2 ) .", "For example , the filamentation phenotype of fts- , rpl- and dna- genes was reflected in the enrichment of cell cycle control ( D ) , Translation ( J ) and DNA replication and repair ( L ) COGs with increases in length ( means and CVs ) .", "In a complementary approach , we visualized the mean population Z-scores for key morphological features by COG ( Figure 4 ) .", "Again , consistent with the cell atlas and enrichment results , specific COGs ( Supplementary file 3 ) produced distinct morphological signatures ( or ‘phenoprints’ ) .", "Together , these results highlighted the utility of multi-parameter morphological descriptors for characterizing mutants and , moreover , signaled the potential to link genes according to whole-cell phenotype independent of known or predicted ( annotated ) function .", "Based on prior cytological profiling studies in eukaryotic ( Caicedo et al . , 2017 ) and prokaryotic ( Nonejuie et al . , 2013; Huang , 2015; Campos et al . , 2018 ) systems , we applied an unsupervised learning approach that combined dimensionality reduction with clustering of our multidimensional dataset .", "For this , we utilized uniform manifold approximation and projection ( UMAP ) ( Lel et al . , 2018 ) and optimized hyper-parameters to produce consistent visual clusters of our control strains in combination with density-based clustering using hdbscan ( Lel et al . , 2017; Figure 5 ) .", "This process produced a two-dimensional UMAP space consisting of three reproducible clusters ( Figure 5A ) .", "The scale of different morphological features varied across the 2-dimensional space with visible patterns , such as regions of decreased length , or increased width ( Figure 5D ) .", "To explore the three clusters , we tested for COG enrichment and visualized mean feature Z-scores for each cluster ( Figure 5B and C ) .", "Cluster 1 , which was enriched for lipid metabolism ( I ) and cell-wall metabolism ( M ) , was associated with increases in width and curvature and decreases in area and aspect ratio .", "Conversely , Cluster 3 , which was associated with translation ( J ) and DNA replication and repair ( L ) , was characterized by increases in area and length .", "Cluster 2 , which contained the control , non-targeting strains , was enriched for energy production and conversion ( C ) and nucleotide metabolism ( F ) , highlighting the comparatively small impact that silencing of these genes has on cellular morphology .", "To analyze further the spatial arrangements within the three large clusters , and across UMAP space , we adapted a spatial analysis of functional enrichment ( SAFE ) approach ( Baryshnikova , 2016; Campos et al . , 2018 ) to identify sub-clusters of either COG or KEGG enrichment .", "This yielded distinct sub-clusters that were not originally apparent ( Figure 5E and F ) .", "For example , Cluster 1 contained regions enriched for either lipid metabolism ( I ) or the cell wall ( M ) , while cluster three could be subdivided into a region enriched for DNA metabolism ( L ) , ribosomal genes ( K ) and genes involved in aminoacyl-tRNA biosynthesis ( by KEGG enrichment ) .", "Using this approach , even cluster two could be subdivided into functionally enriched regions despite the apparent similarity of these strains ( on visual inspection ) to wild-type morphologies .", "As UMAP maintains the essential topological structure of data , we wondered if biological connections would be reflected in the distances between genes in UMAP space .", "Given the acknowledged propensity of CRISPRi to produce polar effects ( Rock et al . , 2017 ) , we predicted that these would be reflected in the Euclidean distance between operonic genes .", "Consistent with our hypothesis , genes in verified operons ( Martini et al . , 2019 ) were located more proximally than random pairs of genes ( Figure 5—figure supplement 1 ) .", "Similarly , predicted protein–protein interaction pairs ( Cong et al . , 2019 ) were found to be non-randomly proximate ( Figure 5—figure supplement 1 ) .", "To demonstrate further the utility of UMAP space for understanding the link between gene function and morphology , we performed more granular analysis of manually annotated functional groupings .", "Again , consistent clustering of many genes with similar biological functions was observed ( Figure 6 ) , a striking finding given that the close associations in UMAP space were driven by similarities in cytological characteristics ( phenoprints ) independent of annotated gene function .", "The observation that subtle morphological changes appeared to associate with particular genetic functions implied the potential to utilize the pipeline and mutant library to elucidate biological relationships and as an additional tool in MOA studies .", "Four examples are presented below which illustrate the application of this approach to explore", "( i ) putative genetic function ,", "( ii ) pathway-specific phenotypes ,", "( iii ) macromolecular biosynthetic phenotypes , and", "( iv ) chemical-genetic approaches to antimycobacterial MOA identification .", "Morphological profiling has the capacity to identify mutants with unexpected phenotypes , providing a preliminary phenotypic characterization which can guide focused downstream investigations toward assigning gene function .", "An example illustrating this possibility is MSMEG_3213 , which is annotated as a putative DNA methylase but lacks biological validation .", "The M . tuberculosis homolog , Rv3263 , has been implicated in bacillary fitness during hypoxia ( Shell et al . , 2013 ) but , unlike its M . smegmatis homolog , is not included in the essential gene set in the pathogen ( DeJesus et al . , 2017 ) .", "In our imaging pipeline , MSMEG_3213 produced a marked filamentation phenotype , clustering with components of DNA replication and repair in UMAP space ( Figure 7A and B ) .", "Utilizing the ParB-mCherry marker , we noted a significant disturbance in normal cell-cycle progression , consistent with the interpretation that knockdown of MSMEG_3213 disrupted DNA replication ( Figure 7C ) .", "Moreover , Nanopore-based RNA-Seq confirmed the specificity of MSMEG_3213 knockdown , eliminating potential off-target effects on downstream or upstream genes ( Figure 7D ) .", "Notably , the whole-genome transcriptional profile on MSMEG_3213 knockdown showed marked overlap with the well-characterized DNA damage ( SOS ) regulon ( Davis et al . , 2002; Boshoff et al . , 2003 ) induced by treatment with genotoxins such as the DNA crosslinking agent , mitomycin C ( MMC , Müller et al . , 2018; Figure 7E ) .", "Based on this intriguing observation , we hypothesized that , since depletion of the methylase appeared to trigger a DNA damage response , MSMEG_3213 might be part of a restriction-modification ( R-M ) system ( Loenen et al . , 2014 ) .", "Following an extensive bioinformatic search , this conclusion gained further credence by the observation that REBASE ( Roberts et al . , 2015 ) lists MSMEG_3213 as putative DNA methylase of a type II R-M system , with MSMEG_3214 the associated restriction endonuclease ( Figure 7F ) .", "We reasoned that , if MSMEG_3213-MSMEG_3214 does constitute a cryptic R-M system , knockdown of MSMEG_3214 should render MSMEG_3213 non-essential .", "To test this , a dual-targeting CRISPRi construct was generated to enable simultaneous silencing of both genes .", "Knockdown of MSMEG_3214 alone produced no growth phenotype whereas dual silencing of MSMEG_3213 and MSMEG_3214 alleviated the lethal impact of MSMEG_3213 depletion ( Figure 7G ) .", "Further biochemical and/or functional characterization is required before MSMEG_3213 can be definitively assigned as methylase; however , the evidence derived here from morphological profiling , transcriptomic profiling and combinatorial CRISPRi strongly support the identification of a predicted Type II R-M system in M . smegmatis .", "Other examples supporting the utility of morphological profiling to inform single-gene functional analyses arose during the course of this work ( Figure 7—figure supplement 1 ) .", "For example , the C . glutamicum homolog of MSMEG_0317 is required for lipomannan maturation and lipoarabinomannan ( LM/LAM ) synthesis ( Cashmore et al . , 2017 ) .", "We observed that MSMEG_0317 clustered closely with genes involved in arabinogalactan synthesis and localized to the cell wall ( Figure 7—figure supplement 1B ) .", "It was pleasing , therefore , when a separate study emerged suggesting that MSMEG_0317 was involved in the transport of LM/LAM ( Gupta et al . , 2019 ) .", "In another example , the transcriptional regulator , whiA , which is involved in sporulation in Streptomyces ( Bush et al . , 2013 ) , appears to play a key role in the mycobacterial cell cycle , clustering with other components of cell division ( Figure 7—figure supplement 1C ) .", "Furthermore , MSMEG_6276 – a putative mur ligase – clusters closely with the peptidoglycan synthesis protein , mviN ( Gee et al . , 2012 ) , and exhibits strong homology to murT/gatD from S . pneumoniae ( Morlot et al . , 2018; Figure 7—figure supplement 1D ) .", "In combination , these additional examples support the utility of image-based profiling for informing or validating hypothetical or predicted gene function – particularly those involved in cell-wall metabolism or DNA metabolism and cell-cycle regulation .", "A feature of the library is that many metabolic pathways are represented by multiple mutants , allowing for pathway-level analyses of metabolic function .", "M . smegmatis possesses a full complement of genes for the biosynthesis of the essential amino acid , L-histidine ( Figure 8A and B ) .", "In the absence of histidine supplementation , the majority of the his- prefix genes are predicted to be essential , but most have not been validated individually ( Lunardi et al . , 2013 ) .", "Surprisingly , CRISPRi-mediated knockdown of histidine biosynthesis genes produced a filamentation phenotype that was tightly clustered in UMAP space ( Figure 8C ) and , among all the amino acid knockdowns , appeared unique to histidine .", "The possibility existed that the operonic arrangement of some genes in the pathway ( hisB , hisC1 , hisH , hisA , hisF , hisI; hisE , hisG ) had confounded interpretation of the CRISPRi phenoprints as a result of polar effects .", "However , our transcriptional data and those of others ( Martini et al . , 2019 ) appeared to eliminate this possibility: hisD , hisC , hisB , hisH and hisF possess nested promoters and are therefore likely to be impervious to CRISPRi-mediated silencing of upstream genes .", "Moreover , CRISPRi knockdown of hisG and hisS , which are distally located in the M . smegmatis genome , produced the same phenotype , suggesting that filamentation might be a consistent , and previously unreported , consequence of disruptions to histidine biosynthesis .", "To test the specificity of this phenotype , we supplemented ATc-containing media with L-histidine .", "The reversal of the growth inhibitory phenotype verified all his knockdowns as histidine auxotrophs , with the notable exceptions of hisA and hisS .", "Previous reports indicate that hisA likely encodes a bifunctional HisA/TrpF enzyme ( Due et al . , 2011 ) .", "Consistent with this annotation , growth of the hisA CRISPRi mutant was rescued in medium supplemented with tryptophan ( Figure 8D ) .", "Intriguingly , for all his knockdowns other than hisS ( encoding the histidine tRNA synthetase ) , histidine supplementation reversed the filamentation phenotype ( Figure 8E ) .", "This was also the case for hisA , even when the lack of tryptophan impaired growth .", "Morphologically , the filaments observed under histidine starvation differ from those which result from depletion of components of the divisome: that is , the his mutants display minimal branching .", "Moreover , staining of peptidoglycan with the D-alanine analogue , NADA ( Botella et al . , 2017 ) , failed to resolve any clear septa ( Figure 8—figure supplement 1 ) .", "The mechanism underlying the filamentation response to histidine deficiency remains unclear , but likely reflects translation defects resulting from uncharged tRNAs .", "Further work is required to establish this definitively , but the observations reported here nevertheless support the potential utility of the CRISPRi-imaging pipeline to discover , and then validate , pathway-specific phenotypes .", "Mycobacteria possess a multilayered cell envelope , the outer mycolic acid layer of which is synthesized via a well-characterized pathway containing a number of established and experimental drug targets ( Jankute et al . , 2015; Figure 9A ) .", "CRISPRi-mediated inhibition of the components of this pathway produced consistent changes in cellular morphology ( Figure 9B and C ) , with the majority of genes implicated in mycolic acid synthesis clustering tightly together in 2-dimensional UMAP space ( Figure 9B ) .", "Notably , the fact that clustering was observed despite the location of these genes throughout the chromosome appeared to eliminate polar effects as a potential confounder .", "By superimposing our imaging results and quantitative analyses of morphology on the mycolic acid biosynthetic pathway , we noted highly analogous morphotypes irrespective of the gene targeted for silencing ( Figure 9C and D ) .", "It was also evident that knockdown of components involved in early steps produced similar results to inhibition of the final step , involving the flippase , MmpL3 ( Xu et al . , 2017; Su et al . , 2019 ) .", "Given the consistency observed following genetic disruption of mycolic acid synthesis , we wondered whether chemical inhibition would produce a comparable morphological change .", "To test this possibility , we treated cells with isoniazid ( INH ) , a frontline anti-TB drug which inhibits the essential enoyl-ACP reductase , InhA ( Timmins and Deretic , 2006; Vilchèze and Jacobs , 2007 ) .", "Exposure to 1X minimum inhibitory concentration ( MIC90 ) INH triggered increases in cell width and morphological alterations which were closely similar to those produced by genetic silencing of inhA ( Figure 9C and D ) .", "This was an important observation since it suggested the possibility that , by analogy with bacterial cytological profiling ( Nonejuie et al . , 2013; Nonejuie et al . , 2016 ) , drug-induced morphological changes could be utilized to inform antimycobacterial MOA more broadly; that is , by exploiting the CRISPRi-imaging database of correlated genotype-phenotype connections with chemical ( drug-induced ) phenotype .", "To ascertain if the CRISPRi dataset could be used to determine antimycobacterial MOA , we investigated a panel of compounds from different classes that inhibit various essential mycobacterial processes ( Figure 10A ) .", "For proof-of-concept assays , the initial selection was limited to five clinically used anti-TB drugs: the first-line agents , INH and ethambutol ( EMB ) , which disrupt cell-wall biosynthesis ( Abrahams and Besra , 2018 ) ; RIF , which binds the DNA-dependent RNA polymerase subunit , RpoB ( Koch et al . , 2014 ) ; the second-line fluoroquinolone , moxifloxacin ( MOXI ) , which inhibits DNA gyrase ( Kumar et al . , 2014 ) and bedaquiline ( BDQ ) , an inhibitor of mycobacterial ATP biosynthesis recently approved for the treatment of MDR-TB ( Sarathy et al . , 2019 ) .", "Thereafter , the panel was expanded to include an additional 12 known and experimental antimycobacterial compounds ( Figure 10—figure supplements 1–3 ) .", "Morphotypes observed following drug treatment were often positioned in regions of UMAP space consistent with their known MOAs .", "For example , cells exposed to BDQ at 2X and 4X MIC clustered tightly with components of the mycobacterial ATP synthase and mapped to a region enriched for genes involved in energy metabolism ( Figure 10A ) ; the nearest neighbor for both 2X and 4X BDQ-treated cells was atpA ( Figure 10B ) .", "The cell-wall targeting compounds similarly produced closely correlating profiles: EMB-treated cells clustered tightly together , irrespective of applied concentration , and were positioned in lipid metabolism-enriched UMAP space .", "The nearest neighbor at 2X and 4X MIC was pks16 , a gene involved in mycolic acid synthesis; moreover , consistent with inhibition of arabinogalactan synthesis , aftA , ubiA , dprE1 , glfT2 were proximal to the EMB-exposed cells at all three applied drug concentrations .", "INH-treated cells were situated in lipid metabolism-enriched space at 1X MIC but , at higher concentrations , fell in a region associated with energy production and conversion .", "Notably , however , the nearest neighbor at 4X MIC was fabD , a component of mycolic acid synthesis .", "In contrast , compounds targeting peptidoglycan , including vancomycin and D-cycloserine , did not yield definitive profiles ( Figure 10—figure supplements 1–3 ) .", "Manual inspection of the data indicated that this was probably due to the induction of lysis , highlighting a limitation inherent in applying a single , 18 hr endpoint for analysis of all drug treatments .", "Compounds targeting DNA replication generally produced filamentation , and clustered accordingly .", "Notably , MOXI-treated cells did not co-localize with gyrA and gyrB knockdowns ( the DNA-gyrase subunits , and targets of MOXI ) in UMAP space and were instead associated with DNA metabolism , an observation common to all fluoroquinolones .", "In contrast , novobiocin ( NVB ) , a gyrase B inhibitor ( Chatterji et al . , 2001 ) , did not trigger filamentation , instead positioning closely with gyrA and gyrB genetic knockdowns .", "Like the fluoroquinolones , the experimental agents nargenicin ( putative DnaE1 inhibitor; Painter et al . , 2015 ) and griselimycin ( which binds to the β-clamp , DnaN; Kling et al . , 2015 ) were associated with DNA metabolism pathways , in this case mapping much closer to their known or predicted targets .", "While compounds disrupting DNA replication and cell envelope biogenesis were readily associated with a target class , this was not universally true for drugs with other MOAs .", "At 4X MIC , RIF was associated with rpoB .", "However , at the two lower concentrations , RIF-treated cells were positioned at dramatically different locations in UMAP space .", "It was noticeable , too , that compounds targeting mycobacterial protein synthesis – for example , linezolid ( Leach et al . , 2011 ) , streptomycin or kanamycin ( Riska et al . , 2000 ) – similarly failed to produce responses analogous to their cognate gene knockdowns .", "Like RNA polymerase , the protein translation machinery operates as a large , multi-protein complex , perhaps exposing a deficiency in this approach when applied to the macromolecular machines responsible for transcription and translation .", "Overall , however , the results supported the utility of CRISPRi-enabled cytological profiling to inform compound MOA , especially for agents targeting DNA replication , cell-wall biosynthesis , and energy metabolism ." ], [ "We have described the construction and validation of an arrayed library of CRISPRi mutants targeting essential M . smegmatis homologs of M . tuberculosis genes .", "By coupling the library with a high-throughput imaging pipeline , we confirmed the utility of quantitative methodologies to describe morphological changes and , moreover , established the capacity to harness morphotypic information to construct a database of cytological phenotypes , or mycobacterial phenoprints .", "Then , in demonstrating the potential utility of the resulting phenoprint atlas , we presented four examples illustrating its use in enabling preliminary characterization of single-gene function ( DNA methylase ) , the identification of distinct morphotypes associated with biosynthetic ( histidine ) and macromolecular ( mycolic acid ) pathway disruptions , and the potential to exploit CRISPRi morphotypes as a chemical-genetic tool to illuminate antimycobacterial MOA determination .", "Advances in imaging technologies ( Camsund et al . , 2020 ) and sample preparation ( Shi et al . , 2017 ) , together with the availability of software tools such as MicrobeJ ( Ducret et al . , 2016 ) , have enabled the application of imaging in bacterial systems biology ( Huang , 2015 ) .", "Our pipeline was optimized for medium-throughput imaging of mycobacteria; however , increasing the throughput of imaging , for example with the Strain Library Imaging Protocol ( Shi et al . , 2017 ) , would be advantageous for larger collections of mutants .", "To ensure completeness in our online ‘Morphotypic Landscape’ database , we plan in future to expand the library to include all 423 predicted essential M . smegmatis genes from our pooled screening data ( de Wet et al . , 2018 ) .", "There appears to be significant value , too , in establishing an equivalent library in M . tuberculosis , a challenging undertaking but one which would benefit from the lessons learnt from this study .", "In this context , we note with interest key recent advances in mycobacterial cytological profiling ( Smith et al . , 2020 ) which suggest the potential to combine these complementary approaches in applying CRISPRi-imaging to M . tuberculosis .", "In developing our analytical pipeline , we relied heavily on the pioneering work of Jacobs-Wagner and colleagues ( Campos et al . , 2018 ) .", "In particular , we adapted a machine learning-based cleanup that leverages the power of MicrobeJ but adds a Mycobacterium-specific post-processing step .", "This classifier should have downstream utility for future imaging studies in M . smegmatis but will require retraining if utilized for M . tuberculosis .", "For analysis , we applied an unsupervised learning approach that combined UMAP dimensionality reduction with density-based clustering of our multidimensional dataset .", "UMAP is a generally applicable algorithm , but has enjoyed especially rapid uptake for the visualization of single-cell sequencing results ( Becht et al . , 2018 ) .", "In our pipeline , we also used UMAP for data visualization and as a preprocessing step prior to the application of density-based clustering approaches , analogous to the use of t-SNE by Jacobs-Wagner and colleagues ( Campos et al . , 2018 ) .", "While non-linear dimensionality reduction techniques must be used with caution , our extensive validation supports the utility of the approach .", "Additionally , our dataset should be amenable to further analysis using alternative analytic approaches , including deep-learning ( von Chamier et al . , 2019 ) .", "Using data obtained from our CRISPRi screen , we generated a quantitative atlas of morphological changes associated with essential gene silencing in M . smegmatis .", "For ~90% of the strains in our library , no prior morphological data were available at the outset of the study .", "Moreover , approximately 40% of the genes and their protein products had not been characterized at all , either functionally ( biochemically or microbiologically ) or structurally .", "It was notable , therefore , that almost 78% of the essential genes assayed here resulted in at least one dramatically aberrant morphological feature on CRISPRi knockdown .", "This number exceeds the ~60% of B . subtilis mutants that produce clear terminal phenotypes ( Peters et al . , 2016 ) and may reflect increased sensitivity of our analytic approach and/or the differential compositions of the respective libraries .", "However , given that only 20% of the non-essential E . coli Keio Collection displays significant morphological changes ( Campos et al . , 2018 ) , it is evident that silencing of essential genes is more likely to disrupt core processes affecting maintenance of general bacillary morphology .", "A number of CRISPRi mutants produced morphological alterations that were unexpected .", "For example , while filamentation is a well-described phenomenon consequent on interference in cell-division ( Dziadek et al . , 2003; Campos et al . , 2018; Wu et al . , 2018 ) , DNA replication ( Greendyke et al . , 2002; Justice et al . , 2008 ) and depletion of ClpP protease components ( Li et al . , 2010 ) , it is not typically associated with genes involved in protein synthesis or export .", "Nevertheless , we recorded filamentation responses following silencing of ribosomal subunits , the Sec translocase , and histidine biosynthesis .", "These phenotypes contrast with B . subtilis where filamentation is not observed on silencing of the corresponding genes ( Peters et al . , 2016 ) .", "In Salmonella typhimurium ( Murray and Hartman , 1972 ) and E . coli ( Frandsen and D'Ari , 1993 ) , mutants overexpressing hisH and hisF are known to produce filaments , possibly through a scarcity of PBP3 substrates required for cell division ( Cano et al . , 1998 ) .", "To our knowledge , though , filamentation of histidine auxotrophs has not been reported in any bacterial species , including mycobacteria .", "The mechanism underlying the filamentation phenotype is unclear , though potentially attributable to interference in cell division through an unknown mechanism .", "Moreover , the fact that this phenotype manifests on knockdown of the L-histidine-tRNA ligase , hisS , suggests the involvement of a regulatory mechanism dependent on the presence of his-tRNAs ( Raina and Ibba , 2014 ) .", "However , further work is required to explain this observation .", "While interfering in cell envelope biogenesis might be expected to cause aberrations in morphology , the increases in mycobacterial cell width observed on silencing essential steps in mycolic acid synthesis have limited precedent in the literature .", "One example is the association of temperature-sensitive mutants of inhA with analogous phenotypes ( Vilchèze et al . , 2000 ) .", "The phenotypic consistency across the pathway was nevertheless surprising; again , however , the precise mechanism remains elusive .", "It is possible that the cell-wall synthetic machinery acts together in a spatiotemporally related complex that requires the presence of all proteins for function .", "It might be instructive , for example , that fluorescently-tagged versions of InhA and MabA co-localize ( Vilchèze et al . , 2000 ) and may be present in the same specialized membrane domain ( Hayashi et al . , 2018 ) .", "Knockdown of individual components of the pathway might therefore impact multiple protein–protein interactions , collapsing numerous components of cell-wall synthesis to produce the same phenotypic outcome .", "Alternatively , it is possible that biosynthetic precursors accumulate , producing consistent alterations in cell structure .", "A further possibility is that mycolic acids might play a more important structural role in the maintenance of mycobacterial cell width – an intriguing prospect considering the absence of the prokaryotic actin homolog , MreB , in mycobacteria ( Singh et al . , 2010 ) .", "Further work is needed to resolve these possibilities and should benefit from the collection of CRISPRi mutants described here .", "In general , the CRISPRi-induced morphological changes appeared consistent within pathways and following exposure to the various anti-mycobacterial drugs .", "This was especially true of genes involved in DNA replication , cell division , cell-wall metabolism , and energy metabolism – a property which allowed us to leverage the platform to assign functional predictions for a number of genes , and to infer antimicrobial MOA .", "An example is MSMEG_3213 , which morphological profiling , transcriptomic profiling and combinatorial CRISPRi identified as methylase of a predicted Type II R-M system in M . smegmatis .", "The biological function of this system is currently unclear; however , one practical implication is that , if involved as expected in intrinsic phage defense , it might inadvertently eliminate phages , which could be otherwise useful for clinically relevant mycobacteria such as M . tuberculosis or M . abscessus ( Dedrick et al . , 2019 ) .", "It is tempting to consider , too , if the differential complement of R-M systems among mycobacteria might determine the apparent species-selectivity of some mycobacteriophages – for example , DS6A ( Mayer et al . , 2016 ) – and whether the MSMEG_3213 methylase , which is conserved in M . tuberculosis , functions in cell-cycle regulation ( Wion and Casadesús , 2006 ) in addition to its role in self-defense .", "Cytological profiling has been extensively exploited to determine antimicrobial MOA ( Nonejuie et al . , 2013; Huang , 2015; Nonejuie et al . , 2016 ) , with a very recent report providing the first evidence of its utility in mycobacteria ( Smith et al . , 2020 ) .", "To our knowledge , all approaches have consistently utilized whole-cell drug treatment to create profiles of known mechanisms .", "In this work , we instead applied a genetic approach , generating a database of phenoprints based on essential gene knockdown which were then compared with profiles produced by drug exposure of whole cells .", "Notwithstanding the fundamental difference between genetic ( transcriptional ) knockdown and pharmacological ( small molecule ) inhibition , we observed strong overlap in the resulting phenotypes , especially for compounds targeting energy metabolism ( BDQ ) , the mycolic acid ( INH ) or arabinogalactan ( EMB ) components of the cell wall , as well as DNA metabolism ( fluoroquinolones and griselimycin ) .", "In some cases , we noted , too , that different concentrations of compound could produce disparate morphological profiles , suggesting a possible dependence of MOA on drug concentration ( Bernier and Surette , 2013 ) .", "Furthermore , not all compounds produced defining phenotypes .", "For example , compounds targeting peptidoglycan synthesis ( DCS and VAN ) led to rapid lysis , while aminoglycosides did not cluster with the majority of ribosomal knockdowns .", "While these exceptions expose limitations in cytological profiling ( discussed below ) , the approach nevertheless appears useful as rapid pre-screen of compound MOA in drug discovery pipelines , prior to more in-depth investigation .", "Moreover , the pioneering development by Aldridge and colleagues ( Smith et al . , 2020 ) of the MorphEUS ( morphological evaluation and understanding of stress ) system for mycobacterial cytological profiling suggests the potential to replicate the CRISPRi approach described here in M . tuberculosis , a daunting but tractable challenge .", "It was notable that MOXI-treated cells did not sit closely with gyrA and gyrB knockdowns and were instead associated with DNA metabolism , an observation common to other fluoroquinolones .", "While this separation in UMAP space might be deemed surprising , two factors are salient to the interpretation of this result: firstly , the role of mycobacterial DNA gyrase in the removal of torsional stress and the maintenance of template topology during RNA transcription ( Ahmed et al . , 2017 ) is consistent with the dominant effect of gyrA and gyrB knockdown manifesting as more similar to RNA polymerase inhibition ( here , the close association with rpoB in the UMAP analysis is potentially telling ) ; and , secondly , the filamentation observed on fluoroquinolone exposure is SOS-dependent ( Drlica et al . , 2008 ) and involves the formation of lethal double-strand DNA breaks following drug-mediated trapping of the DNA-gyrase complex – an effect which ATc-induced CRISPRi knockdown does not produce .", "Supporting this interpretation , the gyrase B inhibitor , NVB , did not trigger filamentation , and was instead closely associated with gyrA and gyrB knockdowns , a key observation given previous work noting the lack of SOS induction in NVB-treated cells ( Boshoff et al . , 2004 ) .", "A question which commonly arises is how the apparently discrepant gyrA/gyrB and moxifloxacin phenoprints can be reconciled with recent data from a pioneering high-throughput chemical-genetic screen which reported the identification of novel gyrase inhibitors by utilizing a library which included gyrA and gyrB hypomorphs ( Johnson et al . , 2019 ) .", "In addressing this dilemma , it is important to remember that hypomorphs provide a powerful means to identify hypersusceptibilities consequent on bacillary exposure to chemicals which inhibit the same ( cognate ) gene or pathway targeted by the knockdown system ( i . e . the genetic and antibiotic targets are identical ) ; in contrast , whole-cell morphotypes report on the physiological response to ( or manifestation of ) lethal stress induced by essential gene silencing .", "Importantly , a key element distinguishing antibiotic-mediated target inhibition or corruption from transcript depletion is time: antibiotic treatment inhibits ( or corrupts ) active processes , triggering intracellular catastrophe ( in the case of MOXI treatment , an SOS response ) , whereas gene silencing is much more gradual , or ordered , avoiding the impact of instantaneous loss of function .", "As for all large-scale assays , the approach detailed here inevitably carries inherent limitations which must be borne in mind when analyzing and applying the data:", "( i ) Although motivated primarily by pragmatism , the decision to apply an 18 hr endpoint for all analyses might inadvertently have biased the ‘strongest’ phenotypes to genes involved in DNA replication , cell division , and cell-wall metabolism .", "One obvious complication pertains to the drug MOA analyses , and is evident in the striking frequency with which the data points for 1X , 2X and 4X concentrations map differently in UMAP space for different compounds .", "Intuitively , increasing drug concentration might be expected to have two principal effects – increasing the rate of cell death while retaining the primary MOA ( put simply , cells treated at 4X MIC will be further along the ‘path to death’ at 18 hr than those treated at 1X MIC ) or increasing the likelihood of hitting secondary targets .", "This issue has been noted previously in transcriptional profiling of the M . tuberculosis drug-exposure response ( Boshoff et al . , 2004 ) in which optimizing the drug concentration and exposure time were critical in avoiding convergence on a common stress pathway , obscuring the informative , drug-specific transcriptional signature .", "Similarly , the propensity for the rate of gene-product depletion to impact qualitatively the terminal phenotype has been elegantly demonstrated in B . subtilis ( Peters et al . , 2016 ) .", "Notwithstanding the significant resources required , future work might therefore envisage the use of time-lapse imaging of all mutants to identify optimal gene-specific knockdown durations .", "( ii ) This proof-of-concept study employed only aerobically grown mycobacteria cultivated at 37°C in media comprising Middlebrook 7H9 broth base , with glycerol as primary carbon source .", "There is ample precedent in the literature indicating that gene essentialities , metabolic vulnerabilities and drug susceptibilities can alter as a function of growth conditions; therefore , the phenotypes presented here are necessarily condition-specific , and could benefit significantly from expanding these analyses to other , ‘disease-relevant’ culture systems .", "By extension , there is also the potential to perform equivalent analyses in different genetic backgrounds , avoiding the well-described pitfalls of ‘wild-type’ laboratory strains .", "( iii ) Morphological profiling appears to offer a rapid means of preliminary gene function assignment or compound MOA; however , definitive validation is required – via further biochemical and/or functional analysis – to ratify the functional assignments predicted using this tool .", "( iv ) Future iterations should also aim to resolve the large cluster ( Cluster 2 ) of genes exhibiting minimal morphological effects on knockdown into more informative sub-clusters , perhaps through the incorporation of additional features and/or by applying alternative fluorescent reporters ( possibly in addition to the ParB reporter ) .", "Some approaches to consider include the use of sentinel genes and transcription factors involved in different aspects of macromolecular biosynthesis and/or metabolic remodeling ( Naran et al . , 2016; Boot et al . , 2018 ) .", "In addition to the potential for improved MOA delineation , such refinements are necessary to address the large number of hypothetical proteins whose functions were not significantly informed by the current morphological profiling algorithm .", "In conclusion , we are hopeful that the library of M . smegmatis mutants – and the associated online database – will offer a potentially valuable resource for the mycobacterial research community , particularly given the utilization of the ParB-mCherry reporter background which , to our knowledge , is unique in coupling whole-cell morphological information with a measure of replicative status and ploidy .", "While a large collection of inducible protein-degradation mutants was recently described in M . tuberculosis ( Johnson et al . , 2019 ) , there is not yet an equivalent in the faster-growing model mycobacterium , M . smegmatis .", "Moreover , in addition to the validated strains , the plasmids used to establish our library should enable relatively simple construction of either single mutants or mutant libraries in alternative genetic backgrounds and reporter strains ." ], [ "We limited our initial selection to 294 genes .", "In addition to the 288 genes found universally essential in both Tn-seq and CRISPRi-Seq ( de Wet et al . , 2018 ) , we added six components of the ATP synthase which were found to be non-essential in Tn-seq owing to gene duplication in M . smegmatis ( Dragset et al . , 2019 ) .", "While CRISPRi is a reliable technology for producing knockdown , it is subject to variation in knockdown efficacy , depending on the selected guide RNA .", "Moreover , when targeting a gene with CRISPRi , the efficacy of a particular guide is not known de novo , and activity prediction algorithms are still in their infancy , particularly for bacteria ( Wang et al . , 2017 ) .", "As a result , it is generally best practice to target a gene with more than one sgRNA to account for differences in activity .", "However , in the case of arrayed mutant collections , this requirement becomes unwieldy; as such , previous arrayed CRISPRi mutant collections in bacteria have utilized one predicted sgRNA per gene ( Liu et al . , 2017; Wang et al . , 2017 ) .", "Our previous CRISPRi-Seq data ( de Wet et al . , 2018 ) allowed us to identify essential genes that were growth-suppressed by multiple sgRNAs , but additionally provided empirical data on sgRNA knockdown efficiency .", "As a result , we were able to identify the most efficient sgRNAs for any gene .", "For each of the 294 selected genes , two oligonucleotides ( Supplementary file 1 ) were synthesized at 50 nmol and annealed by LGC Biosciences .", "For five genes , an additional set of oligonucleotides was used to validate the observed phenotypes ( Figure 2—figure supplement 2 ) .", "Oligonucleotides were delivered in 96-well plates , and were resuspended in water to a final concentration of 100 µM .", "Cloning was performed as previously described ( Rock et al . , 2017 ) but adapted for scale .", "Briefly , pJR962 ( Rock et al . , 2017 ) was digested overnight with BsmBI-FastDigest ( Thermo ) and gel purified .", "Ligations were performed in 96-well plates with 1U T4 Ligase ( NEB ) and incubated at room temperate overnight .", "Ligation reactions were transformed into 5 μl High-Efficiency DH5α cells ( NEB ) with heat-shock , rescued in TY Broth , and plated on LB agar prepared in 6-well plates .", "Following overnight growth at 37°C , single colonies were picked into 800 μl of LB broth , in 2 ml deep 96-well plates , and grown overnight at 37°C with vigorous shaking .", "All E . coli cultures were supplemented with Kanamycin ( Roche ) at a final concentration of 50 µg/ml .", "Plasmids were extracted with a Zyppy-96 Plasmid Miniprep kit ( Zymo ) according to manufacturer instructions and quantified via Nanodrop .", "Approximately 100 ng of plasmid was electroporated into the M . smegmatis ParB-mCherry reporter strain ( Santi and McKinney , 2015 ) and rescued in 7H9 broth supplemented with OADC and 0 . 05% Tween 80 , for 3 hr at 37°C .", "Electroporation reactions were plated on 7H10 agar supplemented with OADC and Kanamycin at 20 µg/ml , prepared in 6-well plates , and grown at 37°C until visible colonies formed .", "Single colonies were picked into 400 μl 7H9 supplemented with kanamycin ( 20 µg/ml ) and grown with shaking until stationary phase .", "Glycerol stocks of all transformants were stored at −80°C .", "Any failed ligations or transformations were repeated .", "PCR Primers ( Supplementary file 1 ) designed to target the sgRNA-containing region of plasmid pJR962 were synthesized by Inqaba Biotech .", "PCR reactions were prepared with OneTaq Master-mix ( NEB ) in 96-well plates to a final volume of 20 μl .", "Using pipette tips , scrapings of glycerol stocks were taken and mixed with the PCR reaction .", "The PCR was performed with the settings described ( Supplementary file 4 ) .", "PCR reactions were purified and sequenced by the Stellenbosch Central Analytic Facility using the same forward primer as the PCR reaction .", "Sequencing results were analyzed using a custom written R script .", "For PCR amplification , we opted not to use a high-fidelity polymerase owing to the number of reactions performed .", "Since this risked introducing artificial mismatches or ambiguities into the sequencing results , a sequence was considered correct if the mutant had a high-quality alignment with its expected sequence .", "We accepted ambiguities in the sequencing trace if the strain functionally validated in downstream growth analysis .", "PCR reactions that did not produce sequencing results were repeated; ultimately , sequence-validation was obtained for 238 mutants from the initial cloning workflow , a success rate of 83% .", "Of the invalidated mutants , 15 wells contained plasmid backbone , 15 were cross-contaminants which occurred at some point during the cloning process , and the remaining 20 failed to sequence .", "We maintained the plasmid-backbone strains in our library and downstream workflow as these represented useful empty vector sequences , so that control strains were nested within the arrayed library .", "Cloning was repeated for invalidated mutants .", "Square plates containing standard Middlebrook 7H10 OADC agar were prepared and supplemented with kanamycin ( Roche , 20ug/ml ) , with or without anhydrotetracycline ( ATc , Sigma , 100 ng/ml ) .", "Fresh cultures were inoculated from glycerol freezer stocks into 400 μl Middlebrook 7H9 liquid medium supplemented with kanamycin ( Roche , 20 µg/ml ) in deep 96-well plates .", "Cultures were grown with shaking until saturated .", "Stationary-phase cultures were diluted 1:10 000 into fresh 7H9 before spotting onto prepared 7H10 plates using a 96-well replicator pin ( Sigma ) .", "All spotting was performed in triplicate .", "Plates were grown until colonies formed and photographed using a lightbox .", "Colony sizes were quantified using Iris ( Kritikos et al . , 2017 ) .", "Fresh cultures were inoculated from glycerol stocks into 400 μl 7H9 with kanamycin ( Roche , 20 µg/ml ) and grown until saturated .", "Saturated cultures were diluted 1:800 into fresh 7H9 with kanamycin ( Roche , 20 µg/ml ) and grown for 24 hr until exponential phase .", "Exponential-phase cultures were inoculated 1:40 into 7H9 supplemented with kanamycin ( Roche , 20 µg/ml ) and ATc ( Sigma 100 ng/ml ) and grown for 18 hr , with shaking .", "Large-format agarose pads were prepared with water to a final concentration of 2% .", "Briefly , 2 ml of molten low-melt agarose was sandwiched between two rectangular coverslips ( No . 1 . 5 , 24 x 60 mm ) .", "The bottom coverslip was marked using a laser-cut stencil to indicate sample placement .", "Agarose pads were left to dry , and 1 μl of induced culture spotted onto the pads , using a multichannel pipette .", "Each pad contained eight samples .", "Twenty-four strains were imaged per day , and replicate imaging was performed for 137 samples to validate the reproducibility of the imaging workflow .", "Large-format agarose pads were imaged using a Zeiss Axio Observer Z1 and ZEN 2 ( blue edition ) software , with the ZEN Tiles and Positions and ZEN Autofocus Modules installed .", "Images were captured using a Zeiss Axiocam 503 with 3X analogue gain .", "Using a low magnification 10X objective , each sample was localized on the pad .", "Approximately 24 fields-of-view were selected with a 100X Phase Contrast Objective ( 1 . 4NA ) , before capturing images using bright-field and fluorescent imaging .", "Fluorescence was excited using a Colibri Green LED ( 555/30 nm ) and filtered at 590–650 nm .", "Exposure times were maintained across imaging sessions for Brightfield Images , and for fluorescent images .", "A software autofocus regimen was used before each field-of-view was captured .", "In the case of low-density samples , fields-of-view were manually chosen and increased in number .", "Raw images were saved as CZI files prior to processing and data extraction .", "Time-lapse imaging was performed on 1 . 5% agarose pads embedded with 7H9 OADC medium containing ATc ( Sigma , 100 ng/ml ) and kanamycin ( Roche , 20 µg/ml ) , in glass-bottomed dishes ( NEST Biotechnology ) .", "Cells were maintained in an incubated chamber at 37°C and imaged every 15 min with a software autofocus regimen implemented between each frame .", "All image processing was performed in FIJI ( Schindelin et al . , 2012 ) .", "CZI Images were converted to TIFF images and were manually inspected and out-of-focus fields removed .", "To enhance foci in the fluorescent channel , a Gaussian blur filter was applied to the fluorescent channel and subtracted from the original image .", "Furthermore , overall intensity of each fluorescent channel was normalized throughout the imaging dataset , to a mean pixel intensity of 15 .", "ImageJ processing scripts are available at https://osf . io/pdcw2/ .", "Pre-processed images were analyzed using MicrobeJ ( Ducret et al . , 2016 ) with limited constraints on cell and foci detection .", "MicrobeJ output ( cell contours , shape descriptions and fluorescent localization – see Figure 2—figure supplement 1 ) was exported as CSV files .", "Quantitation of microcolony growth rates was performed in ImageJ and R with a custom script .", "Briefly , images were thresholded in ImageJ and the microcolony area extracted per frame .", "Time-lapse plots were produced in R using a combination of ggplot2 ( Wickham , 2016 ) and gganimate .", "To curate the output of MicrobeJ , a classifier was built in R . Two fields-of-view were sampled from each mutant and replica dataset and processed and analyzed as described above .", "All detected objects were initially exported .", "To create a reference of correctly identified cells , cells were inspected with the interface built into MicrobeJ , and misidentified cells removed from the dataset .", "Thus , two datasets were exported: the full dataset of all identified objects , and the dataset of classified cells .", "In total , approximately 22 , 000 objects were manually classified .", "The classified data were imported into R and utilized to build a classification model .", "Briefly , 20% of the dataset was reserved as a test sample , and the remaining 80% was used to train a variety of models , using the Caret package ( Kuhn , 2008 ) .", "For model training , fivefold cross-validation was used , and resampling performed across the default tuning parameters .", "ROC was used to select the optimal tuning parameters for each model .", "A selection of models was tested and compared based on ROC AUC .", "An Averaged Neural Network was empirically chosen as the best-performing model ( Figure 2—figure supplement 3 ) .", "The trained classifier was used to classify the entire dataset and remove misidentified objects .", "Data were input through the curation pipeline and labeled based on sequencing results and growth validations .", "Reproducibility of the imaging workflow was tested by comparing length of the replica image sets .", "The scripts used to develop and test the models , and all input data , are available at https://osf . io/pdcw2/ .", "For each imaged mutant strain , we derived the mean values for a selection of 13 morphological features – Angularity , Area , Aspect Ratio , Circularity , Curvature , Feret , Length , Perimeter , Sinuosity , Solidity , Roundness , Width and Width Variation – and counts of ParB maxima .", "We utilized the approximate nearest neighbor approach in the R package , RANN , to identify a single cell that best approximated the mean values of the mutant .", "The cell contour , derived from MicrobeJ , was plotted using ggplot , and maxima identified by MicrobeJ were superimposed on the plotted contour .", "Visualizing scripts are available at https://osf . io/pdcw2/ .", "For data processing , we adapted a previously described approach ( Campos et al . , 2018 ) .", "For each mutant , we calculated the mean of each measured feature , and the CV ( mean/standard deviation ) .", "We transformed each feature into a Z-score , relative to the distribution of means derived from the empty vector strains utilizing the equation:Z= ( Fi-mean ( FiWT ) ) /sd ( FiWT ) The normalized Z-Score therefore represents the number of standard deviations away from the mean of the wild-type distribution ( Figure 4—figure supplement 1 ) .", "All subsequent analyses utilized Z-Score adjusted data , unless otherwise described .", "Each gene in our dataset was assigned to a Cluster of Orthologous Groups using annotations obtained from eggNOG ( Huerta-Cepas et al . , 2016 ) .", "We utilized both M . smegmatis and M . tuberculosis annotations to assign COGs to as many genes as possible .", "In addition , we manually annotated a number of genes with recently identified function ( Wu et al . , 2018 ) .", "For phenoprint visualization , variables were chosen for their interpretability and potential biological relevance .", "Variables included angularity , area , circularity , curvature , length , sinuosity , roundness , width , width variation and number of ParB foci ( maxima ) .", "For visualization , the mean and CV Z-scores for each variable were displayed in a circular coordinate system .", "Where visualizations represented groups of mutants ( for example COGs ) means of the mutant Z-scores were used for visualization .", "Phenoprint visualization code is available at https://osf . io/pdcw2/ .", "For each feature , we utilized a cut-off of 3 standard deviations above or below the mean of the control distribution , and tested for enrichment of each COG category utilizing Fisher’s Exact Test .", "We corrected for multiple testing using the Benjamini-Hochberg Procedure and utilized a significance cut-off of p<0 . 05 .", "For display of data , we opted to replicate prior work ( Campos et al . , 2018 ) in displaying all genes found above or below the threshold .", "COG categories that were enriched for a particular feature were highlighted to differentiate them from the background .", "We only displayed plots where at least one COG was found to be statistically enriched ( Figure 4—figure supplement 2 ) Scripts are available at https://osf . io/pdcw2/ .", "Dimensionality reduction allows visualization of multidimensional data in two-dimensional space .", "Utilizing our normalized Z-score data , we performed dimensionality reduction in R . We tested a number of approaches for dimensionality reduction , including a Principle Component Analysis ( PCA ) , t-SNE ( Maaten and Hinton , 2008 ) and UMAP ( Maaten and Hinton , 2008 ) algorithms .", "We superimposed these dimensionality reduction techniques with a hierarchical density-based spatial clustering of applications with noise ( hdbscan ) ( Lel et al . , 2017 ) .", "For each technique , we tested a variety of optimization parameters to produce distinct clusters of points , while still maintaining a consistent cluster of wild-type samples .", "On comparison , UMAP produced the best separated clusters , while maintaining a relatively uniform wild-type cluster .", "UMAP is characterized by a degree of stochasticity during initialization ( Lel et al . , 2017 ) ; therefore , we generated 100 maps which were each clustered with hdbscan ( minPts = 12 ) .", "Consistently present clusters across initializations were determined with hierarchical clustering .", "To explain the features that assigned points to particular clusters , we superimposed a color-gradient based on the mutant Z-Scores for a chosen set of features .", "Additionally , we generated heatmaps describing mean Z-scores of chosen features , for each cluster .", "We performed COG enrichment on each cluster , utilizing Fisher’s Exact Test , and adjusted for multiple testing using the Benjamini-Hochberg Procedure .", "For visualizing sub-clusters within the UMAP , we adapted an approach inspired by the previously described spatial analysis of functional enrichment ( SAFE ) approach ( Baryshnikova , 2016; Campos et al . , 2018 ) , to identify regions of the UMAP output that were enriched for particular functions .", "Briefly , for each point on our 2-dimensional UMAP projection , we examined surrounding points , within a predefined radius ( the 10th percentile of the distribution of pairwise distances between all points ) and tested for enrichment of either COG or KEGG ontologies using a hypergeometric test .", "We combined enrichment testing with a k-nearest neighbor interpolation approach to identify spatial regions of our UMAP manifold that were enriched for particular functions .", "For each drug , twofold serial dilutions were prepared in 400 μl of 7H9-OADC medium in deep 96-well plates .", "Exponential-phase M . smegmatis ParB-mCherry cells were inoculated into prepared media at a 1:40 dilution and cultured with shaking for 18 hr .", "Prior to imaging , ODs were measured and a dose-response curve fitted to the results with the DRC package in R ( Ritz et al . , 2015 ) .", "The MIC90 was derived from the fitted curve and defined as 1X MIC .", "We validated that the MIC90 was consistent with results from a fluorescent resazurin based assay .", "To this end , 10 μl of resazurin ( Sigma ) was added to 50 μl of cell culture , incubated for one hour at 37°C and color change determined visually .", "For imaging , cultures were spotted on agarose pads , and imaged and analyzed as previously described .", "A population of cells at a single-timepoint contains a degree of temporal information , as the population contains cells at different stages of the cell cycle ( Campos et al . , 2018 ) .", "To visualize movement of ParB through the cell cycle , we utilized extracted maxima-localization data and cell length from MicrobeJ and processed the data further utilizing a bespoke R script .", "For each mutant , we arranged cells by length , and produced consensus heatmaps of ParB localization relative to cell length using ggplot2 .", "Scripts are available at https://osf . io/pdcw2/ .", "Cultures of M . smegmatis were grown , with shaking , to exponential phase ( OD600 ~0 . 9–1 . 0 ) in 15 ml of 7H9 OADC at 37°C prior to RNA extraction .", "For CRISPRi knockdowns , the strain of interest was inoculated from an exponential-phase culture into 15 ml of 7H9-OADC supplemented with kanamycin ( Roche , 20 µg/ml ) and ATc ( Sigma , 100 ng/ml ) and cultured for 18 hr at 37°C with shaking .", "RNA was extracted using a FastRNA Blue Kit ( MP Biomedicals ) and ethanol-precipitated , according to manufacturer instructions .", "Extracted RNA was quantified by Nanodrop .", "Ten µg of RNA was treated with 2U DNase ( NEB ) at 37°C for 10 min , prior to cleanup with a Zymo RNA Clean and Concentrator-5 kit ( Zymo Research ) according to manufacturer instructions .", "The purified DNAse-treated RNA was poly-A tailed using E . coli Poly ( A ) Polymerase ( NEB ) , according to manufacturer instructions , prior to clean-up with a Zymo RNA Clean and Concentrator-5 kit ( Zymo Research ) .", "Ribosomal RNA was depleted with a Bacterial Ribo-Zero rRNA Removal Kit ( Illumina ) according to manufacturer instructions , prior to clean-up with a Zymo RNA Clean and Concentrator-5 kit ( Zymo Research ) .", "Library preparation for Nanopore sequencing was performed using either a Nanopore Direct RNA Sequencing Kit ( SQK-RNA001 , Oxford Nanopore Technologies ) or Nanopore Direct cDNA ( SQK-DCS109 , Oxford Nanopore Technologies ) according to manufacturer protocols , and sequenced on MinION sequencers ( Oxford Nanopore Technologies ) .", "Base-calling was performed by MinKNOW software ( Oxford Nanopore Technologies ) , and data stored as FASTQ files for downstream analysis .", "Reads were aligned to the M . smegmatis mc2155 genome ( NC_008596 ) with Minimap2 ( Li , 2018 ) , utilizing the map-ont command .", "Produced SAM files were sorted and indexed with samtools ( Li et al . , 2009 ) .", "For visualization , coverage was calculated using the bamCoverage command of deepTools2 ( Ramírez et al . , 2016 ) with normalization by Reads Per Kilobase per Million mapped reads ( RPKM ) .", "featureCounts ( Liao et al . , 2014 ) was utilized for assigning read counts to genes , and DESeq2 ( Love et al . , 2014 ) was used for comparisons of upregulated transcripts .", "All visualizations were produced in R , using ggplot2 .", "sgRNAs were chosen to target MSMEG_3214 and cloned into pJR962 as previously described ( Love et al . , 2014 ) .", "Golden Gate ( Supplementary file 3 ) primers were designed to amplify the promoter-sgRNA-terminator region of the plasmid , with SapI restriction sites included at the 5’ region of the primers , and synthesized by Inqaba Biotech .", "The PCR reaction was performed with Q5 high-fidelity polymerase master mix ( NEB ) and the product column purified with a QIAquick PCR purification kit ( Qiagen ) and quantified by Nanodrop .", "PCR primers and reaction settings are available in Supplementary file 4 .", "Golden Gate cloning was performed utilizing the purified MSMEG_3213-targeting plasmid cloned during establishing the library as a backbone .", "Briefly , a single-pot reaction was set up containing T4 ligase ( 10 000U ) and buffer ( NEB ) , SapI ( NEB , 10Units ) , 75 ng of backbone , and purified PCR product at a molar ratio of 2:1 .", "The reaction was incubated at 37°C for one hour , and at 55°C for 5 min , prior to transformation into E . coli DH5α ( NEB ) .", "Colonies were screened by PCR using the sequencing primers , plJR962_Seq_F and plJR962_Seq_R , and OneTaq polymerase ( NEB ) , as described previously , and positive clones sequenced by the Stellenbosch Central Analytic Facility utilizing plJR962_Seq_F to confirm the insertion .", "The sequence-verified plasmid was electroporated into M . smegmatis mc2155 and tested for growth rescue by spotting assays ." ] ]
[ "Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function , undermining fundamental understanding of pathogenicity and drug susceptibility .", "To address this challenge , we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen , M . smegmatis .", "Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library , we derive robust , quantitative descriptions of bacillary morphologies consequent on gene silencing .", "Leveraging statistical-learning , we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function .", "Exploiting this observation , we infer the existence of a mycobacterial restriction-modification system , and identify filamentation as a defining mycobacterial response to histidine starvation .", "Our results support the application of large-scale image-based analyses for mycobacterial functional genomics , simultaneously establishing the utility of this approach for drug mechanism-of-action studies ." ]
[ "Caused by the microorganism Mycobacterium tuberculosis , tuberculosis kills more people around the world than any other infectious disease .", "M . tuberculosis is also becoming increasingly resistant to treatments , which are particularly difficult for patients to complete .", "The M . tuberculosis genome carries about four thousand genes , with several hundred being vital for survival .", "Finding new ways to fight tuberculosis relies on understanding the exact role of these essential genes , but they are difficult to study in living bacteria .", "To investigate this question , de Wet et al . used the related , fast-dividing bacterial species called M . smegmatis as a model .", "Microscopic imaging was combined with CRISPR-interference – a method that temporarily disrupts expression of a specific gene – to examine how blocking an essential gene would affect the shape of the living microorganism .", "Experiments were conducted on a collection of 270 mutants , capturing single-cell data for hundreds of thousands of live bacteria .", "To analyze the data , a computational pipeline was built , which automatically clustered similar-shaped bacteria .", "These groups , or ‘phenoprints’ , brought together genes of known and unknown roles; this indicated that these genes participate in similar biological networks – and , if unknown , hinted at their function .", "Finally , targeting essential genes with CRISPR-interference often yielded the same shape changes as blocking their encoded proteins with antibiotics .", "This suggests that phenoprints could be useful to understand the mode of action of potential new tuberculosis treatments .", "When applied to M . tuberculosis and other deadly bacteria , the approach developed by de Wet et al . might speed up drug development ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "immunology and inflammation" ]
The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold
elife-06925-v2
[ [ "B lymphocytes are responsible for the protective antibody responses arising from the recognition of the pathological antigens by the surface expressed B cell receptor ( BCR ) ( Kurosaki et al . , 2010 ) .", "The BCR is composed of a membrane-bound immunoglobulin ( mIg ) and a non-covalently associated heterodimer of Igα and Igβ in a 1 mIg: 1 Igα–Igβ heterodimer stoichiometry ( Schamel and Reth , 2000; Tolar et al . , 2005 ) .", "BCRs are distinguished from other types of receptors by their ability to recognize a wide range of antigen molecules .", "In addition to the BCR's ability to recognize antigen diversity , the activation of BCR signaling is also efficiently regulated by the presentation of variable forms of antigens .", "These forms include antigen density ( Fleire et al . , 2006; Liu et al . , 2010a ) , antigen affinity ( Fleire et al . , 2006; Liu et al . , 2010a ) , antigen valency ( Bachmann et al . , 1993; Liu et al . , 2004; Liu and Chen , 2005 ) , the Brownian mobility feature of the antigen ( Wan and Liu , 2012 ) , and the stiffness feature of the substrates presenting the antigen ( Wan et al . , 2013; Zeng et al . , 2015 ) .", "All these results suggest that the BCR is an extraordinary receptor which can efficiently discriminate the chemical and physical features of an antigen ligand .", "Numerous early studies have investigated how the chemical cues from the antigen determine the strength of the signaling cascade mediated by the BCR ( Harwood and Batista , 2010; Pierce and Liu , 2010 ) .", "However , chemical cues are not the only type of external information that is delivered to the BCRs by antigens .", "In fact , physical cues are another layer of important information derived from the antigens for the purposes of regulating B cell activation and subsequent responses ( Liu et al . , 2015 ) .", "For example , a recent study by Tolar and his colleagues demonstrated that B cells used the mechanical forces to rupture the bonds between BCRs and membrane-bound antigens .", "The authors found that only the high affinity BCR and antigen microclusters would be internalized for antigen processing , giving a new mechanism to explain B cell affinity discrimination ( Natkanski et al . , 2013 ) .", "Physiologically , the physical cues can also come from the stiffness of the substrates presenting the antigens .", "Stiffness usually describes the extent that an object can resist deformation in response to an applied force .", "Stiffness is quantified by Young's modulus with a measurable unit of Pascal ( Pa or N/m2 or m−1·kg·s−2 ) ( Discher et al . , 2005 ) .", "B cells could encounter the antigens presented on substrates with various levels of stiffness in vivo ( Bachmann and Jennings , 2010 ) .", "For example , viral capsid substrates presenting antigen exhibited a high degree of stiffness ( 45 , 000–1 , 000 , 000 kPa ) ( Mateu , 2012 ) , while substrates presenting antigen on the membrane of the host cell infected by virus showed a medium level of stiffness ( 0 . 01–1000 kPa ) ( Nemir and West , 2010 ) .", "Additionally , B cells can also efficiently acquire antigens that are associated with extracellular matrix ( ECM ) in the tissue ( Ciechomska et al . , 2014 ) , which is well documented to exhibit an extraordinary range of stiffness from 0 . 012 to 20 kPa ( Bao and Suresh , 2003; Engler et al . , 2004; Paszek et al . , 2005; Nemir and West , 2010 ) .", "Soluble antigen in plasma has also been detected and displayed a remarkably low level of stiffness ( several Pa ) ( Araujo Gde et al . , 2012 ) .", "More recently , Kam and his colleagues quantified the traction forces between T cell receptors ( TCRs ) and the pillar substrates presenting antigens ( Bashour et al . , 2014 ) .", "Indeed , significant shape changes of the substrates were observed due to the bending of the pillars by TCRs after TCR and antigen recognition ( Bashour et al . , 2014 ) .", "Since stiffer substrates are known to be more resistant to shape changes ( Trappmann et al . , 2012 ) , it is reasonable to expect that antigens presented on stiffer or softer substrates will inevitably produce and deliver different mechanical forces to the BCRs .", "Indeed , our recent study showed that the activation of a B cell is very sensitive to the stiffness of the substrates presenting the antigens ( Wan et al . , 2013; Zeng et al . , 2015 ) .", "Thus , all these studies suggest that physical cues , such as mechanical force , comprise important external information delivered to the BCRs by antigens for the purposes of regulating B cell activation and subsequent responses .", "However , the mechanism regulating the strength of the BCR activation in response to mechanical forces that are delivered to the BCR by the antigens on the grounding substrates remains unknown ( Pierce and Liu , 2010 ) .", "There are several important questions which need to be addressed .", "First of all , does the BCR itself have mechanosensing capability , or do B cells perform mechanosensing through the conventional mechanosensors such as lymphocyte function-associated antigen 1 ( LFA-1 ) ( Chen et al . , 2010 , 2012 ) .", "Second , is there a threshold for BCR activation from the mechanical forces that are delivered to the BCR by the antigen ?", "Third , how sensitive will the BCR be toward different mechanical forces above that threshold in the initiation of BCR activation ?", "Fourth , B cells use different isotypes of BCRs to recognize antigens and initiate transmembrane activation signaling ( McHeyzer-Williams and McHeyzer-Williams , 2005; Pierce and Liu , 2010 ) .", "Mature naive B cells use IgM-BCRs , while memory B cells mainly use IgG-BCRs along with a small fraction that use IgE-BCRs .", "We do not know how IgM-BCRs expressed by naive B cells and IgG-BCRs ( or IgE-BCRs ) expressed by memory B cells show differences in terms of the sensitivity and threshold toward mechanical forces in their activation .", "A major obstacle to answer all of these questions is that it is technically challenging to set up an experimental system with predefined mechanical forces between BCR and antigen , which shall then be linked to an assay to accurately measure BCR activation .", "This is especially challenging since it is known that BCR activation may begin seconds after the recognition of the BCR and the antigen ( Pierce and Liu , 2010 ) .", "To overcome these technical difficulties , we took advantage of a platform utilizing a dsDNA-based tension gauge tether ( TGT ) that was recently developed by Ha and his colleagues ( Wang and Ha , 2013 ) .", "DNA is an excellent molecule showing force application geometry that has been accurately calculated and validated by in vitro single molecule force measurements ( Albrecht et al . , 2003; Lang et al . , 2004 ) .", "Ha and his colleagues used these TGT sensors to probe the tension on a single integrin–ECM ligand ( cyclic RGDfK ) bond required for cell adhesion .", "Here , we modified this TGT system by conjugating the B1-8 BCR-specific antigen , 4-Hydroxy-3-nitrophenylacetyl ( NP ) , to the ligand chain of the TGT system ( NP-TGT ) .", "By doing so , we acquired a series of 8 TGT sensors with predefined mechanical force of 12 , 16 , 23 , 33 , 43 , 50 , 54 , and 56 pN respectively .", "We analyzed the activation of BCRs in response to these TGT molecules using high resolution high speed live cell imaging techniques via total internal reflection fluorescence microscopy ( TIRFM ) .", "This allowed us to determine the sensitivity and threshold for the mechanical force signal in the activation of IgM-BCR or isotype-switched IgG-BCR and IgE-BCR ." ], [ "To further our understanding of how mechanical forces influence BCR activation , we constructed the NP-TGT sensors by modifying a dsDNA-based TGT system ( Wang and Ha , 2013 ) .", "Each NP-TGT molecule is composed of two single-stranded DNA ( ssDNA ) molecules with different modifications ( Figure 1A , B ) .", "The first ssDNA molecule is biotin-conjugated at different positions to provide a defined range of rupture force anchoring positions as illustrated in Figure 1B .", "In the original version of the TGT system utilized by Ha and his colleagues ( Wang and Ha , 2013 ) , the second ssDNA molecule was conjugated with a well-characterized integrin ligand , cyclic RGDfk peptide , to provide an integrin binding site for quantifying the mechanical force spectrum ( 12 , 16 , 23 , 33 , 43 , 50 , 54 , and 56 pN ) in the activation of integrin molecules .", "As stated by the authors ( Wang and Ha , 2013 ) , TGT molecules with modifications can provide an experimental system for the study of many other types of receptors .", "Here , we conjugated the B1-8-BCR-specific antigen , NP , to the second ssDNA molecule ( Figure 1A , B , Figure 1—figure supplement 1A ) .", "Since we just exchanged the cyclic RGDfk peptide with the NP hapten antigen and did not change the sequence and design of these dsDNA-based TGT sensors , our NP-TGT system shall have the same tension gauge scales as the one used by Ha and his colleagues ( Wang and Ha , 2013 ) .", "In our experimental system , each NP-TGT molecule can be recognized by the B1-8-IgM-BCRs ( Figure 1A ) .", "Thus , the NP-TGT sensor that is immobilized on the surface of coverslip would be ruptured if the mechanical force applied by the B1-8-IgM-BCR is larger than the predefined tension force of a certain NP-TGT sensor .", "A key feature of dsDNA-based mechanical force sensor is that the specific force value of each sensor is not an absolute value but presents a distribution with a full width at half maximum ( FWHM ) of 5 pN for unzipping rupture mode and 15 pN for shearing rupture mode ( Lang et al . , 2004 ) .", "So , precisely , the actual range of any given NP-TGT sensor represents a distribution with the most possible value of rupture force at 12 , 16 , 23 , 33 , 43 , 50 , 54 , and 56 pN , respectively . 10 . 7554/eLife . 06925 . 003Figure 1 . The construction of B1-8-BCR-specific NP-TGT mechanical force sensor system .", "( A ) Schematic representation of the NP-TGT and NP-specific B1-8-BCR expressing B cells .", "NP-TGT molecule is immobilized on the surface of coverslip , which will get ruptured if the mechanical force applied by the B1-8-BCR is larger than the predefined tension force of a certain NP-TGT ( for example 56 pN is depicted in the figure ) .", "FITC-conjugated anti-NP antibody is used to quantify the molecule density of each different type of NP-TGT sensors tethered on coverslip .", "( B ) The dsDNA geometries and predefined tension force of all eight NP-conjugated TGT sensors and one control TGT without NP conjugation .", "( C ) Representative TIRFM images showing the dynamics of the synaptic accumulation of BCRs from J558L cells expressing B1-8-IgM-BCR in contact with coverslip presenting 56 pN NP-TGT sensor or control TGT ( NC ) at the indicated time points .", "Scale bar is 1 . 5 μm .", "( D ) Comparisons of averaged traces showing the dynamic accumulation of BCRs as demonstrated in ( C ) in a 13 min TIRFM imaging time course .", "Bars represent mean ±SEM .", "Data were from at least 20 cells over three independent experiments .", "( E ) Primary mature naive B cells from wild-type C57BL/6 mice expressing non-NP-specific IgM-BCR did not initiate the activation when encountering 56 pN NP-TGT sensor compared to the response of the same B cells encountering 56 pN TGT sensor without NP conjugation .", "Biotin-conjugated goat anti-mouse IgM surrogate antigens were used as a positive control to efficiently drive the synaptic accumulation of IgM-BCRs in B cell activation .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were from at least 30 cells over three independent experiments .", "( F ) Quantification of the mean fluorescence intensity ( MFI ) of FITC-conjugated NP-specific antibodies on the surface of coverslip tethering the same amount of NP-TGT sensors .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "The surface density is 29 . 0 molecule/µm2 , seeing more in Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 00310 . 7554/eLife . 06925 . 004Figure 1—figure supplement 1 . The quality control of NP-TGT sensor based experimental system .", "( A ) The quality control of the purified NP-ssDNA by mass spectrum .", "( B , C )", "No obvious dissociation of the neutravidin was detected in a 10 min incubation time course .", "In B , two-color TIRF images showing the formation of a typical immunological synapse ( IS ) of a single B cell ( BCR , red color ) and the corresponded Alexa488-conjugated neutravidin within the B cell IS .", "Also given are control neutravidin TIRF images from an area without B cells .", "Scale bar is 5 μm .", "In C , the statistical analyses of the MFI of Alexa488-conjugated neutravidin from the area on coverslip without B cells ( no cell region ) vs the case within the area of B cell IS ( beneath the cell ) that were induced by 12 pN , 43 pN or 56 pN NP-TGT sensors and a negative control 56 pN TGT without NP conjugation ( NC ) .", "( D ) Statistical analyses for the MFI of FITC-conjugated NP-specific antibodies to show that NP-TGT can only be coated to coverslip in a neutravidin-dependent manner .", "( E ) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR to show that non-specific NP-TGT that were tethered on coverslip in a neutravidin-independent manner cannot activate B cells .", "J558L cells expressing naive B1-8-IgM-BCR were either placed on neutravidin-coated coverslip that were incubated with 56 pN NP-TGT sensor or placed on neutravidin-non-coated coverslip that were incubated with indicated types of NP-TGT sensors .", "NC represents 56 pN TGT sensor without NP conjugation .", "In figure C , D , and E , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were from at least 30 cells or 20 measurements in each group of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 004 We first determined whether the NP-TGT sensor can trigger the activation of the B cells expressing B1-8-IgM-BCR .", "We used a similar protocol as reported ( Wang and Ha , 2013 ) to tether the highest force NP-TGT molecule ( mean rupture force 56 pN in the condition they used ) with NP-conjugated ssDNA on coverslip pre-coated with neutravidin .", "We also used the same TGT molecule without NP conjugation as a negative control ( NC ) ( Figure 1B ) .", "DyLight 649 AffiniPure Fab Fragment Goat Anti-Mouse IgM , µ chain specific antibodies was used to pre-label the B1-8-IgM-BCRs on J558L cells ( J558L cells expressing B1-8-IgM-BCR ) before TIRFM imaging experiment as reported in our previous studies ( Liu et al . , 2010a ) .", "We found that J558L cells expressing B1-8-IgM-BCR initiated the activation responses as quantified by the dramatic accumulation of BCRs into the contact interface of B cells with coverslip presenting 56 pN NP-TGT sensor and formed a typical B cell immunological synapse ( IS ) as illustrated by the time lapse TIRFM images ( Figure 1C , D , Video 1 ) .", "These results were not observed with the negative control ( NC ) 56 pN TGT without NP conjugation ( Figure 1C , D , Video 1 ) .", "Additional experiments showed that neutravidin would not dissociate from the coverslip during the 10-min time course of our experiments ( Figure 1—figure supplement 1B , C ) , and NP-TGT can only be attached to coverslip in a neutravidin-dependent manner ( Figure 1—figure supplement 1D ) .", "Further experiments also indicated that the non-specific attachment of NP-TGT molecules if any on the coverslip without pre-coated neutravidin cannot induce the synaptic accumulation of BCRs ( Figure 1—figure supplement 1E ) . 10 . 7554/eLife . 06925 . 005Video 1 . Time lapse images showing the dynamics of the synaptic accumulation of BCRs from J558L cells expressing naive B1-8-IgM-BCR in contact with coverslip presenting 56 pN NP-TGT or control TGT ( NC ) sensor . Scale bar is 1 . 5 μm .", "The video was recorded with a 4-s time interval and is shown at 30 frames per second .", "Related to Figure 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 005 Since TGT is a dsDNA-based molecule , we were concerned that dsDNA may directly stimulate B cells in a BCR-independent manner .", "To check it , we examine the NP-TGT-triggered responses of primary mature naive B cells from wild-type C57BL/6 mice , which do not express NP-specific B1-8-IgM-BCRs ( Figure 1E ) .", "These primary B cells did not accumulate BCRs into the B cell IS in response to coverslip presenting NP-TGT sensor compared to the case of coverslip alone , although a dramatic accumulation was readily observed in the case of encountering coverslip that was coated with anti-mouse IgM surrogate antigens as a positive control ( Figure 1E ) .", "The B cell activation is known to be very sensitive to the density of antigen on the surface of the antigen-presenting substrates ( Fleire et al . , 2006; Liu et al . , 2010a ) .", "To exclude such a variant , we then used NP-specific antibodies to quantify the density of each NP-TGT sensor ( Figure 1F ) .", "Thus , we established the B1-8-BCR-specific NP-TGT sensor system which showed a predefined mean mechanical force gauge ranging from 12 to 56 pN .", "We compared the response of naive B1-8-IgM-BCR expressing B cells after immune recognition of these series of NP-TGT molecules .", "Numerous early studies showed that after BCR-antigen recognition , B cells immediately begin to spread over the antigen-containing surfaces to form the B cell IS and attempt to acquire the antigens by actively accumulating BCRs and antigens into the B cell IS ( Fleire et al . , 2006; Liu et al . , 2010a , 2010b , 2010c; Seeley-Fallen et al . , 2014 ) .", "Thus , we first examined the B cell spreading response and the concomitant accumulation of BCRs into the B cell IS by TIRFM imaging using J558L cells expressing B1-8-IgM-BCR in NP-TGT-based experimental system ( Figure 2A ) .", "The results suggested that NP-TGT sensors with higher mechanical force generally triggered more aggressive spreading response and enhanced BCR synaptic accumulation than NP-TGT molecules with lower mechanical force ( Figure 2A–C ) .", "More strikingly , a careful examination of the response of B cells toward these series of NP-TGT sensors with mechanical forces ranging from 12 to 56 pN suggested that there are three levels of thresholds .", "The low-force ( 12–16 pN ) NP-TGT sensors triggered a weak activation , whereas the middle-force ( 23–43 pN ) NP-TGT sensors initiated a medium-level activation , and the high-force ( 50–56 pN ) NP-TGT sensors accounted for a strong activation ( Figure 2A ) .", "This unique pattern of the dependence on mechanical forces suggested that IgM-BCR activation exhibits a multi-threshold effect .", "Specifically , an increase of the mechanical force from one threshold to another threshold dramatically enhanced the activation of the naive IgM-BCRs .", "However , within these three threshold barriers , increasing the mechanical force , such as from 23 pN to 33 pN or to 43 pN ( the medium level threshold ) , will not result in dramatically enhanced activation responses of IgM-BCRs . 10 . 7554/eLife . 06925 . 006Figure 2 . The synaptic accumulation of the IgM-BCRs is dependent on mechanical forces and exhibits a multi-threshold effect .", "( A , B )", "Statistical quantification of the synaptic recruitment of IgM-BCR in J558L cells expressing naive B1-8-IgM-BCR ( A ) and primary naive B cells expressing B1-8-IgM-BCR ( B ) .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data are from at least 40 cells over three independent experiments .", "( C ) Representative TIRFM images showing the dynamics of the synaptic accumulation of IgM-BCRs from J558L cells expressing naive B1-8-IgM-BCR in contact with coverslip presenting 12 pN , 43 pN or 56 pN NP-TGT sensors at the indicated time points .", "Scale bar is 1 . 5 μm .", "( D , E )", "Comparisons of averaged traces showing the dynamic accumulation of naive IgM-BCRs into the immunological synapse ( D ) and the growing features of the size of contact area ( E ) for J558L cells expressing naive B1-8-IgM-BCR as demonstrated in ( C ) in a 10 min TIRFM imaging time course .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data are from at least 20 cells over two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 00610 . 7554/eLife . 06925 . 007Figure 2—figure supplement 1 . The contact area after IgM-BCR activation is dependent on mechanical forces with multi-threshold effects and such a pattern is still evident at low density of NP-TGT sensor .", "( A ) Statistical analyses of the size of the contact area of primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice when encountering of the indicated types of NP-TGT sensors .", "( B ) The representative image of NP-TGT molecule indicated by FITC-conjugated NP-specific antibodies within a counting area ( 473 . 1 μm2 ) .", "Scale bar is 1 . 5 μm .", "( C ) The conversion and strong linear correlation between the MFI and the density of FITC-conjugated NP-specific antibody on coverslip .", "NP-specific antibody was used to indicate the density of NP-TGT sensor on coverslip .", "The surface density is quantified at the appropriate incubation concentration of NP-TGT sensor to achieve well-separated and approximately round spots in TIRF imaging ( B ) , which were subsequently analyzed by a Matlab supported 2D Gaussian fitting code ( Source code", "1 ) to perform the counting as reported in our previous studies ( Liu et al . , 2010a ) .", "The equation of the fitted linear regression is: Surface density ( per counting area , about 473 . 1 μm2 ) = 2 . 42 × MFI , R square value for the linear fitting is 0 . 99 .", "( D ) Quantification of the MFI of NP-specific antibody on the coverslip at different incubation concentration of NP-TGT sensor .", "( E ) Surface density of NP-TGT sensors at different incubation concentration when were coated on coverslip as calculated by combining the data in C and D . ( F , G ) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR ( F ) or primary naive B cells expressing B1-8-IgM-BCR ( G ) that were placed on coverslip coated with surface density of 4 . 0 molecule/μm2 of 12 pN , 43 pN , and 56 pN NP-TGT sensors .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were from at least 30 cells over three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 00710 . 7554/eLife . 06925 . 008Figure 2—figure supplement 2 . The patterned dependence on the mechanical forces of IgM-BCR activation does not rely on BCR internalization .", "( A ) Representative confocal images showing the efficient internalization of BCR and antigen molecules in primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice that were interacted with soluble NP8-BSA for 10 min .", "Scale bar is 1 . 5 μm .", "( B ) Side view confocal images showing the lack of internalization of the pre-stained BCR molecules in primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice that were placed on coverslip presenting 12 pN , 43 pN or 56 pN NP-TGT sensors for 10 min .", "B cells were pre-stained with DyLight 649 AffiniPure Fab Fragment Goat Anti-Mouse IgM , µ Chain Specific antibodies before the imaging experiment .", "Scale bar is 2 μm .", "( C ) Statistical quantification of the percentage of B cells with internalization of BCRs as represented in A and B . ( D ) Statistical quantification to show that B cell internalization can be blocked by MDC inhibitor in the condition that the B cells were pretreated by MDC before were activated for 10 min by soluble NP8-BSA as represented in A . ( E , F ) The synaptic accumulation of IgM-BCRs in primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice in contact with the indicated types of NP-TGT sensors .", "In this experiment , B cells were pretreated with either DMSO or MDC following a protocol as detailed in ‘Materials and methods’ section .", "Cross comparison strategy were used in these figures .", "In all of these plots , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were at least from 30 cells or 15 measurements in each group of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 008 We also validated these observations by analogously examining the activation response of primary mature naive B cells expressing B1-8-IgM-BCR from IgHB1-8/B1-8 Igk−/− transgenic mice ( referred to as primary naive B cells expressing B1-8-IgM-BCR ) ( Hauser et al . , 2007 ) upon the recognition of these NP-TGT sensors ( Figure 2B , Figure 2—figure supplement 1A ) .", "Furthermore , we were concerned that the density of the NP-TGT sensors on coverslip might affect the pattern of the activation response of the naive IgM-BCRs .", "Thus , we quantified the density ( number of molecules/µm2 ) of NP-TGT sensors on the coverslip , which was coated with different incubation concentration ( 2 , 5 , 10 , 50 nM ) of NP-TGT sensor ( Figure 2—figure supplement 1B–E ) following a published protocol ( Liu et al . , 2010a; Wang and Ha , 2013 ) .", "At the density of 4 . 0 NP-TGT molecule/µm2 ( 5 nM incubation concentration ) , we can capture the same pattern of the activation response of IgM-BCRs ( Figure 2—figure supplement 1F , G ) .", "At the very low density of 0 . 3 NP-TGT molecule/µm2 ( 2 nM incubation concentration ) , we found that only 56 pN NP-TGT sensor can very mildly trigger the BCR accumulation , while both 12 pN and 43 pN NP-TGT failed to trigger B cell activation ( data not shown ) , suggesting that we cannot use the incubation concentration of 2 nM for these experiments .", "Thus , it is clear that the activation of the IgM-BCR is extremely sensitive to the changes in mechanical forces exceeding the thresholds such as the increase in the forces from 12 pN to 23 pN instead of 16 pN , or from 23 pN to 56 pN instead of 43 pN .", "In the following experiments , we chose the 12 pN , 43 pN , and 56 pN NP-TGT molecules in each of the three threshold barriers ( low , medium , and high ) for further analyses .", "To investigate the temporal dependence of BCR accumulation into the B cell IS on mechanical forces , we took advantage of high speed time lapse TIRFM imaging to test the dynamics of BCR accumulation into the B cell IS and the growing size of contact area starting with the initial immune recognition of BCRs with 12 pN , 43 pN , and 56 pN NP-TGT sensors ( Figure 2C–E , Video 2 ) .", "As expected , we found that the synaptic accumulation of the BCRs in the entire 10-min time lapse was dependent on mechanical forces with high-force NP-TGT sensor ( 56 pN ) exhibiting the highest accumulation , medium force NP-TGT sensor ( 43 pN ) showed medium-level accumulation , and low-force NP-TGT sensor ( 12 pN ) had the lowest level of accumulation , consistent with the results from the above mentioned end point experiments .", "Using these NP-TGT molecules , we also showed that there were no obvious internalization during the 10-min time course of our experiments ( Figure 2—figure supplement 2A–C ) , consistent with the published studies ( Fleire et al . , 2006; Natkanski et al . , 2013 ) .", "These results suggest that the internalization of BCR and antigen molecules did not contribute to the different levels of the accumulation of the IgM-BCRs into the B cell IS in response to different NP-TGT sensors with different mean rupture forces .", "Indeed , B cells that were pretreated with monodansylcadaverine ( MDC ) , an inhibitor to block B cell internalization ( Tolar et al . , 2005; Liu et al . , 2010a ) , also exhibited the dependent on mechanical forces in the synaptic accumulation of the IgM-BCRs ( Figure 2—figure supplement 2D–F ) . 10 . 7554/eLife . 06925 . 009Video 2 . Representative time lapse TIRFM images showing the dynamics of the synaptic accumulation of IgM-BCRs from J558L cells expressing naive B1-8-IgM-BCR in contact with coverslip presenting 12 pN , 43 pN , or 56 pN NP-TGT sensor . Scale bar is 1 . 5 μm .", "The video was recorded with a 4-s time interval and is shown at 30 frames per second .", "Related to Figure 2C . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 009 BCR microclusters have been demonstrated to serve as the most basic platform for the investigation of the initiation of BCR signaling by our studies and those of others ( Harwood and Batista , 2010; Pierce and Liu , 2010 ) .", "We thus assessed the volume of the BCR microclusters produced by the series of NP-TGT sensors by quantifying the fluorescence intensity ( FI ) of each individual BCR microcluster .", "We mathematically fitted each BCR microcluster using a 2D Gaussian function as reported earlier ( Source code", "1 ) ( Liu et al . , 2010a ) .", "We similarly observed that the volume of the BCR microclusters is dependent on the strength of the mechanical force with 56 pN NP-TGT sensors producing much bigger and brighter BCR microclusters than 43 pN and 12 pN NP-TGT molecules ( Figure 3A , B ) .", "The multi-threshold effect was also observed when we analyzed the volume of the generated BCR microclusters .", "NP-TGT molecules with low mean rupture force ( 12–16 pN ) triggered the formation of small BCR microclusters , whereas NP-TGT sensors with medium mean rupture force ( 23–43 pN ) initiated medium-level volume BCR microclusters , and NP-TGT molecules with high mean rupture force ( 50–56 pN ) accounted for bigger and brighter BCR microclusters ( Figure 3A , B ) .", "Thus , the volume of the IgM-BCR microcluster produced by different NP-TGT sensors is dependent on mechanical forces and exhibits a similar multi-threshold effect . 10 . 7554/eLife . 06925 . 010Figure 3 . The volume of the IgM-BCR microcluster produced by different NP-TGT sensors is dependent on mechanical forces and exhibits a similar multi-threshold effect .", "( A ) Representative original ( top panel ) , pseudo-colored 2D ( middle panel ) , and 2 . 5D Gaussian images ( bottom panel ) of typical BCR microclusters induced by 12 , 16 , 23 , 33 , 43 , 50 , 54 , and 56 pN NP-TGT sensors .", "Scale bar is 1 . 5 μm .", "( B ) Statistical comparison of FI of hundreds of BCR microclusters in the immunological synapse in J558L cells expressing naive B1-8-IgM-BCR encountering NP-TGT sensors with indicated tension force .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data are from at least 30 cells over three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 010 Next , we examined the initiation of BCR signaling in B cells when encountering the 12 pN , 43 pN , and 56 pN NP-TGT molecules by quantifying the accumulation of pSyk , pPLCγ2 , and pTyr molecules into the B cell IS using the TIRFM image analysis method as reported in our earlier studies ( Liu et al . , 2010b ) and those of others ( Fleire et al . , 2006; Liu et al . , 2012a ) .", "All of these signaling molecules play essential roles in the transmembrane signaling transduction of BCR .", "For example , Syk binds to the phosphorylated Immunoreceptor tyrosine-based activation motif ( ITAM ) on the cytoplasmic tail of Igα and Igβ , and subsequently Syk will undergo auto-phosphorylation at multiple tyrosine sites within its linker regions to be converted to a signaling active form ( Saouaf et al . , 1994 ) .", "The phosphorylated Syk also provides docking sites for PLCγ2 ( Weber et al . , 2008 ) .", "We found that the membrane proximal recruitment of each of these signaling molecules is dependent on mechanical forces with the 56 pN NP-TGT sensors showing the highest recruitment , while the 43 pN NP-TGT sensors had a medium level of recruitment and the 12 pN NP-TGT sensors had the lowest level of recruitment ( Figure 4A–C ) , consistent with the results from the above mentioned experiments quantifying the synaptic accumulation of the BCR molecules ( Figure 2A , B ) .", "We also analyzed the volume of these signaling molecule microclusters by quantifying the FI of the microclusters and confirmed that the FI of pSyk , pPLCγ2 , and pTyr microclusters produced by different NP-TGT molecules is dependent on mechanical force with a similar multi-threshold effect ( Figure 4D–F ) .", "Thus , it was clear that the strength of the initiated IgM-BCR signaling is dependent on mechanical forces . 10 . 7554/eLife . 06925 . 011Figure 4 . The strength of IgM-BCR signaling is dependent on mechanical forces .", "( A–C )", "Statistical quantification of the synaptic recruitment of pSyk ( A ) , pPLCγ2 ( B ) , and pTyr ( C ) in primary naive B cells expressing B1-8-IgM-BCR that were placed on coverslip presenting 12 pN , 43 pN or 56 pN NP-TGT sensors .", "( D–F )", "Statistical comparison of the volume of pSyk ( D ) , pPLCγ2 ( E ) , or pTyr ( F ) microclusters in J558L cells expressing naive B1-8-IgM-BCR that were produced by 12 pN , 43 pN , or 56 pN NP-TGT molecules .", "In all of these plots , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were at least from 30 cells of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 011 We explored whether the conventional mechanosensor integrin LFA-1 expressed on the surface of B cells influences the patterned dependence on the mechanical forces in B cell activation .", "To address this question , we co-tethered both NP-TGT sensors and the adhesion molecule , intercellular adhesion molecule 1 ( ICAM-1 , which is a ligand for integrin LFA-1 ) on the surface of coverslip and similarly performed TIRFM imaging experiments .", "The addition of ICAM-1 dramatically enhanced the synaptic accumulation of the BCRs ( Figure 5A ) , suggesting the increased sensitivity to antigen stimulation in the initiation of B cell activation , consistent with the published studies in both the BCR ( Carrasco et al . , 2004 ) and TCR system ( Bachmann et al . , 1997; Dustin , 2009 ) .", "However , it was clear that the outside-in activation of integrin did not change the fact that B cell activation is dependent on mechanical forces with a similar multi-threshold effect ( Figure 5B ) .", "To further confirm this conclusion , we inactivated the function of focal adhesion kinase ( FAK ) , a member of the non-receptor protein-tyrosine kinase family , that is known to play a key role in the activation of integrin signaling pathways ( Slack-Davis et al . , 2007; Yu et al . , 2012; Bashour et al . , 2014 ) .", "We pretreated B cells with FAK-specific inhibitor PF573-228 ( Slack-Davis et al . , 2007 ) .", "We found that the pretreated B cells still maintained the general patterned dependence on mechanical forces with a multi-threshold effect ( Figure 5C , D , Figure 5—figure supplement 1A ) .", "However , it was clear that the high-end ( 56 pN ) and medium-level ( 43 pN ) but not low-end ( 12 pN ) mechanical force threshold was more influenced by the inactivation of integrin , suggesting that the breakthroughs of the medium-level and high-end threshold of mechanical forces are partially supported by the inside-out activation of integrin . 10 . 7554/eLife . 06925 . 012Figure 5 . The patterned dependence on the mechanical forces of IgM-BCR activation does not rely on LFA-1 mediated adhesion and dynein , and is only partially dependent on myosin IIA .", "( A , B )", "The synaptic accumulation of IgM-BCRs in primary naive B cells expressing B1-8-IgM-BCR in contact with the indicated types of NP-TGT sensors with or without ICAM-1 co-tethering .", "Cross comparison strategy were used in these figures .", "( C–H )", "The synaptic accumulation of IgM-BCRs in primary naive B cells expressing B1-8-IgM-BCR in contact with the indicated types of NP-TGT probes .", "In this experiment , primary naive B cells expressing B1-8-IgM-BCR were pretreated with DMSO as a control in combination with FAK inhibitor ( C , D ) , myosin IIA inhibitor ( E , F ) or dynein inhibitor ( G , H ) following a protocol that was detailed in ‘Materials and methods’ section .", "Cross comparison strategy were used in these figures .", "In all of these plots , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were at least from 30 cells in each group of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 01210 . 7554/eLife . 06925 . 013Figure 5—figure supplement 1 . Functional test of pharmaceutical inhibitors .", "( A–C )", "Positive control experiments were performed to show that each inhibitor used in Figure 5 is working .", "These experiments showed that FAK inhibitor significantly reduced the size of the contact area of B cells that were placed on coverslip presenting antigens , consistent with the reported function of FAK inhibitor ( Mlinaric-Rascan and Yamamoto , 2001 ) ( A ) ; Myosin IIA inhibitor dramatically reduced the proportion of B cells with internalized Alexa488-conjugated NP8-BSA antigen in the soluble format after reaction for 30 min , consistent with the reported function of Myosin IIA inhibitor ( Vascotto et al . , 2007 ) ( B ) ; Dynein inhibitor dramatically reduced the formation of cSMAC structure in B cell that were placed on planar lipid bilayers presenting antigens for 20 min , consistent with the reported function of Dynein inhibitor ( Schnyder et al . , 2011 ) ( C ) .", "In all of these plots , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were at least from 30 cells or at least 15 measurements in each group of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 013 Motor proteins including myosin IIA and dynein are known to play important roles in B cell activation .", "There are recent studies showing that B cells utilize the contraction forces generated by myosin IIA to rupture the interaction between the BCR and antigen molecules ( Natkanski et al . , 2013 ) , while dynein is required for the retrograde motile feature of the BCR microclusters into the center of B cell IS ( Schnyder et al . , 2011 ) .", "Thus , we assessed the contribution of these two motor proteins to the patterned dependence on mechanical forces during IgM-BCR activation .", "Unexpectedly , the inactivation of myosin IIA did not dramatically change the dependence on mechanical forces , however we observed a significant drop in the strength of the synaptic accumulation of the BCRs for the high threshold NP-TGT sensor at 56 pN only , and no drop was observed for the medium-level threshold NP-TGT sensor at 43 pN , nor the low threshold NP-TGT sensor at 12 pN ( Figure 5E , F , Figure 5—figure supplement 1B ) .", "On the other hand , when inactivating dynein , we did not observe significant changes in the BCR accumulations compared to DMSO controls ( Figure 5G , H , Figure 5—figure supplement 1C ) .", "All of these results suggest that the breakthroughs of the high-end but not the medium-level and low-end thresholds of mechanical forces are supported by myosin IIA .", "B cells use different isotypes of BCRs to recognize antigens and to initiate transmembrane activation signaling .", "Mature naive B cells use IgM-BCRs ( termed naive IgM-BCR thereafter ) , while memory B cells mainly use IgG-BCRs ( termed memory IgG-BCR thereafter ) with a fraction of these use IgE-BCRs ( termed memory IgE-BCR thereafter ) .", "Memory B cells are responsible for the rapid antigen recall humoral responses upon vaccine immunization ( McHeyzer-Williams and McHeyzer-Williams , 2005; Pierce and Liu , 2010 ) .", "Here , we explore the sensitivities and thresholds toward mechanical forces in the activation of memory B cells expressing isotype-switched IgG-BCRs or IgE-BCRs .", "We addressed this question using J558L cells expressing memory B1-8-IgG-BCR in the same experimental system that has been used for the naive B1-8-IgM-BCR-expressing J558L cells as described in detail above .", "Surprisingly , we observed a totally different pattern in terms of the dependence on mechanical forces for the activation of memory IgG-BCRs .", "Each of the three NP-TGT sensors producing mechanical forces at 12 , 43 , or 56 pN similarly drove the cells to undergo spreading responses and the synaptic accumulation of BCRs or pSyk signaling molecules into the IS ( Figure 6A–D ) .", "This suggested that the activation of memory IgG-BCRs requires either no tension or a mechanical force below the mean rupture force of the lowest-force NP-TGT we used ( 12 pN ) .", "Similar patterned activation of memory IgG-BCR was observed at different surface density of NP-TGT sensors on coverslip ( Figure 6—figure supplement 1A–C ) .", "Furthermore , a similar phenomenon was observed when examining the activation of B1-8 primary B cells expressing memory IgG-BCRs that were derived from an in vitro class-switch response following our published protocol ( Liu et al . , 2010b ) ( Figure 6E , F ) .", "We also used high speed TIRFM imaging to test the dynamics of memory IgG-BCR accumulation into the B cell IS starting with the initial immune recognition of memory IgG-BCRs with 12 pN , 56 pN and NP non-conjugated control TGT molecules within a 13-min time course ( Figure 6G , H , Video 3 ) .", "It was clear that 12 pN and 56 pN NP-TGT molecules similarly drove the accumulation of BCRs into the B cell IS .", "All of these results show that the activation of the memory IgG-BCR is independent on mechanical forces ranging from 12 to 56 pN as tested in our NP-TGT experimental system . 10 . 7554/eLife . 06925 . 014Figure 6 . The activation of isotype-switched IgG-BCRs or IgE-BCRs on memory B cells requires either no tension or a mechanical force below 12 pN .", "( A , B )", "Statistical quantification of the synaptic accumulation of IgG-BCR and the volume of the contact area of J558L cells expressing memory B1-8-IgG-BCR encountering 12 pN , 43 pN , or 56 pN NP-TGT sensors .", "( C , D )", "Statistical analyses of synaptic accumulation of pSyk accumulation ( C ) and the volume of pSyk microcluster ( D ) in response to 12 pN , 43 pN , or 56 pN NP-TGT sensors .", "( E , F )", "Quantification of the synaptic accumulation of IgG-BCRs ( E ) or pSyk ( F ) in memory B cells expressing isotype-switched B1-8-IgG-BCR from B1-8 Tg mice that were placed on coverslip presenting 12 pN , 43 pN , or 56 pN NP-TGT probes .", "( G ) Representative TIRFM images showing the dynamics of the synaptic accumulation of IgG-BCRs from J558L cells expressing memory B1-8-IgG-BCR in contact with coverslip presenting 12 pN , 56 pN NP-TGT sensor , or control TGT ( NC ) molecule at the indicated time points .", "Scale bar is 1 . 5 μm .", "( H ) Comparisons of averaged traces showing the dynamic accumulation of memory IgG-BCRs as demonstrated in ( G ) in a 13 min TIRFM imaging time course .", "Bars represent mean ±SEM .", "Data were from at least 20 cells over three independent experiments .", "( I–L )", "Statistical analyses of the synaptic accumulation of two types of chimeric BCRs and pSyk , Human mIgE heavy chain ( I , J ) , or Human mIgM heavy chain ( K , L ) with mouse B1-8 variable region in human Ramos B cells encountering 12 pN , 43 pN , or 56 pN NP-TGT sensors .", "In all of these plots , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were from at least 30 cells in each group of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 01410 . 7554/eLife . 06925 . 015Figure 6—figure supplement 1 . Quantification of the accumulation of IgG-BCR in recognition of NP-TGT sensors at different surface density .", "( A–C )", "Quantification of the synaptic accumulation of IgG-BCRs in J558L cells expressing memory B1-8-IgG-BCR that were placed on coverslip coated with NP-TGT sensors at a density of 29 . 0 molecule/μm2 ( A ) , 19 . 0 molecule/μm2 ( B ) , and 0 . 3 molecule/μm2 ( C ) .", "12 pN NP-TGT , 56 pN NP-TGT , and 56 pN TGT molecule without NP conjugation ( NC ) were used in the experiment .", "Bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were from at least 30 cells over three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 01510 . 7554/eLife . 06925 . 016Video 3 . Time lapse images showing the dynamics of the synaptic accumulation of IgG-BCRs from J558L cells expressing memory B1-8-IgG-BCR in contact with coverslip presenting 12 pN , 56 pN NP-TGT or control TGT ( NC ) sensor at the indicated time points . Scale bar is 1 . 5 μm .", "The video was recorded with a 4-s time interval and is shown at 30 frames per second .", "Related to Figure 6G . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 016 Next , we examined the dependence on mechanical forces in terms of the activation of memory IgE-BCRs in Ramos human B cells expressing memory B1-8-IgE-BCRs .", "Here , we similarly observed that the activation of memory IgE-BCRs require either no tension or a mechanical force below 12 pN ( Figure 6I , J ) .", "As a system control , it was clear that the activation of the naive B1-8-IgM-BCRs expressed in Ramos B cells was also dependent on mechanical force with a multi-threshold effect ( Figure 6K , L ) .", "Conclusively , it was clear that the activation of the memory IgG-BCRs or IgE-BCRs requires either no mechanical force or a force lower than that provided by the 12 pN NP-TGT , highlighting the significant low threshold for the activation of memory IgG-BCR or IgE-BCR that are typically expressed on memory B cells compared to the case of the naive IgM-BCR on mature naive B cells .", "All these results provide new explanations for the rapid and high-titered IgG antibody responses upon re-encounter with antigen by memory B cells .", "The unexpected extremely low mechanical force threshold ( might be less than 12 pN or even lower ) for the memory IgG-BCR but not the naive IgM-BCR activation drove us to ask the next question: why does the memory IgG-BCR behave differently from the naive IgM-BCR ?", "This question is especially intriguing as both memory IgG-BCR and naive IgM-BCR use the exact same BCR component , the Igα and Igβ heterodimer , to initiate BCR signaling .", "In contrast , the mIgG and the mIgM are the BCR components to recognize the antigens ( Schamel and Reth , 2000; Tolar et al . , 2005 ) .", "In addition to the constant region of the Ig , mIgG and mIgM also differ greatly in their cytoplasmic domain .", "In fact , mIgM has only three-amino acids in its cytoplasmic tail ( KVK ) , while all mIgG subtypes have 28-amino acid cytoplasmic tails , which are extremely conserved across species ( Reth , 1989; Tarlinton , 1997; Liu et al . , 2010a , 2012b ) .", "Early mice model studies utilizing biochemical assays and live cell imaging demonstrated that the cytoplasmic tail of mIgG is both necessary and sufficient to confer an enhanced activation of IgG-BCR expressing memory B cells compared to the case of IgM-BCR expressing naive B cells ( Kaisho et al . , 1997; Martin and Goodnow , 2002; Wakabayashi et al . , 2002; Horikawa et al . , 2007; Waisman et al . , 2007; Engels et al . , 2009; Liu et al . , 2010b , 2012b; Engels et al . , 2014 ) .", "To examine the contribution of the cytoplasmic tail of mIgG in the low mechanical force threshold for the activation of the memory IgG-BCR , we took advantage of the four types of J558L cells expressing naive B1-8-IgM-BCR and memory B1-8-IgG-BCR with cytoplasmic tail swapped forms as reported in our previous study ( Liu et al . , 2010b ) : ( 1 ) memory B1-8-IgG-BCR; ( 2 ) mIgG swapped with a mIgM cytoplasmic tail ( memory B1-8-IgG-BCR equipped with a mIgM cytoplasmic tail , termed GGM thereafter ) ; ( 3 ) naive B1-8-IgM-BCR; ( 4 ) mIgM swapped with a mIgG cytoplasmic tail ( naive B1-8-IgM-BCR equipped with a mIgG cytoplasmic tail , termed MMG thereafter ) ( Figure 7A ) .", "We found that the J558L cells expressing GGM showed the force dependent activation by accumulating more BCRs and pSyk into the IS with the higher mean rupture force NP-TGT molecules , similar to the case of J558L cells expressing naive B1-8-IgM-BCR ( Figure 7B–D ) .", "In contrast , it was apparent that the J558L cells expressing MMG behaved similarly to the case of J558L cells expressing memory B1-8-IgG-BCR in a force-independent manner ( Figure 7E–G ) .", "All of these results suggested that the lower mechanical force threshold of memory IgG-BCR activation depends on its cytoplasmic tail . 10 . 7554/eLife . 06925 . 017Figure 7 . The lower mechanical force threshold of IgG-BCR activation is dependent on its cytoplasmic tail .", "( A ) Schematic illustration of the strategy of swapping the cytoplasmic tail of B1-8-IgG- or B1-8-IgM-BCRs .", "( B–G )", "Quantification of the synaptic accumulation of BCRs in J558L cells expressing memory B1-8-IgG-BCR ( B ) , J558L cells expressing memory B1-8-IgG-BCR equipped with a mIgM cytoplasmic tail , termed GGM ( C ) , J558L cells expressing naive B1-8-IgM-BCR ( E ) and J558L cells expressing naive B1-8-IgM-BCR equipped with a mIgG cytoplasmic tail , termed MMG ( F ) .", "Also given are the synaptic accumulations of pSyk in GGM ( D ) and MMG cells ( G ) .", "In all of these plots , bars represent mean ±SEM .", "Two-tailed t tests were performed for the statistical comparisons .", "Data were from at least 30 cells in each group of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06925 . 017" ], [ "B cells produce protective antibodies against pathogens .", "Antibody response is originated from the activation of B cells upon BCR and antigen recognition .", "As mentioned earlier , the antigens recognized by B cells in vivo exhibit great diversities including antigen density ( Fleire et al . , 2006; Liu et al . , 2010a ) , antigen affinity ( Fleire et al . , 2006; Liu et al . , 2010a ) , antigen valency ( Bachmann et al . , 1993; Liu et al . , 2004; Liu and Chen , 2005 ) , Brownian mobility feature of the antigen ( Wan and Liu , 2012 ) , and the stiffness of the substrates presenting the antigen ( Wan et al . , 2013; Zeng et al . , 2015 ) .", "All these studies demonstrated that BCR is an extraordinary receptor which can efficiently discriminate the chemical and physical features of an antigen ligand .", "That raises a long-standing question in immunological studies that how possible the strength of BCR activation can be so complicated and efficient to combine sensitivity , threshold , speed , specificity , affinity , and substrate stiffness discriminatory power ?", "In this report , we modified the dsDNA-based TGT developed by Ha and his colleagues ( Wang and Ha , 2013 ) to acquire a series of 8 NP-TGT sensors with predefined mean rupture force from low to high , respectively ( 12 , 16 , 23 , 33 , 43 , 50 , 54 and 56 pN at the condition in the original study ) .", "The use of these NP-TGT molecules in combination with the high resolution high speed live cell imaging technique through TIRFM allows us to investigate several intriguing questions: ( 1 ) Does the BCR itself possess any mechanosensing properties ?", "( 2 ) Is there a threshold for BCR activation from the mechanical forces that are delivered to BCR by antigen ?", "( 3 ) How sensitive the BCR will be toward different mechanical forces above that threshold in BCR activation ?", "Supported by NP-TGT system , for the first time , we demonstrated that IgM-BCR activation is in a mechanical force dependent manner by providing evidence that high mean rupture force NP-TGT sensors which can stand for high-force load-triggered more aggressive spreading response and the enhanced BCR signaling than low mean rupture force NP-TGT sensors .", "Since such a patterned dependence on mechanical force in the initiation of IgM-BCR does not strictly rely on integrin or adhesion molecule , we propose that IgM-BCR alone could function as a mechanosensor .", "The performance of IgM-BCRs in TGT-based mechanical force experimental system is mostly consistent with the reported findings on integrin molecules ( Wang and Ha , 2013 ) .", "However IgM-BCR and integrin also exhibit some obvious differences in their respective responding pattern to the same mechanical force spectrum ( 12 pN–56 pN ) produced by TGT sensors .", "First of all , 12 pN NP-TGT sensor , the one enduring the smallest mechanical force , can induce the mild but consistent activation of B1-8-naive IgM-BCR when was compared to the control TGT without the conjugated NP antigen .", "Though , it is a fact that the strength of such activation produced by 12 pN NP-TGT sensor is dramatically weaker than those produced by other NP-TGT molecules bearing higher force ( such as 56 pN NP-TGT ) .", "In contrast , integrin does not seem to get activated under 12 pN or even 16 pN TGTs ( Wang and Ha , 2013 ) .", "These results suggested that the initiation of BCR signaling could be extremely sensitive to mechanical forces .", "BCR might rely on such fine sensitivity to initiate its signaling cascade during the surveillance for non-self-antigens that could be of very low density .", "Subsequently , those initially weak signals could be further boosted by other well-characterized mechanisms through activating co-receptors ( such as CD19 ) ( Depoil et al . , 2008 ) or co-stimulatory factors ( such as LFA-1 ) ( Spaargaren et al . , 2003; Carrasco et al . , 2004; McLeod et al . , 2004; Dustin et al . , 2006; Tseng et al . , 2008; Arana et al . , 2008a , 2008b ) .", "Secondly , IgM-BCR exhibits an obvious multi-threshold effect in response to the NP-TGT sensors enduring different mechanical forces from 12 pN to 56 pN .", "The low-force 12–16 pN NP-TGT sensors triggered a weak activation , whereas the middle-force 23–43 pN NP-TGT sensors initiated a medium-level activation , and the high-force 50–56 pN NP-TGT sensors accounted for a strong activation .", "Within these three thresholds , there is a quite obvious barrier effect as an increase in mechanical force , such as from 23 pN to 33 pN or to 43 pN within the medium-level mechanical force barrier , will not result in any dramatically enhanced activation responses of IgM-BCRs .", "Instead , the activation of IgM-BCR is extremely sensitive to the changes of mechanical forces exceeding these barriers , such as the increase of the mechanical force from 12 pN to 43 pN or from 43 pN to 56 pN .", "We speculate that these observations might reflect some key features of dsDNA-derived mechanical force sensor that each sensor represents a distribution of force value with an FWHM of 5 pN for unzipping rupture mode and 15 pN for shearing rupture mode ( Lang et al . , 2004 ) .", "Thus , the actual rupture force of any given TGT sensor shall not be viewed as a fixed value , but rather a distribution with ‘shoulder overlap’ to the nearby TGT sensors .", "We also speculate that these findings would potentially deliver key meanings with biological significance .", "Different from many other receptors , IgM-BCR composes a mIgM and a heterodimer of Igα and Igβ in a 1 mIg: 1 Igα–Igβ stoichiometry with mIgM , the biosensor for antigens , and Igα–Igβ heterodimer , the BCR signaling initiator ( Schamel and Reth , 2000; Tolar et al . , 2005 ) .", "The antigenic signal needs to be delivered from the antigen binding region on the extracellular domain of mIgM to the signaling active ITAM on the cytoplasmic domain of both Igα and Igβ .", "It is still a big open question in terms of the molecular mechanism of how exactly BCR transduces physical and chemical signals .", "Here , we tried to interpret our data in a way that such multi-threshold effect of the activation of IgM-BCRs reflects the multi-threshold progress of delivering these mechanical forces induced potential conformational changes from mIgM to Igα and Igβ heterodimer complex .", "It is also our speculation that such multi-threshold likely provides a chance for the proof-testing of the mechanical forces applied to IgM-BCR during its activation .", "All these speculations are under our further extensive investigation .", "These observed differences that were induced by different NP-TGT sensors cannot exclude the contribution of lifetime of the bonds between BCR and NP-TGT sensors .", "Although , the contribution from mechanical forces and the lifetime of the BCR with the NP-TGT molecules cannot be absolutely separated .", "This especially intrigues a question when considering the recent study by Zhu and his colleagues ( Liu et al . , 2014 ) , showing that the mechanical force prolongs lifetime of catch bond between TCR and agonist pMHC , but decreases the lifetime of slip bond between TCR and antagonist pMHC antigen .", "The authors showed that both the value of the force and the lifetime of the force are important factors in TCR activation .", "The situation with BCR shall be more complicated as different from TCR , BCR is a bivalent binding receptor , stating that one BCR can bind to two NP-TGT molecules .", "Additionally , the recent advance of the super-resolution imaging studies demonstrated that BCRs form obvious nanoclusters with 20–50 IgM-BCRs per nanocluster on quiescent B cells ( Mattila et al . , 2013 ) .", "All these features of BCR demonstrated the high possibility of multivalent bonds based mechanical forces in regulating the initiation of B cell activation .", "Additionally , Tolar and his colleagues recently used AFM to examine the rupture forces between B1-8-IgM-BCR and NP antigen ( Natkanski et al . , 2013 ) .", "Their study provides useful reference information to better understand the relationship between rupture forces and lifetime of a single bond B1-8-IgM-BCR and NP antigen .", "It shall be noted that the values of the rupture forces of TGT sensors were all calculated based on the data measured by magnetic tweezers system with a loading rate of 0 pN/s ( constant force with a time under force mode of 2 s during the measurements ) .", "Rupture force measuring systems using AFM-based vs magnetic tweezers-based approaches show many differences including but not limited to different spring constants , different loading rate and different time under load ( Albrecht et al . , 2003; Lang et al . , 2004; Hatch et al . , 2008 ) .", "Thus , all these differences prevented the feasibility of a direct comparison of the mechanical force value that is acquired from different kind of measurements ( constant force vs constant loading ) .", "From this point of view , it would be of great importance to use the constant force measurements to examine the interaction of B1-8-BCR with NP antigen , and to investigate how the magnitude and duration of mechanical forces will influence B cell activation .", "During the measurement , the time of the force under load shall be carefully picked up because it is known that such a parameter greatly influence the measured value of the rupture forces ( Lang et al . , 2004; Natkanski et al . , 2013; Wang and Ha , 2013 ) .", "As mentioned above a constant force mode with a time under force of 2 s was used to measure the rupture force of these TGT sensors by magnetic tweezers ( Wang and Ha , 2013 ) .", "The relevant timescale of rupturing NP-TGT sensors in our experimental system shall be at the seconds to dozens of seconds level with consideration from the recent studies by Tolar and his colleagues ( Natkanski et al . , 2013 ) .", "In this report , we examined the molecule requirements of the patterned dependence on mechanical forces in IgM-BCR activation .", "The source accounting for the applied force would be quite diverse in a live cell .", "In the case of BCR , motor proteins including myosin IIA and dynein molecules are known to be the mechanical force provider .", "Myosin IIA executes the vertical forces to BCR microclusters to rupture the bonds between BCRs and membrane-bound antigens , and such event is used by B cells to discriminate antigen affinity ( Natkanski et al . , 2013 ) .", "Dynein is shown to be required for the retrograde motile feature of BCR microclusters into the center of the B cell IS , suggesting dynein would more likely execute a lateral force ( Schnyder et al . , 2011 ) .", "We assessed the contribution of myosin IIA and dynein in the patterned dependence on mechanical forces of IgM-BCR activation .", "Unexpectedly , the inactivation of either myosin IIA or dynein did not dramatically changed the patterned dependence on mechanical forces in IgM-BCR activation , suggesting the formation of the basic barrier for the patterned multi-threshold effect does not strictly reply on myosin IIA or dynein .", "However , in primary naive B cells expressing B1-8-IgM-BCR expressing B cells with inactivated myosin IIA , we observed a significantly decreased accumulation of BCR in response to high-end 56 pN NP-TGT sensor only , and such drop was not observed in the case of medium-level threshold 43 pN NP-TGT sensor or low-end 12 pN NP-TGT sensor .", "No such drop was observed in each of these multi-threshold NP-TGT molecules after inactivating dynein .", "All these data drive us to speculate that the breakthrough of the high-end but not the medium-level or low-end mechanical force threshold might be contributed by myosin IIA .", "In contrast , dynein does not seem to play an obvious role in the establishment of the barriers of mechanical forces .", "The unique multi-threshold pattern in IgM-BCR activation is not strictly mediated by the outside-in or inside-out activation of the integrin molecules as addition of neither ICAM-1 nor FAK inhibitor changed that patterned dependence on mechanical forces in IgM-BCR activation .", "However , in FAK inactivated B cells , we observed a significantly decreased accumulation of BCR in response to high-end 56 pN NP-TGT sensor and medium-level threshold 43 pN NP-TGT sensor , while no such drop was observed in the case of low-end 12 pN NP-TGT sensor .", "We speculate from these data that the breakthrough of the high-end and medium-level threshold but not low-end mechanical force barriers might be contributed by the inside-out activation of integrin .", "This is in consistent with the role of myosin IIA as discussed above .", "Myosin IIA bound actin filaments are physically connected with integrin through the key focal adhesion molecules talin and vinculin and was shown to be important for the function of TCR microclusters ( Ilani et al . , 2009 ) .", "Inactivation of either myosin IIA or FAK affects the high-end mechanical force barrier for IgM-BCR activation seems to suggest that these two molecules may regulate B cell activation through similar mechanism at least partially .", "The most striking observation of this report is that the activation of isotype-switched IgG-BCR and IgE-BCR on memory B cells only requires either no tension or a mechanical force below 12 pN since 12 pN , 43 pN or 56 pN NP-TGT sensors similarly drove the activation of IgG-BCR ( or IgE-BCR ) .", "All these results highlighted the significant low-force requirement to initiate the activation of IgG-BCR or IgE-BCR that are expressed on memory B cells compared to the case of IgM-BCR on mature naive B cells , implicating a new possible mechanism to explain the rapid and high-titered IgG antibody responses upon re-encounter with antigen of memory B cells .", "It is worth noting that the recent study by Ha and his colleagues showed that the activation of Notch receptor also requires either no tension or a single molecule force smaller than 12 pN ( Wang and Ha , 2013 ) .", "It is of great interest to address the underlying fundamental mechanism of the different mechanical force requirements for different receptors .", "In this report , we investigated which domain of mIgG resulted in the low or none requirements on mechanical forces for IgG-BCRs compared to the case of IgM-BCRs since both of these two forms of BCRs use the same signaling initiation component , Igα and Igβ heterodimer .", "By a systemic swap experiment we demonstrated that the lower threshold on mechanical forces of memory IgG-BCR activation is dependent on its cytoplasmic tail .", "At this moment , we are addressing the question of why an evolutionarily conserved cytoplasmic tail confers such low threshold on mechanical forces for the activation of memory IgG-BCR .", "We hypothesize that the cytoplasmic tail of mIgG ( or mIgE ) might have some capabilities to facilitate the conformational changes of the ‘umbrella-opening like’ cytoplasmic domain of Igα and Igβ complex during the transmembrane signaling transduction of BCR activation ( Tolar et al . , 2005 ) .", "In conclusion , all these results define the sensitivity and threshold for mechanical force that is required to activate IgM-BCRs and isotype-switched IgG-BCR and IgE-BCR , highlighting the significant contribution of mechanical force signals in the enhanced activation of isotype-switched IgG-BCR and IgE-BCR expressing memory B cells in rapid and high-titered antigen recall responses ." ], [ "Mouse J558L cells stably expressing Igα-YFP and high affinity version of NP-specific B1-8-mIgM-CFP or mIgG-CFP were constructed and maintained as previously described ( Liu et al . , 2010a ) .", "J558L B cells and Human Ramos B cells were cultured in RPMI 1640 medium containing 10% FBS , penicillin and streptomycin antibiotics ( Invitrogen , Carlsbad , CA ) as described ( Liu et al . , 2010c; Sohn et al . , 2011 ) .", "Both of these B cell lines were gifts from Dr Susan K Pierce ( NIAID , NIH , USA ) .", "Primary B1-8 specific mature naive B cells were negatively isolated from spleens of the IgHB1-8/B1-8 Igk−/− transgenic mice as reported previously ( Liu et al . , 2010a ) .", "Following our published protocol ( Liu et al . , 2010b ) , the spleen B cells from IgHB1-8/B1-8 Igk−/− transgenic mice were incubated with 40 µg/ml LPS and 20 ng/ml recombinant mouse IL-4 ( rm-IL-4 ) for 3 days to induce the isotype-switch to IgG1-BCR .", "DyLight 649 AffiniPure Fab Fragment Goat Anti-Mouse IgM , µ Chain Specific ( Jackson ImmunoResearch , West Grove , PA ) and Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Mouse IgG ( H + L ) ( Jackson ImmunoResearch , West Grove , PA ) were used for IgM and IgG staining accordingly .", "In short , the cells were stained with 100 nM antibody on ice for 10 min , after washing three times with PBS , the cells are ready for further experiments .", "FITC-conjugated mouse IgG2a anti-NP antibody named Mouse IgG2a isotype control antibody was purchased from Miltenyi Biotec ( Auburn , CA ) .", "NP-TGTs were prepared following the published protocols ( Wang and Ha , 2013 ) with modifications .", "To specifically activate B cells , we conjugate NP ( 4-Hydroxy-3-nitrophenylacety ) hapten to one DNA strand ( single strand DNA , ssDNA ) of the double strand DNA ( dsDNA ) of TGTs .", "Briefly , NP-e-Aminocaproyl-OSu ( Biosearch Technologies , Petaluma , CA ) were conjugated to ssDNA with NH2 group modification .", "The sequence is as below: 5′-CAC AGC ACG GAG GCA CGA CAC-NH2/-3′ The other strand of the TGT has a biotin tag at a pre-designed position for performing different rupture force points and binding to the coverslip through biotin–neutravidin bond .", "The sequence is as below: 5′-GTG TCG TGC CTC CGT GCT GTG-3′ with biotin label at position 1 , 2 , 4 , 7 , 11 , 15 , 18 , and 21 base , which form 12 , 16 , 23 , 33 , 43 , 50 , 54 , and 56 pN , respectively .", "NP-ssDNA and biotin-ssDNA were further annealed in the annealing buffer following the protocol from Invitrogen .", "Coverslip ( VWR International ) were pretreated with stripping buffer ( H2SO4:H2O2 = 7:3 ) , washed and dried before were glued to the disposable 8-well chamber frame ( Nunc Lab-Tek chamber ) .", "And then 200 µg/ml neutravidin were added to the coverslip , after incubation for 30 min , extensive washing was performed .", "NP-TGTs were then loaded to the coverslip at the concentration of 50 nM for 30 min at room temperature for the purpose of tethering NP-TGTs to the coverslip .", "After carefully washing with PBS , the coverslip was blocked with 1% casein ( wt/vol ) in PBS for 30 min .", "And then , the NP-TGT conjugated coverslip was ready for use .", "The cells were then loaded on the surface for reaction at 37°C for 10 min , if no specific indications .", "PLBs were prepared following our published protocol ( Liu et al . , 2010a; Wan and Liu , 2012 ) , which biotinylated NP8-BSA were attached to biotin lipid through streptavidin .", "In brief , biotin liposomes were prepared by sonication of 1 , 2-Dioleoyl-sn-Glycero-3-phosphocholine and 1 , 2-Dioleoyl- sn-Glycero-3-phosphoethanolamine-cap-biotin ( Avanti Polar Lipids , Alabaster , AL ) in a 25:1 molar ratio in PBS at a lipid concentration of 2 . 5 mM .", "The PLBs were prepared in 8-well chambers ( Nunc Lab-Tek ) with the coverslip cleaned by nanostrip buffer .", "The coverslip was incubated with 0 . 1 mM biotin liposomes in PBS for 20 min at room temperature .", "After washing with 10 ml PBS , the PLB was incubated with 40 nM streptavidin for 15 min and excessive streptavidin was washed away with 10 ml PBS .", "And then the streptavidin-containing PLBs were incubated with 30 nM biotinylated , NP8-BSA ( pre-mixed Alexa488-conjugated NP8-BSA with non-fluorescent conjugated NP8-BSA at 1:10 molar ratio ) for 15 min .", "After washing , PLBs were blocked with 1% ( wt/vol ) Casein in PBS for 30 min at 37°C and washed thoroughly for further use .", "TIRFM images were acquired by an Olympus IX-81 microscope equipped with a TIRF port , Andor iXon+ DU-897D electron-multiplying CCD camera , Olympus 100× 1 . 49 N . A . objective lens .", "The acquisition was controlled by Metamorph software ( Molecular Devices ) .", "The excitation area is about 11 , 600 μm2 .", "Laser power was measured at the head of the objective lens by placing the laser beam at a perpendicular orientation to the imaging plane .", "The laser power of 647 nm laser in BCR imaging is about 7 . 61 μW ( 2% output power only in the configuration of Metamorph supported operating panel; 647 nm laser is 10 mW if using 100% output power ) .", "The laser power of 488 nm laser for the imaging of NP-TGT sensors by FITC-conjugated NP-specific antibodies is about 22 . 9 μW ( 5% output power only in the configuration of Metamorph supported operating panel ) , and the laser power is about 377 . 1 μW in single molecule imaging based experiment for the quantification of surface density of NP-TGT sensors ( 20% output power only in the configuration of Metamorph supported operating panel; 488 nm laser is 3 mW if using 100% output power ) .", "For the imaging options , the exposure time was 100 ms for 512 × 512 pixels image , unless specially indicated .", "All the images are confirmed not over exposed by the software .", "Confocal images were acquired by the Olympus FLUOVIEW FV1000 confocal laser scanning microscope with a 100× oil objective lens .", "The reconstructed side view images were processed by Bitplane Imaris .", "All the images were analyzed and processed with Image J ( NIH , U . S . ) software as indicated .", "The mean fluorescence intensity ( MFI ) and the total fluorescence intensity ( Total FI ) , as arbitrary unit , of BCRs and signaling molecules accumulated to the IS were calculated based on the intensity and area analysis as described ( Lakadamyali et al . , 2004; Liu et al . , 2010b , 2010c , 2012b ) .", "The recruitment of signaling molecules into the IS of B cells stimulated by NP-TGTs was imaged by TIRFM , and the detection of internalized BCR is imaged by Confocal following our previously published protocol ( Liu et al . , 2010a , 2010b , 2010c , 2012b ) .", "In brief , BCRs were pre-stained and then B cells were loaded to the chambers for reaction with NP-TGTs for 10 min followed by 4% paraformaldehyde fixation .", "After washed with 10 ml PBS , the B cells were permeabilized with 0 . 1% Triton X-100 and then blocked with 100 μg/ml goat non-specific IgG ( Jackson ImmunoResearch Laboratory , West Grove , PA ) .", "Subsequently , cells were stained with different primary antibodies including phospho-Zap-70 ( Tyr319 ) /Syk ( Tyr352 ) antibody ( Cell Signaling Technology ) , phospho-PLCγ2 ( Tyr759 ) antibody ( BD ) , phospho-PI3K ( Tyr458 ) antibody ( Cell Signaling Technology , Danvers , MA ) , anti-phosphotyrosine ( Millipore Upstate , Billerica , MA ) at 37°C for 1 hr .", "After washed with 10 ml PBS , B cells were stained with secondary antibody Alexa Fluor 568-conjugated F ( ab′ ) 2 goat antibody specific for rabbit or mouse IgG ( Invitrogen , Carlsbad , CA ) as previously described ( Liu et al . , 2012b ) .", "Images were analyzed by software Image J ( NIH , U . S . ) following our published protocols ( Liu et al . , 2010a , 2010b , 2010c , 2012b ) .", "For inhibitor studies , primary mature naive B cells from IgH B1-8/B1-8 Igk−/− transgenic mice were pretreated with myosin IIA inhibitor Blebbistatin ( Hung et al . , 2013 ) , FAK inhibitor PF573-228 ( Sigma–Aldrich , St . Louis , MO ) ( Slack-Davis et al . , 2007 ) , dynein inhibitor HPI-4 ( Sigma–Aldrich , St . Louis , MO ) ( Firestone et al . , 2012 ) , or internalization inhibitor MDC ( Sigma–Aldrich , St . Louis , MO ) ( Liu et al . , 2010a ) before the imaging experiment .", "Briefly , B cells were pretreated with 50 μM Blebbistatin at 37°C for 30 min , 1 µM PF573-228 at 37°C for 30 min , 30 µM HPI-4 at 37°C overnight , or 100 µM MDC for 30 min at room temperature to block the function of myosin IIA , FAK , or dynein , respectively .", "As a control , B1-8 primary B cells were pretreated with DMSO for 30 min at 37°C , or overnight as the control for HPI-4 inhibitor , or 30 min at room temperature as the control for MDC inhibitor ." ] ]
[ "B lymphocytes use B cell receptors ( BCRs ) to sense the physical features of the antigens .", "However , the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown .", "Here , we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN .", "We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect .", "In contrast , the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN , providing an explanation for their rapid activation in response to antigen stimulation .", "Mechanistically , we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold .", "These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs ." ]
[ "The immune system protects us from invading bacteria and other microbes .", "Immune cells called B cells help the immune system to identify the microbes so they can be destroyed .", "These cells make proteins called antibodies that bind to molecules from the microbes known as antigens .", "The B cells only start to produce antibodies when they bind to a specific antigen via a large group or ‘complex’ of proteins on the surface of the B cell called the ‘B cell receptor’ .", "After the body has defeated the microbe , some of the B cells will become memory B cells , which are primed to rapidly respond to the antigen if the same microbe tries to invade again in future .", "Previous work on the B cell receptor has largely focused on the chemical features of the antigens .", "However , recent research suggests that B cell receptors are also influenced by physical cues from the antigen .", "For example , the stiffness of the surface of the host cell or microbe that is displaying the antigen may exert a mechanical force on the B cell receptor as it binds to the antigen .", "However , it is not clear what role these mechanical forces play in triggering B cell activation and antibody production .", "Before a B cell encounters an antigen , it expresses a type of B cell receptor called the IgM-BCR , but memory B cells express different types of B cell receptors .", "Wan et al . investigated how the different B cell receptors are activated using a technique involving a DNA-based tension gauge .", "The experiments show that the activation of IgM-BCRs depends on the amount of mechanical force applied .", "Low levels of mechanical force only weakly activated the receptors , while higher levels of force resulted in more robust activation .", "In contrast , small amounts of mechanical force were sufficient to strongly activate the other two types of B cell receptors , IgG-BCR and IgE-BCR , on memory B cells .", "This may help memory B cells to be activated more quickly than other B cells that haven't encountered an antigen before .", "The next challenge is to understand why the B cell receptors on memory B cells are less dependent on mechanical forces than IgM-BCRs ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Neuromodulation of excitatory synaptogenesis in striatal development
elife-10111-v2
[ [ "In the vertebrate basal ganglia , dopamine performs critical functions in motivated , goal-directed learning and behavior , transmitting signals related to rewarding stimuli and other salient experiences ( Bromberg-Martin et al . , 2010 ) .", "Classical models of dopaminergic signaling in adult animals propose that the activity of dopamine-producing neurons encodes reward prediction errors and that released dopamine regulates the strength of corticostriatal excitatory glutamatergic inputs .", "This process is thought to favor the execution of a specific action that previously led to reward , at the expense of competing motor programs .", "An important element of this model is the distinct action of dopamine on the direct and indirect pathways , comprised of two classes of striatal spiny projection output neurons that express different types of dopamine receptors , project to separate downstream targets , and regulate complementary aspects of motor behavior ( Bateup et al . , 2010; Kravitz et al . , 2010; Cui et al . , 2013 ) .", "Dopamine differentially regulates biochemical signaling ( Nishi et al . , 1999; 1997 ) , neuronal activity and synaptic plasticity in these two classes of striatal neurons , promoting synaptic potentiation and depression through different receptor subtypes ( Shen et al . , 2008 ) .", "Less is known about the function of dopamine in the postnatal development of basal ganglia , when goal-oriented motor programs are first expressed and activity-dependent synapse formation occurs in the striatum .", "Therefore , we investigated whether dopamine modulates activity-dependent striatal plasticity and synapse formation during this time .", "In mice , the second postnatal week of life is a period of rapid growth of excitatory synapses on SPNs , a process driven by the release of glutamate from corticostriatal axons ( Kozorovitskiy et al . , 2012 ) , among other striatal glutamatergic projections .", "Furthermore , the balance of activity in direct and indirect pathway SPNs ( dSPNs and iSPNs , respectively ) influences activity in the neocortex , potentially altering the firing patterns of corticostriatal neurons via recurrent circuit interactions ( Kozorovitskiy et al . , 2012; Oldenburg and Sabatini , 2015 ) .", "Thus , positive feedback in recurrent circuits can amplify early glutamatergic signaling , rapidly changing network connectivity .", "If dopamine modulates glutamatergic synapse function in developing SPNs as it does in mature circuits , it likely interacts with glutamate-dependent neural circuit development during the early postnatal period .", "In dSPNs , dopamine acts on type 1 dopamine receptor ( Drd1 ) , a Gαs-coupled GPCR to activate PKA , a critical enzyme involved in cell growth and plasticity ( Nishi et al . , 2000 ) .", "In contrast , in iSPNs , dopamine activates type 2 dopamine receptors ( Drd2 ) , Gαi-coupled GPCRs , decreasing PKA activity .", "Since PKA activity levels influence spinogenesis and synaptogenesis in cortical pyramidal neurons ( Kwon and Sabatini , 2011 ) , dopamine may directly set the threshold for activity-dependent synaptogenesis in striatal SPNs .", "Dopamine is poised to regulate striatal signaling early in postnatal life , since embryonic striatum receives substantial dopaminergic innervation from substantia nigra pars compacta ( SNc ) and the ventral tegmental area ( VTA ) , as is evidenced by neuroanatomical studies ( Hu et al . , 2004 ) and direct functional assays showing embryonic dopamine release ( Ferrari et al . , 2012 ) .", "Two observations indirectly support the involvement of dopamine in the developmental wiring of the striatum .", "First , dysfunctions of the dopaminergic system are linked to numerous diseases that are postulated to have a developmental origin , including schizophrenia ( Lau et al . , 2013; Laruelle , 2014 ) , obsessive compulsive disorder ( Nikolaus et al . , 2010 ) and Tourette Syndrome ( Buse et al . , 2013 ) .", "Second , embryonic exposure to drugs of abuse that act on the dopaminergic system , such as methamphetamine ( Heller et al . , 2001 ) , induces profound brain and behavioral abnormalities in the offspring .", "Here we tested the hypothesis that neuromodulators , and specifically dopamine , regulate glutamate-dependent synaptogenesis during striatal development .", "We found a potentially general mechanism of developmental synaptogenesis that relies on the coordinated actions of glutamate and the activation of PKA via Gαs-coupled receptors ." ], [ "Consistent with early development of the dopaminergic projection from SNc/VTA to striatum , the dopamine transporter promoter is active and dopaminergic axons are found in the neonatal striatum on postnatal day 1 ( P1 ) ( Figure 1A ) .", "By P10 the axons of midbrain dopaminergic neurons densely innervate the striatum and express tyrosine hydroxylase , a critical enzyme for dopamine synthesis ( Figure 1B ) , indicating that dopaminergic neuromodulation is poised to regulate synapse and cell function during this early developmental stage . 10 . 7554/eLife . 10111 . 003Figure 1 . Gαs GPCR stimulation rapidly increases SPN dendritic spine and excitatory synapse number .", "( A ) Cre recombinase expression driven by dopamine transporter gene ( Slc6a3 ) promoter activates tdTomato expression ( red ) in a reporter mouse , revealing cells bodies in SNc ( left ) and densely innervating fibers within striatum ( right ) at postnatal day 1 ( P1 ) .", "( B ) At P10 , immunohistochemistry for tyrosine hydroxylase shows expression in SNc cell bodies ( left ) and a punctate pattern in the striatum ( right ) .", "( C ) Schematic of the experiment and recording preparation .", "( D ) 2PLSM image of a Drd2-GFP negative SPN filled with Alexa 594 during whole-cell recording .", "( E ) 2PLSM images of dendrites and dendritic spines on dSPNs after saline or SKF 81297 administration in vivo .", "( F ) Example mEPSC recordings and individual events demonstrating enhanced mEPSC frequency in dSPNs with Drd1 stimulation .", "( G ) Summaries of spine density , mEPSC frequency and amplitude in dSPNs following in vivo administration of saline or the Drd1 agonist SKF 81297 to control animals or those treated with the PKA antagonist H-89 .", "( H ) Summaries of spine density , mEPSC frequency and amplitude in iSPNs following in vivo administration of saline , the Drd1 agonist SKF 81297 , the Drd2 agonist quinpirole , the A2aR agonist CGS 21680 , or SKF 81297 and CGS 21680 together . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 00310 . 7554/eLife . 10111 . 004Figure 1—figure supplement 1 . Drd1 agonist treatment of the acute brain slice fails to enhance excitatory synapse numbers on dSPNs .", "( A ) Trace examples of miniature EPSC ( mEPSC ) recordings from dSPNs in slices that were incubated in the presence or absence of Drd1 agonist SKF 81297 ( 10 µM ) for ~1 hr . ( B ) Summary data demonstrating no difference in normalized mEPSC frequency between conditions .", "Error bars indicate SEM .", "C . Summary data demonstrating no difference in mEPSC amplitude between conditions .", "Error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 00410 . 7554/eLife . 10111 . 005Figure 1—figure supplement 2 . Acute Drd1 agonist treatment induces locomotion in immature pups .", "( A ) View from above the arena used to record locomotor activity and quantify grid crossings , with example tracks of individual mice from the noted conditions recorded for 30 min . ( B ) Summary graphs of distance travelled and grid coverage from mice injected with saline , Drd1 agonist SKF 81297 , or Drd2 agonist quinpirole .", "A subset of animals was pre-treated with PKA blocker H-89 thirty minutes before saline/agonist injections .", "Drd1 agonist treatment increased locomotion , an effect that was partially blocked by H-89 pre-administration .", "N=9 , 11 , 7 and 5 mice/group; *p<0 . 05 , p<0 . 1; error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 00510 . 7554/eLife . 10111 . 006Figure 1—figure supplement 3 . Drd1 agonist treatment repeatedly potentiates locomotion in immature pups .", "( A ) View from above the arena used to record locomotor activity , with example tracks of individual mice from the noted conditions recorded for 30 min , on day 3 of the experiment .", "P9 mice received priming injections on day 1 , as noted , and their locomotor behavior in response to a second injection on Day 3 was evaluated .", "( B ) Summary graph of distance travelled from mice injected twice with saline on days 1 and 3 , saline and SKF 81297 , or two rounds of SKF 81297 treatment .", "Drd1 agonist treatment increased locomotion and further potentiated SKF 81297-induced locomotion increase on day 3 .", "N=10 mice/group; one-way ANOVA p<0 . 0001 , post hoc tests *p<0 . 05 , **p<0 . 01 , *** p<0 . 001; error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 006 To address whether neuromodulation regulates synapse formation in developing striatum , we examined the number and strength of glutamatergic synapses on SPNs in acute brain slices prepared from P8-13 mice , age-balanced across groups .", "Comparisons of dendritic spine density and spontaneous miniature excitatory post-synaptic currents ( mEPSCs ) were made between slices prepared from mice injected 1 hr previously with saline or ligands for GPCRs differentially expressed by dSPNs and iSPNs .", "We observed that in acute mouse brain slices prepared 1 hr after systemic injection of the Drd1 agonist SKF 81297 ( 5 mg/kg ) , both dendritic spine density and mEPSC frequency in dSPNs were increased compared to saline-injected littermates ( Figure 1C–G ) .", "The frequency of mEPSCs in Drd1 agonist exposed SPNs was 593 ± 168% of that in saline-treated controls ( N = 7 and 8 neurons from 4 mice/group , Kruskal-Wallis test p = 0 . 0063 , with p<0 . 05 for Dunn’s multiple comparison post hoc test ) .", "mEPSC amplitude was 10 . 8 ± 1 . 6 and 12 . 7 ± 1 . 1 pA for control and Drd1 agonist groups and was not significantly different ( one way ANOVA , p = 0 . 313 ) .", "Dendritic spine density was 0 . 29 ± 0 . 02 spines/µm and 0 . 60 ± 0 . 04 spines/µm for saline control and Drd1 agonist groups respectively ( p<0 . 001 on Dunnett’s multiple comparison test after one-way ANOVA; N = 36 dendrites , 12 neurons from 3 mice and 21 dendrites in 7 neurons from 3 mice ) .", "Pretreating mice with a PKA antagonist H-89 ( 5 mg/kg ) 30 min prior to agonist injection prevented the effects on mEPSC frequency and spine density ( mEPSC frequency , 122 ± 47% of control with Drd1 agonist , N = 6 and 8 neurons from 2 mice/group , p>0 . 05 for Dunn’s multiple comparison post hoc test; spine density , 0 . 27 ± 0 . 02 spines/µm vs . 0 . 29 ± 0 . 02 spines/µm in control and Drd1 agonist treatment conditions , N = 18 dendrites in 6 neurons from 2 mice , and 24 dendrites in 8 neurons from 2 mice ) .", "These results suggest a PKA-dependent increase in the number of AMPA receptor-containing glutamatergic synapses induced by in vivo Drd1 activation .", "However , Drd1 receptor activation alone is insufficient to increase synapse number , as changes in mEPSC frequency or amplitude were not observed with incubation of acute brain slices in SKF 81297 in vitro ( Figure 1—figure supplement 1 . For mEPSC frequency , 107 ± 23% of ACSF control values with Drd1 stimulation; N = 6 neurons from 2 mice/group , t-test p = 0 . 83 . The amplitude of mEPSCs was 12 . 3 ± 1 . 9 and 13 . 8 ± 2 . 2 pA for control and Drd1 stimulation groups respectively , t-test p = 0 . 51 ) .", "Thus , dSPN excitatory synapse enhancement by a single , acute dose of a Drd1 receptor agonist alone requires intact circuitry , as it cannot be induced in the slice .", "In contrast to the effects seen in dSPNs , in vivo administration of SKF 81297 had no effects on excitatory synapses in iSPNs ( Figure 1H ) , consistent with the lack of expression of Drd1 receptor on these cells ( Gerfen et al . , 1990 ) ( mEPSC frequency , 73 . 3 ± 30 . 1% of control values in presence of Drd1 agonist; N = 6 neurons from 4 mice and 8 neurons from 2 mice ) .", "iSPNs express Gαs-coupled Adenosine 2a ( A2a ) receptor , which also enhances PKA activity ( Higley and Sabatini 2010 ) – nevertheless , administration of an agonist of A2aRs ( CGS 21680 , 0 . 1 mg/kg ) had no significant effect on iSPN dendritic spine density or mEPSC rate , within the rapid time-frame of 1 hr ( 140 ± 63% of control , p>0 . 05 for control vs . A2aR agonist post-hoc comparison; N = 11 neurons from 3 mice ) .", "Similarly , administering an agonist of Gαi-coupled Drd2 receptors ( quinpirole 0 . 5 mg/kg ) also resulted in no significant differences from controls ( 132 ± 17 . 0% of control , p>0 . 05 for control vs . Drd2 agonist post hoc comparison; N = 8 neurons from 3 mice ) .", "However , co-administration of Drd1 and A2a receptor agonists increased mEPSC frequency and dendritic spine density in iSPNs , with no change in mEPSC amplitude , suggesting an increase in the number of functional excitatory synapses ( Figure 1H ) ( mEPSC frequency , 470 ± 124% of control values with CGS 21680/SKF 81297 , one-way ANOVA p<0 . 0008 , Bonferroni post hoc p<0 . 05; N = 6 neurons from 4 mice and 5 neurons from 2 mice for this comparison . Dendritic spine density , 0 . 29 ± 0 . 02 spines/µm and 0 . 63 ± 0 . 05 spines/µm , for control vs . CGS 21680/SKF 81297 , one-way ANOVA p<0 . 0001 , Bonferroni post hoc p<0 . 0001; 31 dendrites , 9 neurons , 2 mice , and 7 dendrites , 4 neurons in 2 mice , for this comparison ) .", "These results indicate that neither the activation of dopamine receptors , nor of Gαs-coupled GPCRs , is sufficient to cell-autonomously enhance glutamatergic synapses on SPNs .", "Instead , neuromodulatory GPCR signaling may be a permissive , or facilitating , signal that acts in concert with other stimuli to induce synaptogenesis in vivo .", "We hypothesized that GPCR activation modulates glutamate-induced , activity-dependent synaptogenesis .", "Since Drd1 activation also increases dSPN excitability , which in turn may increase recurrent corticostriatal glutamatergic transmission onto SPNs by altering cortical activity ( Oldenburg and Sabatini , 2015 ) , then activation of this receptor alone in vivo will ( 1 ) activate PKA and ( 2 ) enhance glutamate release .", "This may be sufficient to drive synapse formation .", "Such stimulatory effects of dopamine on basal ganglia circuitry are consistent with the observation of Drd1 receptor activation-induced locomotion observed in pups , which normally show limited mobility at P8-13 ( Dehorter et al . , 2011; Wills et al . , 2014 ) ( Figure 1—figure supplement 2 ) .", "This acute locomotion induction has lasting consequences for behavior and brain development , since priming with Drd1 receptor agonist administration at P9 potentiated agonist-evoked locomotion at P11 ( Figure 1—figure supplement 3 ) .", "In contrast to dSPNs , iSPNs would be expected to have more complex requirements for synaptogenesis , since iSPN activity is negatively coupled to intrastriatal glutamate release in the adult , via regulation of cortical activity ( Oldenburg and Sabatini , 2015 ) .", "We hypothesize that the cooperation of presynaptic glutamate release and postsynaptic PKA signaling explains why coincident activation of Drd1 receptors ( to recurrently drive glutamate release ) and A2aRs ( to enhance PKA signaling ) is required in vivo to increase synaptogenesis in iSPNs .", "We directly tested this hypothesis ex vivo by examining the ability of exogenous glutamate exposure , through focal photolysis of MNI-L-glutamate , to trigger synaptogenesis in SPNs under different states of GPCR activation .", "Spatio-temporally controlled glutamate release can focally and rapidly induce de novo formation of a synapse-bearing dendritic spine on young cortical pyramidal neurons ( Kwon and Sabatini , 2011 ) .", "Cre-dependent AAV was used to induce GFP expression in Cre-expressing dSPNs or iSPNs ( Figure 2A–C ) , allowing visualization of the cellular and dendritic morphology of sparsely labeled neurons of either pathway .", "Repeated glutamate uncaging ( 40 pulses at 2 Hz ) with a variety of pulse durations ( 0 . 5–4 ms ) was applied near a GFP-positive dendrite under conditions that maximally activate NMDA-type glutamate receptors ( 0 mM added extracellular Mg2+ ) .", "In a subset of cases , this protocol induced de novo growth of a dendritic spine-like protrusion ( Figure 2C ) .", "As a control for non-specific , photodamage-induced changes in tissue fluorescence , in a subset of trials , an interleaved stimulus of identical strength was delivered to a control site >5 µm away from any labeled dendrite ( Figure 2B , Figure 2—figure supplement", "1 ) ( 0 . 5 vs . 0 success probability at proximal and distal uncaging locations; 82 trials from 2 mice ) . 10 . 7554/eLife . 10111 . 007Figure 2 . Drd1 or A2a receptor activation facilitates spinogenesis on dSPNs and iSPNs , respectively .", "( A ) Schematic of experimental setup .", "AAVs encoding Cre-dependent GFP expression are used with appropriate transgenic mice to induce sparse GFP labeling of dSPNs or iSPNs .", "( B ) Schematic illustrating de novo dendritic spine formation triggered by focal 2-photon glutamate uncaging .", "In a subset of experiments , uncaging was rapidly alternated between two sites , located close to ( <2 µm ) and far from ( >5 µm ) the GFP-positive dendrite .", "De novo spinogenesis occurred in a subset of trials in locations near the dendrite .", "( C ) Example of a GFP+ neuron imaged at low magnification ( left ) and higher magnification images of a dendrite before ( middle ) and after ( right ) successful induction of new spine formation ( arrow ) .", "( D ) Probability of successful spinogenesis across all induction attempts in dSPNs ( left ) and iSPNs ( right ) as a function of uncaging pulse width in control conditions ( black ) or in the presence of the indicated Gαs GPCR agonist ( grey , Drd1 agonist SKF 81297; burgundy , A2aR agonist CGS 21680 ) .", "( E ) Average probability of spinogenesis for dSPNs and iSPNs analyzed in control conditions or in the presence of the indicated Gαs GPCR agonist .", "Error bars indicated SEM of the probability across mice .", "( F ) Analysis of spinogenesis kinetics using line scans to measure fluorescence in the emerging spine head ( left ) indicates that although the actual process of spinogenesis is fast ( right ) , some spines are generated early ( burgundy ) and others late ( black ) during the stimulation protocol .", "( G ) Gαs GPCR agonist treatment reduces the number of uncaging pulses needed for detection of an emergent dendritic spine .", "Error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 00710 . 7554/eLife . 10111 . 008Figure 2—figure supplement 1 . Dual site uncaging de novo spinogenesis induction .", "( A ) Experimental schematic .", "For sparse expression of GFP-expressing AAV in striatal SPNs , mice were injected with a combination of two AAVs , mCherry-Cre and Cre-dependent DIO-EGFP AAV .", "( B ) At P8-13 , acute slices were prepared and GFP+ dendritic segments were imaged with one laser tuned to 840 nm before , during and after delivering a two-site MNI-L-glutamate uncaging protocol .", "One site was near the dendrite ( <2 µm ) , while the second one was at least 5 µm away .", "Distal sites ( 2 ) were selected either on the same or different side of the dendrite , relative to the proximal ( 1 ) uncaging location .", "( C ) Fluorescence image illustrating the uncaging sites selected for one trial ( left ) .", "Before ( middle ) and after ( right ) images , demonstrating successful induction of a spiny protrusion .", "( D ) Summary data for 82 trials .", "Success rate for proximal uncaging site spinogenesis is consistent with that from previous studies ( ~50% success rate with no added Mg2+ ACSF; Figure 2 ) .", "No trials were associated with appearance of a protrusion or an isolated fluorescence spot at the distal uncaging site .", "To further evaluate dynamics of signal change in the locations of induction attempts , we tracked line-scan intensity changes across time and uncaging pulses .", "( E ) Examples of individual successful and unsuccessful inductions of spinogenesis showing line-scan tracking at both uncaging locations .", "( F ) Summary graph of line-scans before and after uncaging showing normalized intensity levels for trials at proximal and distal sites . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 008 As was previously described for layer 2/3 pyramidal neurons , P8-13 SPNs of both pathways were capable of de novo spinogenesis in response to glutamate uncaging ( Kozorovitskiy et al . , 2012; Kwon and Sabatini , 2011 ) .", "The basal rate of spinogenesis ( the probability of induction of a structure shaped like a dendritic spine ) , as well as its dependence on uncaging pulse width and proximity to the dendrite were similar for striatal SPNs and previously examined cortical neurons ( Kwon and Sabatini , 2011 ) .", "For both dSPNs and iSPNs , the success rate for inductions with stimulation 1–2 µm away from a labeled dendrite was ~20–50% , with longer uncaging pulses associated with a greater probability of spinogenesis ( dSPNs , N = 146 and 174 trials in 10 mice for control group and in presence of GPCR agonist , respectively , balanced across conditions; iSPNs , N = 132 and 164 trials in 11 mice ) .", "The probability of spinogenesis increased in the presence of SKF 81297 for dSPNs , and CGS 21680 for iSPNs , respectively ( the proportion of success trials was 0 . 23 , 0 . 40 , and 0 . 57 in the control condition , for varied length glutamate uncaging pulses , and 0 . 44 , 0 . 62 , and 0 . 76 in presence of Drd1 receptor agonist for dSPNs; for iSPNs , the respective proportions were 0 . 19 , 0 . 46 , and 0 . 49 in the control condition vs . 0 . 73 , 0 . 74 , and 0 . 70 for iSPNs with A2aR stimulation; two-tailed Z tests for proportions , p = 0 . 040 , 0 . 015 , and 0 . 018 for dSPNs , and p<0 . 0001 , p = 0 . 018 , and 0 . 008 for iSPNs ) .", "This enhancement was observed for all 3 uncaging pulse-widths tested ( Figure 2D ) , and is also evident in across-cell averages within each mouse , for the 4 ms-long uncaging pulse condition ( Figure 2E; proportion of successful trials , 0 . 48 vs . 0 . 80 for dSPNs in control and with Drd1 agonist treatment , and 0 . 48 vs . 0 . 72 for iSPNs; dSPNs , N = 3 and 4 mice/group , 68 and 75 trials; iSPNs , N = 3 and 4 mice/group , 61 and 103 trials ) .", "Analysis of the time course of spine formation for successful inductions reveals that Gαs-coupled receptor stimulation decreased the number of glutamate pulses necessary to induce spinogenesis ( Figure 2F ) .", "Whereas spine growth occurs rapidly in all conditions , as observed in pyramidal neurons ( Kwon and Sabatini , 2011 ) , SKF 81297 and CGS 21680 accelerated the onset of spinogenesis for dSPNs and iSPNs , respectively .", "We compared the number of photoactivation pulses to threshold , defined as 50% fluorescence increase over baseline in the uncaging location , leading to an appearance of a spine-like extension from the dendrite .", "At 2 Hz stimulation , for successful trials , threshold pulse number was 21 . 3 without drug and 6 . 0 in the presence of Gαs agonist for dSPNs , and 17 . 4 vs . 8 . 7 pulses for iSPNs ( Mann-Whitney test , p = 0 . 0025 and p = 0 . 0048 for d- and iSPN comparisons , respectively; N = 12 and 13 induction trials for dSPN control vs . in the presence of agonist , with 16 and 17 induction trials for iSPNs , respectively ) .", "Given that glutamate was exogenously controlled and Gαs receptors were stimulated by agonist bath application , these findings suggest the presence of cell-autonomous mechanisms for neuromodulatory regulation of glutamate-dependent spinogenesis .", "If a similar interaction of neuromodulation and glutamate release controls synapse formation in vivo , it should be possible to bias rapidly developing networks towards increased synaptogenesis by briefly enhancing either PKA signaling or glutamate release .", "To test this prediction for PKA signaling , we used a pharmacogenetic approach based on Rs1 , a modified Gαs-coupled GPCR sensitive to a small , exogenous , blood-brain-barrier permeable ligand ( Srinivasan et al . , 2003 ) .", "The efficacy of this GPCR coupling to Gαs was examined in engineered HEK293 cells , in which GPCR coupling to Gαs elevates cyclic AMP and activates a transcriptional cAMP response element , leading to the production of secreted alkaline phosphatase ( SEAP ) ( Liberles and Buck , 2006 ) .", "Transfection of HEK293 cells with Rs1 , followed by incubation in the agonist RS 67333 and chemi-luminescent analysis of SEAP levels revealed a sub-micromolar EC50 of Rs1 activation ( Figure 3A ) . 10 . 7554/eLife . 10111 . 009Figure 3 . Chemogenetic Gαs GPCR activation is sufficient to rapidly enhance corticostriatal innervation .", "( A ) Secreted alkaline phosphatase ( SEAP ) -based in vitro test of the efficacy of PKA activation following activation of the Gαs-targeted RASSL with RS 67333 .", "( B ) Experimental design showing unilateral neonatal virus injection of DIO-Rs1-EGFP AAV and mCherry-Cre AAV into striatum of Rbp4-Cre; Ai32 mice .", "At P8-13 , pups were systemically injected with RS 67333 and acute slices were prepared 1 . 5 hr after the injection .", "Light-evoked EPSCs were recorded in SPNs and compared across injected and uninjected hemispheres .", "( C ) Confocal image showing striatal expression of a Cre-dependent Gαs targeted Rs1-EGFP AAV in a Cre-expressing mouse .", "( D ) YFP fluorescence image illustrating the expression ChR2-YFP in layer 5 pyramidal neurons in coronal brain section of an Rbp4-Cre; Ai32 mouse .", "( E ) Images mCherry-Cre ( left , red ) and NeuN ( middle , green ) expression in control ( top ) or Cre-mCherry encoding AAV injected ( bottom ) striatum .", "The proportion of striatal neurons identified immunohistochemically by NeuN expression that co-express Cre in infected or uninfected hemispheres .", "( F ) Examples of light-evoked EPSCs from SPNs in control and virally injected hemisphere of the same mouse .", "Ten overlaid consecutive acquisition traces are shown for each neuron .", "( G ) Summary data showing larger amplitudes of ChR2-evoked EPSCs for SPNs from the injected side of the brain .", "Circles are average responses of single neurons , whereas lines reflect individual mouse averages . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 00910 . 7554/eLife . 10111 . 010Figure 3—figure supplement 1 . Age dependence of optogenetically evoked corticostriatal responses .", "( A ) Examples of individual ( grey ) and average ( black , 5 consecutive traces ) light-evoked , pharmacologically isolated AMPAR-dependent responses in SPNs from Rbp4-Cre; Ai32 mice of different ages .", "More individual response failures in responsive SPNs and non-responsive neurons are observed at the younger ages .", "( B ) Summary data across age groups indicating expansion of connectivity from P8 through eye opening at P13-14 .", "Individual neuron peak response amplitude averages are shown as clear circles , with group averages in red .", "Error bars reflect SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 010 We tested the sufficiency of Gαs-coupled GPCR activation in SPNs for enhancing corticostriatal transmission , using a combination of pharmacogenetic activation in vivo and optogenetic analysis of evoked corticostriatal transmission ex vivo ( Figure 3B–G ) .", "We compared the magnitude of EPSCs evoked by channelrhodopsin ( ChR2 ) -mediated activation of Rbp4-Cre expressing neurons , which include a dense corticostriatal projection , in SPNs in acute brain slices from animals with and without Rs1 expression .", "Rbp4-Cre; Ai32 mice were unilaterally injected in the striatum with a Cre-expressing AAV and Cre-dependent AAV encoding Rs1 ( Hsiao et al . , 2008 ) ( Figure 3C–D ) .", "This co-injection resulted in broad expression in dorsal striatum SPNs of the injected hemisphere in both dSPNs and iSPNs ( Figure 3E ) ( 0% vs . 98% co-labeling , N = 750 and 728 neurons in control and injected hemispheres , from 3 mice ) .", "P8-13 infected mice were injected with the Rs1 ligand RS 67333 ( 3 mg/kg ) and acute brain slices were prepared 1 . 5 hr later for analysis .", "Whole-cell voltage clamp recordings were used to monitor pharmacologically isolated , AMPAR-mediated blue light-evoked corticostriatal EPSCs , which were recorded from SPNs in dorsal striatum , located either in the control or in the infected hemisphere .", "Although this strategy results in ChR2 expression in SPNs , the brief time of expression from a single floxed ChR2 allele yielded negligible direct ChR2-evoked currents in SPNs under whole-cell voltage clamp configuration ( e . g . , Figure 3F ) .", "With this strategy , both the control and infected striatum were exposed to RS 67333 and comparisons were made across SPNs from control and infected hemispheres in the same animal .", "In the control condition , there was a strong age dependence of peak amplitude of EPSCs , ranging from an average of 10 pA to an average of 196 pA per age group , in a cohort of P8-P13/14 mice ( Figure 3—figure supplement 1; light evoked EPSC amplitude across age , N = 14 neurons from 3 mice , 30 neurons from 6 mice and 14 neurons from 3 mice/group . Kruskal-Wallis test p<0 . 0001 , for main effect and Dunn’s post hoc tests ) .", "Consistent with our hypothesis , light-evoked EPSC amplitude was increased in virally transduced SPNs , compared to control hemisphere SPNs ( Figure 3G; light-evoked EPSC amplitude , 68 . 6 ± 29 . 9 pA control vs . 150 . 3 ± 55 . 8 pA injected hemisphere; N = 21 and 23 neurons from 4 mice/group , t-test p = 0 . 039 ) .", "Thus , in vivo transient activation of a Gαs-coupled GPCR in SPNs is sufficient to rapidly enhance corticostriatal connectivity , in the presence of otherwise normal corticostriatal transmission .", "The observed sufficiency of pharmacogenetic PKA activation to enhance corticostriatal connectivity for iSPNs ( in contrast to stimulating iSPNs with an A2aR agonist , as in Figure 1H ) stems from the likelihood that the concurrent pharmacogenetic activation of Gαs cascades in dSPNs enhances corticostriatal glutamatergic drive via recurrent connections and regulating cortical activity ( Oldenburg and Sabatini , 2015 ) .", "Conversely , to examine whether increased glutamate release in vivo is able to enhance corticostriatal transmission during this developmental period , a small blue light LED was mounted over somatosensory cortex on one hemisphere and used to activate ChR2-expressing Rbp4-Cre positive neurons in cortex ( Figure 4 ) .", "Mice were awake and freely moving within an enclosure , when a mild stimulus was delivered , consisting of 300 pulses of light over the course of 1 hr , given in bursts of 5 pulses at 20 Hz every minute ( Figure 4A ) .", "This treatment induced moderate c-fos expression in the stimulated cortical region ( Figure 4B ) .", "Acute slices were prepared immediately after the stimulation protocol and AMPAR-mediated light-evoked EPSC were recorded from dorsal striatum SPNs , located in the control or stimulated hemisphere .", "Both EPSC peak amplitude and charge transfer were increased on the stimulated side ( Figure 4D , Figure 4—figure supplement", "1 ) ( Light-evoked EPSC amplitude , 30 . 5 ± 4 . 8 pA vs . 50 . 4 ± 6 . 6 pA for control and stimulated hemisphere comparison; charge transfer , 0 . 32 ± 0 . 14 pC and 0 . 89 ± 0 . 21 pC; N = 29–33 neurons/group , 4 mice/group ) .", "Paired analysis of EPSC amplitude within subjects across the two hemispheres is consistent with individual cell data ( Figure 4D ) , confirming the successful induction of corticostriatal rewiring following cortical optogenetic stimulation ( EPSC amplitude , control 16 . 2 ± 3 . 2 vs . 27 . 3 ± 3 . 2 pA in the stimulated hemisphere , paired t-test p = 0 . 026 , 4 mice/group ) . 10 . 7554/eLife . 10111 . 011Figure 4 . Rapid activity-dependent in vivo corticostriatal plasticity depends on PKA and Gαs-coupled receptor activation .", "( A ) Experimental schematic .", "A mouse pup expressing ChR2-YFP in corticostriatal fibers ( Rbp4-Cre; Ai32 ) receives 300 light pulses over the course of 1 hr via an extracranially mounted LED located over somatosensory and motor cortices .", "Immediately after in vivo optogenetic stimulation , acute slices are prepared and ChR2-mediated EPSCs in SPNs are recorded from stimulated and control hemispheres .", "( B ) Immunohistochemical labeling of immediate early gene c-fos product shows increased labeling on the optogenetically stimulated side .", "( C ) Schematic of the experiment and example traces showing ten sequentially evoked EPSCs from example SPNs of the control and stimulated hemisphere in one mouse .", "( D ) Average amplitudes of optogenetically evoked EPSCs showing successful induction of corticostriatal plasticity under baseline conditions .", "Group averages are shown on the left ( error bars reflect SEM ) and single neuron ( circles ) as well as individual mouse ( lines ) averages are on the right .", "( E ) As in D , repeated in in striatum-only slices , prepared by removing cortex before recording from SPNs .", "( F , G )", "As in D , showing in vivo pre-administration of PKA blocker H-89 30 min before optogenetic stimulation ( F ) or antagonism of Drd1 and A2a receptors with SKF 83566 and KW-6002 ( G ) prevent optogenetically induced corticostriatal plasticity . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 01110 . 7554/eLife . 10111 . 012Figure 4—figure supplement 1 . Light-evoked response properties in SPNs of Rbp4-Cre; Ai32 mice .", "( A ) Examples of individual pharmacologically isolated AMPAR-dependent light-evoked EPSCs , recorded from SPNs in Rbp4-Cre; Ai32 mice , either in a standard coronal preparation ( left ) , or in a striatum-only slice with the neocortex removed ( right ) .", "Individual acquisition sweeps are shown in grey and the average of 5 consecutive traces is in black .", "( B ) Classification of light-evoked responses into silent , unitary ( single-peak ) and complex responses , in control and in optogenetically stimulated hemispheres .", "( C ) Charge transfer was similar for standard slices and striatum only preparations . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 012 Because stimulating layer 5 pyramidal neurons may alter intra-cortical connectivity , which could indirectly account for the observed differences in SPN responses , we tested whether this form of plasticity is striatally expressed by trimming off cortex from acute coronal brain slices with a fine scalpel prior to recording ( Figure 4E , Figure 4—figure supplement 1 ) .", "Both peak EPSC amplitude and charge transfer were increased on the stimulated side ( light-evoked EPSC amplitude , 9 . 6 ± 2 . 4 pA vs . 23 . 4 ± 4 . 3 pA for control vs . stimulated hemisphere; charge transfer , 0 . 64 ± 0 . 14 and 1 . 2 ± 0 . 26 pC for the same comparison; N = 19 and 26 neurons from 4 mice/group ) .", "These data demonstrate that a major component of the functional reorganization induced through brief in vivo stimulation of corticostriatal afferents in young mice is locally expressed in the striatum .", "We further probed the pharmacological dependence of this form of plasticity and found that it was blocked by either pre-administration of PKA inhibitor H-89 ( 5 mg/kg ) , or by a combination of Drd1 and A2aR blockers SKF 83566 ( 0 . 5 mg/kg ) and istradefylline/KW-6002 ( 2 . 5 mg/kg ) , administered 30 min before optogenetic stimulation ( Figure 4F–G ) .", "Light-evoked EPSC amplitude , in presence of H-89 , was 18 . 8 ± 4 . 4 pA vs . 13 . 8 ± 3 . 3 pA in neurons from control and stimulated hemispheres ( t-test p = 0 . 376 , N = 22 and 19 neurons from 3 mice/group ) .", "In presence of Drd1 and A2aR blockers , it was 34 . 2 ± 9 . 3 pA and 26 . 5 ± 5 . 3 pA , respectively ( t-test p = 0 . 458 , N = 12 and 15 neurons from 3 mice/group ) .", "Thus , SPN changes induced by glutamatergic stimulation require PKA signaling , which in turn is activated via Drd1 and A2aR for dSPNs and iSPNs , respectively .", "Increasing either pre-synaptic glutamatergic drive or enhancing striatal PKA signaling is sufficient to rapidly alter SPN synaptogenesis , supporting the idea of cooperative , neuromodulation-dependent circuit remodeling during striatal development .", "In order to directly evaluate whether PKA activation and glutamate release within striatum are sufficient for strengthening corticostriatal synapses , we performed ex vivo plasticity induction using optogenetic stimulation of corticostriatal afferents in the presence of drugs activating PKA in SPNs ( Figure 5 ) .", "We found that the same optogenetic stimulation parameters that evoked PKA-dependent enhancements in evoked responses in vivo were sufficient to produce a moderate enhancement in light-evoked corticostriatal responses in vitro .", "In experiments using within-mouse controls , the amplitude of evoked responses was 528 . 8 ± 69 pA and 694 . 4 ± 79 pA , for neurons in control and stimulated slices respectively ( Figure 5B–C ) ( Mann-Whitney test , p = 0 . 037 , N = 64 and 61 neurons from 4 mice ) .", "In contrast , incubation in PKA agonists alone was insufficient to alter evoked response amplitude ( Mann-Whitney test , p = 0 . 6 , N = 19 and 14 neurons ) .", "These data directly confirm the existence of a mechanism for cooperative , neuromodulation-dependent circuit remodeling during striatal development . 10 . 7554/eLife . 10111 . 013Figure 5 . Glutamate release and Gαs-coupled receptor activation are sufficient to rapidly enhance corticostriatal connectivity in brain slices .", "( A ) Experimental schematic for optogenetic stimulation parameters .", "( B ) Graphic representation of stimulation conditions in the acute slice .", "( C ) Example traces showing average evoked EPSCs for representative neurons in control , stimulation ( light+Gαs-coupled receptor agonists ) and drugs only conditions .", "( D ) Average amplitudes of optogenetically evoked EPSCs .", "Left , cumulative distribution of responses in control and stimulated groups .", "Group averages are shown on the middle and right graphs .", "Single neurons are represented by circles , error bars reflect SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 013" ], [ "We examined the hypothesis that neuromodulation interacts with glutamate-dependent circuit wiring in the developing striatum .", "Instead of a privileged action of dopamine in striatal synapse development , we discovered a mechanism of regulated synaptogenesis requiring cooperative action of glutamatergic drive and neuromodulation .", "Our data reveal that neuromodulation , through the activity of PKA , enhances the probability of dendritic spine formation and lowers the amount of glutamate necessary to trigger spinogenesis in the developing striatum .", "These findings must be integrated into the context of basal ganglia circuitry , where inhibitory outputs of the direct and indirect pathways form functionally complementary loops that provide polysynaptic recurrent positive and negative feedback for excitatory striatal synaptogenesis ( Kozorovitskiy et al . , 2012 ) .", "Because dopamine tends to increase dSPN PKA activity and decrease iSPN PKA activity ( models , Figure 6A–B ) , iSPN spinogenesis may be effectively yoked to dSPN development , as described below .", "Dopamine is abundant in the developing striatum , and it enhances PKA activity in dSPNs .", "This allows dSPNs to strongly respond to glutamate release from the first invading axons , becoming integrated into early basal ganglia circuits .", "On the other hand , iSPN activity and PKA levels are decreased by dopamine ( Hernandez-Lopez et al . , 2000 ) and their excitatory wiring may rely either on the positive feedback through direct pathway ( Kozorovitskiy et al . , 2012 ) to increase glutamate release in the striatum , or on striatal adenosine levels that may be driven by local activity .", "This scenario predicts a transient developmental bias in pathway innervation towards dSPNs , which we have previously observed in the frequency of mEPSCs , as well as in dendritic spine density at P14-15 ( Figure 6C ) ( mEPSC frequency , 0 . 61 ± 0 . 1 and 0 . 35 ± 0 . 1 Hz for d- and iSPNs , t-test p = 0 . 019; mEPSC amplitude , 14 . 3 ± 0 . 5 and 14 . 0 ± 0 . 5 pA , p = 0 . 679; dendritic spine density , 0 . 74 ± 0 . 025 and 0 . 52 ± 0 . 013 spines/µm , p<0 . 0001; N = 55 neurons from 12 mice and 56 neurons from 11 mice for electrophysiology; a distributed subset of recorded neurons was imaged for dendritic spine density analyses , 67 dSPN dendrites from 22 neurons and 93 iSPN dendrites from 31 neurons ) .", "Thus , the palette of neuromodulators and their postsynaptic coupling in a given brain region or developmental time window orchestrate the integration of each neuronal population into functional circuitry . 10 . 7554/eLife . 10111 . 014Figure 6 . Pathway bias and neuromodulatory control of SPN excitatory synaptogenesis during development .", "( A ) Schematic summarizing the key result of this study .", "Neuromodulation , acting via PKA , regulates glutamate-dependent developmental synaptogenesis .", "( B ) These results fit into the larger framework of the basal ganglia model , which indicates that through polysynaptic recurrent loops involving the neocortex , dSPN activity is positively coupled to striatal glutamate release onto SPNs of both pathways , whereas indirect pathway activity provides negative feedback .", "( C ) Together the two models can account for a transient developmental bias towards the earlier innervation of dSPNs over iSPNs .", "During development , dSPNs have greater dendritic spine density and miniature EPSC frequency but not amplitude , compared to iSPNs .", "These graphs represent re-analyses of control data from a previously published study ( Kozorovitskiy et al . , 2012 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10111 . 014 The observation that two types of Gαs-dependent modulation appear to regulate synaptogenesis in d- and iSPNs raises the possibility that PKA activity , via Gαs GPCRs , generally serves to potentiate activity-dependent synaptogenesis during the time of rapid synapse production .", "The array of Gαs GPCRs expressed in the central nervous system is substantial and includes abundant receptors such as 5-HT4 and 5-HT7 serotonin receptors ( Giulietti et al . , 2014 ) , as well beta-adrenergic ( Nomura et al . , 2014; Arriza et al . , 1992 ) and corticotropin-releasing factor receptors ( Fu and Neugebauer , 2008; Riegel and Williams , 2008 ) .", "A general role for neuromodulators acting on Gαs GPCRs during neural circuit development would transform our understanding of core principles in developmental neurobiology , greatly expanding the list therapeutic targets for neurodevelopmental and neurodegenerative disorders ." ], [ "Animals were handled according to protocols approved by the Harvard Standing Committee on Animal Care , in accordance with the guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals .", "In order to identify direct- and indirect-pathways SPNs in experiments described in Figure 1 and related supporting data , we used Drd2-EGFP transgenic mice ( GENSAT , #RP23-161H15 ) .", "For de novo spinogenesis experiments , Drd1a-Cre ( GENSAT , founder line EY262 ) ( Gong et al . , 2003 ) and Adora2A-Cre ( Gong et al . , 2007 ) ( GENSAT , founder line KG139 ) mouse pups were generated by crossing a parent carrying a single Cre-positive allele to C57BL6 wild-type mice .", "Genotyping primers and PCR protocols have been previously described ( Kozorovitskiy et al . , 2012 ) .", "Experiments were also performed on mice that carried the Rbp4-Cre transgene ( GENSAT , #RP24-285K21 ) crossed to floxed TdTomato reporter mice ( Ai14 , Jackson Lab , #007914 ) ( Madisen et al . , 2010 ) or floxed ChR2 ( H134R ) mice ( Ai32 , Jackson Lab , Bar Harbor , ME , #012569 ) .", "For detecting dopamine transporter ( DAT ) activity at a young age , knock-in mice expressing Cre recombinase under DAT promoter , Slc6a3-ires-Cre ( Jackson Lab , # 006660 ) ( Bäckman et al . , 2006 ) , were crossed to Ai14 mice .", "To evaluate locomotor activity in mouse pups , P8-13 mice were videotaped from above in square or rectangular enclosures , for 30–45 min , 15 min following a single or repeat i . p . administration of saline or receptor agonists ( doses are described below ) .", "Euclidean distance traveled was calculated from body center positions in x , y space over time .", "Conditional expression of EGFP in Cre-containing neurons was achieved using recombinant adeno-associated viruses ( AAVs ) encoding a double-floxed inverted open reading frame ( DIO ) of EGFP , as described previously ( Kozorovitskiy et al . , 2012 ) .", "Double-floxed Rs1-EGFP AAV ( Srinivasan et al . , 2003 ) was co-delivered with an AAV expressing Cre fused to mCherry , in order to drive expression in most striatal neurons .", "Vector DNA was amplified in recombination deficient bacteria ( Stbl3 , Invitrogen , Thermo Fisher Scientific , Waltham , MA ) and packaged in the vector core of University of North Carolina .", "P0-3 day old mice were cryoanesthetized , received ketoprofen for analgesia , and were placed on a cooling pad .", "Virus was delivered at a rate of 100 nl/minute using an UltraMicroPump ( World Precision Instruments , Sarasota , FL ) .", "Dorsal striatum was targeted by directing the needle approximately 1 mm anterior to midpoint between ear and eye , 1 . 5 mm from midline and 1 . 8 mm ventral to brain surface .", "Coordinates were slightly adjusted based on pup age and size .", "After 200–300 nl injections and wound closure , mice were warmed on a heating pad and returned to home cages .", "Coronal striatal slices were prepared as described previously ( Kozorovitskiy et al . , 2012 ) .", "Animals were deeply anesthetized by inhalation of isoflurane .", "Cerebral hemispheres were removed and placed in cold choline-based artificial cerebrospinal fluid ( choline-ACSF ) containing 25 mM NaHCO3 , 1 . 25 mM NaH2PO4 , 2 . 5 mM KCl , 7 mM MgCl2 , 25 mM glucose , 1 mM CaCl2 , 110 mM choline chloride , 11 . 60 mM ascorbic acid , and 3 . 10 mM pyruvic acid , and equilibrated with 95%O2/5%CO2 .", "Tissue was blocked and transferred to a slicing chamber containing choline-ACSF .", "Two hundred and seventy-five or three hundred μm-thick slices were cut on a Leica VT1000s ( Leica Instruments , Buffalo Grove , IL ) and transferred into a holding chamber with ACSF containing ( in mM ) 127 NaCl , 2 . 5 KCl , 25 NaHCO3 , 1 . 25 NaH2PO4 , 2 . 0 CaCl2 , +/-1 . 0 MgCl2 , and 25 glucose , equilibrated with 95% O2/5% CO2 .", "Slices were incubated at room temperature , or at 34°C , for 20–30 min prior to imaging or electrophysiological recording , respectively .", "In a subset of experiments , cortex was removed from the striatum in coronal sections using a microsurgical knife ( EMS , Hatfield , PA ) .", "Whole-cell recordings were obtained from striatal SPNs visualized under infrared differential interference contrast ( IR-DIC ) using patch pipettes with pipette resistance of ~2–4 MΩ .", "Epifluorescent illumination enabled identification of infected or GFP-positive SPNs .", "For miniature excitatory postsynaptic current ( mEPSC ) recordings and for light-evoked responses , the internal solution consisted of 120 mM CsMeSO4 , 15 mM CsCl , 8 mM NaCl , 10 mM TEACl , 10 mM HEPES , 2 mM QX314 , 4 mM MgATP , 0 . 3 mM NaGTP ( pH 7 . 4 ) .", "Alexa Fluor 594 ( 10–20 µM ) was added to internal to visualize cell morphology and confirm cell identity as SPN .", "Recordings were made from cells held at -70 mV using Axoclamp 200B or 700B amplifiers ( Axon Instruments , Union City , CA ) at room temperature .", "Data were filtered at 3 kHz and sampled at 10 kHz .", "Series resistance , measured with a 5 mV hyperpolarizing pulse in voltage clamp , was under 20 MΩ and was left uncompensated .", "All responses were examined for light-evoked EPSC analysis .", "Miniature EPSC amplitude cut-off for analysis was 6 pA and cells that had no mEPSCs during the recording were included .", "Neurons with high root-mean-square current noise values ( ~IRMS>2 . 5 pA ) were excluded , regardless of series resistance and holding current .", "Several minutes following breaking in , ~5 min of continuously acquired 30 second long sweeps were collected and analyzed offline per neuron .", "The amplitude of mEPSCs amplitude reflects absolute value of inward currents for neurons held at -70 mV in all figures .", "For normalized mEPSC frequency calculation , the mean of the entire control group was considered 100% .", "Ages of mice were balanced across drug treatment groups and were always P8-13 , except for P14-15 mEPSC and dendritic spine data reanalyzed from a previously published paper in Figure 6C ( Kozorovitskiy et al . , 2012 ) , as well as light-evoked EPSC data in Figure 3—figure supplement 1 , which includes a P13-14 group of mice after eye-opening .", "A bare Cree XPE emitter blue LED was used for in vivo unilateral optogenetic stimulation .", "P8-14 mice were anesthetized and received analgesia .", "The emitter surface was flattened and attached directly to the skull over somatosensory cortex on one side .", "Skin was closed over the emitter , and upon recovery from anesthesia , awake mice were transferred into the stimulation chamber where they could move freely .", "Three hundred 10 ms-long pulses ( ~15 mW outside the cranium ) were delivered through the skull .", "Pulses were delivered in a train of 5 stimuli at 20 Hz , once per minute .", "Brain slices were prepared immediately after completion of stimulation .", "For ex vivo whole cell recording of light-evoked EPSCs in SPNs , light from a 473 nm laser ( Optoengine , Midvale , UT ) was focused on the back aperture of the microscope objective to produce wide-field illumination .", "Brief pulses of light ( 10 ms duration , 2–3 mW under the objective ) were delivered at the recording site at 30 second intervals .", "Epifluorescence illumination was used sparingly to minimize ChR2 activation prior to recording and was never used with a GFP filter cube .", "For ex vivo optogenetic stimulation of acute brain slices we used a custom built chamber with blue ( 470 nm ) LEDs set perpendicular to the plane of slices .", "Slices were incubated in ACSF with 5 µM CGP 55845 , 10 µM SR 95531 , 10 µM SKF 81297 , 10 µM CGS 21680 at room temperature and stimulated with 10 mW . mm-2 light pulses ( 5 ms ) for one hour .", "Light pulses were triggered with a Master-8 stimulator and the pulse pattern used for stimulation was 5 pulses at 20 Hz every minute .", "Dendritic imaging and uncaging of MNI-glutamate for spinogenesis induction were accomplished on a custom-built microscope combining two-photon laser-scanning microscopy ( 2PLSM ) and two-photon laser photoactivation ( 2PLP ) , as previously described ( Kwon and Sabatini , 2011 ) .", "Two mode-locked Ti:Sapphire lasers ( Chameleon lasers , Coherent , Santa Clara , CA ) were tuned to 840 and 725 nm for exciting GFP or Alexa 594 fluorescence and uncaging , respectively .", "The intensity of each laser was independently controlled by Pockels cells ( Conoptics , Danbury , CT ) .", "A modified version of ScanImage software was used for data acquisition ( Pologruto et al . , 2003 ) ( https://github . com/bernardosabatinilab ) .", "For glutamate uncaging , 2 . 5 mM MNI-caged-L-glutamate was perfused into the slice chamber , and 10–15 mW of 725 nm light at the specimen plane ( 60X objective , Olympus , Waltham , MA ) was used to focally release the caging group .", "Secondary and tertiary dendritic branches were selected for dendritic imaging and spinogenesis induction .", "MNI-glutamate was uncaged near the dendrite ( ~0 . 5 or >5 µm away from the edge ) at 2 Hz using up to forty 0 . 5 , 2 or 4 ms-long pulses .", "Images were continually acquired during the induction protocol at 1 Hz , and uncaging was stopped if an apparent spinehead was visible before 40 uncaging pulses were delivered .", "For analysis of the spinogenesis timecourse and the 2-site uncaging comparisons , the intensity of pixels along a line crossing the middle of the spinehead ( or uncaging spot if no detectable spine appeared ) was measured across consecutive images ( ImageJ , NIH ) and expressed as percentage of maximum fluorescence reached in the uncaging locus 1–2 min following induction .", "For dendritic spine density analyses , stacks through secondary and tertiary dendrites were acquired , coded and analyzed in MATLAB ( MathWorks , Natick , MA ) and ImageJ .", "Pharmacological agents were acquired from Tocris ( Bristol , UK ) or Sigma-Aldrich ( St . Louis , MO ) .", "For mEPSC recordings , ACSF contained 1 μM TTX , 50 μM SR 95531/Gabazine , 10 μM CPP , and 10 μM Scopolamine hydrobromide .", "For light evoked EPSC recordings , SR 95531 and CPP were used at the same concentrations as for mEPSC recordings .", "Acute slices were treated with 10 μM SKF 81297 or CGS 21680 .", "In vivo treatment of mice included intraperitoneal or subcutaneous injections of SKF 81297 ( 2 . 5–5 mg/kg ) , CGS 21680 ( 0 . 1 mg/kg ) , quinpirole ( 0 . 5 mg/kg ) , H-89 ( 5 mg/kg ) , istradefylline/KW-6002 ( 2 . 5 mg/kg ) , SKF 83566 ( 0 . 5 mg/kg ) , and RS 67333 ( 3 mg/kg ) .", "Mice were transcardially perfused with 4% paraformaldehyde and brains were post-fixed for 1–5 days , prior to sectioning at 40–50 µm on a Vibratome .", "No immunoenhancement was used to increase the signal of virally transduced fluorescent proteins .", "For tyrosine hydroxylase ( TH ) , NeuN and c-fos immunofluorescence , 1:4 series of coronal sections through the striatum or SNc were incubated overnight at 4ºC with rabbit anti-TH , mouse anti-NeuN , or rabbit anti-c-fos antibody in TBS with 0 . 5% Tween-20 ( 1:500–1000 , all from EMD Millipore , Billerica , MA ) .", "The following day tissue was rinsed in TBS , reacted with goat anti-rabbit or anti-mouse Alexa Fluor 594 ( 1:500 , Molecular Probes , Thermo Scientific ) for 1 hr at RT , rinsed , mounted onto superfrost slides , dried and coverslipped under ProLong antifade reagent with DAPI ( Molecular Probes ) .", "Images were acquired with a Zeiss LSM 510 META or a Leica SP8 confocal microscope ( Harvard NeuroDiscovery Center ) .", "For quantitative estimates of mCherry-Cre and NeuN overlap , slides were coded for confocal microscopy and data analysis .", "Z-stacks matching the dorsal targeting of electrophysiological recordings and viral injections were selected for analysis .", "Two-dimensional 1 µm-thick optical sections were analyzed in ImageJ ( FIJI ) ( Schindelin et al . , 2012 ) .", "The percentage of Cre+ and Cre- among NeuN+ neurons was quantified in both hemispheres .", "HEK293 cells were grown in DMEM containing 5% FBS and 500 µg/ml G-418 ( all Invitrogen ) and maintained at 37°C in an atmosphere of 5%CO2 .", "Cells were plated in 96-well plates at 30 , 000 cells/well and transfected with the SEAP reporter plasmid using Lipofectamine® and PLUS® reagent ( Invitrogen ) .", "The transfection media was replaced with ligand-containing DMEM ( 200 µl/well ) and cells were incubated in the dark for 24 hrs at 37°C with 5%CO2 .", "After transferring 100 µl aliquots from each well to a fresh 96-well plate , 100 µl of an aqueous buffer containing 2 M diethanolamine bicarbonate and 1 . 2 mM methylumbelliferone phosphate , pH 10 . 0 , was added to each well .", "Plates were imaged on a Perkin Elmer Envision plate reader using optical settings for methylumbelliferone fluorescence ( Perkin Elmer , Waltham , MA ) .", "Data reflect an average of 6 condition replicates , run in parallel during two independent runs of 6 replicates each , done on different days .", "Offline analysis of electrophysiology data was performed using Igor Pro ( Wavemetrics , Portland , OR ) and MATLAB .", "Statistical analyses were done using GraphPad PRIZM 5 software ( GraphPad , LaJolla , CA ) .", "All data except for probabilities are expressed as mean +SEM .", "Probabilities are expressed as aggregate probabilities across experiments of the same type .", "For two-group comparisons , statistical significance was determined by two-tailed Student’s t-tests or two-sample Z test for proportions .", "For multiple group comparisons , one-way analysis of variance ( ANOVA ) tests were used for normally distributed data , followed by post hoc analyses .", "For non-normally distributed data , non-parametric tests for the appropriate group numbers were used , such as Mann-Whitney and Kruskal-Wallis .", "p<0 . 05 was considered statistically significant ." ] ]
[ "Dopamine is released in the striatum during development and impacts the activity of Protein Kinase A ( PKA ) in striatal spiny projection neurons ( SPNs ) .", "We examined whether dopaminergic neuromodulation regulates activity-dependent glutamatergic synapse formation in the developing striatum .", "Systemic in vivo treatment with Gαs-coupled G-protein receptors ( GPCRs ) agonists enhanced excitatory synapses on direct pathway striatal spiny projection neurons ( dSPNs ) , whereas rapid production of excitatory synapses on indirect pathway neurons ( iSPNs ) required the activation of Gαs GPCRs in SPNs of both pathways .", "Nevertheless , in vitro Gαs activation was sufficient to enhance spinogenesis induced by glutamate photolysis in both dSPNs and iSPNs , suggesting that iSPNs in intact neural circuits have additional requirements for rapid synaptic development .", "We evaluated the in vivo effects of enhanced glutamate release from corticostriatal axons and postsynaptic PKA and discovered a mechanism of developmental plasticity wherein rapid synaptogenesis is promoted by the coordinated actions of glutamate and postsynaptic Gαs-coupled receptors ." ]
[ "The brain is composed of intricate circuits of connected neurons that communicate via a combination of electrical and chemical signals .", "Some signals ( referred to as excitatory signals ) increase the probability that the neuron receiving the chemical message will produce an electrical impulse .", "On the other hand , inhibitory messages decrease the likelihood of this activity .", "Both of these kinds of signals are fast , and act over milliseconds .", "There is also a diverse set of slower signals , referred to as neuromodulation , which regulates the faster signals .", "A signaling chemical called dopamine is involved in neuromodulation and is essential for rewarding behavior and complex motor actions .", "The importance of dopamine is clear from the profound lack of movement seen in individuals with Parkinson’s disease , which is caused by the death of dopamine producing brain cells .", "Many nerve endings from dopamine-releasing neurons connect to a part of the brain’s reward system called the striatum .", "The neurons in this region are organized into two pathways that have opposing impacts on behavior .", "Dopamine activates different kinds of receptors called “G protein-coupled dopamine receptors” on neurons from each pathway .", "This allows dopamine to alter the activity of a protein called Protein Kinase A ( or PKA ) and alter the signaling state of these neurons .", "The impact of dopamine on neural circuits in adults has been extensively studied .", "However it was unknown whether dopamine might influence how neural circuits are wired during brain development .", "Because the nerve endings from dopamine-releasing neurons reach the striatum before most excitatory connections between the neurons are formed , dopamine stands to influence the development of connections in the striatum .", "Kozorovitskiy et al . have now investigated the role of neuromodulation in brain development in young mice .", "This involved measuring the formation of excitatory connections or synapses and the electrical activity of different striatal neurons during the maturation of brain circuits that occurs after birth .", "This analysis revealed that turning on dopamine receptors that increase PKA activity rapidly enhances the number of excitatory synapses on the neurons that express this receptor .", "Kozorovitskiy et al . then used a variety of approaches to investigate whether there is cooperation between G protein-coupled receptors , PKA activity and a signaling molecule called glutamate in striatal development .", "This revealed a more general mechanism by which the activation of G-protein-coupled receptors interacts with glutamate ( the primary excitatory signal sent between neurons ) in order to produce new synapses .", "These results reveal a previously unknown role for neuromodulation in “wiring up” the brain and open the possibility of new therapies to treat neurodevelopmental and neurodegenerative disorders ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "immunology and inflammation" ]
High mTOR activity is a hallmark of reactive natural killer cells and amplifies early signaling through activating receptors
elife-26423-v2
[ [ "Natural killer ( NK ) cells are group 1 innate lymphoid cells characterized by their ability to kill target cells and to secrete cytokines such as IFN-γ ( Spits et al . , 2013 ) .", "Thereby , they take part in the early response against infected and neoplastic cells .", "Target cell recognition and NK cell activation are controlled by the balance between positive and negative signals arising from the engagement of an array of NK activating receptors ( NKar ) and NK inhibitory receptors ( NKir ) .", "While normal cells express an excess of NKir ligands , stressed cells , such as tumor and infected cells , may lose expression of NKir ligands ( ‘missing-self’ ) or acquire expression of NKar ligands ( ‘modified-self’ ) , thus tilting the balance towards activation .", "NKirs , which mostly recognize classical or non-classical MHC-I molecules , are stochastically expressed , resulting in a variegated expression pattern .", "Depending on the species , three types of NKir interact with MHC-I: Killer Immunoglobulin-like Receptors ( KIR ) in primates , Ly49 receptors of the lectin-like family in rodents and the heterodimer formed by CD94 and NKG2A in these species ( Vivier et al . , 2008 ) .", "In addition , considerable functional heterogeneity is observed in the NK cell population .", "Such cell intrinsic differences led to the proposition that NK cell reactivity and consequently their ability to discriminate self from non-self is the result of an education process ( Anfossi et al . , 2006; Fernandez et al . , 2005; Kim et al . , 2005 ) .", "There was however considerable debate over the molecular process leading to education .", "Two theories were crafted to account for these observations: the first one proposing that a priming ( or arming ) signal was required to confer reactivity to otherwise hyporesponsive cells , the second positing that responsiveness is a default state that is lost upon unopposed chronic stimulation of NKar ( disarming ) ( Höglund and Brodin , 2010 ) .", "The data accumulated so far are in favor of the latter model , suggesting that intrinsic reactivity is lost upon chronic engagement of NKar unless this is opposed by concomitant engagement of NKir .", "Indeed , there is no evidence so far that priming signals are a prerequisite for acquisition of responsiveness .", "In contrast , disarming is the simplest explanation to account for the tolerance to self of NK cells raised in a mosaic or chimeric environment ( Johansson et al . , 1997; Wu and Raulet , 1997 ) .", "Moreover , the loss of reactivity consequent to exposure of NK cells to activating ligands functionally demonstrates the possibility to disarm reactive NK cells ( Oppenheim et al . , 2005; Tripathy et al . , 2008 ) .", "At the molecular level , physical interaction between NKirs and their ligands is required to maintain responsiveness as ( 1 ) only NK cells expressing NKir engaged by MHC-I at the surface of surrounding cells are reactive and as ( 2 ) NK cells expressing NKirs but developing in MHC-I deficient humans or animals are functionally impaired ( Fernandez et al . , 2005; Kim et al . , 2005; Zimmer et al . , 1998 ) .", "In addition , the inhibitory signaling module acting downstream of NKirs is required to maintain reactivity .", "Indeed , mutation of the immunoreceptor tyrosine-based motifs ( ITIM ) of inhibitory Ly49 molecules or deficiency in the phosphatase SHP-1 , recruited to NKirs upon ligation , decreases responsiveness ( Kim et al . , 2005; Viant et al . , 2014 ) .", "Inhibition of the activating signal by NKir thus serves two-distinct but related purposes: it counters inappropriate NK cell activation and it prevents the desensitization induced by chronic stimulation thereby preserving NK cell reactivity .", "In inbred C57BL/6 mice , Ly49C ( specific for H2-Kb ) , Ly49I ( specific for H2-Kb ) and the CD94/NKG2A receptor ( specific for a Db peptide presented by Qa-1 ) have been shown to interact with substantial affinity with self-MHC class I molecules , while other receptors show no or marginal affinity ( Hanke et al . , 1999; Michaëlsson et al . , 2000; Vance et al . , 1998 ) .", "Consequently , NK cell populations expressing these receptors are educated in C57BL/6 mice , that is , they are more reactive than their non-educated counterparts ( Fernandez et al . , 2005; Joncker et al . , 2009; Kim et al . , 2005 ) .", "Education is a dynamic process tuned by the number of engaged NKirs and the strength of each interaction in a rheostat-like manner ( Brodin et al . , 2009a; Johansson et al . , 2005; Joncker et al . , 2009 ) .", "It is also reversible in as little as one or two days as shown in different experimental set-ups ( Ebihara et al . , 2013; Elliott et al . , 2010; Joncker et al . , 2010 ) .", "This suggests the existence of a potent cellular process integrating activating and inhibitory educating signals of variable strength ( i . e . the strength of the NKar or NKir-ligand interaction and number of different interactions over time ) and controlling the display of effector functions in response to NKar stimulation .", "Previous studies have shown that reactive NK cells are characterized by stronger calcium flux and LFA-1 integrin activation upon NKar stimulation ( Guia et al . , 2011; Thomas et al . , 2013 ) .", "However , the nature of the molecular process conditioning NK cell reactivity and negatively affected by chronic engagement of NKar is unknown .", "To address this question , we systematically compared phosphorylation levels of key molecules involved in immunoreceptor tyrosine-based activating motif ( ITAM ) signaling in reactive vs . hyporesponsive NK cells at steady-state and following NKar stimulation .", "We discovered that NK cell reactivity is associated with a higher basal activity of the mammalian target of rapamycin ( mTOR ) pathway .", "Our genetic and pharmacological approaches collectively demonstrate a prominent role of mTOR signaling in controlling steady-state NK cell responsiveness ." ], [ "Seeking to identify molecular pathways involved in NK cell education , we systematically screened the basal levels of 20 phosphorylations on 16 proteins involved in ITAM signaling between reactive and hyporesponsive NK cells by flow cytometry ( complete list in Table 1 ) .", "This flow-cytometry based approach allowed us to combine the advantages of single-cell analysis and comparison of equivalent cell subset thanks to electronic gating .", "In C57BL/6 mice , the main educating NKirs are NKG2A and Ly49C , defining four subsets of which the double-negative display the lowest , the double-positive the highest and the single positives an intermediate responsiveness ( Joncker et al . , 2009 ) .", "We also analyzed B2m−/− NK cells that are uniformly unreactive .", "Most of these phosphorylations are developmentally regulated ( Figure 1—figure supplement 1 ) , thus , to exclude any developmental bias , we compared similar developmental stages defined by CD11b and CD27 ( Figure 1—figure supplement 2 ) .", "Strikingly , all analyzed phosphorylations in the Akt/mTOR pathway correlated positively with the level of NK cell reactivity ( Figure 1A ) .", "This was true when comparing C57BL/6 and B2m−/− NK cells as well as reactive and unreactive populations in C57BL/6 mice , regardless of the maturation stage .", "In C57BL/6 populations , absence of either NKG2A or Ly49C had a measurable negative effect , the absence of both leading to further decrease in the phosphorylation level .", "We also noted a significant correlation between education status and the level of pNFκB S529 and S468 as well as pLck Y505 and pItk Y180 ( Figure 1A ) .", "However , as the most consistent differences lied in the Akt/mTOR pathway , we decided to focus our analysis on this pathway .", "The phosphatase SHP-1 is required to maintain an optimal NK cell reactivity ( Viant et al . , 2014 ) .", "To test its involvement in the maintenance of the basal activity of the Akt/mTOR pathway , we measured the phosphorylation levels of the ribosomal S6 protein and Akt in NK cells deficient in Ptpn6 , the gene encoding SHP-1 .", "As a control , we also measured the level of phosphorylation of STAT5 in these cells .", "The basal activity of the Akt/mTOR pathway was specifically decreased in NK cells from Ncr1iCre/+ Ptpn6lox/lox mice compared to control NK cells while pSTAT5 levels were unchanged ( Figure 1B ) .", "Thus , basal activation of the Akt/mTOR pathway is correlated with NK cell reactivity and controlled by SHP-1-dependent signaling downstream of NKirs .", "We next compared mTOR-related signaling events arising from NKar stimulation in reactive versus hyporesponsive NK cells .", "To this end , we stimulated splenocytes from C57BL/6 ( around 85% of NK cells are reactive in these mice ) and B2m−/− mice by crosslinking NK1 . 1 and we measured phosphorylation events over time .", "Phosphorylation of Akt on T308 and S473 and phosphorylation of the ribosomal protein S6 were induced at higher levels in reactive NK cells compared to hyporesponsive NK cells ( Figure 1C , D ) .", "By contrast , other signaling events not linked to the mTOR pathway were induced at similar levels ( Figure 1C , D and Figure 1—figure supplement 3 ) .", "In summary , high activity of the Akt/mTOR pathway is a hallmark of reactive NK cells both at steady-state and following stimulation through NKars .", "Importantly , considering that education is not a discrete but rather a continuous process , absence of one or two of the educating NKir in C57BL/6 resulted in a commensurate loss in mTOR activity .", "Education is rapidly reverted by unopposed chronic stimulation .", "Indeed , transfer of reactive NK cells into a host devoid of MHC-I leads to their rapid loss of reactivity and to their tolerance to MHC-I negative cells ( Joncker et al . , 2010 ) .", "We thus sought to test whether chronic NKar stimulation decreased the activity of the Akt/mTOR pathway in parallel with the decrease of reactivity .", "To this purpose , we transferred reactive C57BL/6 NK cells into control C57BL/6 or B2m−/− mice and measured basal Akt/mTOR phosphorylation levels and their reactivity 3 days after transfer .", "To quantify the intensity of NKar signaling , we took advantage of a transcriptional reporter of the TCR signaling ( Moran et al . , 2011 ) .", "This reporter consists of a GFP under the control of the promoter sequence of Nur77 , an orphan nuclear receptor strongly induced in response to TCR stimulation .", "The signaling pathways triggered by TCR or NKar engagement mobilizing the same signaling adaptors , we reasoned that the Nur77GFP construct might also report NKar triggering .", "Indeed , in vitro stimulation with an NK1 . 1 agonist antibody or YAC-1 cells , a lymphoblastic cell line detected as foreign by C57BL/6 NK cells , resulted in an increase in the GFP fluorescence ( Figure 2—figure supplement 1 ) .", "Moreover , this increase was commensurate with reactivity so that higher GFP levels were reached in reactive NKG2A+Ly49C+ NK cells , thus validating the expression of GFP as a reporter of NKar stimulation .", "Transfer of Nur77GFP cells into B2m−/− mice resulted in a transient increase in the GFP level in the reactive subsets one day after transfer indicative of ongoing NKar signaling ( Figure 2A ) .", "Interestingly , this was followed , 3 days after transfer , by a significant decrease in steady-state GFP level indicative of a loss of the cell capacity to signal following NKar stimulation .", "As previously reported , NK cells transferred into B2m−/− mice lost their reactivity while reactivity was maintained upon transfer into C57BL/6 host ( Figure 2B , anti-NK1 . 1 stimulation and Figure 2—figure supplement 2 , anti-NKp46 or YAC1 stimulation ) .", "Importantly , this was paralleled by a decrease in the phosphorylation of S6 and Akt S473 and a loss of the gradient observed between the different subsets expressing Ly49C and NKG2A ( Figure 2C ) .", "Collectively , these results demonstrate that the basal activity of the Akt/mTOR pathway is negatively affected by persistent and unopposed NKar stimulation .", "This suggests that engagement of Ly49C and NKG2A in C57BL/6 mice preserves Akt/mTOR basal activity resulting in higher basal phosphorylation in the NK cell population expressing these NKir .", "To test if high mTOR activity was required for NK cell reactivity , we stimulated NK cells from Ncr1iCre/+ Mtorlox/lox or control mice with plate-bound anti-NK1 . 1 antibody or YAC-1 cells and measured NK cell degranulation relative to the expression of the major educating receptors Ly49C and NKG2A .", "Control NK cells responded significantly better than mTOR-deficient NK cells , irrespective of the subset analyzed ( Figure 3A ) .", "Moreover , within control NK cells , reactive Ly49C+NKG2A+ degranulated more than the other subsets , while mTOR deficiency resulted in equally hyporesponsive subsets .", "These results suggested a major role of mTOR in NK cell reactivity .", "However , mTOR deficiency leads to a severe NK cell developmental block that may confound the interpretation of the results ( Marçais et al . , 2014 ) .", "To address this issue we took advantage of Torin2 , a highly selective ATP-competitive mTOR inhibitor targeting both mTORC1 and mTORC2 ( Liu et al . , 2011 ) .", "We stimulated mature NK cells from C57BL/6 and B2m−/− mice with plate-bound anti-NK1 . 1 in the presence or absence of the inhibitor .", "Torin2 significantly decreased the capacity of C57BL/6 NK cells to produce IFN-γ and to degranulate upon stimulation , regardless of the subset analyzed ( Figure 3B ) .", "Moreover , treatment of C57BL/6 NK cells with Torin2 abrogated the differences between highly reactive ( Ly49C+NKG2A+ ) and hyporesponsive ( Ly49C-NKG2A- ) cells .", "Treatment of hyporesponsive B2m−/− NK cells led to a further decrease in their capacity to degranulate while their production of IFN-γ was unaffected .", "Similar results were obtained upon NKp46 stimulation ( Figure 3—figure supplement 1 ) .", "Torin2 treated C57BL/6 NK cells thus functionally behaved like B2m−/− hyporesponsive NK cells .", "Similarly , Torin2 inhibited C57BL/6 NK cells from triggering YAC-1 lysis at a similar level seen in hyporesponsive B2m−/− NK cells ( Figure 3C ) .", "Torin2 treatment had no effect on the lytic capacity of B2m−/− NK cells .", "Education conditions the phenomenon of missing-self recognition .", "A classical readout to highlight this property is to measure the rate of rejection of MHC-I negative target cells in vivo .", "To test whether basal activity of the Akt/mTOR pathway was involved in this process , we transferred a mix of C57BL/6 and NK-sensitive MHC-I negative ( B2m−/− ) target cells into C57BL/6 mice , previously treated or not with Torin2 .", "While injection into control mice led to the disappearance of 50% of the target cells , this rejection was abrogated in Torin2 treated animals , underlining the importance of mTOR activity in NK cell recognition of missing-self under steady-state conditions ( Figure 3D ) .", "Altogether , these results demonstrate that mTOR is required for NK cell reactivity .", "The ‘rheostat’ model of education proposes that the strength of the MHC-I input translates into a quantitative modification of NK cell responsiveness ( Brodin et al . , 2009b ) .", "Indeed , several studies reported that the higher the number of self–MHC-I receptors expressed by NK cells interacting with their ligands , the stronger their responsiveness ( Brodin et al . , 2009a; Johansson et al . , 2005; Joncker et al . , 2009 ) .", "As shown in Figure 1 , the level of mTOR activity was tightly correlated with the number of educating NKirs in NK cells , suggesting that mTOR could serve as the molecular rheostat translating the MHC-I input into quantitative tuning of the responsiveness .", "To directly test this point , we analyzed how the ex vivo modulation of mTOR activity by pharmacologic mTOR inhibitors changed NK cell responsiveness .", "We took advantage of four different inhibitors of graded mTOR inhibitory potential: the macrolide Rapamycin that primarily inhibits mTORC1 and three ATP-competitive inhibitors targeting both mTORC1 and mTORC2 to a varying extent: AZD2014 , KU-0063794 ( KU ) and Torin2 ( García-Martínez et al . , 2009; Guichard et al . , 2015; Liu et al . , 2011; Sabatini et al . , 1994; Yang et al . , 2013 ) .", "The use of different concentrations of those compounds allowed us to modulate mTOR activity in NK cells over a dynamic range of 10-fold for mTORC1 or 2-fold for mTORC2 as measured by phosphorylation of S6 and Akt S473 respectively ( Figure 4A ) .", "Of note , we confirmed that Rapamycin acted specifically on mTORC1 while AZD , KU and Torin2 inhibited both complexes .", "Importantly , at these concentrations no significant changes in STAT5 phosphorylation or specific toxicity over a 24 hr incubation period were noted ( Figure 4—figure supplement 1A and B ) .", "We then correlated the S6 and Akt phosphorylation levels to the IFN-γ production and degranulation induced by NK1 . 1 crosslinking .", "S6 phosphorylation was positively correlated with the effector functions in all conditions tested ( Figure 4B ) .", "Similar correlations were found between Akt phosphorylation and effector function upon AZD , KU or Torin2 treatment ( Figure 4C ) .", "However , this correlation was lost upon Rapamycin treatment , suggesting that mTORC2 activity alone is not sufficient to sustain effector functions ( Figure 4B , C ) .", "In addition , effector functions were not correlated to STAT5 phosphorylation levels ( Figure 4—figure supplement 1B , C ) .", "Similar results were obtained upon stimulation of NK cells from Ncr1iCre and Ncr1iCre Mtorlox/lox mice and measure of the phosphorylation levels of the S6 and Akt proteins in parallel thus genetically confirming the results ( Figure 4—figure supplement 1D ) .", "Overall , these results demonstrate that mTOR acts as a molecular rheostat of NK cell responsiveness .", "Together with results in Figures 1 and 2 , they demonstrate that NK cell education relies on the modulation of mTOR activity that in turn controls NK cell responsiveness through NKars .", "Next , we asked whether mTOR activity could regulate signaling via NKar .", "Previous studies established that reactive NK cells display higher calcium flux ( Guia et al . , 2011 ) and higher integrin activation than hyporesponsive NK cells ( Thomas et al . , 2013 ) .", "Hence we sought to test the impact of mTOR activity on these cardinal events in lymphocyte activation .", "We first measured the calcium flux in real time by flow cytometry following NK1 . 1 stimulation using fluorescent calcium probes and we quantified the intensity of the fluorescence peak .", "When we challenged Ncr1iCre/+ ( control ) and Ncr1iCre/+ Mtorlox/lox NK cells , NK1 . 1 cross-linking resulted in a detectable calcium flux in NK cells of both genotypes ( Figure 5A ) .", "However , the peak was lowered ( 15–20% ) in the absence of mTOR .", "We next applied the same protocol to control C57BL/6 NK cells in the presence or absence of Torin2 to acutely inhibit mTOR .", "As shown in Figure 5B , mTOR inhibition resulted in a decreased calcium flux characterized by a 20%-decrease in the peak intensity , thus phenocopying the impact of mTOR deficiency .", "Next , we assessed the effect of mTOR deficiency on LFA-1 integrin activation following NKar triggering of inside-out signaling .", "For this purpose , we incubated NK cells from Ncr1iCre/+ and Ncr1iCre/+ Mtorlox/lox mice with beads coated with ICAM-1 , the ligand of LFA-1 , in the presence or absence of NK1 . 1 cross-linking .", "At different times , we measured by flow-cytometry the percentage of beads-associated NK cells as an indicator of LFA-1 activation in NK cells .", "As shown in Figure 5C , NK1 . 1 cross-linking failed to induce LFA-1 activation in mTOR-deficient NK cells contrary to control NK cells .", "In parallel , we also tested the effect of acute mTOR inhibition on LFA-1 activation in mature educated NK cells .", "As shown in Figure 5D , addition of Torin2 resulted in significant inhibition of LFA-1 activation induced by NK1 . 1 stimulation .", "Thus , using genetic and pharmacological tools , we showed that the mTOR pathway lies upstream of two signaling events , calcium flux and LFA-1 integrin activation , which are elevated in reactive NK cells .", "mTOR is a well-known regulator of the cell metabolism .", "We thus asked whether the higher activity of mTOR measured in reactive NK cells resulted in detectable changes in metabolic activity .", "We first measured cell size and granularity using the FSC and SSC flow-cytometry parameters .", "Reactive NK cells from C57BL/6 control mice presented a slight but significant increase of both morphological indicators when compared to hyporesponsive NK cells of B2m−/− mice ( Figure 6A ) .", "Similarly , their mitochondrial content as well as glucose and fatty-acid uptake capacities estimated by measure of the uptake of the glucose fluorescent analog 2-NBDG or the fatty-acid fluorescent analog Bodipy FL-C16 were significantly higher ( Figure 6B ) .", "In contrast , mitochondrial ROS production , lipid droplet content or lipid peroxidation were comparable in both cell types ( data not shown ) .", "Differences were also detectable for FSC and SSC values as well as fatty-acid uptake when comparing reactive and hyporesponsive NK cell subsets present in the most mature CD27low population of C57BL/6 mice ( Figure 6C ) .", "In summary , the higher activity of the Akt/mTOR pathway observed in reactive cells increased their metabolic activity compared to hyporesponsive NK cells , which may also contribute to their enhanced responsiveness .", "Several studies have demonstrated that hyporesponsive NK cells can be rendered reactive ( Ebihara et al . , 2013; Elliott et al . , 2010; Joncker et al . , 2010; Sun and Lanier , 2008 ) .", "The underlying molecular mechanism has however remained elusive .", "We reasoned that if the mTOR pathway was really a key determinant of NK cell reactivity , acute activation of this pathway should immediately restore reactivity of hyporesponsive cells .", "To test this hypothesis , we stimulated NK cells from C57BL/6 or B2m−/− mice with plate-bound antibodies stimulating NK1 . 1 or NKp46 and we simultaneously added IL-2 , a cytokine known to potently activate mTOR ( Marçais et al . , 2014 ) .", "To test the requirement for the mTOR pathway in this process , cells were also treated or not with Torin2 .", "IL-2 resulted in an increase of the cell capacity to produce IFN-γ and to degranulate as measured by CD107a exposure ( Figure 7A ) .", "This acute treatment was sufficient for hyporesponsive cells to acquire a level of reactivity equal or even higher than that of reactive NK cells from C57BL/6 , regardless of the stimulating antibody .", "mTOR activity was required for this effect since the increase in reactivity was suppressed by mTOR inhibition ( Figure 7A ) .", "Similar results were obtained when using IL-15 instead of IL-2 ( Figure 7—figure supplement 1 ) .", "Acute IL-15 stimulation also restored the cytotoxic activity of hyporesponsive NK cells against YAC-1 cells while further enhancing cytotoxicity of C57BL/6 cells ( Figure 7B ) .", "Again , this effect was completely reversed upon concomitant Torin2 treatment .", "Taken together , these results show that induction of responsiveness in NK cells upon cytokine exposure is a rapid phenomenon acting via mTOR activation .", "In order to decipher the mechanism required for NK cell re-education , we next tested whether acute IL-15 treatment restored early signaling in hyporesponsive cells .", "We first investigated the impact of IL-15 treatment on the calcium flux triggered by NK1 . 1 stimulation in control or hyporesponsive NK cells .", "As expected , NK1 . 1 stimulation of hyporesponsive NK cells resulted in a very poor calcium flux compared to reactive NK cells ( Figure 7C ) .", "Strikingly , treatment with IL-15 increased the calcium flux ability of reactive and hyporesponsive NK cells in an mTOR-dependent way ( Figure 7C and Figure 7—figure supplement 2 ) .", "We then measured the impact of IL-15 treatment on LFA-1 activation following NK1 . 1 stimulation .", "The presence of IL-15 in the assay rendered hyporesponsive NK cells able to activate LFA-1 upon NK1 . 1 stimulation and bind ICAM-1 coated beads ( Figure 7D ) .", "This effect was strongly decreased upon Torin2 treatment , underlying the non-redundant role of mTOR in this process .", "Altogether , these results show that acute stimulation of the mTOR pathway can restore the ability of hyporesponsive NK cells to induce calcium flux and activate LFA-1 upon NKar engagement , thereby re-establishing their reactivity ." ], [ "Here , to gain mechanistic insight into the phenomenon of NK cell education , we explored signal transduction pathways downstream NKars in reactive and hyporesponsive NK cells .", "We found that the activity of the Akt/mTOR pathway was selectively higher in reactive NK cells .", "This was characterized by higher basal phosphorylation of direct and indirect targets of both mTOR complexes ( mTORC1 and 2 ) in strict correlation with the reactivity level .", "In addition , this pattern was lost concomitantly with the loss of reactivity observed upon transfer of reactive cells in B2m−/− hosts .", "Our screen also revealed that two out of the three NFκB p65 phosphorylations investigated ( S468 and S529 ) correlated with reactivity .", "This could be the result of the heightened Akt/mTOR pathway activity , as mTORC2 has been involved in NFκB activation during CD4 T cell stimulation ( Lee et al . , 2010 ) .", "Alternatively , this could reveal the involvement of other pathways in the control of NK cell reactivity .", "What is the extracellular signal and the signaling pathway responsible for the maintenance of mTOR basal activity in reactive NK cells specifically ?", "An obvious candidate would be IL-15 as this cytokine is a privileged activator of this pathway ( Marçais et al . , 2014 ) .", "However , pSTAT5 levels were identical between reactive and hyporesponsive NK cells ( data not shown ) .", "Moreover , in vivo treatment with antibodies blocking IL-15 signaling did not alter NK cell education ( data not shown ) .", "Finally , it is difficult to envisage how reactive cells would get preferential access to IL-15 .", "Instead , in line with the disarming hypothesis , we would favor a model in which basal mTOR activity is set independently of education signals .", "This initial activity would then be decreased by disarming signals .", "How mTOR activity is decreased by chronic NKar stimulation is still an open question .", "We hypothesize that in the absence of surrounding MHC-I or in NK cells lacking functional NKirs , unopposed NKar signaling could lead to shut-down of the Akt/mTOR pathway due to depletion of necessary intermediates or establishment of negative feedbacks as it has been demonstrated in the case of induction of resistance to insulin ( Um et al . , 2006 ) .", "Engagement of NKirs would prevent this desensitization and maintain an optimal activity of the pathway .", "In favor of this hypothesis , we show that SHP-1 , the phosphatase triggered by NKir ligation and necessary to maintain NK cell reactivity ( Viant et al . , 2014 ) , was required to maintain an optimal activity of the mTOR pathway .", "Furthermore , transfer of Nur77GFP cells into B2m−/− hosts was accompanied by an increase in the GFP level , evidence of active NKar signaling , and followed by the loss of mTOR basal activity concomitant with the loss of reactivity of NKG2A+Ly49C+ NK cells .", "Previous studies have conclusively shown that NK cell education is not an on-off switch but rather a variation on a continuous axis ( Brodin et al . , 2009a; Joncker et al . , 2009 ) .", "We propose that the mTOR pathway is the long-sought molecular rheostat able to both respond to educating signals and control effector functions in return .", "Indeed , we showed that activity of the Akt/mTOR pathway is regulated commensurate with the level of NKir engagement by MHC-I molecules .", "Furthermore , we demonstrated that modulation of mTOR activity by exogenous cytokine or pharmacologic treatments was directly correlated with NK cell responsiveness .", "Furthermore , mTOR could also regulate NK cell responsiveness by integrating signals beyond NKir ligands .", "Considering the concept of the extended rheostat model as described initially by Höglund and colleagues ( Brodin et al . , 2009b ) , we envision extracellular inputs in an extended sense , including immunological as well as purely metabolic inputs .", "Interestingly , a number of environmental conditions , such as the presence of inflammatory ( Sun and Lanier , 2008 ) or anti-inflammatory cytokines ( Sungur et al . , 2013 ) , but also the presence of nutrients ( Keppel et al . , 2015 ) , impact on NK cell responsiveness .", "All these stimuli positively or negatively affect mTOR activity ( Efeyan et al . , 2015; Marçais et al . , 2014; Sinclair et al . , 2013; Viel et al . , 2016 ) .", "mTOR activity could thus be the nexus targeted by these different stimuli which would explain their impact on NK cell responsiveness .", "Thus , considering mTOR as the rheostat of NK cell responsiveness would help to build a common conceptual framework in which these observations could be ordered .", "Finally , we also present evidence on how mTOR activity affects NK cell effector functions .", "We demonstrated that mTOR activity controls two distinct events characterizing reactive NK cells and required for the triggering of effector functions: Ca2+ flux and integrin activation .", "How could mTOR activity control such apparently unrelated signaling events ?", "Depending on the relative involvement of mTORC1 or mTORC2 , several possibilities can be considered .", "First , the fact that Rapamycin which specifically inhibits mTORC1 is sufficient to decrease responsiveness unmasks the non-redundant role of this complex .", "In line with the role of mTORC1 in the control of cellular metabolism , we described that higher basal mTOR activity in educated cells translated into higher basal metabolism as measured by morphological parameters as well as glucose and fatty-acid uptake and mitochondrial content .", "We and others have described the necessary role of the mTORC1-dependent metabolism in the development of NK cell effector functions ( Donnelly et al . , 2014; Marçais et al . , 2014 ) .", "In addition to improving the cellular fitness , metabolism could directly modulate signaling by controlling the availability of key intermediates as recently described for Th17/Treg differentiation ( Araujo et al . , 2017 ) .", "Another possibility would be through the regulation of the actin cytoskeleton .", "Indeed , an emerging mode of lymphocyte signaling regulation is through cytoskeleton-dependent regulation of membrane receptors compartmentalization ( Mattila et al . , 2016 ) , a process that has been proposed to explain the reactivity of educated NK cells ( Guia et al . , 2011 ) .", "mTORC2 has been shown to regulate the cytoskeletal organization ( Huang et al . , 2013; Sarbassov et al . , 2004 ) and could therefore prime reactive NK cells by cytoskeletal modifications .", "An interesting parallel can also be drawn with T cell anergy .", "Indeed , TCR stimulation in the absence of CD28 co-stimulation results in T cell hyporesponsiveness to further re-stimulation .", "Numerous studies have shown that the precise control of mTOR activity is at the heart of this phenomenon ( Chappert and Schwartz , 2010; Marcais et al . , 2014; Zheng et al . , 2007; 2009 ) .", "Interestingly , this state is characterized by defective Ca2+ flux ( Dubois et al . , 1998 ) .", "Further resembling hyporesponsive NK cells , treatment of anergic T cells with IL-2 restores their responsiveness , an event that relies on mTOR activation ( Dubois et al . , 1998; Zheng et al . , 2007 ) .", "Ca2+ flux is classically triggered by IP3-induced release of endoplasmic reticulum stores which , upon detection by the STIM1/2 sensors , leads to opening of the ORAI channels present on the plasma membrane and extracellular Ca2+ entry ( Hogan and Rao , 2015 ) .", "In addition , an underestimated Ca2+ store is the endo-lysosomal compartment ( Morgan et al . , 2011 ) , which constitutes a further link with mTOR since mTORC1 is activated on the lysosomal surface and positively regulated by lysosomal nutrients ( Efeyan et al . , 2015 ) as well as by calcium release from lysosomal stores ( Li et al . , 2016 ) .", "Concerning regulation of integrin activation , a putative link would be through the inhibition of GSK3β .", "Indeed , this kinase is inhibited by Akt following mTORC2 activation ( Hagiwara et al . , 2012 ) , and a recent study showed that its inhibition leads to better ability of NK cells to form conjugate via integrin activation ( Parameswaran et al . , 2016 ) .", "In addition , PKCθ , a target of mTORC2 ( Lee et al . , 2010 ) , activates WIP via S488 phosphorylation in lymphocytes ( Fried et al . , 2014 ) .", "Since a macro-complex involving WIP , WASp , actin and myosin IIa has been defined in NK cells ( Krzewski et al . , 2006 ) , WIP activation could explain better interaction with ICAM-1-coated beads in our assay and ultimately better docking to target cell .", "In summary , these findings identify the activity of the mTOR pathway as the molecular rheostat responsible for the control of basal NK cell reactivity in response to NKir ligation .", "In addition , this provides a molecular basis for a number of previous experiments showing that NK cell education can be overcome by cytokine treatment .", "Finally , our data underline the extreme versatility of the regulation of NK cell responsiveness and further point to mTOR as a valid target for the manipulation of NK cells for therapeutic purposes ." ], [ "Wild-type C57BL/6 mice were purchased from Charles River Laboratories ( L’Arbresle ) .", "B2m−/− ( Koller et al . , 1990 ) , Ncr1iCre/+ Mtorlox/lox ( Marçais et al . , 2014 ) and Ncr1iCre/+ Ptpn6lox/lox mice ( Viant et al . , 2014 ) were previously described , littermate control mice were used as controls .", "Nur77GFP mice were previously described ( Moran et al . , 2011 ) .", "Female mice 8 to 24 week-old were used .", "Nur77GFP splenocytes were injected i . v . in C57BL/6 or B2m−/− host .", "Each host received 25 × 106 splenocytes labeled with CTV ( 1 µM , Molecular Probes ) to allow subsequent identification .", "Host mice were sacrificed one or 3 days after for analysis of the spleen .", "This study was carried out in accordance with the French recommendations in the Guide for the ethical evaluation of experiments using laboratory animals and the European guidelines 86/609/CEE .", "All experimental studies were approved by the bioethic local committee CECCAPP .", "Mice were bred in the Plateau de Biologie Expérimentale de la Souris , our animal facility .", "Single cell suspensions of spleens were obtained and stained .", "Intracellular stainings for phosphorylated proteins were done using Lyse/Fix and PermIII buffers ( BD Bioscience ) .", "Measurement of glucose uptake was performed as described ( Marçais et al . , 2014 ) .", "Mitochondrial content was measured using Mitotracker Green ( Molecular Probes , 1 µM ) incubated for 10 min at 37°C in PBS .", "Lipid uptake was measured using BodipyFL C16 ( Molecular Probes , 1 µM ) incubated for 30 min at 37°C in complete medium .", "Surface staining were then performed to identify the different populations .", "Flow cytometry was carried out on a FACS LSR II or on a FACS Fortessa ( Becton-Dickinson ) .", "Data were analysed using FlowJo ( Treestar ) .", "The following mAbs from eBioscience , BD Biosciences or Biolegend were used: anti-CD19 ( ebio1D3 ) , anti-CD3 ( 145–2 C11 ) , anti-NK1 . 1 ( PK136 ) , anti NKp46 ( 29A1 . 4 ) , anti-CD49b ( DX5 ) , anti-CD11b ( M1/70 ) , anti-CD27 ( LG . 7F9 ) , anti-Ly49I ( YLI90 ) , anti-NKG2A/C/E ( 20d5 ) , anti-IFN-γ ( XMG1 . 2 ) , anti-CD107a ( 1D4B ) .", "The mAb 4LO3311 recognizing Ly49C was purified on protein A column from supernatant of the 4LO3311 hybridoma generously provided by Pr .", "Suzanne Lemieux ( Institut Armand Frappier , Québec ) .", "NKG2A positive cells were identified using the 20d5 clone which also recognizes NKG2C and NKG2E , however , since mouse resting NK cells only express NKG2A , we considered 20d5 reactive cells as NKG2A positive ( Vance et al . , 1998 ) .", "1 . 5 × 106 splenocytes were cultured on antibody coated plates ( anti-NKp46 ( Goat polyclonal , R&D ) , anti-NK1 . 1 ( PK136 , BioXCell ) at 10 µg/ml on Immulon 2HB or Nunclon plates ) with Golgi-stop ( BD Biosciences ) in the presence of anti-CD107a for 4 hr .", "Cytokines and mTOR inhibitors were used at the following concentrations unless otherwise stated: rmIL-15 ( Peprotech; 100 ng/ml ) , IL-2 ( muIL-2 supernatant; 200 U/ml ) , Rapamycin ( Calbiochem; 25 nM ) , KU-0063794 ( Stemgent; 3 µM ) , AZD2014 ( Selleckchem; 5 µM ) and Torin2 ( Tocris; 250 nM ) .", "Surface and intracellular stainings were then performed and IFN-γ production as well as CD107a exposure was measured by flow cytometry .", "In some experiments , cell viability was determined using 7AAD ( Invitrogen , 250 nM ) .", "For phospho-flow stainings following short-term NK1 . 1 stimulation , 3 × 106 splenocytes were stimulated using biotinylated NK1 . 1 ( PK136 , 5 µg/ml ) followed 1 min 30 s later by streptavidin ( Life Technologies , 10 µg/ml ) and fixed by addition of 10 volumes of Lyse/Fix at the indicated time point .", "Recipient mice were treated by daily i . p . injection of Torin2 ( 10 mg/kg , vehicle: 40% H2O , 40% PEG400 ( Sigma ) , 20 % N methyl two pyrrolidone ( Sigma ) ) for 6 days prior to target transfer .", "Splenocytes from C57BL/6 or B2m−/− mice were labeled respectively with CellTraceViolet ( 1 µM ) or CFSE ( 5 µM ) ( both from Life Technologies ) , and 10 × 106 cells ( 5 × 106 of each genotype ) were transferred by i . v . injection .", "60 hr after transfer , splenocytes were isolated and analyzed by FACS .", "Percentage of remaining B2m−/− cells was calculated using the following formula: % remaining cells = 100 x ( number B2m−/− cells/number C57BL/6 cells ) at 60 h / ( number B2m−/− cells/number C57BL/6 cells ) in input mix .", "NK cells were first enriched by negative depletion prior to killing assay .", "Briefly , splenocytes suspension were incubated with biotinylated mAb against: CD3 ( 14–2 C11 ) , TCRβ ( H57-597 ) , TCRγδ ( GL3 ) , CD19 ( ebio1D3 ) , TER-119 ( ter119 ) ( eBioscience ) , followed by incubation with anti-biotin microbeads ( Miltenyi ) , and enrichment by magnetic separation on an AutoMACS .", "Enriched NK cells were co-cultured for 4 hr with YAC-1 cells labeled with CFSE ( Life Technologies ) at different Effector to target ( E/T ) ratios calculated based on the cell number and the percentage of NK cells after purification .", "The percentage of dead cells within CFSE positive YAC-1 cells was measured by flow cytometry after staining with 7AAD .", "Calcium flux was measured essentially as described ( Guia et al . , 2011 ) .", "Briefly , RBC-lysed splenocytes suspension in RPMI/0 . 2% BSA/25 mM HEPES were stained at RT with the following mAb: anti-CD3/CD19 PEeFluor610 , anti-CD49b APC , anti-CD11b APCCy7 , anti-CD27 PE .", "They were then stained at 1 × 107 cells/ml with Indo-1 ( 1 µM , Life Technologies ) for 30 min at 37°C and washed two times at 4°C .", "They were resuspended in the above medium and placed at 37°C for 30 min prior acquisition in the presence or absence of rmIL-15 ( 100 ng/ml ) or Torin2 ( 250 nM ) .", "Samples were acquired on a LSRII ( BD ) as follow: 15 s baseline acquisition , addition of anti-NK1 . 1 biotin ( PK136 , 5 µg/ml ) , acquisition for 1 min 30 s , addition of Streptavidin ( Life Technologies , 10 µg/ml ) and , acquisition for another 3–5 min .", "One mg Protein G-coated 4–4 . 9 µm beads ( Spherotec ) was incubated for 30 min with 3 . 5 µg ICAM1-hIgG1Fc ( R&D ) on a rotating wheel at RT in PBS .", "Beads were then pelleted by centrifugation and washed two times with complete medium , counted on a FACS Accuri ( BD ) and resuspended at 1 × 107 beads/ml .", "In parallel , NK cells were purified ( 80–90% purity ) using biotinylated antibodies directed against CD3 , CD19 , CD5 , CD24 , F4/80 and Ly6G and anti-biotin beads .", "They were then incubated with anti-NKp46-PE ( 29A1 . 4 , BD ) and purified anti-NK1 . 1 ( PK136 , BioXCell ) .", "100 , 000 purified NK cells in 10 µl were placed in a U-bottom well and 100 , 000 ICAM-1 coated beads were added .", "To cross-link NK1 . 1 and measure the effect of inside-out signaling , a Goat F ( ab ) ’2 anti-mouse IgG ( 10 µg/ml , Life Technologies ) was added to the wells .", "Interaction was fixed at the indicated time-point by addition of 100 µl Cytofix/Cytoperm ( BD ) .", "The percentage of interaction ( i . e . percentage of NKp46 positive cells attached to beads ) was measured by flow cytometry .", "Statistical analyses were performed using Prism 5 ( Graph-Pad Software ) .", "Two tailed unpaired t-test , and ANOVA tests with Bonferroni correction were used as indicated in the figure legends .", "Significance is indicated as follows: *p<0 . 05; **p<0 . 01; ***p<0 . 001 .", "The heatmap presented in Figure 1A was established as follow: we first selected the phosphoepitopes for which the MFI ( Mean Fluorescence Intensity ) was significantly above the one of the FMO control ( Student T-test ) .", "The MFI of the 15 selected phosphoepitopes for the 4 NC sub-populations defined in Figure 1—figure supplement 2 was then normalized to the MFI value of the NKG2A+Ly49C+ populations in the C57BL/6 mice and the values obtained were averaged to calculate the means for each populations .", "These values were used to establish the Heatmap using the Multiple Experiment Viewer application .", "We used the R statistical language to manage our database and carry out the statistical analysis ( R version 3 . 3 . 2 ) .", "We splited the database into six datasets ( 2 Mouse strains * Differentiation subsets ) , each containing the 15 phospho-epitopes .", "We performed an ANOVA for each phospho-epitope to test for the phosphorylation difference between the 4 NC sub-populations .", "The parameters of the ANOVA Type I SS were adapted to control for the experiment effect .", "The Bartlett Homogeneity of Variances Test was applied first , when it failed to reject its H0 , then the phospho-epitope was retained for the ANOVA test .", "The normality of the residuals of the ANOVA model was checked graphically and numerically with the Shapiro-Wilk Normality Test .", "When this test failed to reject its H0 then the adjusted P values for multiple comparisons were extracted with the Tukey's 'Honest Significant Difference' method ." ] ]
[ "NK cell education is the process through which chronic engagement of inhibitory NK cell receptors by self MHC-I molecules preserves cellular responsiveness .", "The molecular mechanisms responsible for NK cell education remain unclear .", "Here , we show that mouse NK cell education is associated with a higher basal activity of the mTOR/Akt pathway , commensurate to the number of educating receptors .", "This higher activity was dependent on the SHP-1 phosphatase and essential for the improved responsiveness of reactive NK cells .", "Upon stimulation , the mTOR/Akt pathway amplified signaling through activating NK cell receptors by enhancing calcium flux and LFA-1 integrin activation .", "Pharmacological inhibition of mTOR resulted in a proportional decrease in NK cell reactivity .", "Reciprocally , acute cytokine stimulation restored reactivity of hyporesponsive NK cells through mTOR activation .", "These results demonstrate that mTOR acts as a molecular rheostat of NK cell reactivity controlled by educating receptors and uncover how cytokine stimulation overcomes NK cell education ." ]
[ "The cells of the immune system patrol the body to detect and destroy harmful microbes and diseased cells .", "Natural killer cells are immune cells with a natural capacity to kill infected or cancerous cells , as their name suggests .", "Importantly , they do so while sparing the surrounding healthy cells .", "As natural killer cells mature they go through an “education” process to learn to distinguish between normal and abnormal cells .", "During education , the natural killer cells interact continuously with nearby healthy cells .", "However , it remains unknown how these interactions change the natural killer cells , or how these changes control their killing activity .", "Marçais et al . now show that a protein called mTOR is essential to the education of natural killer cells .", "Comparing natural killer cells that had or had not completed the education process revealed that mTOR is more active in the educated cells .", "Moreover , inhibiting the activity of mTOR caused educated natural killer cells to lose their ability to identify diseased cells , while stimulating mTOR activity in uneducated natural killer cells mimicked the education process , allowing them to recognize and eliminate diseased host cells .", "Certain nutrients are known to control the activity of mTOR , which suggests these nutrients could also affect how natural killer cells develop .", "In addition , manipulating the activity of mTOR could be used to control the response of natural killer cells to diseased host cells , and so could form part of treatments for cancer and infectious diseases .", "However , given that mTOR plays numerous roles within different body cells , any potential therapies that are developed would need to be able to manipulate mTOR specifically in natural killer cells ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "evolutionary biology" ]
The developmental relationship between teeth and dermal odontodes in the most primitive bony fish Lophosteus
elife-60985-v1
[ [ "A tooth is a particular type of ‘odontode’ ( Figure 1A ) : an exoskeletal structure that forms at an interface between an epithelial fold and the underlying mesenchyme , by dentine growing inwards from the epithelial contact surface .", "A mature odontode consists of dentine , in some cases covered with enamel or other hypermineralized surface tissue , irrigated through a central pulp cavity or pulp canals , and attached by a vascularized bone-like tissue ( Ørvig , 1977; Smith and Hall , 1993; Huysseune and Sire , 1998; Karatajute‐Talimaa , 1998 ) .", "Although teeth are the only odontodes to persist in tetrapods ( Reif , 1982 ) , various forms of dermal odontodes covered the entire body surface of many extinct jawless vertebrates , before jaws and teeth evolved ( Janvier , 1996 ) , and persist in some extant groups .", "Probably , the most familiar examples of dermal odontodes in extant vertebrates are the placoid scales of sharks , which are commonly called ‘skin teeth’ .", "Besides this form of individual pointed denticles , multiple dermal odontodes can be fused to a basal plate , like in the growing scales of primitive chondrichthyans ( Reif , 1978b ) , or anchored on a dermal bone , like on the skull bones of primitive osteichthyans , where the odontodes are usually accreted into patterned tubercles or ridges , referred to as ‘ornament’ .", "Dermal odontodes have long been regarded as an independent developmental module distinct from teeth , because of their supposed lack of the temporo-spatial regulation that , in teeth , is provided by a specific epithelial structure such as a dental lamina or odontogenic band ( Reif , 1982; Fraser and Smith , 2011 ) .", "However , shark placoid scales were recently shown to be patterned by a Turing-like mechanism ( Maisey and Denton , 2016; Cooper et al . , 2018 ) .", "Nevertheless , current knowledge about the growth patterns of dermal odontodes is basically limited to modern sharks .", "The evolutionary developmental relationship between teeth and dermal odontodes is pivotal for understanding the origin of teeth .", "The classic ‘outside-in’ hypothesis is currently in favor after decades of debate ( Donoghue and Rücklin , 2014; Haridy et al . , 2019 ) , but the developmental continuum between teeth and dermal odontodes , which is one of its central premises , still lacks unequivocal evidence .", "Extant gnathostomes with dermal odontodes ( sharks , rays , and some bony fishes such as Polypterus ) always display a sharp demarcation between teeth and ornament .", "Even though they can provide data of all ontogenetic stages , they are not informative about the evolution of the developmental relationship between teeth and dermal odontodes .", "For that we must turn to the fossil record of the earliest jawed vertebrates , in particular to the jawed stem gnathostomes and the stem osteichthyans , which form the common ancestral stock of Chondrichthyes + Osteichthyes and of Actinopterygii + Sarcopterygii , respectively ( Figure 1B ) .", "This study presents the marginal dentition of the Late Silurian ( 422 million years old; https://stratigraphy . org/timescale/ ) stem osteichthyan Lophosteus superbus , based on investigation by propagation phase-contrast synchrotron microtomography ( PPC-SRμCT ) , which allows the dentition to be digitally dissected in 3D with sub-micrometer resolution and the dental ontogeny to be reconstructed ( Figure 2 ) .", "The same technique has revealed the earliest osteichthyan-style tooth replacement , in the 424 million year old stem osteichthyan Andreolepis ( Chen et al . , 2016 ) , and the most phylogenetically basal gnathostome dentitions ( Vaškaninová et al . , 2020 ) .", "The latter occur in the Early Devonian armored fish known as ‘acanthothoracids’ , including Radotina , Kosorapis , and Tlamaspis ( Figure 1B ) .", "They all have non-shedding dentitions with lingual tooth addition , carried by marginal dermal bones , suggesting that these are the ancestral conditions of teeth ( Vaškaninová et al . , 2020 ) .", "Chondrichthyans and osteichthyans both inherited the lingual tooth addition , but evolved tooth shedding independently , while marginal jawbones ornamented by dermal odontodes were only kept by osteichthyans .", "The jawbones of Kosoraspis and Tlamaspis consist of multiple short pieces ( Vaškaninová et al . , 2020 , Figs . 3 and 4 ) , a condition strikingly similar to that in Lophosteus ( Figure 3A , Figure 3—figure supplement 1 ) , but unknown in other taxa .", "Stellate ( star-shaped ) dermal odontodes , which are characteristic of acanthothoracids , are also present in Lophosteus ( Figure 3—figure supplement 1; Figure 4A , e . g . O3g-3-6 ) but not in Andreolepis or other described early osteichthyans .", "Lophosteus further resembles a stem gnathostome in completely lacking enamel , whereas Andreolepis has enamel on its scales ( Qu et al . , 2015 ) .", "In fact , the only dental character of Lophosteus that unambiguously distinguishes it from acanthothoracids and identifies it as an osteichthyan is the presence of tooth shedding by partial and basal resorption ( Chen et al . , 2017 ) .", "This character distribution suggests that Lophosteus is the least crownward of known stem osteichthyans ( Figure 1B ) , making it uniquely informative about the evolution of the osteichthyan dentition ." ], [ "The oldest odontodes in the entire system are two rows of first-generation odontodes , fused into longitudinal ridges with confluent pulp cavities , at the level of the probable ossification center ( Figures 2A , 3C and 4B; Figure 3—figure supplement 2 , FRla , FRlin ) .", "We designate them as ‘founder ridges’ .", "On the lingual founder ridge , the main cusps are tall , conical , lingually recurved and widely spaced; on the labial founder ridge they are blade-like , labially inclined and united by small cusps .", "Side-cusps are more numerous on the labial founder ridge .", "The labial flanks of both ridges carry more side-cusps than the lingual flanks ( Figures 4B and 5F ) .", "There is no overlap between the two ridges; instead , their bases join as a continuous sheet ( Figure 3—figure supplement 2A ) , implying they formed simultaneously .", "Following the establishment of the founder ridges , more isolated odontodes were added sequentially in both lingual and labial directions , overlapping the lingual and labial edges of the founder ridges , respectively ( Figure 3—figure supplement 2 , O1g-2–1 and TF5-3 ) .", "These new odontodes are unicuspid , conical teeth on the lingual side of the lingual ridge ( Figure 5F ) , but are multicuspid and quickly take on the stellate morphology with crenulated ridges on the labial side of the labial ridge ( Figure 4B ) .", "Simply put , as the odontode skeleton spreads away from the two founder ridges , it turns into teeth lingually and into ornament labially .", "In the rows that follow lingually from the lingual founder ridge , all the main cusps become isolated and sharp .", "Although variable in size , they are arranged in semi-regular alternate files , and the lingual founder ridge can be regarded as a union of two alternate rows ( Figure 4B , C; Figure 5A , TP3-1 , TP5-1 , TP7-1 and TP4-2 , TP6-2 ) .", "These unicuspid odontodes are considered as the first-generation teeth .", "Only the most labial ( and thus oldest ) first-generation teeth are complete .", "More lingually , the first-generation teeth are resorbed semi-basally , and their remaining bases overlap considerably ( Figures 2B and 5F ) , just like the first-generation teeth of the tooth cushions that form the inner dental arcades ( Chen et al . , 2017 ) .", "The resorption surfaces are wide open , not only to the next first-generation tooth to be added to the file , but also to the replacement tooth buds that formed immediately above them ( Figure 5C–F ) .", "The first-generation teeth thus establish the tooth positions for the cyclic replacement teeth ( Figure 2C , D; Video 1 ) .", "The pulp cavities of the first-generation teeth lie directly on the basal compact bone , coinciding with the territory of the basal feeder vessels ( Figure 5A ) .", "This indicates that the most lingual row of first-generation teeth was once located at the jaw margin and that the oral lamina at this early developmental stage was much narrower ( Figure 2B; Figure 4—figure supplement 1 , TF ) .", "Following labially from the labial founder ridge is a single row of isolated spiny odontodes ( Figures 2B , 4B and 5A ) .", "They overlap the labial edge of the labial founder ridge substantially , without partially resorbing it .", "As a result , their pulp cavities are constrained by the space available ( Figure 3—figure supplement 2 , O1g-2–1; Figure 4—figure supplement 1B and Figure 5A , OP0-1 , OP2-1 , OP4-1 , OP6-1 ) .", "As the facial lamina extends labially , younger generations of odontodes are initiated to cover the new bone; as the jawbone rotates labially , they also begin to invade the oral lamina ( Figure 2C , D; Figure 4—figure supplement 1 , O2g , O3g; Video 1 ) .", "Unlike the teeth , no resorption occurs in the dermal odontodes .", "Younger generations of larger odontodes thus simply overgrow , rather than replace , the preexisting dermal odontodes .", "There are two levels of overgrowth and the dermal odontodes are divided into three generations , inferred from their size and distribution .", "All generations of dermal odontodes follow a global morphologic gradation .", "The further they are from the biting margin at the time they are initiated , the more side-cusps , ridgelets , and crenulations are associated with the main cusp , and the more ascending canals are attached to the flattened pulp cavities .", "The largest and youngest stellate odontodes have the most elaborate appearance ( Figure 4A , O3g-3–6 , O3g-7 ) and the most osteodentine-like tissue ( Figure 4—figure supplements 2–5 ) , which probably reflects their location furthest from the jaw margin , rather than their size or age .", "We surmise that this pattern reflects a morphogenetic signal gradient ( Figure 4 ) between the jaw margin and the ‘generic dermal surface’ of the face , represented here by the facial lamina beyond the lateral line canal .", "Where the dermal odontodes invade the tooth-bearing oral lamina , a subtle but important morphologic variability affects the labial surface of some shedding teeth .", "The replacement teeth at the marginal positions , like the first-generation teeth , always have a smooth labial surface , but teeth close to the invading ornament often carry side-cusps on the labial face ( Figure 4A , arrowhead; Figure 6C , D , TR1-9 ( 2 ) , TR3-7 , TR3-10 , TR6-9 , TR7-3 , TR7-7 ) .", "The dentine of the side-cusps tends to include some cell spaces .", "In other words , these teeth show ornament-like characteristics; there appears to be a degree of ‘morphologic cross-contamination’ between the two odontode sets ( Figure 4A , bars in lavender and mint ) .", "At the invasive front line , both overgrowing odontodes and replacement teeth have a half-tooth half-ornament morphology ( Figure 6C , compare O2g-1 and O2g-2 with TR7-3 and TR7-7 ) , though the replacement teeth are recognizable by their possession of basal resorption surfaces ." ], [ "The marginal dentitions of the stem osteichthyan Andreolepis ( Late Silurian , Gotland , Sweden ) and the ‘acanthothoracid’ stem gnathostome Kosoraspis ( Early Devonian , Prague Basin , Czech Republic ) are of particular interest as comparators for Lophosteus because they show a similar combination of transverse alternate tooth files with lingual tooth addition , carried on marginal bones that also bear dermal odontodes on their facial laminae ( Chen et al . , 2016; Vaškaninová et al . , 2020 ) .", "Lophosteus aligns with Andreolepis and differs from stem gnathostomes in showing resorptive tooth shedding , a unique osteichthyan characteristic ( Chen et al . , 2016 ) .", "Indeed , this process is of fundamental importance in shaping the dentition of Lophosteus , with some tooth positions showing as many as twenty replacement cycles .", "This has important implications for understanding the ontogeny underlying the final adult morphology .", "Kosoraspis has a much simpler ontogenetic history without resorption-replacement cycles , essentially corresponding to the first-generation odontodes of Andreolepis .", "Kosoraspis shows a unidirectional change from small dermal odontodes at the external margin of the jawbone , through gradually larger and progressively more tooth-like dermal odontodes , to teeth ( Vaškaninová et al . , 2020 ) .", "This indicates the ossification center is located at the external margin .", "The gradient between teeth and dermal odontodes of the first generation appears to be unidirectional in Andreolepis too , from a morphology with elongate bases to a more prominently tooth-like morphology with round bases as they approach the jaw margin ( Chen et al . , 2016 , Figure 2a , b ) .", "Any equivalents of the founder ridges of Lophosteus , if present , have not been captured by the high-resolution scan that only covers approximately a quarter of the height of the facial lamina ( Chen et al . , 2016 , Figure 1b ) .", "Nevertheless , it is certain that the location of the odontode founder region and the bone ossification center is considerably external to the original oral-dermal boundary in Andreolepis .", "By contrast , the three developmental landmarks overlap in Lophosteus , which thus displays a bidirectional morphologic gradation in the initial odontode skeleton .", "The dermal odontodes and teeth of Lophosteus are initiated simultaneously in the form of two parallel and closely spaced founder ridges ( Figures 2A , B and 4B ) .", "The cusps of the lingual founder ridge incline lingually , and those of the labial founder ridge , labially; subsequent odontodes are added to the lingual flank of the lingual founder ridge and the labial flank of the labial founder ridge .", "This geometric layout strongly suggests that the initiation site for odontode formation is the boundary between two patterning domains: a labial domain where the odontodes become ornament and a lingual domain where they become teeth .", "Because the development of an odontode is always initiated by an epithelium , we infer that the two domains are covered with two distinct epithelia , which we refer to respectively as the dermal and oral epithelium .", "The cusps of the two founder ridges are already morphologically distinct , and the odontodes that are subsequently produced at their labial and lingual sides quickly acquire the full characteristics of ornament and teeth , respectively .", "This must represent the establishment of their complete regulatory cascades , and possibly relates to the initiation of new odontodes on each side moving away from the boundary between the dermal and oral epithelia into the presumably more homogeneous signaling environment of a single epithelium ( Figure 4 , 1g ) .", "The strict , linear separation described above only characterises the first-generation odontodes .", "Already in the second-generation , stellate odontodes have overgrown some of the oldest teeth , in a considerably more lingual position than the original boundary .", "The third generation penetrates even further into the territory of teeth ( Figure 4 , 2g and 3g; Figure 4—figure supplement 1 , O2g , O3g ) .", "This suggests an irregular expansion of the dermal epithelium into the region previously occupied exclusively by the oral epithelium .", "A similar invasion of the tooth field by dermal ornament has been observed in Andreolepis ( Chen et al . , 2016 ) and an unnamed acanthothoracid from the Canadian Arctic , specimen CPW . 9 ( Smith et al . , 2017; Vaškaninová et al . , 2020 ) .", "In Lophosteus , the ornament invasion produces an effect that is highly informative about the relationship between these two odontode sets .", "Essentially , the deposition of odontodes remains characteristic for the two sets – teeth continue to be replaced cyclically until overgrown by ornament , and the dermal odontodes are never shed – but the morphology of each set seems to become influenced by the other .", "The teeth nearest to the ornament bear side-cusps that tend to form a labial ridgelet .", "Conversely , in the most lingual dermal odontodes , the conical main cusp tends to stand out and point lingually , the side-cusps being restricted to the labial side ( Figures 4A , B and 6C , D ) .", "That is to say , in the invasion zone , the teeth are ornament-like and the dermal odontodes are tooth-like .", "The simplest explanation for this phenomenon is that the invasion zone provides a mixed set of morphogenetic signals , because it is a patchwork of dermal and oral epithelium , and that both dermal odontodes and teeth respond to both signals .", "Note , however , that this ‘regulatory cross-contamination’ only affects the morphology , not the deposition and ( if present ) resorption cycles .", "In Andreolepis , the lingualmost odontodes of any generation of invading ornament can be tall and bear biting damage , in contrast to the characteristic flat-topped ornament morphology ( Chen et al . , 2016 , Extended Data Figure 3c ) .", "In many basal actinopterygians with acrodin-capped teeth , such as Birgeria and Boreosomus , the dermal odontodes labial to the jaw teeth also bear an acrodin cap ( Ørvig , 1978a , PP . 38 , Figs . 3 , 4 , 7–9 ) .", "The zone of the acrodin-bearing odontodes varies in size , being , for example , narrower in Colobodus and wider in Nephrotus and others ( Ørvig , 1978c , pp . 307 ) , but is invariably restricted to the vicinity of dentition , regardless of the type of jawbone ( Ørvig , 1978b , pp . 41–42 ) .", "Similar phenomena can be seen in stem chondrichthyans .", "The labial face of the blade-like teeth on the tooth whorls of Ptomacanthus can be ornamented , like the tesserae that they are interlocked with ( Miles , 1973 ) .", "The toothless Obtusacanthus displays a morphological gradation from stellate head scales , via fan-shaped scales , to tooth-like lip scales ( Blais et al . , 2011 ) , comparable to the superficial morphological gradation from stellate facial ornament with crenulated ridgelets , via multicuspid invasive ornament and ornament-like replacement teeth , to unicuspid marginal teeth in Lophosteus .", "The labio-lingual rows of ‘extra-oral teeth’ on the whorl-like cheek scales , pointed lip scales , platelets or tesserae labial to the jaw margin of ischnacanthid acanthodians ( Gross , 1971 , Tafel 4 , Fig . 24-29; Ørvig , 1973 , Text-fig . 1C; Blais et al . , 2011 , Blais et al . , 2015; Burrow et al . , 2018 ) also suggests the presence of a mixed dermal-oral signaling environment extending beyond the mouth .", "Together with these observations , the tooth-like ornament and the ornament-like teeth of Lophosteus call into question the demarcation between dermal and oral developmental domains .", "The description of ornament invasion presented above incorporates the assumption that the position and orientation of the jawbone is static relative to the edge of the mouth .", "In fact , the bone appears to have rotated labially during growth ( Figure 2 ) , so that the location of the mouth margin shifted during ontogeny from the lingual founder ridge , via each lingual row of the first-generation teeth , to each successive row of replacement teeth at the current lingual margin of the oral lamina .", "This implies that the oral-dermal epithelial boundary does not drift lingually to any great degree; rather , the rotation of the bone causes the labial tooth rows to move onto the face , where they get covered by dermal epithelium and overgrown by ornament .", "This is comparable to the rotation of the tooth whorls at the jaw margin of primitive chondrichthyans , with the post-functional teeth slid beneath the skin ( Smith and Coates , 2001 , Figure 14 . 3 ( a ) ; Williams , 2001 ) .", "In Lophosteus , the first-generation teeth are added sequentially toward the growing lingual margin , with the tooth families arranged in horizontal alternate files .", "In the next stage , each successor , if not overgrown by invasive odontodes , turns into an initiator and sets up its own tooth family by cyclic replacement .", "The replacement teeth hence inherit the pattern of the first-generation teeth , generation after generation , forming vertical alternate columns .", "The replacement columns , including those inserted later and those disturbed by overgrowing odontodes , follow the same transverse alignment as the first-generation teeth ( Figures 4B , 5 and 6 ) .", "Longitudinally , the exposed tooth rows appear somewhat irregular , which is due to two reasons .", "Firstly , new positions that are not established by the first-generation teeth are inserted whenever space is available along the marginal replacement column ( e . g . Figure 5B , RC3-10 ) .", "Secondly , different labial positions are terminated randomly by different generations of overgrowing odontodes , and the total number of replacement cycles is different in each tooth position .", "As a consequence , the final replacement teeth drifted lingually from their first-generation teeth for a variable distance , reflected by the variable length of replacement columns ( Figure 6 , compare TR7-3 with TR 6–4 and TR4-4 ) .", "Nevertheless , the marginal positions that maintain the original pattern throughout the growth of bone are invariably aligned in a row .", "Sequential addition along the files established by the first generation also applies to the stellate odontodes on the facial lamina .", "Such an alternate arrangement is constant throughout the teeth and tooth-like odontodes on the marginal jawbone of Andreolepis and Kosoraspis as well , irrespective of whether they will be overgrown by younger generations of odontodes ( Chen et al . , 2016; Vaškaninová et al . , 2020 ) .", "The alternate pattern of teeth and ornament of Lophosteus is already established by the founder ridges .", "The ornament differs from teeth in the fact that every other alternate position of ornament is suppressed as the ornament extends labially ( Figure 4—figure supplement 1B ) ; this is because consecutive generations of dermal odontodes increase in length more quickly than the bone .", "The replacement of two teeth by a single larger successor at a crowded site has been observed in sharks , frogs and lizards , and considered as a common phenomenon ( Gillette , 1955; Edmund , 1960; Cooper , 1963; Osborn , 1971; Reif , 1976 ) .", "In alligator embryos , the reduction of jaw growth can cause the suppression of a tooth family during ontogeny ( Westergaard and Ferguson , 1987 ) .", "The suppression does not represent an irregularity; instead , it may reflect the fundamental mechanism producing the alternate pattern .", "The hexagonal pattern is the most efficient form of close packing of rounded objects .", "The close packed teeth of myliobatid rays , which have turned into short hexagonal prisms ( Edmund , 1960; Underwood et al . , 2015 ) , is an extreme example of a dentition imitating the structure of a honeycomb .", "A regular pattern , which was thought to be unique to teeth and reflect the spatio-temporal regulation of the dental lamina ( Smith , 2003; Underwood et al . , 2016 ) , is actually not uncommon in dermal odontodes , as well as in bony denticles .", "Ordered tubercles can be seen covering the armor of jawed stem gnathostomes as long as there is only one generation , irrespective of whether they are dentinal units on the gnathal plates and spines , or bony units on the postbranchial lamina ( Bystrow , 1957 , Fig . 2; Burrow , 2003; Johanson and Smith , 2003; Johanson and Smith , 1999; Young , 2003 ) .", "In the polyodontode scales of the earliest known chondrichthyans , odontodes are often organized in parallel or radial rows by sequential addition ( Andreev et al . , 2020 ) .", "An alternate pattern can be produced by odontodes that are laid down directly on the bony plate , simply through filling the gaps between the odontodes in the previous row , even if the previous row is from the older generation ( Figure 4—figure supplement 1B ) .", "But it is difficult for the enlarged overgrowing odontodes to find and fit such gaps , by reason of the preexisting odontodes , so the pattern will be disturbed .", "This effect is clearly visible in a digitally dissected spine of the jawed stem gnathostome Romundina , which carries three generations of odontodes ( Jerve et al . , 2017 , Fig . 2D1–D3 ) ; the first generation shows an ordered pattern , but this breaks down in the second and third generations .", "The single-file arrangement of the first-generation odontodes of Andreolepis scale is also obscured by the overgrowing odontodes ( Qu et al . , 2013 ) .", "All these examples agree with a fundamental embryologic mechanism of odontode patterning shared among the skeletons of vertebrates , which had evolved prior to the origin of teeth , as already proposed by Osborn , 1971 .", "The alternate pattern can be self-generating , as long as the size of inhibitory zones is equivalent or in a smooth gradient ( Osborn , 1977 ) .", "Therefore , the regularity of organization should not be considered as a criterion of true teeth .", "The claim that the ectoderm ( dermal epithelium ) lacks patterning capacity ( Fraser and Smith , 2011; Smith and Johanson , 2015 ) , which has been used to support the idea of a fundamental difference between dermal odontodes and teeth , is biased by the derived adult condition of modern chondrichthyans .", "Actually , skin denticles of chondrichthyans embryos are added sequentially in regular rows , including the caudal denticles , dorsal denticles and the first-generation general denticles ( Grover , 1974; Reif , 1976; Ballard et al . , 1993; Miyake et al . , 1999; Johanson et al . , 2007; Debiais-Thibaud et al . , 2015; Maisey and Denton , 2016; Martin et al . , 2016; Cooper et al . , 2018 ) .", "The oro-pharyngeal denticles also emerge in rows in embryos , even if frequently interrupted by the oral papillae and undulated by the uneven surface of the oropharynx ( Rangel et al . , 2017 , Fig . 4F , G ) .", "All the skin or oral denticles are likely to be self-organized by Turing’s reaction-diffusion system , a patterning mechanism probably common to epithelial appendages ( Maisey and Denton , 2016; Cooper et al . , 2018 ) .", "Later in ontogeny , these denticles may display a random organization , because the regular rows have been obscured by the mix of denticles in variable sizes , which may be due to the loss and repair of original denticles at different developmental stages .", "Even so , both skin and oropharyngeal denticles retain their alternating pattern in adulthood ( Rangel et al . , 2016 ) .", "Denticle files with regular spacing line up the rear border of the pharyngeal pads in some requiem sharks , remarkably resembling the tooth files lining the length of jaw and probably functioning like osteichthyan pharyngeal dentitions ( Nelson , 1970 , fig . 13 , 15 , 16B ) .", "Wound-healing experiments on sharks show that the loss of the diagonal rows and the rostro-caudal polarity as well as the regular size and shape of the scales in repaired squamations is actually caused by disturbance of the original diagonal arrangement of the anchoring collagen fibers ( Reif , 1978a ) .", "By contrast , the orderly shedding and replacing of chondrichthyan teeth preserves the embryonic pattern .", "Our data from Lophosteus are in two respects uniquely informative about the relationship between teeth and ornament .", "Firstly , as a Silurian stem osteichthyan , Lophosteus represents a very short phylogenetic branch in a basal part of the gnathostome crown group , and is thus likely to present primitive characters for the Osteichthyes and maybe for the Gnathostomata as a whole; secondly , we can trace the developing relationship between teeth and dermal odontodes through the life history of the animal , whereas all the dermal odontodes and teeth that have been compared in previous studies are fully differentiated forms in adults .", "The unified arrangement of the teeth and ornament of Lophosteus challenges the currently popular idea that teeth and dermal denticles have fundamentally different patterning regimes ( Fraser and Smith , 2011 ) .", "In Lophosteus , teeth and ornament are never starkly different .", "The replacement teeth can have an ornament-like appearance , and can be partially shed and overlapped .", "The dermal odontodes can be added sequentially and alternately , and organized in rows and files .", "New tooth positions can be inserted once a gap appears , obliterating the original addition sequence .", "Looking further afield , it is noteworthy that the ‘extra-oral teeth’ that occur in some teleosts also display a regular organization , development in epithelial invaginations , and shedding-replacing by basal resorption of the attachment bone and supporting bone ( Sire and Huysseune , 1996; Sire et al . , 1998; Sire , 2001; Sire and Allizard , 2001 ) .", "These structures reveal the potential plasticity of odontodes and suggest the conventional criteria of ‘true teeth’ ( Burrow , 2003; Smith and Johanson , 2003a; Smith and Johanson , 2003b ) are in fact not unique to oral teeth .", "Therefore , teeth and ornament are not merely homologs ( Reif , 1982; Huysseune and Sire , 1998; Donoghue , 2002 ) , as supported by a common gene regulatory network ( Fraser et al . , 2010; Debiais-Thibaud et al . , 2011 ) .", "More importantly , they develop from the same developmental module , modified through a simple mechanism of heterotopy ( Debiais-Thibaud et al . , 2011 ) , which can be evidenced by the developmental continuum , as on the marginal jawbone of Lophosteus .", "However , contrary to the scale-to-teeth scenario of the ‘outside-in’ theory , we argue that teeth did not evolve from ontogenetically mature ornament , but rather from a primordial type of founder odontode , when covered by the oral epithelium .", "The primordial odontodes might be similar all over the body , outside or inside the oropharyngeal cavity , with later deposited odontodes differentiated according to their location and function .", "For example , the primordial odontodes in the ganoid scales of Andreolepis or the cosmoid scales of Psarolepis , revealed by PPC-SRµCT , have a more pointed and tooth-like morphology than the overgrowing odontodes ( Qu et al . , 2013; Qu et al . , 2017 ) , resembling the relationship between the founder ridges and ornament in Lophosteus .", "This earliest developmental stage of the odontode skeleton may have been lost in derived taxa or missed by the conventional investigative techniques .", "Direct comparison between the substantially modified mature subsets of odontodes , could lead to the conclusion that teeth and dermal odontodes are two wholly separate systems .", "Crucially , Lophosteus shows us that only ontogenetic data going back to the earliest stages of development are able to reveal the original patterning relationships within the odontode skeleton ." ], [ "The marginal jawbone fragments of Lophosteus were collected from fallen blocks of limestone at Ohesaare cliff , Saaremaa Island , Estonia , the type locality of Lophosteus .", "Acetic acid dissolution and extraction of the microremains were carried out at the Department of Earth Sciences of Lund University and the Department of Organismal Biology of Uppsala University , Sweden .", "The specimens are registered to the Geological Institute , Tallinn , Estonia GIT 760–12 ~ 28 .", "All specimens in Figure 3—figure supplement 1 were photographed under an Olympus SZX10 microscope-camera setup , with ImageView imaging software , at the Swedish Museum of Natural History , Stockholm .", "The specimen was imaged at beamline ID19 of the European Synchrotron Radiation Facility ( ESRF ) in Grenoble , France , using propagation phase-contrast synchrotron radiation microtomography ( PPC-SRµCT ) adapted to fossil mineralized tissues histology ( Tafforeau and Smith , 2008; Sanchez et al . , 2012 ) .", "With an isotropic voxel size of 0 . 696 µm , the scan was obtained with an objective 10× , NA0 . 3 coupled with a 2 × eyepiece .", "The optics , associated with a gadolinium gallium garnet crystal of 10 µm thickness ( GGG10 ) scintillator , is coupled to a FreLoN 2K14 detector [fast readout low noise camera; Labiche et al . , 2007] used in full frame mode with a fast shutter .", "The specimen was set up 15 mm from the optics .", "The gap of the undulator U17 . 6 was set to 20 mm and provided a pink beam ( direct beam with a single main narrow harmonic ) at an energy of 19keV , filtered by 0 . 7 mm of aluminum .", "A total of 4998 projections of 0 . 3 s each were taken over 360° by half-acquisition of 600 pixels .", "Reconstruction was done with a modified version of a single-distance phase retrieval approach ( Paganin et al . , 2002; Sanchez et al . , 2012 ) .", "The virtual thin sections of the sample in the form of stacks of images were segmented into three- dimensional sub-volumes through the software VG Studio 3 . 1 .", "Embedded subtle structures , such as the surfaces of resorption and dentine , were traced manually ." ] ]
[ "The ontogenetic trajectory of a marginal jawbone of Lophosteus superbus ( Late Silurian , 422 Million years old ) , the phylogenetically most basal stem osteichthyan , visualized by synchrotron microtomography , reveals a developmental relationship between teeth and dermal odontodes that is not evident from the adult morphology .", "The earliest odontodes are two longitudinal founder ridges formed at the ossification center .", "Subsequent odontodes that are added lingually to the ridges turn into conical teeth and undergo cyclic replacement , while those added labially achieve a stellate appearance .", "Stellate odontodes deposited directly on the bony plate are aligned with the alternate files of teeth , whereas new tooth positions are inserted into the files of sequential addition when a gap appears .", "Successive teeth and overgrowing odontodes show hybrid morphologies around the oral-dermal boundary , suggesting signal cross-communication .", "We propose that teeth and dermal odontodes are modifications of a single system , regulated and differentiated by the oral and dermal epithelia ." ]
[ "Human teeth are an example of odontodes: hard structures made of a material called dentine that are sometimes coated in enamel .", "Teeth are the only odontodes humans have , but other vertebrates ( animals with backbones ) have tooth-like scales on their skin .", "These structures are called dermal odontodes , and sharks and rays , for example , are covered with them .", "How these structures evolved , and whether teeth or dermal odontodes developed first , continues to spark great discussion among palaeontologists .", "Some researchers think that teeth evolved from dermal odontodes , a theory known as the ‘scales-to-teeth’ hypothesis .", "Others think dermal odontodes are distinct from teeth because they lack the same spatial organization .", "To investigate this further , palaeontologists are looking at the earliest examples of odontodes they can find: fossils of early vertebrates that carry both teeth and dermal odontodes .", "Here , Chen et al . have studied Lophosteus , one of the earliest bony fishes that lived more than 400 million years ago , to explore early tooth evolution and growth patterns .", "Chen et al . digitally dissected a fossilized Lophosteus jawbone using submicron X-ray imaging , a technique with resolution to less than one millionth of a metre .", "Imaging thin sections of the specimen , found in Estonia , Chen et al . reconstructed the entire sequence of odontode development in the bony fish in 3D .", "The analysis showed that teeth and dermal odontodes initially take shape together but differentiate as they grow , presumably instructed to do so by various developmental signals .", "However , at a later stage , the two types of odontodes become similar in appearance again , suggesting that they respond to each other’s signals .", "For example , as the jawbone grows , dermal odontodes overgrow the earliest formed teeth .", "These younger odontodes resemble teeth , while the new teeth developing near the dermal odontodes take after dermal odontodes .", "These findings suggest that teeth and dermal odontodes are not wholly separate systems but , instead , are closely related on a molecular level .", "The results also show that contrary to the ‘scale-to-teeth’ hypothesis , teeth do not evolve from fully formed dermal odontodes , rather the two types of odontodes form out of one founder .", "This research builds on our knowledge from modern sharks and points to a previously unrecognised evolutionary relationship between teeth and dermal odontodes .", "It also furthers our understanding of how molecular regulation controls development ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "microbiology and infectious disease" ]
Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery
elife-26910-v2
[ [ "Influenza virus infections initiate with a burst of gene expression from pre-formed RNPs deposited by the incoming viral particles .", "Primary transcription is followed by replication of the genome and subsequent transcription of the replicated genome , further increasing gene expression .", "This transition from transcription to replication requires the de novo assembly of RNPs and is absolutely required for successful infection and the production of infectious progeny .", "Viral product have been proposed to regulate this transition: NEP has been shown to shift the viral polymerase towards replication; svRNAs are thought to associate with the viral polymerase and promote copying of full-length genomic RNA; and newly synthesized polymerase proteins have been proposed to stimulate replication in trans ( Jorba et al . , 2009; Perez et al . , 2012 , 2010; Robb et al . , 2009; York et al . , 2013 ) .", "Nonetheless , the mechanisms regulating upstream events of RNP assembly and the host factors contributing to this coordinated shift from transcription to RNP assembly and genome replication are largely unknown .", "The influenza virus RNP is a double helical structure containing the viral polymerase and repeating NP subunits coating each of the eight genomic RNAs ( Arranz et al . , 2012; Klumpp et al . , 1997; Moeller et al . , 2012; Pons et al . , 1969 ) .", "The viral polymerase , a heterotrimer composed of the subunits PB1 , PB2 and PA , is located at one end of the RNP where it binds both the 5’ and 3’ genomic termini .", "This RNP performs both transcription and replication .", "Transcription of viral mRNAs occurs via a ‘cap-snatching’ mechanism , beginning immediately following nuclear import of the incoming RNPs and continuing throughout infection ( Bouloy et al . , 1978; Plotch et al . , 1981 ) .", "Replication occurs at later time points when RNPs direct synthesis of a positive-sense complementary RNA ( cRNA ) intermediate that templates replication of the negative-sense viral RNA genome ( vRNA ) ( Hay et al . , 1977 ) .", "Importantly , this replication requires the assembly of RNPs containing newly synthesized polymerase , NP , and either cRNA ( cRNPs ) or vRNA ( vRNPs ) ( Barrett et al . , 1979; Vreede et al . , 2004 ) .", "To fully coat the genome , NP forms homo-oligomers and binds RNA in a sequence-independent fashion .", "These same properties cause NP to oligomerize spontaneously and bind non-specifically to cellular RNAs ( Baudin et al . , 1994; Prokudina-Kantorovich and Semenova , 1996; Zhao et al . , 1998 ) .", "Therefore , control of NP oligomerization and RNP assembly are key regulatory steps as the infectious cycle progress towards genome replication .", "Influenza virus NP oligomerizes by inserting a small ‘tail loop’ ( aa 402–428 ) into the binding groove of a neighboring protomer ( Ng et al . , 2008; Ye et al . , 2006 ) .", "NP binds RNA via a large basic surface and is thought to encapsidate the nascent RNA genome concomitant with its synthesis , hence a continuous supply of RNA-free monomeric NP is required for assembly into RNA-bound RNPs and replication of the viral genome ( Beaton and Krug , 1986; Ng et al . , 2008; Shapiro and Krug , 1988; Vreede et al . , 2004 ) .", "We and others reported that phosphorylation at the homotypic interface inhibits NP oligomerization during both influenza A and B virus replication ( Chenavas et al . , 2013; Hutchinson et al . , 2012; Mondal et al . , 2015; Turrell et al . , 2015 ) .", "Specifically , phosphorylation or phospho-mimetics at residue S165 in the groove or S407 in the tail loop drives influenza A NP towards a monomeric state , prevents RNP assembly , and severely impairs viral replication ( Mondal et al . , 2015 ) .", "NP mutants lacking key phospho-sites are also defective in supporting influenza polymerase activity and virus replication , and in some cases result in NP hyper-oligomerization ( Mondal et al . , 2015; Turrell et al . , 2015 ) .", "Thus , both hyper- and hypo-phosphorylation of NP is deleterious suggesting that the reversible phosphorylation of NP must be carefully balanced to enable recruitment of oligomerization-competent NP to sites of genome replication and ultimately incorporation into growing RNPs .", "Influenza virus does not encode a kinase , therefore the phospho-regulation of NP must be performed by host enzymes .", "Here we identify the protein kinase C ( PKC ) family , and PKCδ in particular , as host kinases that control RNP assembly by phospho-regulating NP oligomerization and subsequently impact the transition from gene expression to genome replication .", "We show that PKC activity disrupts influenza virus polymerase function and that polymerase-associated PKCδ specifically phosphorylates NP .", "PKCδ is recruited by the polymerase subunit PB2 and targets key residues at the tail loop:groove interface to regulate NP oligomerization .", "Knockout of PKCδ in human lung cells decreased NP phosphorylation during infection and significantly reduced viral gene expression and production of infectious progeny .", "As de novo formation of RNPs is required for genome replication and the amplification of viral gene expression , these findings predict that PKCδ is important at late stages of infection .", "Indeed , primary transcription at early time points was unaffected in PKCδ knockout cells whereas the transition to genome replication at later time points was severely impaired .", "Thus , influenza virus exploits host PKCδ to regulate the ordered assembly of RNPs enabling the resultant transition from gene transcription to genome replication ." ], [ "Both activators and inhibitors of the PKC family have been shown to modulate influenza virus replication ( Hoffmann et al . , 2008; Kistner et al . , 1989 ) .", "More recently we demonstrated that activating PKCs with phorbol-12-myristate-13-acetate ( PMA ) stimulates NP phosphorylation and inhibits its oligomerization ( Mondal et al . , 2015 ) .", "As NP oligomerization and RNP assembly are required for replication of the viral genome , we undertook a targeted approach to investigate the role of PKCs in regulating influenza polymerase activity .", "The PKC family consists of at least eleven different members which can be divided into classical ( α , β1 , β2 , γ ) , novel ( δ , ε , η , θ ) and atypical ( ι/λ , ζ ) isoforms based on their structure and co-factor requirements .", "To test the ability of PKC isoforms to phosphorylate NP and impact polymerase function , we performed polymerase activity assays in cells co-expressing a panel of PKC variants .", "Influenza polymerase activity was reconstituted in cells by expressing the trimeric polymerase , NP , and a vRNA-like reporter encoding luciferase .", "The viral reporter is replicated and transcribed only in the presence of a functional polymerase and NP , and serves as a proxy for RNP formation ( Figure 1—figure supplement 1 ) .", "PKCs were expressed as constitutively active truncations containing the C-terminal catalytic domain ( PKC-CAT ) , but lacking the regulatory domains ( Soh and Weinstein , 2003 ) .", "In a cell , PKCs are synthesized in an inactive conformation and are activated upon binding phosphatidylserine , and in most cases also require binding to diacylglycerol , Ca2+ or phosphatidylinositol 4 , 5-bisphosphate ( Antal and Newton , 2014 ) .", "Using the constitutively active forms eliminated variability that may arise from the distinct second messenger activators required by different PKC family members .", "Expressing active PKCβ2 , PKCδ , PKCθ and PKCη considerably reduced polymerase activity with respect to the empty vector control .", "While PKCβ2 and PKCη showed 60–80% decrease , PKCδ and PKCθ abrogated polymerase activity completely to background levels ( Figure 1A ) .", "PKCε and PKCα showed moderate , but statistically significant reductions , whereas the remaining isoforms caused minimal changes in polymerase activity , or even minor increases in activity .", "Western blotting showed that slower migrating forms of NP appeared in conditions where polymerase activity was inhibited .", "Phosphatase treatment confirmed that these slower migrating species resulted from NP hyper-phosphorylation and quantification revealed a strong correlation between increased NP phosphorylation and decreased polymerase activity ( Figure 1—figure supplement 2A–B ) .", "Blotting also showed comparable expression of all PKC isoforms and detected previously described minor bands due to differential post-translational modifications ( Soh and Weinstein , 2003 ) .", "Subsequent experiments were focused on PKCβ2 , PKCδ , and PKCη , but not PKCθ as its expression is heavily restricted to skeletal muscle and cells of the immune system and not the lung epithelial cells where influenza virus primarily replicate ( Zhang et al . , 2013 ) .", "To determine if kinase activity from different PKC isoforms drives polymerase activity inhibition , polymerase activity assays were repeated in the presence of inactive PKC mutants with single amino acid changes in their catalytic domain .", "Whereas the catalytic domains of PKCβ2 , PKCδ and PKCη inhibited polymerase activity , this phenotype was significantly reduced for the inactive mutants ( Figure 1B and Figure 1—figure supplement 1 ) .", "Moreover , NP was not hyper-phosphorylated in the presence of the inactive mutants , suggesting that specific PKC isoforms inhibit influenza polymerase activity by causing the phosphorylation of NP .", "PKCs can function directly by phosphorylating a target or indirectly by activating downstream kinases that then phosphorylate the target protein .", "To determine if the effects of PKCs on polymerase activity were direct or indirect , we performed binding assays to test if inhibitory isoforms of PKC associate with proteins in the viral RNP .", "Whereas NP is phosphorylated when PKCs are expressed and this correlates with the inhibitory phenotype , co-immunoprecipitations failed to detect stable interactions between NP and PKC when co-expressed in 293T cells ( Figure 2A ) .", "This was consistent with the transient interactions frequently observed between kinase and substrate .", "Surprisingly , NP was efficiently co-precipitated with catalytic fragments of PKCδ and PKCη when they were co-expressed with the other components of the RNP ( i . e . viral polymerase and vRNA ) ( Figure 2A ) .", "PKCβ2 co-precipitated only limited amounts of NP .", "Notably , NP co-precipitated by PKCδ and PKCη was highly enriched for the hyper-phosphorylated form relative to its abundance in total cell lysate .", "To ensure specificity of these interactions , experiments were repeated using full-length PKC isoforms ( Figure 2B ) .", "Again , NP was co-precipitated by full-length PKC when the polymerase and vRNA were co-expressed .", "Differences in NP co-precipitation were not due to differences in immunoprecipitation of the different PKC isoforms , as each PKC immunoprecipitated with equivalent efficiency relative to its expression in the cell lysate ( Figure 2A–B ) .", "Additionally , NP showed a clear preference for interaction with PKCδ , although PKCη also co-precipitated minor amounts of NP .", "Interactions between NP and PKC isoforms were enhanced in the presence of the viral polymerase and vRNA ( Figure 2A–B ) .", "This enhanced interaction was still observed when the vRNA template was excluded from transfections or when RNaseA was included during the immunoprecipitation ( not shown ) , suggesting that the viral polymerase is sufficient to mediate the interaction between NP and PKC .", "We determined which proteins of the heterotrimeric polymerase are essential for NP-PKCδ interactions ( Figure 2C ) .", "Reconstituting the complete polymerase by expressing PB1 , PB2 and PA enabled strong co-precipitation of NP by PKCδ .", "Only minor amounts of NP were co-precipitated in the absence of the polymerase .", "Interestingly , co-expression of PB2 alone was sufficient to ensure significant co-precipitation of NP by PKCδ , whereas co-expression of PB1 and PA resulted in limited co-precipitation similar to that observed in the absence of the polymerase .", "Furthermore , the oligomerization defective NP E339A was co-precipitated by PKCδ in the presence of PB2 , suggesting NP monomers also participate in this interaction .", "To obtain further evidence for this interaction and identify the most relevant PKC isoforms , we performed a co-immunoprecipitation experiment in cells expressing NP , PB2 and either catalytic or full-length PKC isoforms ( Figure 2D ) .", "PB2 bridged interactions between NP and the catalytic domains of PKCδ and PKCη .", "In the context of full length protein , PKCδ showed the strongest interactions with NP and PB2 .", "Our data suggested that PB2 anchors a hetero-oligomeric NP:PB2:PKCδ complex .", "NP present in this immuno-precipitated complex was significantly enriched for the hyper-phosphorylated form , suggesting that PB2 facilitates a functional interaction between NP and activated PKC resulting in NP phosphorylation .", "Phosphorylation of NP at the homotypic interface inhibits oligomerization ( Chenavas et al . , 2013; Mondal et al . , 2015; Turrell et al . , 2015 ) .", "Our data implicate PKCs as the host kinases that phospho-regulate NP oligomerization .", "We therefore assessed the ability of PKC isoforms to directly phosphorylate NP .", "PKC catalytic domains were immunopurified from cell lysates and used in in vitro kinase assays with recombinant NP ( Figure 3A ) .", "All three PKC isoforms that inhibited polymerase activity , i . e . PKCβ2 , PKCδ and PKCη , phosphorylated NP .", "By contrast , PKCε , which exhibited modest inhibition of polymerase activity , showed no specific kinase activity and its activity was similar to that in the negative control .", "To demonstrate specificity , the PKC inhibitor 1- ( 5-Isoquinolinesulfonyl ) −2-methylpiperazine ( H7 ) was included in kinase reactions .", "H7 almost completely eliminated NP phosphorylation ( Figure 3B ) , compared to the vehicle-only control that did not impact NP phosphorylation .", "These kinase assays showed that select PKC isoforms , and not other fortuitously co-precipitating kinases , directly phosphorylated NP in vitro .", "If PKC regulates NP:NP interactions , phospho-sites at this interface are likely PKC targets .", "But , our in vitro kinase assays were performed with purified recombinant NP , which is a complex mixture of monomeric and multimeric species in equilibrium where many of the key phospho-regulatory sites may be concealed by NP:NP interactions ( Figure 3—figure supplement 1A ) ( Mondal et al . , 2015 ) .", "To gain additional insight into the impact of PKC on NP oligomerization , we performed in vitro kinase assays with either wild-type ( WT ) NP or the oligomerization-defective mutant NP E339A .", "NP E339A purified exclusively as a monomer with the phospho-sites S165 and S407 solvent exposed compared to NP oligomers where they are concealed at the protein:protein interface ( Figure 3—figure supplement 1A ) ( Chenavas et al . , 2013; Ye et al . , 2013; Ye et al . , 2006 ) .", "Kinase assays again demonstrated that NP is phosphorylated by the catalytic domains of PKCβ2 , PKCδ and PKCη .", "Compared to the WT NP control , phosphorylation of NP E339A was markedly increased by all three PKC isoforms , supporting a model where PKC targets sites at the NP:NP interface ( Figure 3C ) .", "Multiple phospho-sites have recently been mapped to the NP:NP interface , including S165 in the groove and S402 , S403 , S407 , S413 in the tail loop ( Hutchinson et al . , 2012; Mondal et al . , 2015 ) .", "Our prior mutagenesis showed that NP S165 and S407 are the crucial phospho-sites that regulate homo-oligomerization and RNP formation ( Mondal et al . , 2015 ) .", "We asked whether PKCs selectively phosphorylate NP S165 and S407 and whether this regulates oligomerization .", "A double mutant eliminating both key phospho-sites , NP S165A/S407A , was purified and used as substrate in in vitro kinase assays .", "NP S165A/S407A purifies as a monomer due to the loss of important inter-subunit hydrogen bonds involving these serines ( Figure 3—figure supplement 1A ) ( Mondal et al . , 2015 ) .", "Phosphorylation was significantly reduced for NP S165A/S407A compared to the high levels detected for the monomer NP E339A ( Figure 3D ) .", "This defect was most pronounced for PKCδ and PKCη , where mutation of NP S165 and S407 reduced phosphorylation by almost 50% ( Figure 3—figure supplement 1B ) , confirming that these sites are major targets for PKC-mediated phosphorylation .", "Residual phosphorylation detected on the NP S165A/S407A mutant may reflect phosphorylation at one of the other previously identified phospho-sites ( Hutchinson et al . , 2012; Mondal et al . , 2015 ) , and our quantitative mass spectrometry suggest that some of these sites could also be targets of PKCs ( see below ) .", "We subsequently tested the functional consequences of PKC-mediated phosphorylation in cells .", "Each NP protomer contains a tail loop and binding groove , and can thus both insert into a growing NP chain and then receive the next incoming subunit .", "To understand the specific steps of NP self-association impacted by PKC phosphorylation , the complex multivalent nature of NP oligomerization was simplified to a binary interaction in our tail loop:groove interaction assay ( Mondal et al . , 2015 ) .", "Binding was measured between an NP deletion lacking the tail loop ( NPΔTL ) , which cannot oligomerize on its own , and the NP tail loop fused to green fluorescent protein ( GFP-TL ) ( Figure 3—figure supplement 2 ) .", "Binding partners were expressed in cells either with constitutively active PKC isoforms or controls ( Figure 3E ) .", "In control experiments , GFP-TL co-precipitated NPΔTL , recapitulating NP:NP interactions .", "Expression of PKCδ or PKCη severely reduced NP:NP interactions and co-precipitation of NPΔTL .", "Moreover , this loss of function in the presence of PKCδ and PKCη was again associated with the appearance of minor hyper-phosphorylated forms of NP .", "Whereas PKCβ2 can phosphorylate NP in an isolated kinase assay , expression of PKCβ2 in cells did not change the amount of co-precipitated NPΔTL .", "This disparity perhaps reflects the enhanced specificity of substrate:kinase interactions in cells or the involvement of other cellular co-factors that affect the functional impact of NP phosphorylation .", "Finally , we assessed the impact of PKCδ on RNP assembly by measuring the amount of NP co-precipitated with PA during a polymerase activity assay ( Figure 3—figure supplement 2 ) .", "As NP does not interact directly with PA , co-precipitation can only occurs in the context of an RNP .", "NP was specifically co-precipitated by PA-FLAG , yet these interactions were nearly eliminated when PKCδ was co-expressed ( Figure 3F ) .", "Notably , co-precipitation of the other polymerase subunits PB1 and PB2 was unaffected by PKCδ expression , suggesting that phosphorylation by PKCδ selectively impairs RNP assembly but not polymerase trimer formation .", "Together these results provide strong evidence that PKCδ and PKCη disrupt influenza polymerase activity by phospho-regulating NP tail loop:grove interactions , and thus have the potential to control NP oligomerization and RNP formation in cells .", "To confirm the biological importance of the NP:PB2:PKC complex , we examined its formation and activity during infection in the presence of endogenous levels of PKC .", "We focused on PKCδ given that NP exclusively interacts with full-length PKCδ ( Figure 2B , D ) , PKCδ phospho-regulates NP oligomerization and RNP assembly ( Figure 3 ) , and that PKCδ is abundantly expressed in human lung tissue , typical sites of influenza virus replication , and the human lung epithelial A549 cells used here ( Goldberg and Steinberg , 1996 ) .", "A549 cells were infected with influenza virus , influenza virus encoding FLAG-tagged PB2 , or mock treated .", "PB2-containing complexes were purified by anti-FLAG immunoprecipitation and blotted with anti-PKCδ antibody .", "Endogenous PKCδ specifically co-precipitated with PB2-FLAG ( Figure 4A ) .", "The reciprocal immunoprecipitation performed on infected cell lysates showed a similar interaction; PB2 specifically co-precipitated with endogenous PKCδ , compared to background amounts precipitated by a non-specific control ( Figure 4B ) .", "These data demonstrated interactions between PKCδ and PB2 during infections and validate results from our transfection assays showing that PB2 anchors interactions between NP and active PKC .", "This conclusion raised the possibility that PB2 isolated from infected cells contained PKC-specific kinase activity .", "To test this , PB2 complexes purified from infected cell lysate were used as a source of both kinase and substrate for in vitro phosphorylation .", "In vitro kinase assays performed with immuno-precipitated PB2 complexes revealed specific phosphorylation of NP , which was eliminated when the PKC inhibitor H7 was included in the reaction ( Figure 4C ) .", "Moreover , treatment of the immunoprecipitated complex with RNase A markedly increased phosphorylation , suggesting that the lower-order NP released by RNase A treatment rather than the oligomerized RNA-associated form was a better substrate for the kinase activity associated with the complex .", "This parallels our prior results showing enhanced phosphorylation of monomeric NP ( Figure 3C ) .", "Note that these kinase reactions did not include any exogenous second messenger activators of PKC , suggesting that PB2 is associated with activated PKCδ .", "Phospho-proteomics provided further evidence that PKCδ is activated during influenza .", "We identified peptides from PKCδ containing phosphorylated serines at residues 302 and 304 ( Table 1 ) , an autocatalytic marker of PKCδ activation ( Durgan et al . , 2007 ) .", "Combined , these data provide multiple lines of evidence that the viral polymerase protein PB2 anchors active PKCδ during infection .", "We generated PKCδ-deficient human lung A549 cells to study NP phospho-regulation during infection .", "Two independent clonal cell lines with nonsense mutations in both PRKCD alleles were created using the CRIPSR-Cas9 system ( Figure 5—figure supplement 1A–B ) .", "PKCδ protein expression was completely abolished in the knockout cells , while very low levels of PKCβ2 were detected by the cross-reactive antibody ( Figure 5A , Figure 5—figure supplement 1C ) .", "Despite the loss of PKCδ , both lines showed regular morphology and grew similar to the parental A549 cells .", "Regardless , care was taken to use early passage cells to avoid compensatory changes in gene expression or phenotypic drift that may arise over time .", "To test if PKCδ is responsible for NP phosphorylation during infection , we employed quantitative mass spectrometry to measure the extent of NP phosphorylation in the knockout cell lines ( Merrill and Coon , 2013; Richards et al . , 2015 ) .", "NP was purified from infected WT or PRKCD−/− cell lines and analyzed by targeted mass spectrometry ( Mondal et al . , 2015; Peterson et al . , 2012 ) .", "Phosphorylation was detected at sites in the tail loop ( S402 , S403 , S407 , S413 ) , binding groove ( S165 ) , and the body domain ( T378 ) of NP , in agreement with prior results ( Figure 5B–C ) ( Hutchinson et al . , 2012; Mondal et al . , 2015 ) .", "Compared to the WT cells , NP phosphorylation was considerably reduced in the PRKCD−/− cells ( Figure 5C ) .", "This was especially true at the key regulatory positions of NP S165 and S407 , where phosphorylation levels decreased by 60–80% .", "In two out of three replicates , pS407 was barely detected in samples from knockout cells .", "By contrast , the relative abundance of phosphorylation at T378 in the body was only marginally affected .", "These data illustrate that PKCδ is an important kinase targeting phospho-regulatory sites at the site of NP:NP interface .", "However , as phosphorylation was not completely ablated in the absence of PKCδ , these results also suggest at least some functional redundancy for NP phosphorylation , possibly fulfilled by other PKC isoforms .", "Given the reduction of NP phosphorylation at S165 and S407 in PKCδ knockout cell lines and the importance of these phosphorylation sites in virus replication , we predicted that viral gene expression and replication should be impaired in these cells .", "To test this , WT or PRKCD−/− A549 cells were infected with influenza reporter viruses ( PASTN ) based on the lab-adapted strain WSN or a primary isolate from the 2009 pandemic ( CA04 ) ( Karlsson et al . , 2015; Tran et al . , 2013; Tran et al . , 2015 ) .", "Gene expression was reduced for both viruses by ~60% in the PRKCD−/−−1 cell line and ~30% in the PRKCD−/−−2 line ( Figure 5D ) .", "This PKCδ-dependent defect was not simply a generic reduction in viral entry or gene expression , as gene expression from a West Nile virus replicon did not differ significantly between WT and knockout cells ( Figure 5D ) .", "PKCs have been previously implicated in the process of influenza virus entry , especially PKCβ2 ( Root et al . , 2000; Sieczkarski et al . , 2003 ) .", "To separate this role in entry from our functional analysis , we bypassed normal HA-mediated attachment and entry by performing infections with an influenza reporter virus pseudotyped with the vesicular stomatitis virus glycoprotein ( FVG-R ) ( Watanabe et al . , 2003 ) .", "Flow cytometry confirmed that entry of the pseudotyped influenza virus or bona fide vesicular stomatitis virus was similar in the presence or absence of PKCδ ( Figure 5—figure supplement 2A ) .", "Even with the pseudotyped FVG-R virus , influenza gene expression was impaired in the knockout cell lines , indicating that PKCδ is important post-entry for gene expression and genome replication independent of its role in influenza virus entry ( Figure 5D ) .", "Chemical inhibitor experiments have also suggested that PKCs play a role in the nucleo-cytoplasmic shuttling and localization of NP ( Bui et al . , 2002; Neumann et al . , 1997 ) .", "Nonetheless , immunofluorescence assays showed that the kinetics of NP nuclear import and subsequent export to the cytoplasm were unchanged in infected PRKCD−/− cells , although total NP levels might be reduced ( Figure 5—figure supplement 2B ) .", "We next tested whether PKCδ is important for production of infectious progeny .", "Compared to WT cells , production of normal influenza virus in the knockout cell lines was reduced by up to 1000 fold in a single-cycle infection , consistent with the decreases in viral gene expression observed with our reporter viruses ( Figure 5E ) .", "We created clonal A549 cell lines expressing a non-targeting control sgRNA to control for any effects that constitutive Cas9 expression might have on virus replication .", "Viral replication in these cells was indistinguishable from the pooled parental A549 cells ( Figure 5—figure supplement 3 ) .", "However , viral titers were reduced by over 1 . 5 logs in both PRKCD−/− cell lines compared to the sgRNA control line in a multi-cycle replication assay ( Figure 5F ) .", "Together , these data establish that phosphorylation of NP by endogenous PKCδ is crucial for viral gene expression , production of infectious progeny and multi-cycle replication .", "These reductions in replication likely represent combined effects of PKCδ knockout on both viral gene expression and entry ( see below ) .", "As viral gene expression and replication was not completely abolished in the absence of PKCδ , they also implicate additional PKC family members or other host kinases as playing secondary or redundant roles in regulating NP phosphorylation .", "Loss of PKCδ dramatically reduced NP phosphorylation and impaired viral gene expression ( Figure 5 ) .", "After entry , initial rounds of primary transcription occur on pre-formed viral RNPs deposited by the incoming virion and are dependent on attachment , entry and nuclear import .", "At later time points , the virus transitions to genome replication and secondary transcription in a process that requires NP oligomerization and formation of new RNPs .", "We probed the early steps during infection to identify the precise events impacted by PKCδ .", "Attachment and entry were measured with bioluminescent viral particles created with a reporter virus ( PASN ) that package the luciferase reporter into virions , allowing investigation of entry steps independent of the downstream events of vRNP nuclear import and transcription ( Tran et al . , 2015 ) .", "Virions were bound to cells 4˚C , subsequently shifted to 37˚C to synchronize entry , and cells were then treated with cyclohexamide to ensure any detected reporter activity was derived only from incoming virions .", "Knockout of PKCδ reduced virion attachment or uptake ~2 fold ( Figure 6A ) .", "To determine if this defect arose from attachment or fusion , a classic ‘acid bypass’ assay was performed where the viral membrane is fused to the plasma membrane by a transient low pH treatment causing vRNPs to be deposited directly into the cytoplasm ( Banerjee et al . , 2014; Matlin et al . , 1981 ) .", "Attachment and entry were indistinguishable for all cell lines in the acid bypass assay ( Figure 6A ) , indicating that the lack of PKCδ does not alter attachment or pH-dependent fusion , but rather endosome-mediated uptake .", "Entry assay results agreed with prior reports showing that PKC inhibitors and mutants interfere with entry and trap influenza virions in the late endosome , suggesting that PKCδ is one of the isoforms involved in these early steps during infection ( Root et al . , 2000; Sieczkarski et al . , 2003 ) .", "Our polymerase activity assays ( Figure 1 ) , RNP assembly experiments ( Figure 3E–F ) , and infections with pseudotyped virus ( Figure 5D ) provided multiple lines of evidence that PKCδ is also important post-entry .", "To test this , we used the acid bypass approach to assess the role of PKCδ in viral gene replication independent of its role in entry .", "Infections were initiated via acid bypass with the PASTN reporter virus that requires viral gene expression for reporter activity ( Tran et al . , 2013 ) .", "PKCδ knockout cell lines showed a significant reduction in viral gene expression at both 8 and 24 hr post-inoculation compared to parental A549 cells ( Figure 6B ) .", "Thus , PKCδ directly impacts gene expression and possibly genome replication during infection .", "Finally , we investigated how PKCδ regulates influenza virus RNA synthesis .", "Primary transcription is performed by pre-formed vRNPs deposited by the incoming virions .", "Transcripts from these vRNPs were quantified during infection by treating cells with cycloheximide to prevent the transition to genome replication and secondary transcription ( Barrett et al . , 1979 ) .", "Cells were infected by acid bypass , treated with cycloheximide , and total RNA was then extracted and used in primer extension assays to measure incoming vRNPs ( vRNA ) and primary transcription ( mRNA ) .", "vRNA and mRNA levels were similar in WT and PRKCD−/− cells , providing evidence that PKCδ is not required for nuclear import or primary transcription of incoming vRNPs ( Figure 6C ) .", "This is consistent with the fact that NP is already oligomerized in these incoming vRNPs and need not be further regulated .", "In stark contrast , we detected significant reductions in replication and transcription when infections were allowed to proceed in PRKCD−/− cells ( Figure 6D ) .", "cRNA levels were decreased by over 80% in knockout cells compared to the parental , indicating dramatic defects in genome replication .", "vRNA and mRNA levels were reduced by about 50% .", "The contribution of products from incoming RNPs , which do not require PKCδ , might possibly masking the full extent of the defects observed in replication and transcription .", "Thus , PKCδ plays an important role enabling genome replication and secondary transcription , events that both require formation of new RNPs ." ], [ "Influenza virus infections begin with a pioneering round of transcription from RNPs deposited by the incoming virion and transitions to genome replication and additional transcription at later time points .", "The polymerase copies genomic RNA while NP concomitantly oligomerizes along the length of the nascent product .", "NP changes during this process from an RNA-free monomer to a high-order RNA-bound oligomer in a process regulated by phosphorylation ( Mondal et al . , 2015; Turrell et al . , 2015 ) .", "Here we identify PKCδ as an important host regulator of RNP assembly and the resultant transition from transcription to replication .", "We show that PKCδ is recruited by PB2 to regulate NP oligomerization by phosphorylating both sides of the homotypic interface , targeting key phospho-sites in the binding grove ( S165 ) and tail loop ( S407 ) regions that mediate NP:NP interactions .", "Infections in PKCδ-knockout cells exhibited discrete defects in genome replication , a process that specifically requires NP oligomerization and RNP assembly , but not primary transcription , which is templated by pre-formed RNPs .", "This resulted in an overall reduction in viral gene expression and replication .", "Both hyper-phosphorylation by PKC over-expression or hypo-phosphorylation by PKCδ knockout or NP mutation disrupt the balance of NP oligomerization ( Mondal et al . , 2015 ) , leading us to propose that dynamic phosphorylation establishes a localized pool of monomeric NP that is essential for forming nascent RNPs and the transition to genome replication ( Figure 7 ) .", "This raises the possibility that additional factors , possibly cellular phosphatases , may be required to license NP oligomerization and initiate genome replication .", "Protein phosphatase 6 was reported to directly bind multiple subunits of the viral polymerase and regulate RNA synthesis during infection , although it is not known if it targets any of these proteins for dephosphorylation ( York et al . , 2014 ) .", "It is also possible that PKCδ activity or target specificity changes throughout infection to impact NP oligomerization potential .", "Viral product have also been proposed to regulate this transition , including NEP , svRNAs and trans-activating polymerases ( Jorba et al . , 2009; Perez et al . , 2010; Robb et al . , 2009; York et al . , 2013 ) .", "Thus , influenza virus deploys multiple approaches to ensure temporally regulated transcription and replication of the viral genome .", "Influenza virus infection triggers multiple kinase cascades including phosphoinositide 3 kinase ( PI3K ) , the Raf/MEK/ERK pathway , c-Jun N-terminal kinases ( JNK ) and PKCs ( Planz , 2013 ) .", "These kinases and their associated signaling pathways frequently play fundamental roles in mounting an efficient antiviral response by the host cell .", "However , these same kinases are also exploited by the virus for efficient replication by facilitating viral entry , nuclear import of viral proteins , and the nuclear export of vRNPs ( Planz , 2013 ) .", "We show here that PKCδ is specifically required for regulating RNP assembly and the resultant replication of the viral genome .", "Another family member , PKCα , has also been shown to phosphorylate PB1-F2 at sites that are important for viral replication ( Mitzner et al . , 2009 ) .", "In vitro assays have suggested that PKCα phosphorylates PB1 and NS1 ( Mahmoudian et al . , 2009 ) , although our cell-based assays did not demonstrate any significant regulatory role for PKCα on polymerase function .", "Whereas these specific activities are associated with modifying viral proteins , PKCs play a more general role in viral entry by modifying cellular processes .", "Genetic ablation of PKCδ or PKCβ2 activity reduced viral entry , possibly by perturbing endosomal sorting and maturation ( Figure 6 and ( Sieczkarski et al . , 2003 ) ) .", "The involvement of multiple PKC isoforms at multiple stages during infection suggests that broad-spectrum PKC inhibitors may be especially effective anti-viral compounds , even at concentrations that do not fully block PKC activity or their normal cellular functions .", "This may extend beyond influenza virus , as PKCs have been shown to regulate gene expression and replication of other negative-strand RNA viruses by phosphorylating their P proteins , including human parainfluneza virus 3 , Sendai virus , and rabies virus ( De et al . , 1995; Gupta et al . , 2000; Huntley et al . , 1997 ) .", "NP contains multiple serine and threonine phosphorylation sites throughout the length of the protein ( Hutchinson et al . , 2012; Mondal et al . , 2015 ) .", "Our data show that sites targeted by PKC during infection reside primarily at the tail loop:groove interface ( Figure 5 ) .", "S165 in the NP groove lies within a linear consensus recognition motif for PKCs , RxxS , where arginine at the −3 position facilitates kinase-substrate interactions ( Nishikawa et al . , 1997 ) .", "Both NP R162 and S165 in the PKC recognition motif are highly conserved in influenza A viruses .", "NP S403 also lies within a PKC recognition motif , although the phospho-site and recognition motif are not well conserved .", "In contrast , other residues including S402 , S407 and S413 do not reside within linear motifs and may rely on structure-based recognition motifs formed by non-contiguous regions of the protein ( Duarte et al . , 2014 ) .", "While kinase recognition motifs help to establish a specific kinase-substrate interaction , PKC and PKA family members often rely upon additional co-factors to phosphorylate target proteins ( Mochly-Rosen and Gordon , 1998; Welch et al . , 2010 ) .", "PKCs pair with anchoring proteins , collectively termed receptor of activated C-kinase ( RACKs ) , to enhance kinase activity and stability , increase their affinity and selectivity towards specific substrates , and integrate multiple signaling pathways .", "Our data suggest that PB2 performs an analogous function anchoring PKCδ and NP .", "Like RACKs , PB2 anchors PKCδ and NP through non-overlapping protein-protein interactions; PB2 helps to convey specificity , preferentially interacting with full-length PKCδ compared to full-length PKCβ2 or PKCη; and PB2 anchors activated PKCδ as evidenced by kinase activity in in vitro reactions without the need for exogenous activators ( Figures 3–4 ) .", "It is tempting to speculate that binding to PB2 might activate or stabilize the activated conformation of PKCδ , as has been shown for other RACK:PKC pairings ( Mochly-Rosen and Gordon , 1998 ) , or provide increased recognition of the non-canonical site at S407 .", "It will be important to determine the sites of interaction between PB2 and PKCδ , how this conveys preferential interaction with only one of the eleven different PKC isoforms , and if these interactions affect the activation state of PKC .", "While PKCδ is a major kinase that phospho-regulates NP oligomerization , the RACK-like activity of PB2 suggests that it is the NP:PB2:PKCδ complex that provides specificity and efficient modification of NP .", "It thus appears that the polymerase itself , or perhaps just the PB2 subunit alone , may contribute to the PKC-mediated phospho-regulation of RNP assembly .", "The PB2-phosphoNP complexes may function in cis to regulate the initial stages of RNP assembly or possibly in trans to shuttle monomeric NP to already growing RNPs , analogous to the role of the phosphoprotein P from non-segmented negative-sense RNA viruses that chaperones monomeric nucleocapsid proteins to replicating RNPs ( Masters and Banerjee , 1988 ) .", "It is not known how NP is recruited to the growing oligomer chain , but regardless of the route of recruitment , NP must ultimately be licensed for oligomerization to form an RNP .", "This adds another layer of control , and may involve altered PB2:PKC and PB2:NP interactions , changes to the activation state of PKCδ associated with PB2 , or possibly the involvement of cellular phosphatases .", "As both over expression and knockout of PKCδ perturb RNP formation , it is clear that the activity of PKCs must be finely balanced to enable NP oligomerization when and where it is needed .", "In summary , we have shown that influenza virus exploits the host kinase PKCδ to phospho-regulate NP oligomerization , revealing a complex regulatory pathway controlling RNP formation and the transition from transcription to replication during the viral life cycle ." ], [ "All virus related genes were derived from influenza A/WSN/33 virus .", "NP and polymerase proteins were expressed in cells from the plasmids pCDNA6 . 2-NP-V5 , pCDNA3-PB2-FLAG ( encoding a C-terminal FLAG tag ) or pCDNA3-PA and pCDNA3-PB1 .", "vNA-luc reporter plasmids encode firefly luciferase in the negative sense flanked by UTRs from the NA gene ( Regan et al . , 2006 ) .", "pET28a-NΔ7NP was used for bacterial expression of NP with a C-terminal His tag as described previously ( Mondal et al . , 2015 ) .", "Plasmids expressing full-length PKC isoforms ( β2 , δ , η ) or just the catalytic domains ( PKC-CAT α , β1 , β2 , γ , δ , ε , η , ι ) were previously described ( Table", "2 ) ( Soh and Weinstein , 2003 ) ( Addgene plasmids #21234 , 16380 , 16384 , 21238 , 16388 , 21242 , 21247 , 21254 ) .", "PKCθ-CAT was prepared by cloning the catalytic domain from the full length isoform into pHACE .", "PKC-CAT domains were mutated to catalytically inactive forms replacing specific lysine residues in the catalytic domains to either arginine or to methionine following the approach used for full-length PKC ( Soh and Weinstein , 2003 ) .", "Infections were performed with A/WSN/1933 ( H1N1 ) , WSN stably-encoding PB2 with a C-terminal FLAG tag ( WSN-PB2-FLAG ) ( Dos Santos Afonso et al . , 2005 ) or PA-2A-Swap-Nluc ( PASTN ) reporter viruses based on the strains A/WSN/33 ( H1N1 ) and A/California/04/2009 ( H1N1 ) ( Karlsson et al . , 2015; Tran et al . , 2015; 2013 ) .", "Entry assays were performed with the WSN-based reporter virus PA-SWAP-NLuc ( PASN ) that packages Nanoluc into virions ( Tran et al . , 2015 ) .", "The WSN-based reporter virus encoding VSV-G in place of HA and Renilla luciferase ( FVG-R ) or GFP ( FVG-G ) in place of NA was used as described ( Hao et al . , 2008; Watanabe et al . , 2003 ) .", "The West Nile virus replicon , where structural genes were replaced with Renilla luciferase , was prepared as described ( Lo et al . , 2003; Shi et al . , 2002 ) .", "Antibodies used include: anti-PKCδ ( sc-937 C20 , Santa Cruz Biotech ) , anti-HA clone 3F10 ( Roche ) , anti-V5 ( R961-25 , Invitrogen ) , anti-GFP ( B-2 , Santa Cruz Biotech ) , anti-NP ( H16-L10-4R5 ) ( Yewdell et al . , 1981 ) , anti-FLAG M2 ( Sigma ) , anti-PB1 and anti-PB2 ( Mehle and Doudna , 2008 ) , and anti-influenza virus RNP ( BEI Resources NR-3133 ) .", "293T ( CRL-3216 ) , MDCK ( CCL-34 ) , and A549 ( CCL-185 ) cells were purchased as authenticated stocks from ATCC .", "All cells were maintained in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 10% FBS at 37 ˚C and 5% CO2 .", "Cells are tested for mycoplasma contamination approximately once a month using MycoAlert ( Lonza LT07-218 ) .", "Activity assays were performed following our prior approach by transfecting 293T cells in triplicate with plasmids expressing PA , PB1 , PB2 , NP and negative-sense vNA-luciferase reporter ( Kirui et al . , 2016 ) ( Figure 1—figure supplement 1 ) .", "Where indicated , cells were co-transfected with plasmids expressing PKC catalytic domains or dominant negative versions .", "Polymerase activity was measured using the luciferase assay system ( Promega ) and NP expression was confirmed by western blotting .", "293T cells expressing HA-tagged PKC , V5-tagged NP and polymerase proteins were lysed in radio-immunoprecipitation assay ( RIPA ) buffer ( 50 mM Tris-HCl [pH 7 . 5] , 150 mM NaCl , 2 mMEDTA , 1% NP-40 , 0 . 5% deoxycholate , 0 . 1% SDS ) supplemented with 5 mg/ml of BSA and clarified by centrifugation .", "Lysates were incubated with anti-HA antibody and immunocomplexes were captured on Protein A Dynabeads ( Invitrogen ) , washed extensively and analyzed by western blotting .", "A549 cells were infected with WSN using the low pH fusion buffer as described above at an MOI of 10 to measure primary transcription or at an MOI of 1 to measure replication .", "After attachment and low pH-mediated entry , cells were either incubated in VGM for 8 hr to measure replication or 6 hr with 1 mM cyclohexamide to measure primary transcription or incubated .", "Total RNA was isolated using TRIzol reagent ( Invitrogen ) and primer extensions assays were performed as described using primers using primers to detect viral NA RNAs and host 5S RNA ( Mehle and Doudna , 2008; Robb et al . , 2009 ) .", "Transcription products were resolved via UREA-PAGE , quantified by phosphorimaging , and analyzed using ImageQuant TL software ( GE Healthcare ) .", "Wild type or PKCδ knockout cells grown on coverslips were infected with WSN at an MOI of 2 .", "Virion binding was performed at 4 ˚C for 1 hr and synchronous infections were initiated by shifting cells to 37 ˚C .", "Cells were fixed at different time points post-inoculation with 3% formaldehyde and permeabilized with 0 . 1M Glycine/0 . 1% Triton-X 100 in PBS for 10 min at room temperature .", "After blocking with 4 ˚C with 3% BSA overnight , NP was detected with anti-RNP antibody and Alexa Fluor 488-conjugated donkey anti-goat IgG ( Cell Signaling ) .", "Cells were imaged using Zeiss Axio Imager M2 and post-processed with ImageJ .", "The in vitro kinase assay was adapted from the protocol originally described by Soh et al . ( Soh and Weinstein , 2003 ) .", "Briefly , 293 T cells were transfected with PKC-CAT or control plasmids , cells were lysed in PKC extraction buffer ( 50 mM HEPES [pH 7 . 5] , 150 mM NaCl , 0 . 1% Tween-20 , 1 mM EDTA , 2 . 5 mM EGTA , 10% glycerol , protease and phosphatase inhibitors ) , and PKCs were immunoprecipitated with anti-HA antibody .", "Immune complexes were captured with Protein A Dynabeads , washed in extraction buffer , washed in PKC reaction buffer ( 50 mM HEPES [pH 7 . 5] , 10 mM MgCl2 , 1 mM dithiothreitol , 2 . 5 mM EGTA ) , and finally resuspended in 20 μl of the reaction buffer to provide the source of kinase used in each assay .", "Wild type or mutant recombinant NP was purified from bacteria following our existing protocol and treated with RNaseA for 2 hr prior to use as substrate ( Mondal et al . , 2015 ) .", "2 μg of NP was reacted with equivalent amount of kinase or control complexes in the presence of 10 μCi of γ-32P ATP at 37°C for 1 hr .", "Reactions were terminated by boiling in SDS sample buffer and analyzed by SDS-PAGE gel electrophoresis and autoradiography .", "Where mentioned , the PKC inhibitor 1- ( 5-isoquinolinesulfonyl ) −2-methylpiperazine ( H7 ) ( Sigma ) was added to the reaction .", "For in vitro kinase assays with endogenous PKCδ , A549 cells were infected with WSN-PB2-FLAG virus at an MOI of 5 and harvested at 6 hr of post-inoculation .", "Cells were lysed in PKC extraction buffer and PB2-FLAG was immunoprecipitated with M2 affinity resin ( Sigma ) .", "Immuno-captured complexes were resuspended in PKC reaction buffer and used as a source of both kinase and substrate .", "Samples were further treated with RNaseA , RNaseA and H7 , or left untreated prior to performing a kinase reaction as described above .", "Multicycle replication assays were performed by infecting A549 cells with WSN at an MOI of 0 . 001 .", "Aliquots were collected at the indicated time points and viral titers were measured by plaque assay on MDCK cells .", "Entry assays were performed by incubating WT or PRKCD−/− A549 cells with PASN at an MOI of 5 in virus growth media ( VGM: DMEM , 0 . 2% bovine serum albumin ( BSA ) , 25 mM HEPES buffer , and 0 . 25 μg/ml TPCK-trypsin ) at 4˚C for 1 hr followed by washing with cold PBS to remove unbound virus particles .", "Acid bypass assays were modified from previous approaches ( Banerjee et al . , 2014; Matlin et al . , 1981 ) .", "Following virion binding , cells were incubated in low pH fusion buffer ( 50 mM citrate , pH 5 . 0 , 154 mM NaCl ) or mock-treated with pH 7 buffer ( 20 mM HEPES , pH7 . 4 , 154 mM NaCl ) for 2 min at 37°C and washed with cold PBS .", "Finally , pre-warmed VGM supplemented with 1 mM cycloheximide was added and the cells were incubated at 37˚C for 2 . 5 hr .", "Virion entry was quantified using the Nano-Glo luciferase assay ( Promega ) .", "Gene expression by West Nile virus replicons was measured by inoculating WT or PRKCD-/- A549 cells at an MOI of 0 . 1 .", "Renilla luciferase activity was measured 24 hr post-inoculation .", "PASTN reporter virus replication assays were initiated by acid bypass at an MOI of 0 . 05 .", "Cells were incubated at 37˚C for 8 and 24 hr and viral gene expression was measured using the Nano-Glo luciferase assay ( Promega ) .", "sgRNAs targeting the fourth exon of PRKCD ( aacgatgaaccgccgcggag ) or a non-targeting control ( ACGGAGGCTAAGCGTCGCAA ) were cloned into lentiCRISPR v2 ( Addgene plasmid # 52961 , Feng Zhang ( Sanjana et al . , 2014 ) ) .", "Lentiviral particles were produced with the resulting plasmids and used to transduce A549 cells .", "Transduced cells were selected with puromycin and single-cell sorted by FACS .", "Following outgrowth , clonal lines were screened for homozygous PRKCD gene disruptions by Indel Detection by Amplicon Analysis ( IDAA ) using primers that amplify the edited locus to identify genomic deletions of the target locus ( Yang et al . , 2015 ) ( Figure 5—figure supplement 1A ) .", "Gene disruption was confirmed by Sanger sequencing of IDAA amplicons and identified the clonal lines PRKCD−/−−1 and PRKCD−/−−2 containing homozygous non-sense mutations ( Figure 5—figure supplement 1B ) .", "Knockout was confirmed by western blotting of cell lysate .", "All assays were performed with early passages of knockout cell lines .", "WT or PRKCD−/−−1 and PRKCD−/−−2 A549 cells were infected with WSN at an MOI of 5 .", "Cells were harvested at 4 and 8 hpi , pooled , lysed and NP was purified by immunoprecipitation as described previously ( Mondal et al . , 2015 ) .", "Purified protein samples were lyophilized , dissolved in 8 M urea , reduced and alkylated with 10 mM tris ( 2-carboxyethyl ) phosphine and 40 mM chloroacetamide , diluted to a final concentration of 1 . 5 M urea using 50 mM Tris , and digested with trypsin overnight at room temperature .", "Resultant peptides were desalted using a C18 Strata X column ( Phenomenex ) and enriched for phosphorylation by immobilized metal affinity chromatography using Ni-NTA magnetic agarose beads ( Qiagen ) ( Rose et al . , 2012 ) .", "Both non-phosphorylated and phosphorylated peptide samples were resuspended in 0 . 2% formic acid and analyzed by MS . A 100 min nano-liquid chromatography gradient was used to introduce peptides to an Oribtrap Elite mass spectrometer ( Thermo Scientific ) and peptides were analyzed by data dependent acquisition ( DDA ) using higher-energy collisional dissociation ( HCD ) to fragment them ( Vincent et al . , 2013 ) .", "Spectra obtained from these DDA experiments were searched against a concatenated target-decoy database containing the protein sequences of Homo sapiens and influenza A virus ( Uniprot ) using both Sequest within the Proteome Discoverer software package ( Thermo Fisher ) and MaxQuant ( Cox et al . , 2011; Cox and Mann , 2008 ) .", "For all samples , cysteine carbamidomethylation and methionine oxidation were searched as fixed and variable amino acid modifications , respectively , and phosphorylation of serine , threonine , and tyrosine residues were searched as variable modifications .", "Database searches utilized a precursor mass tolerance of 40 ppm and a fragment ion tolerance of 0 . 02 Da , with peptide identifications filtered to a 1% false discovery rate ( FDR ) .", "Proteome Discoverer searches used PhosphoRS to localize phosphorylation to amino acid residues using a fragment mass tolerance of 0 . 02 Da , automatically considering neutral loss peaks for HCD and a maximum of 500 position isoforms per phosphopeptide ( Taus et al . , 2011 ) .", "Phosphosites were determined as localized if they were scored with a localization probability >75% .", "Influenza NP phosphopeptides were identified from DDA MS runs , including one peptide with phosphorylation localized to the NP T378 .", "Subsequent targeted MS experiments localized phosphorylation to the S165 site on the tryptic NP peptide MCSLMQGSTLPR .", "These targeted MS experiments also monitored the m/z values for this peptide with one or two oxidized methionine residues .", "Four phosphopeptide isoforms for the NP peptide ASSGQISIQPTFSVQR were targeted for relative quantification .", "Using HCD fragmentation , phosphorylation was localized to the S402 , S403 , S407 , and S413 residues on this peptide ( Mondal et al . , 2015 ) .", "Extracted ion chromatograms were generated for the six NP phosphopeptides modified at S165 , S402 , S403 , S407 , S413 , and T378 .", "The peak area for each peptide was normalized to the total NP protein loaded on-column for each lysate , and the relative abundance of each phosphopeptide was compared between the lysates of infected wild-type A549 or mutant PRKCD−/− KO cell lines .", "Data are representative of at least three independent experiments , with each experiment performed in triplicate or greater .", "Multiple comparisons were performed with a one-way ANOVA and statistical significance was indicated when p<0 . 05 ." ] ]
[ "Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points .", "This process requires de novo assembly of the viral replication machinery , large ribonucleoprotein complexes ( RNPs ) composed of the viral polymerase , genomic RNA and oligomeric nucleoprotein ( NP ) .", "Despite the central role of RNPs during infection , the factors dictating where and when they assemble are poorly understood .", "Here we demonstrate that human protein kinase C ( PKC ) family members regulate RNP assembly .", "Activated PKCδ interacts with the polymerase subunit PB2 and phospho-regulates NP oligomerization and RNP assembly during infection .", "Consistent with its role in regulating RNP assembly , knockout of PKCδ impairs virus infection by selectively disrupting genome replication .", "However , primary transcription from pre-formed RNPs deposited by infecting particles is unaffected .", "Thus , influenza virus exploits host PKCs to regulate RNP assembly , a step required for the transition from primary transcription to genome replication during the infectious cycle ." ]
[ "To be able to multiply , the influenza virus needs to enter the cells of its host and trick them into copying the virus’ genetic information and assembling new virus particles .", "The genetic information of the virus is stored in molecules of ribonucleic acid ( RNA ) and encodes several viral proteins that are involved in making the new virus particles .", "These proteins include an enzyme known as the viral polymerase and a “nucleoprotein” .", "The viral polymerase copies the RNA and then the nucleoprotein binds to the new RNA to protect it until it is packaged into new virus particles .", "Many nucleoprotein units assemble into long chains that coat the whole length of the RNA , but it is not yet known exactly how this process is controlled .", "In cells , other enzymes known as kinases are able to alter the activities of many proteins by modifying the structures of proteins by a process called phosphorylation .", "Influenza nucleoprotein was previously shown to be phosphorylated .", "It is therefore possible that the influenza virus may use phosphorylation to control the assembly of nucleoproteins into chains along the RNA .", "However , the virus’ RNA does not encode any kinase enzymes of its own , so it must rely on kinases from its host cell .", "Human cells produce many kinase enzymes that can be grouped into several different protein families .", "Mondal et al . studied the role of the protein kinase C family in making new virus particles .", "The experiments show that modifying the members of this protein family to be permanently active causes the viral nucleoprotein to be phosphorylated at two specific sites on the protein .", "This regulates the assembly of the nucleoproteins into long chains on the RNA , and ultimately promotes the production of new virus particles .", "Closer examination revealed that this effect was primarily down to one specific kinase known as PKCδ .", "The virus was less able to multiply in human lung cells that were missing PKCδ – specifcially because the formation of nucleoprotein chains was no longer regulated – and these cells produced lower quantities of virus proteins .", "Taken together , these findings show that kinases produced by host cells can control the ability of viruses to replicate by modifying the viral nucleoproteins .", "In the future , it may be possible to develop new drugs that target PKCδ and other cellular factors the virus needs to help treat influenza infections ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "microbiology and infectious disease", "cancer biology" ]
A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe
elife-47788-v1
[ [ "Cancers normally arise from mutations in an organism’s own cells , and they either regress or continue to grow until they kill the host organism .", "In a few cases , however , including Tasmanian devils ( Pearse and Swift , 2006; Pye et al . , 2016 ) , dogs ( Murgia et al . , 2006; Rebbeck et al . , 2009 ) , and bivalves ( Metzger et al . , 2016; Metzger et al . , 2015 ) , cancer cells naturally transfer from one animal to another as an infectious allograft , leading to lineages of transmissible cancer that spread through the populations ( Metzger and Goff , 2016 ) .", "In bivalves , disseminated neoplasia ( also called hemic neoplasia ) is a leukemia-like disease that has been found in more than a dozen marine species , characterized by an amplification of rounded , polyploid cells in the hemolymph of animals that infiltrate into tissues .", "This disease often occurs in outbreaks and has led to significant population losses in many bivalve species ( Barber , 2004; Carballal et al . , 2015 ) .", "Pollution and retroviruses had been investigated as suspected causes , but until recently the etiology of disseminated neoplasia in bivalves was unknown .", "In four of the bivalve species affected by disseminated neoplasia ( soft-shell clams , Mya arenaria; cockles , Cerastoderma edule; golden carpet shell clams , Polititapes aureus; and bay mussels , Mytilus trossulus ) , recent investigation has shown that the diseases are bivalve transmissible neoplasias ( BTNs ) , showing that transmissible cancer may , in fact , be a common phenomenon in marine invertebrates .", "Disseminated neoplasia has been reported in marine mussels as early as 1969 ( Farley , 1969 ) , and has been observed consistently throughout Europe in both M . edulis ( Green and Alderman , 1983; Lowe and Moore , 1978; Rasmussen , 1986 ) and M . galloprovincialis ( Ciocan and Sunila , 2005; Carella et al . , 2013; Gombač et al . , 2013 ) , albeit at lower prevalence than has been observed in some M . trossulus populations , which reached >20% in some locations in the 1980s and was associated with population losses ( Barber , 2004; Bower et al . , 1994 ) .", "Mussels in the genus Mytilus form a complex of species , including M . trossulus , M . edulis , and M . galloprovincialis , native to the northern hemisphere , and M . chilensis , M . platensis , and M . planulatus , native to the southern hemisphere ( Zbawicka et al . , 2018 ) .", "These species have an anti-tropical distribution , spanning the temperate waters of the world , and they can hybridize , leading to widespread introgression of allospecific alleles ( Fraïsse et al . , 2016 ) as well as hybrid zones in several locations ( Bierne et al . , 2003 ) .", "Disseminated neoplasia has been reported in four of these Mytilus species across multiple continents ( Barber , 2004; Carballal et al . , 2015 ) , but so far the only lineage of disseminated neoplasia in the genus that has been directly confirmed to be a transmissible cancer is in M . trossulus along the Pacific coast of British Columbia ( BC ) , Canada ( Metzger et al . , 2016 ) .", "A population genetics analysis of M . edulis from France , conducted to detect hybridization and introgression between Mytilus species by detecting species-specific SNPs , identified a few individuals with unexpected ‘chimeric’ signals .", "The DNA samples from these individuals included M . trossulus alleles in a population where that species was absent , and the ratio of the M . edulis and M . trossulus alleles were not consistent with a diploid hybrid genome .", "Instead , this could be better explained by a mixture of two genotypes ( one M . edulis and one M . trossulus in origin ) within a single individual ( Riquet et al . , 2017 ) .", "This suggested the hypothesis that M . edulis in France are affected by a BTN originating from M . trossulus , possibly the same lineage as previously identified in Canada .", "More recently , analysis of a severe mass mortality event ( Benabdelmouna and Ledu , 2016 ) in French mussels that began in 2014 led to the observation of disseminated neoplasia in many populations of M . edulis and M . galloprovincialis ( Benadelmouna et al . , 2018 ) , although it has not yet been determined whether this neoplasia is transmissible .", "M . chilensis mussels are found along the western coast of South America , and disseminated neoplasia has been reported in multiple populations in Chile and Argentina since 1998 ( Lohrmann et al . , 2019; Cremonte et al . , 2015; Campalans et al . , 1998; Cremonte et al . , 2011 ) .", "Prevalence has varied by location , ranging from undetectable in some populations to as high as 12% in the Castro region of Chile and 13% in the Beagle Channel in southern Argentina , although no notable mass mortality events have been associated with neoplasia in these populations .", "We investigated disseminated neoplasia found in M . chilensis from Argentina and Chile , as well as M . edulis from France and the Netherlands , to determine if these cancers are transmissible cancers or conventional cancers and , if transmissible , whether these cancers represent new cases of the same lineage of transmissible cancer previously reported in Canada ( here termed Mytilus BTN1 ) or whether they are novel cancer lineages .", "Mytilus BTN1 was able to be distinguished from normal M . trossulus genotypes at both a nuclear and mitochondrial locus ( Metzger et al . , 2016 ) .", "Through analysis of multiple genetic markers in South American M . chilensis and European M . edulis individuals , we found evidence of transmissible cancer in both populations .", "We found that neither cancer matched the Mytilus BTN1 lineage , previously found in BC M . trossulus , but instead the cancers in both species appear to come from a single , previously unidentified transmissible cancer , here called Mytilus BTN2 or MtrBTN2 .", "This BTN lineage arose from an M . trossulus individual , and it has since crossed into two different Mytilus species and is now affecting animals across both the Atlantic and Pacific Oceans and in both the Northern and Southern Hemispheres ." ], [ "To determine whether the disseminated neoplasia in M . chilensis was due to a conventional cancer or a transmissible cancer lineage , we collected 60 M . chilensis animals from Argentina in 2012 .", "Histological analysis was used to diagnose the animals for the presence of disseminated neoplasia , and we observed a disease prevalence of 10% .", "DNA was extracted from tissues of three diseased animals and three normal animals , and an intron-spanning region in the nuclear gene Elongation Factor one alpha ( EF1α ) was amplified and sequenced .", "A normal individual would be expected to have one or two alleles ( depending on whether it was homozygous or heterozygous at that locus ) , and a cancer would be expected to have its own set of alleles , derived from its original host .", "All three normal animals had one or two alleles , as expected , but two of the diseased animals ( Mch41 and Mch42 ) had four distinct alleles , consistent with a mixture of two host alleles and two cancer-specific alleles ( Figure 1A ) .", "Notably , each diseased sample had two alleles that were unique ( corresponding to host alleles ) , but two alleles from animal Mch41 were nearly exact matches to two alleles from Mch42 ( here notated as G and H ) .", "This is consistent with the presence of a transmissible cancer containing alleles G and H . These cancer-associated alleles were not cloned from one of the three diseased animals ( Mch23 ) , but cloning of PCR products is not very sensitive .", "This individual either does not have this cancer lineage or has a lower level of cancer that was not detectable with this method .", "A second collection of farmed M . chilensis from two locations in Chile confirmed the presence of disseminated neoplasia in these populations ( 9 . 6% in Calbuco and 4 . 5% in Castro ) .", "EF1α alleles cloned from a diseased individual from this location exactly match the G and H cancer-associated sequences from Argentina .", "Phylogenetic analysis of these putative cancer-associated alleles shows that they are likely of M . trossulus or M . chilensis origin ( Figure 1B ) .", "However , both cancer-associated alleles are clearly distinct from normal M . trossulus individuals and distinct from either allele of the previously identified BC transmissible neoplasia , Mytilus BTN1 .", "Additionally , phylogenetic analysis shows that the EF1α S allele from Mytilus BTN1 ( previously termed the ‘minor’ allele ) is closely related to a subset of normal M . trossulus alleles .", "Thus , the tree is incompatible with the cancer-associated G and H alleles arising from the same cancer lineage as the Mytilus BTN1 S allele , although analysis of additional loci is needed to confirm this finding .", "The BTN found in M . chilensis therefore likely represents a separate , independent lineage of transmissible cancer , here called Mytilus BTN2 .", "The first evidence that disseminated neoplasia in European M . edulis was a BTN was the observation of ‘chimeric’ signals in a SNP detection assay , in which SNPs specific for M . trossulus were detected in five M . edulis samples out of 938 .", "Additionally , the M . trossulus SNPs were detected at intermediate fluorescence ratio that could not be explained by an individual that was homozygous or heterozygous at those loci ( i . e . variant allele fractions were not close to 50% or 100% ) ( Riquet et al . , 2017 ) .", "We sequenced the EF1α alleles present in DNA from four of these ‘chimeric’ samples of M . edulis collected from locations on the Atlantic coast of Europe and found more than the normal two alleles in three of them ( Figure 1A ) .", "Only one or two alleles were observed in normal M . edulis from the same populations .", "Two alleles were shared in more than one diseased sample , suggesting that they could be from a transmissible cancer .", "Unexpectedly , these two cancer-associated alleles found in diseased M . edulis did not match the previously reported Mytilus BTN1 lineage , found in M . trossulus in Canada , but instead exactly matched those found in cancer samples from M . chilensis in South America ( Mytilus BTN2 ) ( Figure 1B ) .", "In these European M . edulis cancer samples , we found alleles identical to the G allele from M . chilensis and two nearly identical versions of the H allele , with only one SNP between them ( termed H’ ) .", "Both the H and H’ alleles were represented by multiple clones and found in multiple individuals , so H’ likely represents a true second copy of the H allele within the cancer cells .", "Disseminated neoplasia is polyploid , and this result is consistent with a de novo mutation which occurred in one copy of the H allele after genome duplication ( and likely after divergence of the South American and European strains of this lineage ) .", "To gain more information about identity of the cancer lineage , we sequenced three additional loci using primers which amplify alleles in all three Mytilus species tested here .", "Histone 4 ( H4 ) is a conserved gene present in multiple copies in the nuclear genome ( Eirín-López et al . , 2004 ) .", "We amplified and cloned alleles from M . chilensis and M . edulis samples , as done with EF1α .", "We observed 2–5 alleles in diseased samples and only 1–2 in normal samples ( Figure 2A ) .", "Looking only at normal alleles , there is a clear grouping based on species of origin in a phylogenetic tree ( Figure 2B ) .", "There are three exceptions to this clustering , from one normal M . trossulus ( MW59 ) and two diseased M . chilensis ( Mch41 and Castro26 ) with some distinctly M . edulis-like alleles .", "These may reflect introgression or true hybrid individuals—MW59 also has an EF1α allele corresponding to M . edulis and likely represents a hybrid with this species , which has been introduced into Canada ( Crego-Prieto et al . , 2015 ) .", "Two sets of cancer-associated alleles were identified from both M . chilensis and M . edulis , which group together .", "As with EF1α , the alleles associated with cancer in M . chilensis and M . edulis are distinct from the sequences of Mytilus BTN1 found in M . trossulus from Canada .", "Overall , the H4 locus has less variability than the EF1α locus .", "Notably , there are alleles found in normal M . trossulus individuals that are exact matches to alleles found in both Mytilus BTN1 and Mytilus BTN2 .", "These data again argue that this newly identified cancer lineage has an independent M . trossulus origin and that it arose from a different M . trossulus individual than the one that gave rise to Mytilus BTN1 .", "Mytilus BTN1 likely arose within an M . trossulus individual with a 2A H4 allele and Mytilus BTN2 arose from a different M . trossulus individual which carried both MR-like and KNS-like alleles .", "In addition to the nuclear loci , we also amplified a control region of the mitochondrial genome ( mtCR ) including large subunit ribosomal RNA ( lrRNA ) , a variable domain , and conserved domain ( Burzyński et al . , 2006 ) .", "As Mytilus mussels have double uniparental inheritance of mitochondria ( in which all individuals inherit mitochondria maternally , but a second male-specific lineage of mitochondria is also passed down to male offspring gonads ) , we amplified a female allele from all individuals , and both female and male alleles from about half of the individuals ( 10 of 21 total ) ( Figure 3A ) .", "The sequences of normal M . trossulus and M . edulis are similar to previously published reference sequences from those species .", "In addition , we amplified a M . trossulus-like allele from cancer samples from both M . edulis and M . chilensis .", "This allele ( termed ‘D’ ) is mostly female-like , but it switches to male-like sequence at the 3’ end ( Figure 3B ) .", "This recombinant sequence closely matches a recombinant sequence previously reported in a Baltic M . trossulus individual ( Śmietanka and Burzyński , 2017; Zbawicka et al . , 2014 ) .", "Interestingly , a second M . trossulus-like allele ( C ) was found in all three cancer samples of M . chilensis , which also represents a recombination between female and male sequences , but with a different recombination point than the first .", "This argues for heteroplasmy of the cancer cells in M . chilensis , and either loss of this heteroplasmy in the cancer lineage infecting M . edulis or capture of this new mitochondrial genome in the cancer lineage infecting M . chilensis .", "These unique recombinants between female and male sequences again argue that Mytilus BTN1 and BTN2 represent distinct lineages of cancer .", "Even when considering only the region without any recombination with the male sequence ( Figure 3C ) , both of these mitochondrial alleles are distinct from those found in normal animals and from the previously identified Mytilus BTN1 lineage , and the phylogenetic tree suggests that Mytilus BTN1 and BTN2 arose from different M . trossulus individuals .", "We additionally sequenced a second region in the mitochondrial DNA , the cytochrome c oxidase I gene ( COI ) .", "Standard ‘universal’ molluscan primers used for barcoding ( Folmer et al . , 1994 ) did not amplify the mtCOI allele from Mytilus BTN2 , but by using degenerate primers flanking the region , we were able to amplify and sequence mtCOI from all samples ( Figure 4A , Supplementary file 1 ) .", "The mtCOI of the male-specific mitogenome was not amplified by these primers .", "We identified a clear separation of the mitogenomes of all three species , with the mtCOI from both Mytilus BTN1 and 2 nested within the M . trossulus sequences , as with the three other loci tested ( Figure 4B ) .", "The Mytilus BTN1 allele ( U ) again showed clear differences from the Mytilus BTN2 allele ( B ) .", "As in the mitochondrial control region , the closest published sequence to the Mytilus BTN2 allele is the sequence from 62mc10 , a normal M . trossulus from the Baltic region .", "This again provides evidence that the two lineages of transmissible cancer arose from distinct M . trossulus individuals .", "Unexpectedly , while the B allele was found in Mytilus BTN2 samples from both Europe and Argentina , it was not identified in the M . chilensis samples from Chile .", "In these two samples , two mtCOI alleles were found , and they share a common allele that is likely to be the cancer-associated allele ( Q ) .", "However , this allele is different from allele B , and it is M . chilensis-derived .", "These samples show evidence of the common Mytilus BTN2 alleles at all other nuclear and mitochondrial loci , so this suggests that there has been recombination between cancer and host mitochondria somewhere in the M . chilensis population , resulting in a subset of Mytilus BTN2 cells in Chile with new recombinant mitogenomes .", "While two alleles ( C and D ) were identified in the M . chilensis mtCR region , only one allele was detected at mtCOI .", "The control region alleles C and D were very similar , with the exception of a different recombination site with the male-like sequence , so it is likely that both mitogenomes have the same sequence at the more conserved COI locus .", "Additionally , in Castro26 , a second recombinant sequence was identified that was composed of half M . trossulus sequence and half M . chilensis sequence .", "This could represent the second cancer mitogenome ( i . e . mtCR D and mtCOI Q could be on one mitogenome and mtCR C and the recombinant mtCOI could be the other ) , but it was supported by a single clone , so it is unclear if it was a PCR artifact or a true second allele .", "Further analysis will be required to determine the full recombinant cancer mitogenomes in the Mytilus BTN2 cancers in this region of Chile .", "The cancer-associated alleles identified at these four loci ( EF1α , H4 , mtCR , mtCOI ) were detected through cloning of PCR products from most of the M . chilensis and M . edulis individuals that were diagnosed as diseased , but not all ( 6 of 8 , 5 of 8 , 7 of 8 , and 8 of 9 , respectively ) .", "For example , the cancer-associated EF1α alleles were not cloned from M . chilensis sample Mch23 .", "Quantitative PCR ( qPCR ) was used to determine whether the diseased animals in these cases , such as Mch23 , had the cancer-associated alleles at a level too low to be identified through cloning or whether the transmissible cancer allele was absent from that individual .", "Primers specific for one of the cancer-associated EF1α alleles ( H ) were designed , and amplification was compared to amplification from ‘universal’ Mytilus EF1α primers ( Supplementary file 2 ) .", "Analysis of the M . chilensis samples showed that the cancer-associated allele was found in all three samples from Argentina that were diagnosed as diseased , including Mch23 ( Figure 5 ) , and it was undetectable in all three normal samples .", "In the M . chilensis from the Pacific Ocean , however , we found that two of the four diseased animals had high levels of the cancer-associated allele , but the other two did not .", "They likely have either conventional cancer or a different transmissible cancer lineage .", "Additional samples of all three Mytilus species were analyzed by this method .", "In M . edulis , qPCR showed the presence of the cancer-associated allele in all mussels diagnosed as diseased but not in normal samples .", "To test whether Mytilus BTN2 could be present in the Canadian individuals at a low level , qPCR analysis was conducted on the original samples from which Mytilus BTN1 was identified ( Metzger et al . , 2016 ) .", "The allele associated with Mytilus BTN2 in M . edulis and M . chilensis was undetectable in both normal and diseased M . trossulus samples from Canada .", "qPCR primers specific for both cancer-associated H4 alleles were made , and both were detected in all animals diagnosed as diseased except for the two Chilean samples that were negative for the cancer-associated EF1α allele ( Figure 5 ) .", "One allele was present at a higher level within each diseased animal than the other , but the ratio of the two alleles was not completely consistent across all individuals , suggesting that there has been recombination or copy number variation in some subgroups of the cancer lineage .", "The H4 cancer-associated alleles were also identified in several normal M . trossulus .", "This confirms that H4 alleles found in Mytilus BTN2 are present in some individuals in the normal M . trossulus population .", "This is expected , as each of the two transmissible cancer lineages arose in the past from a different normal M . trossulus individual .", "Analysis of the two cancer-associated mitochondrial control region alleles shows that the C allele ( only cloned from M . chilensis samples ) is present in M . chilensis cancers at a very low level ( <1% of total allele fraction ) .", "It was also undetectable in four of eight diseased M . edulis , again suggesting that this heteroplasmy may be lost in some of the cancer cells spreading in the M . edulis population ( Figure 5 ) .", "Unexpectedly , when comparing the copy number of the D allele to amplification at a conserved locus about 200 bp away ( shown schematically in Figure 3B ) , the neoplastic samples yielded values greater than 100% .", "Further investigation revealed a tandem repeat in the D mitochondrial genome , with multiple copies of the control region that are targeted by allele-specific primers .", "A tandem repeat was confirmed by generating the reverse complement of the qPCR primers specific for allele D and amplifying and sequencing the tandem repeat junction ( marked in Figure 3B ) .", "While it cannot be directly used to estimate the percent of cancer cells in a sample , this locus is a highly sensitive marker of this lineage .", "Analysis of the mtCOI locus by qPCR confirmed that the main Mytilus BTN2 allele was identified in all M . edulis cancer samples , and in the Argentinian M . chilensis , but not the Chilean M . chilensis .", "This suggests that the mitochondria in the Chilean samples ( Castro26 and 52 ) has been completely replaced with one that is a recombinant of the original Mytilus BTN2 M . trossulus-derived sequence and newly acquired M . chilensis-derived sequence .", "Analyses at all loci confirm that two samples from Chile ( Castro49 and 84 ) are not afflicted by Mytilus BTN2 , while it is present at high levels in the other two diseased samples from Chile and all three tested samples from Argentina and eight from Europe .", "These two animals without any of the Mytilus BTN2 markers at any of the four loci thus do not have the Mytilus BTN2 cancer lineage .", "These data confirm that Mytilus BTN2 is distinct from Mytilus BTN1 , and that it can be detected with multiple methods in both M . chilensis and M . edulis affected by disseminated neoplasia .", "Overall , the EF1α qPCR assay is the most highly sensitive and specific assay within these populations , with detection of the cancer-associated allele in all samples known to be affected by Mytilus BTN2 and no detectible amplification in any normal samples .", "In order to confirm the presence of M . trossulus-derived cancer in M . chilensis and M . edulis individuals we tested for the presence of multiple M . trossulus-specific nuclear SNPs in Mytilus BTN2-positive specimens from Europe and Argentina , as well as additional samples of healthy mussels from those populations .", "Following previous studies ( Riquet et al . , 2017 ) , we analysed SNP markers that are diagnostic between M . trossulus and either M . edulis or M . chilensis .", "We used 13 SNPs for each species .", "Five were used for both species , eight were specific to M . edulis hosts , and eight were specific to M . chilensis ( Supplementary file 3 ) .", "We confirm that all six Mytilus BTN2-positive M . edulis and all three positive M . chilensis analysed with the SNP assay have strong fluorescence corresponding to the M . trossulus allele at all diagnostic loci analysed and they are clear outliers in the compound multi-locus average ( Figure 6 ) .", "In contrast , no normal animals of either species have a signal of an M . trossulus allele at more than one locus .", "Therefore , data from multiple independent loci provide further evidence supporting the finding that the Mytilus BTN2 lineage is an M . trossulus derived cancer ." ], [ "Disseminated neoplasia has been previously shown to be due to a transmissible cancer lineage in four bivalve species , including Mytilus trossulus ( Metzger et al . , 2016; Metzger et al . , 2015 ) , and the current study greatly extends both our understanding of cross-species transmission of cancer ( from M . trossulus into two other Mytilus species ) and our understanding of the potential for geographic spread of transmissible cancers ( Figure 7 ) .", "This finding of a single lineage crossing into multiple species across such vast distances is unexpected .", "Phylogenetic evidence from four loci and SNP data from 13 diagnostic nuclear SNPs show that the lineage identified here ( Mytilus BTN2 ) is distinct from Mytilus BTN1 , and that it too arose from a M . trossulus individual before crossing into other Mytilus species , as previously proposed ( Riquet et al . , 2017 ) .", "The gene trees of the four loci are all concordant with each other , and are all consistent with a clonal , asexual cancer lineage derived from an M . trossulus cancer that is independent from Mytilus BTN1 .", "The only exception to this is the observation of recombination with host mitochondrial DNA in the mitogenomes of Mytilus BTN2 in Chile .", "The age of this cancer lineage is unknown , but the other transmissible cancer with world-wide spread ( canine transmissible venereal tumor; CTVT ) was estimated to be thousands of years old ( Murchison et al . , 2014; Baez-Ortega et al . , 2019 ) .", "Our oldest samples are 10 years old , but if the earliest reports of disseminated neoplasia in M . edulis were attributable to this same lineage ( Farley , 1969 ) , it would be at least 50 years old .", "The transmission of cancer cells from the Northern to the Southern Hemisphere and from Atlantic to Pacific Oceans is remarkable , and ocean currents are unlikely to provide a mechanism for travel in either direction ( especially given that anti-tropical distribution prevents a stepping-stone for trans-equatorial propagation ) .", "The most likely route of transmission is human intervention , specifically the transportation of animals harboring neoplastic cells in or on shipping vessels .", "While it is difficult to experimentally prove that a particular disease transmission was due to accidental transport on shipping vessels , examples of anthropogenic introduction of invasive species via shipping transport and aquaculture are common throughout the globe ( Molnar et al . , 2008 ) .", "The accidental anthropogenic introduction of Mytilus species includes M . galloprovincialis introduced in northern Chile ( Daguin and Borsa , 2000 ) and Argentina ( Zbawicka et al . , 2018 ) , M . edulis introduced in BC ( Crego-Prieto et al . , 2015 ) and the Kerguelen Islands ( Fraisse et al . , 2018 ) , and M . trossulus into Scotland ( Dias et al . , 2009 ) .", "Since Mytilus species are known to adhere to ships and spread accidentally across the world , and transmissible cancer can have an incubation period of weeks to months , any diseased animal which adheres to a ship could lead to the spread of a transmissible cancer to new and potentially susceptible populations .", "If transmissible cancers are able to readily travel along shipping lines and infect closely related species , then this and other lineages of transmissible cancer have the potential for world-wide reach .", "When identifying identical or nearly identical sequences in samples from different locations , care must be taken to prevent a small amount of contamination from leading to a false positive .", "In this case , the initial sequencing and amplification of the EF1α loci from M . edulis and M . chilensis samples were conducted by different people at different sites before any plasmids containing the sequence from one site had been transferred to the other .", "The EF1α qPCR assay was independently replicated on European M . edulis infected individuals in two labs , in USA and France .", "Also , the finding of slight differences between the two subgroups of this cancer lineage argue against a single source of contamination .", "Additionally , qPCR verification confirms that a significant fraction of the DNA of each sample contains the cancer-associated allele , and that it is not found in DNA samples from normal animals , which have been treated in the same way .", "Therefore , the data could not be due to contamination with a small amount of DNA and represents genuine identification of a nearly identical cancer from mussels of different continents and species .", "Invertebrates do not possess a vertebrate-like adaptive immune system or a major histocompatibility complex , which vertebrates utilize for discriminating self from nonself .", "However , in most previously identified cases of BTN , the neoplasia has only been identified in the species of origin ( Metzger et al . , 2016; Metzger et al . , 2015 ) , and attempts to experimentally transfer neoplastic cells from M . trossulus or the soft-shell clam Mya arenaria into several bivalve species have only been successful when the transfer is between members of the same species ( Kent et al . , 1991; Mateo et al . , 2016 ) .", "The species in the Mytilus complex are able to hybridize , so the barrier to cross-species transmission may be relatively low .", "However , it has been reported that the disseminated neoplasia found on the Pacific coast of North America ( which was identified as originating from M . trossulus ) was found in M . trossulus at a much higher prevalence than M . edulis in the same region ( Muttray et al . , 2007 ) , which suggested that the M . trossulus cancer preferentially infected M . trossulus hosts .", "It is currently unknown whether Mytilus BTN2 would be able to infect its original host species or if the original host has evolved resistance , as was suggested with the cancer derived from the pullet shell clam Venerupis corrugata ( Metzger et al . , 2016 ) .", "M . chilensis is closely related to M . edulis , and it has even been suggested that it could be considered a subspecies , M . edulis platensis ( Borsa et al . , 2012 ) , although this is controversial ( Zbawicka et al . , 2018 ) , so once the cancer was able to cross into either M . chilensis or M . edulis , the second jump into the other species may have presented a lower barrier .", "Based on analysis of transmissible cancers in devils and dogs it has been hypothesized that transmissible cancers primarily occur in inbred populations , but the finding of this cancer spreading into highly outbred individuals from multiple different species within the same genus shows that this constraint is not universal .", "The evidence for mitochondrial heteroplasmy and recombination observed in this cancer lineage and its loss or gain is a very interesting observation that suggests a dynamic mitochondrial population in BTN , which may not exactly match the nuclear evolutionary path .", "It has been shown that mitochondria in CTVT has been replaced several times by mitochondrial capture of new normal mitochondrial genomes ( Rebbeck et al . , 2011; Strakova et al . , 2016 ) , so a dynamic mitochondrial population may be a common feature among transmissible cancers .", "It is also possible that one of the two cancer-associated mtCR regions could be a nuclear-to-mitochondrial transfer ( numt ) of a copy of the control region instead of mitochondrial heteroplasmy , as this possibility cannot be excluded by PCR of total extracted DNA .", "If this were the case , the sequences would still be evidence of an M . trossulus-derived transmissible cancer that is distinct from Mytilus BTN1 .", "Mitochondrial replacement has not yet been described in any BTN , so further analysis will be required to determine the full recombinant cancer mitogenomes and to test whether there is any selective advantage of this recombinant mitogenome in Mytilus BTN2 .", "Comparison of cancer-associated sequences to those found in normal M . trossulus populations may allow us to identify the location of the origin of these cancer lineages .", "BLAST searches for the neoplasia-associated alleles most closely match M . trossulus from the Baltic and BC .", "The Mytilus BTN2 recombinant mitochondrial sequences closely match the recombinant sequence first identified in the Baltic ( 62mc10 ) , but this haplotype is actually rare in the Baltic , with similar recombinant haplotypes more commonly found on the coast of Norway ( Śmietanka and Burzyński , 2017 ) .", "This suggests that this cancer lineage originated in Northern Europe , but it may not necessarily be from the Baltic .", "However , there are populations of M . trossulus in many locations around the world , and not all have been sequenced at the loci analyzed here .", "Additionally , the mtCR C and D alleles may be directly related to 62mc10 , or they may represent novel recombinations in the same region ( there is a single base difference in the recombination region between the D allele and 62mc10 which suggests that the recombination points are not identical ) .", "Multiple recombination events in which male sequence has recombined into the female mitochondrial lineage in that region ( including several cases with tandem duplications ) have been identified in M . trossulus ( Burzyński et al . , 2006 ) .", "Additionally , while exact matches of a Mytilus BTN2 H4 allele was identified in M . trossulus from BC , exact matches of the alleles at other loci have not been identified in any wild M . trossulus populations .", "Therefore , the exact population of origin of this cancer remains uncertain .", "Overall , these data show that two lineages of cancer from two separate M . trossulus individuals have turned into BTN lineages , and one of these lineages ( Mytilus BTN2 ) has spread world-wide throughout multiple host species .", "This spread also suggests that anthropogenic influence may exacerbate the spread of cancers and may allow access into new environments and new populations , where the pathogenic potential is unknown ." ], [ "60 individual market-sized mussels ( mean , 67 . 6 mm; range , 52–92 mm in the longest axis ) were collected in February 2012 from a culture at Bahía Brown ( 54°52´S , 67°31´W ) , Beagle Channel , Argentina .", "The soft parts of the specimens were carefully removed from their shells and fixed in Davidson’s solution for 24 hr .", "Oblique transverse sections , approximately 5 mm thick , including mantle , gills , gonad , digestive gland , nephridia , and foot were taken from each specimen .", "Tissue samples were embedded in paraffin , and 5 μm sections were stained with hematoxylin and eosin .", "Histological sections were examined using a Leica DM 2500 light microscope for the presence of neoplastic cells .", "Small samples of gill from each mussel were preserved in 100% ethanol for DNA extraction .", "200 individuals were collected from farmed populations at two sites in Chile ( Calbuco and Castro ) .", "Hemolymph was drawn from adductor muscle , centrifuged at low speed and the hemocytes were stored in RNAlater ( ThermoFisher Scientific ) for DNA extraction .", "The animals were opened and the full body with one valve were fixed for at least one week with 10% formaldehyde in PBS .", "Tissue samples were embedded in paraffin , sectioned and stained as above .", "The animals were diagnosed as diseased if infiltration of hemocytes with a high nucleus-cytoplasm ratio and granular chromatin were observed .", "Healthy mussels from other populations of M . chilensis around Punta Arenas , Chile ( n = 46 ) and from islands around Parque Nacional Alberto de Agostini , Chile ( n = 63 ) were analyzed with the SNP assay .", "We used a collection of samples ( gills in 90% ethanol ) accumulated by the ISEM lab for years in their study of hybridization and introgression in the M . edulis complex of species .", "M . trossulus is not naturally found in France and the genetic analysis of thousands of individuals did not identify any M . trossulus individuals even in ports or mussel farms ( Simon et al . , 2019 ) .", "As a consequence , a signal of amplification of trossulus alleles at several diagnostic SNPs was used as a diagnosis for mussels likely infected by a M . trossulus BTN ( Riquet et al . , 2017 ) .", "Here we first used specimens identified by Riquet et al . : two mussels sampled in the Arcachon Bay ( South-West of France ) in 2016 ( AR5 and AR7 ) , one mussel sampled in Barfleur ( Normandy , France ) in 2015 ( BA30 ) , and one mussel sampled in the Wadden Sea ( The Netherlands ) in 2009 ( HO8 ) .", "To these four mussels already described , we added two additional mussels identified in a new extensive genetic survey of >4000 individuals ( Simon et al . , 2019 ) .", "These two mussels were collected from Chausey Island ( English Channel ) in 2009 ( Ch5 and Ch13 ) .", "For comparison , two healthy individuals were randomly chosen from each of the collections from Chausey , Barfleur , and the Wadden Sea ( Ch10 , Ch11 , BA12 , BA24 , HO9 , HO19 ) .", "Additional healthy mussels from the populations of Chausey ( n = 9 ) , Barfleur ( n = 28 ) and Wadden Sea ( n = 21 ) were analyzed with the SNP assay .", "In parallel , a hemocytological analysis was undertaken to identify BTN infected mussels on approximately one hundred individuals from Brittany .", "Briefly , the mussels were anaesthetized by bathing them for 30 min in a solution containing 50 g/L of magnesium chloride dissolved in two-thirds of distilled water and one-third of sea water ( Suquet et al . , 2009 ) .", "40 μl of hemolymph were withdrawn from the adductor muscle with a sterile 1 mL syringe fitted with a 27-gauge needle .", "After the addition of 160 μl of artificial seawater , the hemolymph solution was ‘cytospun’ onto coated slides ( Shandon Cytospin four and Cytoslides , Thermo Scientific; 10 min , 800 rpm ) , fixed , stained with May-Grünwald Giemsa , and observed under light microscopy .", "The whole area of the cell spot on the cytoslide was observed in all the samples in search of neoplastic cells , characterized by an enlarged diameter , elevated nucleus-cytoplasm ratio , and high basophily .", "Two individuals that also showed a signal of M . trossulus allele amplification from gill tissue DNA were chosen for the present study .", "One was sampled in Lannion ( LA84 ) and the other in Saint Brieux ( STBRI43 ) in 2017 .", "An additional normal individual from Saint Brieux ( STBRI28 ) was also analyzed as a control , and additional healthy individuals from Lannion ( n = 4 ) were analyzed with the SNP assay .", "DNA was extracted from ethanol-fixed gill and siphon samples using DNeasy Blood and Tissue Kit ( Qiagen ) with the following additional steps to remove PCR-inhibiting polysaccharides .", "After tissue lysis , 65 µl of P3 Buffer ( Qiagen ) was added for 5 min to precipitate out polysaccharides and spun down at 17k × g at 4°C for 5 min .", "The resulting supernatant was transferred to a new tube , 200 µl of Buffer AL was added , and the manufacturer’s protocol resumed .", "DNA was extracted from hemocyte samples using the standard DNeasy Blood and Tissue Kit protocol .", "Primers and annealing temperatures are listed in Supplementary file", "1 . PCR amplification at the EF1α locus was done using PfuUltra II Fusion HS DNA Polymerase ( Agilent ) with a 15 s extension time .", "Amplification at the H4 , mtCR , and mtCOI loci were achieved using Q5 Hot Start High-Fidelity DNA Polymerase ( NEB ) with a 30 s extension time .", "Primers for H4 were designed to span the full H4 gene ( Eirín-López et al . , 2004 ) .", "Primers for mtCR were adapted from Burzynski et al . and were designed to cover lrRNA , a variable domain , and a conserved domain in the mitochondrial DNA control region ( Burzyński et al . , 2006 ) .", "‘Pan-molluscan’ barcoding primers ( Folmer et al . , 1994 ) did not amplify the Mytilus BTN2 mtCOI allele , so degenerate primers flanking the standard mtCOI were used .", "In all cases , 25–50 ng of genomic DNA was amplified for 35 cycles .", "PCR products were gel extracted using spin columns ( Qiagen , NEB ) and either directly sequenced , or , when multiple alleles at a locus could not be resolved by direct sequencing , were cloned using the Zero Blunt TOPO PCR Cloning Kit ( Invitrogen ) .", "Plasmids were transformed into TOP10 or DH5α competent E . coli ( Invitrogen ) and 6–12 clones were picked for further sequencing using M13F and M13R primers ( Genewiz ) .", "The primer binding regions were excluded from sequence analysis , and all unique alleles were identified .", "In cases where a sequence was found in only a single clone and that clone differed by <0 . 5% ( two differences for EF1α and H4 , three for mtCR ) from another allele that was supported by more than one clone from the same individual , the sequence with higher support was used as the correct allele .", "Additionally , a sequence which was found in only one clone and which is consistent with a recombination event between two alleles from the same sample was discarded .", "For EF1α , every allele presented was found in at least two clones ( two sequences found only in single clones were removed from analysis ) .", "Alleles were named arbitrarily , such that each allele name ( A-2H ) represents the same sequence at that locus .", "Coding sequences had minimal indels and were aligned manually .", "The mtCR locus was aligned with MUSCLE 3 . 8 . 31 ( Edgar , 2004 ) with minor manual adjustment .", "Maximum likelihood phylogenetic trees were generated using PhyML 3 . 0 ( Guindon et al . , 2010 ) , with 100 bootstrap replicates , which treats gaps in the alignment as missing data , with automatic model selection through Akaike Information Criterion .", "Trees were visualized using FigTree version 1 . 4 . 4 .", "All alignments are included as source data , and all sequences available in GenBank ( accession numbers: EF1α , MN546736-MN546756; H4 , MN546757 - MN546792; mtCR , MN546793 - MN546830; mtCOI , MN546831 - MN546858 ) .", "Cancer-associated allele-specific qPCR was performed in M . trossulus , M . edulis , and M . chilensis genomic DNA samples using PowerUp SYBR Green Master Mix ( Applied Biosystems ) .", "For all targets , one primer set was designed to specifically amplify a cancer-associated allele for a given locus ( EF1α: Allele H; H4: Allele RM and KNS; mtCR: Allele C and D: mtCOI: Allele B ) .", "A second pair of universal control primers were designed to amplify all Mytilus alleles at each locus .", "Primers are listed in Supplementary file", "2 . Standards for all targets consisted of a single plasmid containing the relevant cancer-associated allele target , which was linearized by NotI-HF ( NEB ) restriction digest before qPCR .", "Serial dilutions ( 107–101 copies ) of linearized standard plasmid in triplicate were used to generate a standard curve for each qPCR run .", "In all experimental cases , 10–100 ng of each genomic DNA sample was run in triplicate 10 µl reactions .", "All qPCR assays were performed using the Applied Biosystems StepOnePlus Real-Time PCR System ( Applied Biosystems ) and associated software .", "Allele-specific qPCR at the EF1α locus was comprised of a 3-step cycling stage ( 95°C for 15 s , 50°C for 15 s , and 60°C for 30 s , respectively ) for 40 cycles , whereas qPCR at the H4 , mtCR , and mtCOI loci were run using a 2-step cycling stage protocol ( 95°C for 15 s , 60°C for 30 s ) for 40 cycles .", "A melting curve was generated to determine amplification specificity according to the following parameters: an initial denaturation step at 95°C for 15 s , followed by a gradual temperature increase from 60°C to 95°C ( Ramp of +0 . 3°C ) .", "The ratio of the cancer-associated allele to all alleles present at a given locus was calculated for each sample using the average copy number amplified by cancer-associated primers in triplicate reactions divided by the average copy number amplified by the universal primers in triplicate reactions .", "For each sample , the limit of detection for the allele-specific qPCR was set at 10 copies/reaction , and DNA samples were discarded if the universal Mytilus reaction at any of the loci yielded <5000 copies/reaction ( upper limit of detection of 0 . 2% ) .", "The SNPs used were previously described ( Simon et al . , 2018 ) .", "They were identified as being diagnostic ( nearly fixed for alternative alleles ) in a large sequence dataset ( Fraïsse et al . , 2016 ) and were subsequently verified to remain diagnostic with larger sample sizes ( Simon et al . , 2019 ) .", "Non-hybrid individuals can nonetheless occasionally be heterozygous at one , or very rarely at two , of these diagnostic loci because incomplete lineage sorting and introgression is pervasive in the M . edulis complex of species ( Fraïsse et al . , 2018 ) , but never at more than two loci .", "Genotyping was subcontracted to LGC-genomics and performed with the Kompetitive Allele Specific PCR ( KASP ) chemistry ( Semagn et al . , 2014 ) .", "The results are a combination of two fluorescence values , one for allele 1", "( x ) , and another for allele 2", "( y ) , the M . trossulus-specific allele .", "Following a method used for genotyping polyploids ( Cuenca et al . , 2013 ) , we transformed the data to have a single measure of the relative fluorescence of the two alleles , from 0 when the fluorescence of allele one dominates , to one when the fluorescence of allele 2 ( i . e . the M . trossulus allele ) dominates , using the following formula: y’=y/ ( x+y ) .", "The fluorescence of the M . trossulus alleles was averaged over all 13 diagnostic loci for each species to obtain a compound multi-locus estimate ( see Supplementary file 3 ) ." ] ]
[ "Transmissible cancers , in which cancer cells themselves act as an infectious agent , have been identified in Tasmanian devils , dogs , and four bivalves .", "We investigated a disseminated neoplasia affecting geographically distant populations of two species of mussels ( Mytilus chilensis in South America and M . edulis in Europe ) .", "Sequencing alleles from four loci ( two nuclear and two mitochondrial ) provided evidence of transmissible cancer in both species .", "Phylogenetic analysis of cancer-associated alleles and analysis of diagnostic SNPs showed that cancers in both species likely arose in a third species of mussel ( M . trossulus ) , but these cancer cells are independent from the previously identified transmissible cancer in M . trossulus from Canada .", "Unexpectedly , cancers from M . chilensis and M . edulis are nearly identical , showing that the same cancer lineage affects both .", "Thus , a single transmissible cancer lineage has crossed into two new host species and has been transferred across the Atlantic and Pacific Oceans and between the Northern and Southern hemispheres ." ]
[ "Cancer cells can grow and spread in one individual , but they normally do not spread to others .", "There are a few exceptions to this rule .", "For example , there are cancers in Tasmanian devils , dogs and bivalve shellfish that can spread to other members of the same species .", "In these creatures , cancer from one individual evolved the ability to spread throughout the population .", "These cancer cells infect animals like a pathogen .", "A fatal cancer called disseminated neoplasia affects many species of bivalves .", "In four bivalve species , including the marine mussel Mytilus trossulus , scientists have shown that the cancer can spread from one individual to another .", "This transmissible cancer has been found in M . trossulus mussels in British Columbia , Canada; but related species of mussels in other parts of the world also develop disseminated neoplasia .", "It is possible these other cancers are transmissible and have spread from one population of mussels to another .", "Yonemitsu et al . performed genetic analyses to show that cancers found in two other mussel species – Mytilus chilensis in South America and Mytilus edulis in Europe – are transmissible and arose in M . trossulus .", "The cancers in the South American and European mussels were nearly identical genetically , which suggests that they came from a single M . trossulus mussel with cancer at some point in the past .", "Somehow cancer cells spread between the Northern and the Southern Hemispheres and across the Atlantic Ocean , infecting multiple species across the world .", "The analyses also show that this cancer lineage is different from the one previously identified in British Columbia .", "These analyses show that bivalve transmissible neoplasia was able to spread worldwide , most likely through accidental transport of infected mussels on international shipping vessels .", "This suggests that human activities unwittingly introduced the disease to new areas .", "Learning more about transmissible cancers may help scientists understand how cancers evolve with their hosts in extreme situations ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "computational and systems biology", "neuroscience" ]
Neuronal timescales are functionally dynamic and shaped by cortical microarchitecture
elife-61277-v2
[ [ "Human brain regions are broadly specialized for different aspects of behavior and cognition , and the temporal dynamics of neuronal populations across the cortex are thought to be an intrinsic property ( i . e . , neuronal timescale ) that enables the representation of information over multiple durations in a hierarchically embedded environment ( Kiebel et al . , 2008 ) .", "For example , primary sensory neurons are tightly coupled to changes in the environment , firing rapidly to the onset and removal of a stimulus , and showing characteristically short intrinsic timescales ( Ogawa and Komatsu , 2010; Runyan et al . , 2017 ) .", "In contrast , neurons in cortical association ( or transmodal ) regions , such as the prefrontal cortex ( PFC ) , can sustain their activity for many seconds when a person is engaged in working memory ( Zylberberg and Strowbridge , 2017 ) , decision-making ( Gold and Shadlen , 2007 ) , and hierarchical reasoning ( Sarafyazd and Jazayeri , 2019 ) .", "This persistent activity in the absence of immediate sensory stimuli reflects longer neuronal timescales , which is thought to result from neural attractor states ( Wang , 2002; Wimmer et al . , 2014 ) shaped by N-methyl-D-aspartate receptor ( NMDA ) -mediated recurrent excitation and fast feedback inhibition ( Wang , 2008; Wang , 1999 ) , with contributions from other synaptic and cell-intrinsic properties ( Duarte and Morrison , 2019; Gjorgjieva et al . , 2016 ) .", "How connectivity and various cellular properties combine to shape neuronal dynamics across the cortex remains an open question .", "Anatomical connectivity measures based on tract tracing data , such as laminar feedforward vs . feedback projection patterns , have classically defined a hierarchical organization of the cortex ( Felleman and Van Essen , 1991; Hilgetag and Goulas , 2020; Vezoli et al . , 2020 ) .", "Recent studies have also shown that variations in many microarchitectural features follow continuous and coinciding gradients along a sensory-to-association axis across the cortex , including cortical thickness , cell density , and distribution of excitatory and inhibitory neurons ( Huntenburg et al . , 2018; Wang , 2020 ) .", "In particular , gray matter myelination ( Glasser and Van Essen , 2011 ) —a noninvasive proxy of anatomical hierarchy consistent with laminar projection data—varies with the expression of genes related to microcircuit function in the human brain , such as NMDA receptor and inhibitory cell-type marker genes ( Burt et al . , 2018 ) .", "Functionally , specialization of the human cortex , as well as structural and functional connectivity ( Margulies et al . , 2016 ) , also follow similar macroscopic gradients .", "Moreover , in addition to the broad differentiation between sensory and association cortices , there is evidence for an even finer hierarchical organization within the frontal cortex ( Sarafyazd and Jazayeri , 2019 ) .", "For example , the anterior-most parts of the PFC are responsible for long timescale goal-planning behavior ( Badre and D'Esposito , 2009; Voytek et al . , 2015a ) , while healthy aging is associated with a shift in these gradients such that older adults become more reliant on higher-level association regions to compensate for altered lower-level cortical functioning ( Davis et al . , 2008 ) .", "Despite convergent observations of cortical gradients in structural features and cognitive specialization , there is no direct evidence for a similar gradient of neuronal timescales across the human cortex .", "Such a gradient of neuronal dynamics is predicted to be a natural consequence of macroscopic variations in synaptic connectivity and microarchitectural features ( Chaudhuri et al . , 2015; Duarte et al . , 2017; Huang and Doiron , 2017; Huntenburg et al . , 2018; Wang , 2020 ) , and would be a primary candidate for how functional specialization emerges as a result of hierarchical temporal processing ( Kiebel et al . , 2008 ) .", "Single-unit recordings in rodents and non-human primates demonstrated a hierarchy of timescales that increase , or lengthen , progressively along a posterior-to-anterior axis ( Dotson et al . , 2018; Murray et al . , 2014; Runyan et al . , 2017; Wasmuht et al . , 2018 ) , while intracranial recordings and functional neuroimaging data collected during perceptual and cognitive tasks suggest likewise in humans ( Baldassano et al . , 2017; Honey et al . , 2012; Lerner et al . , 2011; Watanabe et al . , 2019 ) .", "However , these data are either sparsely sampled across the cortex or do not measure neuronal activity at the cellular and synaptic level directly , prohibiting the full construction of an electrophysiological timescale gradient across the human cortex .", "As a result , while whole-cortex data of transcriptomic and anatomical variations exist , we cannot take advantage of them to dissect the contributions of synaptic , cellular , and circuit connectivity in shaping fast neuronal timescales , nor ask whether regional timescales are dynamic and relevant for human cognition .", "Here we combine several publicly available datasets to infer neuronal timescales from invasive human electrocorticography ( ECoG ) recordings and relate them to whole-cortex transcriptomic and anatomical data , as well as probe their functional relevance during behavior ( Figure 1A for schematic of study; Tables 1 and 2 for dataset information ) .", "Unless otherwise specified , ( neuronal ) timescale in the following sections refers to ECoG-derived timescales , which are more reflective of fast synaptic and transmembrane current timescales than single-unit or population spiking timescales ( Figure 1A , left box ) , though we demonstrate in macaques a close correspondence between the two .", "In humans , neuronal timescales increase along the principal sensorimotor-to-association axis across the cortex and align with macroscopic gradients of gray matter myelination ( T1w/T2w ratio ) and synaptic receptor and ion channel gene expression .", "Finally , we find that human PFC timescales expand during working memory maintenance and predict individual performance , while cortex-wide timescales compress with aging .", "Thus , neuronal timescales follow cytoarchitectonic gradients across the human cortex and are relevant for cognition in both short and long terms , bridging microcircuit physiology with macroscale dynamics and behavior ." ], [ "Neural time series often exhibit time-lagged correlation ( i . e . , autocorrelation ) , where future values are partially predictable from past values , and predictability decreases with increasing time lags .", "For demonstration , we simulate the aperiodic ( non-rhythmic ) component of ECoG recordings by convolving Poisson population spikes with exponentially decaying synaptic kernels with varying decay constant ( Figure 1B ) .", "Empirically , the degree of self-similarity is characterized by the autocorrelation function ( ACF ) , and ‘timescale’ is defined as the time constant ( τ ) of an exponential decay function ( e−tτ ) fit to the ACF , i . e . , the time it takes for the autocorrelation to decrease by a factor of e ( Figure 1C ) .", "Equivalently , we can estimate timescale in the frequency domain from the power spectral density ( PSD ) .", "PSDs of neural time series often follow a Lorentzian function of the form 1fk2+ f2 , where power is approximately constant until the ‘knee frequency’ ( fk , Figure 1D ) , then decays following a power law .", "This approach is similar to the one presented in Chaudhuri et al . , 2017 , but here we further allow the power law exponent ( fixed at two in the equation above ) to be a free parameter representing variable scale-free activity ( He et al . , 2010; Miller et al . , 2009; Podvalny et al . , 2015; Voytek et al . , 2015c ) .", "We also simultaneously parameterize oscillatory components as Gaussians peaks , allowing us to remove their effect on the power spectrum , providing more accurate estimates of the knee frequency .", "From the knee frequency of the aperiodic component , neural timescale ( decay constant ) can then be computed exactly as τ= 12πfk .", "Compared to fitting exponential decay functions in the time domain ( e . g . , Murray et al . , 2014 ) —which can be biased even without the presence of additional components ( Zeraati et al . , 2020 ) —the frequency domain approach is advantageous when a variable power law exponent and strong oscillatory components are present , as is often the case for neural signals ( example of real data in Figure 1D ) .", "While the oscillatory component can corrupt naive measurement of τ as time for the ACF to reach 1/e ( Figure 1D , inset ) , it can be more easily accounted for and removed in the frequency domain as Gaussian-like peaks .", "This is especially important considering neural oscillations with non-stationary frequencies .", "For example , a broad peak in the power spectrum ( e . g . , ~10 Hz in bandwidth in Figure 1D ) represents drifts in the oscillation frequency over time , which is easily accounted for with a single Gaussian , but requires multiple cosine terms to capture well in the autocorrelation .", "Therefore , in this study , we apply spectral parameterization to extract timescales from intracranial recordings ( Donoghue et al . , 2020 ) .", "We validate this approach on PSDs computed from simulated neural time series and show that the extracted timescales closely match their ground-truth values ( Figure 1E ) .", "Applying this technique , we infer a continuous gradient of neuronal timescales across the human cortex by analyzing a large dataset of human intracranial ( ECoG ) recordings of task-free brain activity ( Frauscher et al . , 2018a ) .", "The MNI-iEEG dataset contains 1 min of resting state data across 1772 channels from 106 patients ( 13–62 years old , 48 females ) with variable coverages , recorded using either surface strip/grid or stereoEEG electrodes , and cleaned of visible artifacts .", "Figure 2A shows example data traces along the cortical hierarchy with increasing timescales estimated from their PSDs ( Figure 2B; circles denote fitted knee frequency ) .", "Timescales from individual channels were extracted and projected from MNI coordinates onto the left hemisphere of HCP-MMP1 . 0 surface parcellation ( Glasser et al . , 2016 ) for each patient using a Gaussian-weighted mask centered on each electrode .", "While coverage is sparse and idiosyncratic in individual patients , it does not vary as a function of age , and when pooling across the entire population , 178 of 180 parcels have at least one patient with an electrode within 4 mm ( Figure 2—figure supplement 1A–F ) .", "Across the human cortex , timescales of fast electrophysiological dynamics ( ~10–50 ms ) predominantly follow a rostrocaudal gradient ( Figure 2C , circles denote location of example data from 2A ) .", "Consistent with numerous accounts of a principal cortical axis spanning from primary sensory to association regions ( Hilgetag and Goulas , 2020; Margulies et al . , 2016; Wang , 2020 ) , timescales are shorter in sensorimotor and early visual areas , and longer in association regions , especially cingulate , ventral/medial frontal , and medial temporal lobe ( MTL ) regions ( Figure 2—figure supplement 1G shows further pooling into 21 labeled macro-regions ) .", "We then compare the timescale gradient to the average T1w/T2w map from the Human Connectome Project , which captures gray matter myelination and indexes the proportion of feedforward vs . feedback connections between cortical regions , thus acting as a noninvasive proxy of connectivity-based anatomical hierarchy ( Burt et al . , 2018; Glasser and Van Essen , 2011 ) .", "We find that neuronal timescales are negatively correlated with T1w/T2w across the entire cortex ( Figure 2D , ρ = −0 . 47 , p<0 . 001; corrected for spatial autocorrelation [SA] , see Materials and methods and Figure 2—figure supplement 2A–C for a comparison of correction methods ) , such that timescales are shorter in more heavily myelinated ( i . e . , lower-level , sensory ) regions .", "Timescales are also positively correlated with cortical thickness ( Figure 2—figure supplement 3 , ρ = 0 . 37 , p=0 . 035 ) —another index of cortical hierarchy that is itself anti-correlated with T1w/T2w .", "Thus , we observe that neuronal timescales lengthen along the human cortical hierarchy , from sensorimotor to association regions .", "While surface ECoG recordings offer much broader spatial coverage than extracellular single-unit recordings , they are fundamentally different signals: ECoG and field potentials largely reflect integrated synaptic and other transmembrane currents across many neuronal and glial cells , rather than putative action potentials from single neurons ( Buzsáki et al . , 2012; Figure 1A , yellow box ) .", "Considering this , we ask whether timescales measured from ECoG in this study ( τECoG ) are related to single-unit spiking timescales along the cortical hierarchy ( τspiking ) .", "To test this , we extract neuronal timescales from task-free ECoG recordings in macaques ( Nagasaka et al . , 2011 ) and compare them to a separate dataset of single-unit spiking timescales from a different group of macaques ( Murray et al . , 2014 ) ( see Figure 2—figure supplement 4 for electrode locations ) .", "Consistent with τspiking estimates ( Murray et al . , 2014; Wasmuht et al . , 2018 ) , τECoG also increase along the macaque cortical hierarchy .", "While there is a strong correspondence between spiking and ECoG timescales ( Figure 2F; ρ = 0 . 96 , p<0 . 001 ) —measured from independent datasets—across the macaque cortex , τECoG are ~10 times faster than τspiking and are conserved across individual sessions ( Figure 2G ) .", "This suggests that neuronal spiking and transmembrane currents have distinct but related timescales of fluctuations , and that both are hierarchically organized along the primate cortex .", "Next , we identify potential cellular and synaptic mechanisms underlying timescale variations across the human cortex .", "Theoretical accounts posit that NMDA-mediated recurrent excitation coupled with fast inhibition ( Chaudhuri et al . , 2015; Wang , 2008; Wang , 1999 ) , as well as cell-intrinsic properties ( Duarte and Morrison , 2019; Gjorgjieva et al . , 2016; Koch et al . , 1996 ) , are crucial for shaping neuronal timescales .", "While in vitro and in vivo studies in model organisms ( van Vugt et al . , 2020; Wang et al . , 2013 ) can test these hypotheses at the single-neuron level , causal manipulations and large-scale recordings of neuronal networks embedded in the human brain are severely limited .", "Here , we apply an approach analogous to multimodal single-cell profiling ( Bomkamp et al . , 2019 ) and examine the transcriptomic basis of neuronal dynamics at the macroscale .", "Leveraging whole-cortex interpolation of the Allen Human Brain Atlas bulk mRNA expression ( Gryglewski et al . , 2018; Hawrylycz et al . , 2012 ) , we project voxel-wise expression maps onto the HCP-MMP1 . 0 surface parcellation , and find that the neuronal timescale gradient overlaps with the dominant axis of gene expression ( i . e . , first principal component of 2429 brain-related genes ) across the human cortex ( Figure 3A , ρ = −0 . 60 , p<0 . 001; see Figure 3—figure supplement 1 for similar results with all 18 , 114 genes ) .", "Consistent with theoretical predictions ( Figure 3B ) , timescales significantly correlate with the expression of genes encoding for NMDA ( GRIN2B ) and GABA-A ( GABRA3 ) receptor subunits , voltage-gated sodium ( SCN1A ) and potassium ( KCNA3 ) ion channel subunits , as well as inhibitory cell-type markers ( parvalbumin , PVALB ) , and genes previously identified to be associated with single-neuron membrane time constants ( PRR5 ) ( Bomkamp et al . , 2019 ) ( all Spearman correlations corrected for SA in gradients ) .", "More specifically , in vitro electrophysiological studies have shown that , for example , increased expression of receptor subunit 2B extends the NMDA current time course ( Flint et al . , 1997 ) , while 2A expression shortens it ( Monyer et al . , 1994 ) .", "Similarly , the GABA-A receptor time constant lengthens with increasing a3:a1 subunit ratio ( Eyre et al . , 2012 ) .", "We show that these relationships are recapitulated at the macroscale , where neuronal timescales positively correlate with GRIN2B and GABRA3 expression and negatively correlate with GRIN2A and GABRA1 ( Figure 3C ) .", "These results demonstrate that timescales of neural dynamics depend on specific receptor subunit combinations with different ( de ) activation timescales ( Duarte et al . , 2017; Gjorgjieva et al . , 2016 ) , in addition to broad excitation–inhibition interactions ( Gao et al . , 2017; Wang , 2020; Wang , 2002 ) .", "Notably , almost all genes related to voltage-gated sodium and potassium ion channel alpha-subunits—the main functional subunits—are correlated with timescale , while all inhibitory cell-type markers except parvalbumin have strong positive associations with timescale ( Figure 3C and Figure 3—figure supplement 2 ) .", "We further test whether single-cell timescale-transcriptomic associations are captured at the macroscale as follows: for a given gene , we can measure how strongly its expression correlates with membrane time constant parameters at the single-cell level using patch-clamp and RNA sequencing ( scRNASeq ) data ( Bomkamp et al . , 2019; Tripathy et al . , 2017 ) .", "Analogously , we can measure its macroscopic transcriptomic-timescale correlation using the cortical gradients above .", "If the association between the expression of this gene and neuronal timescale is preserved at both levels , then the correlation across cells at the microscale should be similar to the correlation across cortical regions at the macroscale .", "Comparing across these two scales for all previously identified timescale-related genes in two studies ( N = 170 [Tripathy et al . , 2017] and 4168 [Bomkamp et al . , 2019] genes ) , we find a significant correlation between the strength of association at the single-cell and macroscale levels ( Figure 3D , horizontal black lines; ρ = 0 . 36 and 0 . 25 for the two datasets , p<0 . 001 for both ) .", "Furthermore , genes with stronger associations to timescale tend to conserve this relationship across single-cell and macroscale levels ( Figure 3D , separated by macroscale correlation magnitude ) .", "Thus , the association between cellular variations in gene expression and cell-intrinsic temporal dynamics is captured at the macroscale , even though scRNAseq and microarray data represent entirely different measurements of gene expression .", "While we have shown associations between cortical timescales and genes suspected to influence neuronal dynamics , these data present an opportunity to discover additional novel genes that are functionally related to timescales through a data-driven approach .", "However , since transcriptomic variation and anatomical hierarchy overlap along a shared macroscopic gradient ( Burt et al . , 2018; Huntenburg et al . , 2018; Margulies et al . , 2016 ) , we cannot specify the role certain genes play based on their level of association with timescale alone: gene expression differences across the cortex first result in cell-type and connectivity differences , sculpting the hierarchical organization of cortical anatomy .", "Consequently , anatomy and cell-intrinsic properties jointly shape neuronal dynamics through connectivity differences ( Chaudhuri et al . , 2015; Demirtaş et al . , 2019 ) and expression of ion transport and receptor proteins with variable activation timescales , respectively .", "Therefore , we ask whether variation in gene expression still accounts for variation in timescale beyond the principal structural gradient , and if associated genes have known functional roles in biological processes ( BP ) ( schematic in Figure 3E ) .", "To do this , we first remove the contribution of anatomical hierarchy by linearly regressing out the T1w/T2w gradient from both timescale and individual gene expression gradients .", "We then fit partial least squares ( PLS ) models to simultaneously estimate regression weights for all genes ( Whitaker et al . , 2016 ) , submitting those with significant associations for gene ontology enrichment analysis ( GOEA ) ( Klopfenstein et al . , 2018 ) .", "We find that genes highly associated with neuronal timescales are preferentially related to transmembrane ion transporter complexes , as well as GABAergic synapses and chloride channels ( see Figure 3F and Supplementary file 1 for GOEA results with brain genes only , and Supplementary file 2 for all genes ) .", "When restricted to positively associated genes only ( expression increases with timescales ) , one functional group related to phosphatidate phosphatase activity is uncovered , including the gene PLPPR1 , which has been linked to neuronal plasticity ( Savaskan et al . , 2004 ) —a much slower timescale physiological process .", "Conversely , genes that are negatively associated with timescale are related to numerous groups involved in the construction and functioning of transmembrane transporters and voltage-gated ion channels , especially potassium and other inorganic cation transporters .", "To further ensure that these genes specifically relate to neuronal timescale , we perform the same enrichment analysis with T1w/T2w vs . gene maps as a control .", "The control analysis yields no significant GO terms when restricted to brain-specific genes ( in contrast to Figure 3F ) , while repeating the analysis with all genes does yield significant GO terms related to ion channels and synapses , but are much less specific to those ( see Supplementary file 3 ) , including a variety of other gene clusters associated with general metabolic processes , signaling pathways , and cellular components ( CC ) .", "This further strengthens the point that removing the contribution of T1w/T2w aids in identifying genes that are more specifically associated with neurodynamics , suggesting that inhibition ( Teleńczuk et al . , 2017 ) —mediated by GABA and chloride channels—and voltage-gated potassium channels have prominent roles in shaping neuronal timescale dynamics at the macroscale level , beyond what is expected based on the anatomical hierarchy alone .", "Finally , having shown that neuronal timescales are associated with stable anatomical and gene expression gradients across the human cortex , we turn to the final question of the study: are cortical timescales relatively static , or are they functionally dynamic and relevant for human cognition ?", "While previous studies have shown hierarchical segregation of task-relevant information corresponding to intrinsic timescales of different cortical regions ( Baldassano et al . , 2017; Chien and Honey , 2020; Honey et al . , 2012; Runyan et al . , 2017; Sarafyazd and Jazayeri , 2019; Wasmuht et al . , 2018 ) , as well as optimal adaptation of behavioral timescales to match the environment ( Ganupuru et al . , 2019; Ossmy et al . , 2013 ) , evidence for functionally relevant changes in regional neuronal timescales is lacking .", "Here , we examine whether timescales undergo short- and long-term shifts during working memory maintenance and aging , respectively .", "We first analyze human ECoG recordings from parietal , frontal ( PFC and orbitofrontal cortex [OFC] ) , and medial temporal lobe ( MTL ) regions of patients ( N = 14 ) performing a visuospatial working memory task that requires a delayed cued response ( Figure 4A; Johnson et al . , 2018a ) .", "Neuronal timescales were extracted for pre-stimulus baseline and memory maintenance delay periods ( 900 ms , both stimulus-free ) .", "Replicating our previous result in Figure 2—figure supplement 1G , we observe that baseline neuronal timescales follow a hierarchical progression across association regions , where parietal cortex ( PC ) , PFC , OFC , and MTL have gradually longer timescales ( pairwise Mann–Whitney U-test , Figure 4B ) .", "If neuronal timescales track the temporal persistence of information in a functional manner , then they should expand during delay periods .", "Consistent with our prediction , timescales in all regions are ~20% longer during delay periods ( Figure 4C; Wilcoxon rank-sum test ) .", "Moreover , only timescale changes in the PFC are significantly correlated with behavior across participants , where longer delay-period timescales relative to baseline are associated with better working memory performance ( Figure 4D , ρ = 0 . 75 , p=0 . 003 ) .", "No other spectral features in the recorded brain regions show consistent changes from baseline to delay periods while also significantly correlating with individual performance , including the 1/f-like spectral exponent , narrowband theta ( 3–8 Hz ) , and high-frequency ( high gamma; 70–100 Hz ) activity power ( Figure 4—figure supplement 1 ) .", "While timescales are consistent with the anatomical and gene expression hierarchy at a snapshot , brain structure itself is not static over time , undergoing many slower , neuroplastic changes during early development and throughout aging in older populations .", "In particular , aging is associated with a broad range of functional and structural changes , such as working memory impairments ( Voytek et al . , 2015c; Wang et al . , 2011 ) , as well as changes in neuronal dynamics ( Voytek et al . , 2015c; Voytek and Knight , 2015b; Wang et al . , 2011 ) and cortical structure ( de Villers-Sidani et al . , 2010 ) , such as the loss of slow-deactivating NMDA receptor subunits ( Pegasiou et al . , 2020 ) .", "Since neuronal timescales may support working memory maintenance , we predict that timescales would shorten across the lifespan , in agreement with the observed cognitive and structural deteriorations .", "To this end , we leverage the wide age range in the MNI-iEEG dataset ( 13–62 years old ) and probe average cortical timescales for each participant as a function of age .", "Since ECoG coverage is sparse and nonuniform across participants , simply averaging across parcels within individual participants confounds the effect of aging with the spatial effect of cortical hierarchy .", "Instead , we first normalize each parcel by its max value across all participants before averaging within participants , excluding those with fewer than 10 valid parcels ( N = 71 of 106 subjects remaining; results hold for a large range of threshold values , Figure 4—figure supplement 2B ) .", "We observe that older adults have faster neuronal timescales ( ρ = −0 . 31 , p=0 . 010; Figure 4E ) , and that timescales shorten with age in most areas across the cortex ( Figure 4F , t = −7 . 04 , p<0 . 001; 114 out of 189 parcels where at least six participants have data , see Figure 4—figure supplement 2C ) .", "This timescale compression is especially prominent in sensorimotor , temporal , and medial frontal regions .", "These results support our hypothesis that neuronal timescales , estimated from transmembrane current fluctuations , can rapidly shift in a functionally relevant manner , as well as slowly—over decades—in healthy aging ." ], [ "We first find that neuronal timescales vary continuously across the human cortex and coincide with the anatomical hierarchy , with timescales increasing from primary sensory and motor to association regions .", "While we use the continuous T1w/T2w gradient as a surrogate measure for anatomical hierarchy , there are multiple related but distinct perspectives on what ‘cortical hierarchy’ means , including , for example , laminar connectivity patterns from tract tracing data ( Felleman and Van Essen , 1991; Vezoli et al . , 2020 ) , continuous ( and latent-space ) gradients of gene expression and microarchitectural features ( Huntenburg et al . , 2018 ) , and network connectivity scales ( see review of Hilgetag and Goulas , 2020 ) —with most of these following a graded sensorimotor-to-association area progression .", "Similarly , it is important to note that there exist many different quantities that can be considered as characteristic neuronal timescales across several spatial scales , including membrane potential and synaptic current timescales ( Duarte et al . , 2017 ) , single-unit spike-train timescales ( Murray et al . , 2014 ) , population code timescales ( Runyan et al . , 2017 ) , and even large-scale circuit timescales measured from the fMRI BOLD signal ( Watanabe et al . , 2019 ) .", "We show here that timescales inferred from ECoG are consistently approximately 10 times faster than single-unit spiking timescales in macaques , corroborating the fact that field potential signals mainly reflect fast transmembrane and synaptic currents ( Buzsáki et al . , 2012 ) , whose timescales are related to , but distinct from , single-unit timescales measured in previous studies ( Dotson et al . , 2018; Ogawa and Komatsu , 2010; Wasmuht et al . , 2018 ) .", "Because field potential fluctuations are driven by currents from both locally generated and distal inputs , our results raise questions on how and when these timescales interact to shape downstream spiking dynamics .", "Furthermore , while we specifically investigate here the aperiodic timescale , which corresponds to the exponential decay timescale measured in previous studies , recent work has shown a similar gradient of oscillatory timescales ( i . e . , frequency ) along the anterior–posterior axis of the human cortex ( Mahjoory et al . , 2020 ) .", "Based on the similarity of these gradients and known mechanisms of asynchronous and oscillatory population dynamics ( e . g . , balance of excitation and inhibition in generating gamma oscillations and the asynchronous irregular state in cortical circuits [Brunel , 2000; Brunel and Wang , 2003] ) , we speculate that timescales of oscillatory and aperiodic neural dynamics may share ( at least partially ) circuit mechanisms at different spatial scales , analogous to the relationship between characteristic frequency and decay constant in a damped harmonic oscillator model .", "Using postmortem gene expression data as a surrogate for protein density , transcriptomic analysis uncovers the potential roles that transmembrane ion transporters and synaptic receptors play in establishing the cortical gradient of neuronal timescales .", "The expression of voltage-gated potassium channel , chloride channel , and GABAergic receptor genes , in particular , are strongly associated with the spatial variation of neuronal timescale .", "Remarkably , we find that electrophysiology-transcriptomic relationships discovered at the single-cell level , through patch-clamp recordings and single-cell RNA sequencing , are recapitulated at the macroscale between bulk gene expression and timescales inferred from ECoG .", "That being said , it is impossible to make definitive causal claims with the data presented in this study , especially considering the fact that several microanatomical features , such as gray matter myelination and cortical thickness , follow similar gradients across the cortex ( Burt et al . , 2018 ) .", "To discover genes specifically associated with timescale while accounting for the contribution of the overlapping anatomical hierarchy , we linearly regress out the T1w/T2w gradient from both timescale and gene expression gradients .", "Although this procedure does not account for any nonlinear contributions from anatomy , gene enrichment control analysis using T1w/T2w instead of timescales further demonstrates that the discovered genes—transmembrane ion transporters and inhibitory synaptic receptors—are more specifically associated with the timescale gradient , over and above the level predicted by anatomical hierarchy alone .", "From these results , we infer that potassium and chloride ion channels , as well as GABAergic receptors , may play a mechanistic role in altering the timescale of transmembrane currents at the macroscopic level .", "However , this interpretation rests on the key assumption that mRNA expression level is a faithful representation of the amount of functional proteins in a given brain region .", "In general , gene expression levels are highly correlated with the percentage of cells expressing that gene within brain regions ( Lein et al . , 2007 ) .", "Therefore , on a population level , the regional density of a particular ion channel or receptor protein is high if bulk mRNA expression is high .", "Furthermore , recent works have shown that neurotransmitter receptor density measured via autoradiography in postmortem brains follows similar cortical gradients ( Goulas et al . , 2020 ) , and that gene expression levels of neurotransmitter receptors ( e . g . , 5HT ) are strongly correlated with ligand binding potential measured via PET ( Gryglewski et al . , 2018 ) .", "Thus , as a first order approximation , receptor gene expression is an adequate surrogate for receptor protein density in the brain at the macroscale , though the relationship between mRNA expression and their transport and translation into channel proteins , the process of incorporating those proteins into membranes and synapses , and how these gene expression maps can be related to other overlapping macroscopic gradients are complex issues ( see e . g . , Fornito et al . , 2019; Liu et al . , 2016 ) .", "Thus , our analyses represent an initial data-mining process at the macroscopic level , which should motivate further studies in investigating the precise roles voltage-gated ion channels and synaptic inhibition play in shaping functional neuronal timescales through causal manipulations , complementary to existing lines of research focusing on NMDA activation and recurrent circuit motifs .", "Finally , we show that neuronal timescales are not static , but can change both in the short and long terms .", "Transmembrane current timescales across multiple association regions , including parietal , frontal , and medial temporal cortices , increase during the delay period of a working memory task , consistent with the emergence of persistent spiking during working memory delay .", "Working memory performance across individuals , however , is predicted by the extent of timescale increase in the PFC only .", "This further suggests that behaviorally relevant neural activity may be localized despite widespread task-related modulation ( Pinto et al . , 2019 ) , even at the level of neuronal membrane fluctuations .", "In the long term , we find that neuronal timescale shortens with age in most cortical regions , linking age-related synaptic , cellular , and connectivity changes—particularly those that influence neuronal integration timescale—to the compensatory posterior-to-anterior shift of functional specialization in healthy aging ( Davis et al . , 2008 ) .", "These results raise further questions regarding contrasting , and potentially complementary , aspects of neuronal timescale: on the one hand , task-free timescales across the cortex are shaped by relatively static macro- and microarchitectural properties ( Figures 2 and 3 ) ; on the other hand , timescales are dynamic and shift with behavioral demand ( Figure 4 ) .", "While long-term structural changes in the brain can explain shifts in neuronal timescales throughout the aging process , properties such as ion channel protein density probably do not change within seconds during a working memory task .", "We speculate that structural properties may constrain dynamical properties ( such as timescale ) to a possible range within a particular brain region and at different spatial scales , while task requirements , input statistics , short-term synaptic plasticity , and neuromodulation can then shift timescale within this range .", "We posit , then , that only shifts in dynamics within the area of relevance ( i . e . , PFC for working memory ) are indicative of task performance , consistent with recent ideas of computation-through-dynamics ( Vyas et al . , 2020 ) .", "Nevertheless , which neuromodulatory and circuit mechanisms are involved in shifting local timescales , and how timescales at different spatial scales ( e . g . , synaptic , neuronal , population ) interact to influence each other remain open questions for future investigation ( Breakspear , 2017; Duarte et al . , 2017; Freeman , 2000; Freeman and Erwin , 2008; Gjorgjieva et al . , 2016; Shine et al . , 2019 ) .", "In summary , we identify consistent and converging patterns between transcriptomics , anatomy , dynamics , and function across multiple datasets of different modalities from different individuals and multiple species .", "As a result , evidence for these relationships can be supplemented by more targeted approaches such as imaging of receptor metabolism .", "Furthermore , the introduction and validation of an open-source toolbox ( Donoghue et al . , 2020 ) for inferring timescales from macroscale electrophysiological recordings potentially allows for the noninvasive estimation of neuronal timescales , using widely accessible tools such as EEG and MEG .", "These results open up many avenues of research for discovering potential relationships between microscale gene expression and anatomy with the dynamics of neuronal population activity at the macroscale in humans ." ], [ "Consistent with previous studies , we define ‘neuronal timescale’ as the exponential decay time constant ( τ ) of the empirical ACF , or lagged correlation ( Honey et al . , 2012; Murray et al . , 2014 ) .", "τ can be naively estimated to be the time it takes for the ACF to decrease by a factor of e when there are no additional long-term , scale-free , or oscillatory processes , or by fitting a function of the form f ( t ) = e−tτ and extracting the parameter τ .", "Equivalently , the PSD is the Fourier Transform of the ACF via Wiener–Khinchin theorem ( Khintchine , 1934 ) and follows a Lorentzian function of the form L ( f ) =Ak+fχ for approximately exponential-decay processes , with χ=2 exactly when the ACF is solely composed of an exponential decay term , though it is often variable and in the range between 2 and 6 for neural time series ( Donoghue et al . , 2020; Miller et al . , 2009; Podvalny et al . , 2015; Voytek et al . , 2015c ) .", "Timescale can be computed from the parameter k as τ= 12πfk , where fk ≈ k1/χ is approximated to be the ‘knee frequency’ , at which a bend or knee in the power spectrum occurs , and equality holds when χ=2 .", "PSDs are estimated using a modified Welch’s method , where short-time windowed Fourier transforms ( STFT ) are computed from the time series , but the median is taken across time instead of the mean ( in conventional Welch’s method ) to minimize the effect of high-amplitude transients and artifacts ( Izhikevich et al . , 2018 ) .", "Custom functions for this can be found in NeuroDSP ( Cole et al . , 2019 ) , a published and open-source digital signal processing toolbox for neural time series ( neurodsp . spectral . compute_spectrum ) .", "For simulated data , Neurotycho macaque ECoG , and MNI-iEEG datasets , we use 1 s long Hamming windows with 0 . 5 s overlap .", "To estimate single-trial PSDs for the working memory ECoG dataset ( CRCNS Johnson-ECoG Johnson et al . , 2018a; Johnson et al . , 2018b ) , we simply apply Hamming window to 900 ms long epoched time series and compute the squared magnitude of the windowed Fourier transform .", "We apply spectral parameterization ( Donoghue et al . , 2020 ) to extract timescales from PSDs .", "Briefly , we decompose log-power spectra into a summation of narrowband periodic components—modeled as Gaussians—and an aperiodic component—modeled as a generalized Lorentzian function centered at 0 Hz ( L ( f ) above ) .", "For inferring decay timescale , this formalism can be practically advantageous when a strong oscillatory or variable power-law ( χ ) component is present , as is often the case for neural signals .", "While oscillatory and power-law components can corrupt naive measurements of τ as time for the ACF to reach 1/e , they can be easily accounted for and ignored in the frequency domain as narrowband peaks and 1/f-exponent fit .", "We discard the periodic components and infer timescale from the aperiodic component of the PSD .", "For a complete mathematical description of the model , see Donoghue et al . , 2020 .", "We simulate the aperiodic background component of neural field potential recordings as autocorrelated stochastic processes by convolving Poisson population spikes with exponentially decaying synaptic kernels with predefined decay time constants ( neurodsp . sim . sim_synaptic_current ) .", "PSDs of the simulated data are computed and parameterized as described above , and we compare the fitted timescales with their ground-truth values .", "Macaque single-unit timescales are taken directly from values reported in Figure 1c of Murray et al . , 2014 .", "Whole-brain surface ECoG data ( 1000 Hz sampling rate ) is taken from the Neurotycho repository ( Nagasaka et al . , 2011; Yanagawa et al . , 2013 ) , with eight sessions of 128-channel recordings from two animals ( George and Chibi , four sessions each ) .", "Results reported in Figure 2E–G are from ~10 min eyes-open resting periods to match the pre-stimulus baseline condition of single-unit experiments .", "Timescales for individual ECoG channels are extracted and averaged over regions corresponding to single-unit recording areas from Murray et al . , 2014; Figure 2F inset and Figure 2—figure supplement 3 , which are selected visually based on the overlapping cortical map and landmark sulci/gyri .", "Each region included between two and four electrodes ( see Figure 2—figure supplement 3B for selected ECoG channel indices for each region ) .", "For each individual recording session , as well as the grand average , Spearman rank correlation was computed between spiking and ECoG timescales .", "Linear regression models were fit using the python package scipy ( Virtanen et al . , 2020 ) ( scipy . stats . linregress ) and the linear slope was used to compute the scaling coefficient between spiking and ECoG timescales .", "The following sections describe procedures for generating the average cortical gradient maps for neuronal timescale , MR-derived T1w/T2w ratio , and gene expression from the respective raw datasets .", "All maps were projected onto the 180 left hemisphere parcels of Human Connectome Project’s Multimodal Parcellation ( Glasser et al . , 2016 ) ( HCP-MMP1 . 0 ) for comparison , described in the individual sections .", "Projection of T1w/T2w and gene expression maps from MNI volumetric coordinates to HCP-MMP1 . 0 can be found: https://github . com/rudyvdbrink/Surface_projection ( van den Brink , 2020 ) .", "All spatial correlations are computed as Spearman rank correlations between maps .", "Procedure for computing statistical significance while accounting for SA is described in detail below under the sections 'Spatial statistics' and 'SA modeling' .", "The MNI Open iEEG dataset consists of 1 min of resting state data across 1772 channels from 106 epilepsy patients ( 13–62 years old , 58 males , and 48 females ) , recorded using either surface strip/grid or stereoEEG electrodes , and cleaned of visible artifacts ( Frauscher et al . , 2018a; Frauscher et al . , 2018b ) .", "Neuronal timescales were extracted from PSDs of individual channels , and projected from MNI voxel coordinates onto HCP-MMP1 . 0 surface parcellation as follows .", "For each patient , timescale estimated from each electrode was extrapolated to the rest of the cortex in MNI coordinates using a Gaussian weighting function ( confidence mask ) , w ( r ) = e− ( r2/α2 ) , where r is the Euclidean distance between the electrode and a voxel , and α is the distance scaling constant , chosen here such that a voxel 4 mm away has 50% weight ( or confidence ) .", "Timescale at each voxel is computed as a weighted spatial average of timescales from all electrodes ( i ) of that patient: i . e . , τvoxel = ∑iw ( ri ) τi∑iw ( ri ) .", "Similarly , each voxel is assigned a confidence rating that is the maximum of weights over all electrodes ( wvoxel ( rmin ) , of the closest electrode ) , i . e . , a voxel right under an electrode has a confidence of 1 , while a voxel 4 mm away from the closest electrode has a confidence of 0 . 5 , etc .", "Timescales for each HCP-MMP parcel were then computed as the confidence-weighted arithmetic mean across all voxels that fall within the boundaries of that parcel .", "HCP-MMP boundary map is loaded and used for projection using NiBabel ( Brett et al . , 2020 ) .", "This results in a 180 parcels-by-106 patients timescale matrix .", "A per-parcel confidence matrix of the same dimensions was computed by taking the maximum confidence over all voxels for each parcel ( Figure 2—figure supplement 1A ) .", "The average cortical timescale map ( gradient ) is computed by taking the confidence-weighted average at each parcel across all participants .", "Note that this procedure for locally thresholded and weighted average is different from projection procedures used for the mRNA and T1w/T2w data due to region-constrained and heterogeneous ECoG electrode sites across participants .", "While coverage is sparse and idiosyncratic in individual participants , it does not vary as a function of age , and when pooling across the entire population , 178 of 180 parcels have at least one patient with an electrode within 4 mm , with the best coverage in sensorimotor , temporal , and frontal regions ( Figure 2—figure supplement 1 ) .", "As a measure of structural cortical hierarchy , we used the ratio between T1- and T2-weighted structural MRI , referred to as T1w/T2w map in main text , or the myelin map ( Burt et al . , 2018; Glasser and Van Essen , 2011 ) .", "Since there is little variation in the myelin map across individuals , we used the group average myelin map of the WU-Minn HCP S1200 release ( N = 1096 , March 1 , 2017 release ) provided in HCP-MMP1 . 0 surface space .", "For correlation with other variables , we computed the median value per parcel , identical to the procedure for mRNA expression below .", "Cortical thickness map was similarly generated .", "We used the Allen Human Brain Atlas ( AHBA ) gene expression dataset ( Hawrylycz et al . , 2015; Hawrylycz et al . , 2012 ) that comprised postmortem samples of six donors ( one female and five males ) that underwent microarray transcriptional profiling .", "Spatial maps of mRNA expression were available in volumetric 2 mm isotropic MNI space , following improved nonlinear registration and whole-brain prediction using variogram modeling as implemented by Gryglewski et al . , 2018 .", "We used whole-brain maps available from Gryglewski et al . , 2018 rather than the native sample-wise values in the AHBA database to prevent bias that could occur due to spatial inhomogeneity of the sampled locations .", "In total , 18 , 114 genes were included for analyses that related to the dominant axis of expression across the genome .", "We projected the volumetric mRNA expression data onto the HCP-MMP cortical surface using the HCP workbench software ( v1 . 3 . 1 running on Windows OS 10 ) with the ‘enclosing’ method and custom MATLAB code ( github . com/rudyvdbrink/surface_projection ) ( van den Brink , 2020 ) .", "The enclosing method extracts for all vertices on the surface the value from enclosing voxels in the volumetric data .", "Alternative projection methods such as trilinear 3D linear interpolation of surrounding voxels , or ribbon mapping that constructs a polyhedron from each vertex's neighbors on the surface to compute a weighted mean for the respective vertices , yielded comparable values , but less complete cortical coverage .", "Moreover , the enclosing method ensured that no transformation of the data ( nonlinear or otherwise ) occurred during the projection process and thus the original values in the volumetric data were preserved .", "Next , for each parcel of the left hemisphere in HCP-MMP , we extracted the median vertex-wise value .", "We used the median rather than the mean because it reduced the contribution of outliers in expression values within parcels .", "Vertices that were not enclosed by voxels that contained data in volumetric space were not included in the parcel-wise median .", "This was the case for 539 vertices ( 1 . 81% of total vertices ) .", "Linear interpolation across empty vertices prior to computing median parcel-wise values yielded near-identical results ( r = 0 . 95 for reconstructed surfaces ) .", "Lastly , expression values were mean and variance normalized across parcels to facilitate visualization .", "Normalization had no effect on spatial correlation between gene expression and other variables since the spatial distribution of gene expression was left unaltered .", "Similar to Burt et al . , 2018; Fagerberg et al . , 2014; Genovese et al . , 2016 , N = 2429 brain-specific genes were selected based on the criteria that expression in brain tissues were four times higher than the median expression across all tissue types , using Supplementary Dataset 1 of Fagerberg et al . , 2014 .", "PC1 result shown in Figure 3A is computed from brain-specific genes , though findings are similar when using all genes ( ρ = −0 . 56 with timescale map , Figure 3—figure supplement 1 ) .", "All correlations between spatial maps ( timescale , T1w/T2w , gene principal component [PC] , and individual gene expressions ) were computed using Spearman rank correlation .", "As noted in Burt et al . , 2020; Burt et al . , 2018; Vos de Wael et al . , 2020 , neural variables vary smoothly and continuously across the cortical surface , violating the assumption of independent samples .", "As a result , when correlating two variables , each with nontrivial SA , the naive p-value is artificially lowered since it is compared against an inappropriate null hypothesis , i . e . , randomly distributed or shuffled values across space .", "Instead , a more appropriate null hypothesis introduces SA-preserving null maps , which destroys any potential correlation between two maps while respecting their SAs .", "For all spatial correlation analyses , we generated N = 1000 null maps of one variable ( timescale map unless otherwise noted ) , and the test statistic , Spearman correlation ( ρ ) , is computed against the other variable of interest to build the null distribution .", "Two-tailed significance is then computed as the proportion of the null distribution that is less extreme than the empirical correlation value .", "All regression lines were computed by fitting a linear regression to log-timescale and the structural feature maps .", "To generate SA-preserving null maps , we used Moran Spectral Randomization ( MSR ) ( Wagner and Dray , 2015 ) from the python package BrainSpace ( Vos de Wael et al . , 2020 ) .", "Details of the algorithm can be found in the above references .", "Briefly , MSR performs eigendecomposition on a spatial weight matrix of choice , which is taken here to be the inverse average geodesic distance matrix between all pairs of parcels in HCP-MMP1 . 0 .", "The eigenvectors of the weight matrix are then used to generate randomized null feature maps that preserves the autocorrelation of the empirical map .", "We used the singleton procedure for null map generation .", "All significance values reported ( Figures 2D and 3A–C ) were adjusted using the above procedure .", "We also compare two other methods of generating null maps: spatial variogram fitting ( VF ) ( Burt et al . , 2020 ) and spin permutation ( Alexander-Bloch et al . , 2018 ) .", "Null maps were generated for timescale using spatial VF , while for spin permutation they were generated for vertex-wise T1w/T2w and gene PC1 maps before parcellation , so as to preserve surface locations of the parcellation itself .", "All methods perform similarly , producing comparable SA in the null maps , assessed using spatial variogram , as well as null distribution of spatial correlation coefficients between timescale and T1w/T2w ( Figure 2—figure supplement 2 ) .", "We used scikit-learn ( Pedregosa , 2011 ) PCA ( sklearn . decomposition . PCA ) to identify the dominant axes of gene expression variation across the entire AHBA dataset , as well as for brain-specific genes .", "PCA was computed on the variance-normalized average gene expression maps , X , an N × P matrix where N = 18 , 114 ( or N = 2429 brain-specific ) genes , and P = 180 cortical parcels .", "Briefly , PCA factorizes X such that X = USVT , where U and V are unitary matrices of dimensionality N × N and P × P , respectively .", "S is the same dimensionality as X and contains non-negative descending eigenvalues on its main diagonal ( Λ ) .", "Columns of V are defined as the PCs , and the dominant axis of gene expression is then defined as the first column of V , whose proportion of variance explained in the data is the first element of Λ divided by the sum over Λ .", "Results for PC1 and PC2-10 are shown in Figure 3A and Figure 3—figure supplement 1 , respectively .", "Single-cell timescale genes were selected based on data from Table S3 of Tripathy et al . , 2017 and Online Table 1 of Bomkamp et al . , 2019 .", "Using single-cell RNA sequencing data and patch-clamp recordings from transgenic mice cortical neurons , these studies identified genes whose expression significantly correlated with electrophysiological features derived from generalized linear integrate and fire ( GLIF ) model fits .", "We selected genes that were significantly correlated with membrane time constant ( tau ) , input resistance ( Rin or ri ) , or capacitance ( Cm or cap ) in the referenced data tables , and extracted the level of association between gene expression and those electrophysiological feature ( correlation ‘DiscCorr’ in Tripathy et al . , 2017 and linear coefficient ‘beta_gene’ in Bomkamp et al . , 2019 ) .", "To compare timescale-gene expression association at the single-cell and macroscale level , we correlated the single-cell associations extracted above with the spatial correlation coefficient ( macroscale ρ ) between ECoG timescale and AHBA microarray expression data for those same genes , restricting to genes with p<0 . 05 for macroscale correlation ( results identical for non-restrictive gene set ) .", "Overall association for all genes , as well as split by quintiles of their absolute macroscale correlation coefficient , are shown in Figure 3D .", "Example ‘single-cell timescale’ genes shown in Figure 3B and C are genes showing the highest correlations with those electrophysiology features reported in Table 2 of Bomkamp et al . , 2019 .", "To remove anatomical hierarchy as a potential mediating variable in timescale–gene expression relationships , we linearly regress out the T1w/T2w map from the ( log ) timescale map and individual gene expression maps .", "T1w/T2w was linearly fit to log-timescale , and the error between T1w/T2w-predicted timescale and empirical timescale was extracted ( residual ) ; this identical procedure was applied to every gene expression map to retrieve the gene residuals .", "SA-preserving null timescale residual maps were similarly created using MSR .", "Due to multicollinearity in the high-dimensional gene expression dataset ( many more genes than parcels ) , we fit a PLS model to the timescale map with one output dimension ( sklearn . cross_decomposition . PLSRegression ) to estimate regression coefficient for all genes simultaneously , resulting in N = 18 , 114 ( or N = 2429 brain-specific ) PLS weights ( Vértes et al . , 2016; Whitaker et al . , 2016 ) .", "To determine significantly associated ( or ‘enriched’ ) genes , we repeated the above PLS-fitting procedure 1000 times but replaced the empirical timescale map ( or residual map ) with null timescale maps ( or residual maps ) that preserved its SA .", "Genes whose absolute empirical PLS weight was greater than 95% of its null weight distribution was deemed to be enriched , and submitted for GOEA .", "The Gene Ontology ( GO ) captures hierarchically structured relationships between GO items representing aspects of biological processes ( BP ) , cellular components ( CC ) , or molecular functions ( MF ) .", "For example , ‘synaptic signaling’ , ‘chemical synaptic transmission’ , and ‘glutamatergic synaptic transmission’ are GO items with increasing specificity , with smaller subsets of genes associated with each function .", "Each GO item is annotated with a list of genes that have been linked to that particular process or function .", "GOEA examines the list of enriched genes from above to identify GO items that are more associated with those genes than expected by chance .", "We used GOATOOLS ( Klopfenstein et al . , 2018 ) to perform GOEA programmatically in python .", "The list of unranked genes with significant empirical PLS weights was submitted for GOEA as the ‘study set’ , while either the full ABHA list or brain-specific gene list was used as the ‘reference set’ .", "The output of GOEA is a list of GO terms with annotated genes that are enriched or purified ( i . e . , preferentially appearing or missing in the study list , respectively ) more often than by chance , determined by Fisher’s exact test .", "Enrichment ratio is defined as follows: given a reference set with N total genes , and n were found to be significantly associated with timescale ( in the study set ) , for a single GO item with B total genes annotated to it , where b of them overlap with the study set , then .", "Statistical significance is adjusted for multiple comparisons following Benjamini–Hochberg procedure ( false discovery rate q-value reported in Figure 3F ) , and all significant GO items ( q < 0 . 05 ) are reported in Figure 3F , in addition to some example items that did not pass significance threshold .", "For a detailed exposition , see Bauer , 2017 .", "Figure 3F shows results using brain-specific genes .", "The GO items that are significantly associated are similar when using the full gene set , but typically with larger q-values ( Supplementary file 1 and", "2 ) due to a much larger set of ( non-brain-specific ) genes .", "Control analysis was conducted using T1w/T2w , with 1000 similarly generated null maps , instead of timescale .", "The CRCNS fcx-2 and fcx-3 datasets include 17 intracranial ECoG recordings in total from epilepsy patients ( 10 and 7 , respectively ) performing the same visuospatial working memory task ( Johnson , 2019; Johnson , 2018c; Johnson et al . , 2018a , Johnson et al . , 2018b ) .", "Subject 3 ( s3 ) from fcx-2 was discarded due to poor data quality upon examination of trial-averaged PSDs ( high noise floor near 20 Hz ) , while s5 and s7 from fcx-3 correspond to s5 and s8 in fcx-2 and were thus combined .", "Together , data from 14 unique participants ( 22–50 years old , five females ) were analyzed , with variable and overlapping coverage in PC ( n = 14 ) , PFC ( n = 13 ) , OFC ( n = 8 ) , and MTL ( n = 9 ) .", "Each channel was annotated as belonging to one of the above macro regions .", "Experimental setup is described in Johnson , 2019; Johnson , 2018c; Johnson et al . , 2018a , Johnson et al . , 2018b in detail .", "Briefly , following a 1 s pre-trial fixation period ( baseline ) , subjects were instructed to focus on one of two stimulus contexts ( ‘identity’ or ‘relation’ information ) .", "Then two shapes were presented in sequence for 200 ms each .", "After a 900 or 1150 ms jittered precue delay ( delay1 ) , the test cue appeared for 800 ms , followed by another post-cue delay period of the same length ( delay2 ) .", "Finally , the response period required participants to perform a 2-alternative forced choice test based on the test cue , which varied based on trial condition .", "For our analysis , we collapsed across the stimulus context conditions and compared neuronal timescales during the last 900 ms of baseline and delay periods from the epoched data , which were free of visual stimuli , in order to avoid stimulus-related event-related potential effects .", "Behavioral accuracy for each experimental condition was reported for each participant , and we average across both stimulus context conditions to produce a single working memory accuracy per participant .", "Single-trial power spectra were computed for each channel as the squared magnitude of the Hamming-windowed Fourier Transform .", "We used 900 ms of data in all three periods ( pre-trial , delay1 , and delay2 ) .", "Timescales were estimated by applying spectral parameterization as above , and the two delay-period estimates were averaged to produce a single delay period value .", "For comparison , we computed single-trial theta ( 3–8 Hz ) and high-frequency activity ( high gamma [Mukamel et al . , 2005] , 70–100 Hz ) powers as the mean log-power within those frequency bins , as well as spectral exponent ( χ ) .", "Single-trial timescale difference between delay and baseline was calculated as the difference of the log timescales due to the non-normal distribution of single-trial timescale estimates .", "All other neural features were computed by subtracting baseline from the delay period .", "All neural features were then averaged across channels within the same regions , then trials , for each participant , to produce per-participant region-wise estimates , and finally averaged across all participants for the regional average in Figure 4B and C . Two-sided Mann–Whitney U-tests were used to test for significant differences in baseline timescale between pairs of regions ( Figure 4B ) .", "Two-sided Wilcoxon rank-sum tests were used to determine the statistical significance of timescale change in each region ( Figure 4C ) , where the null hypothesis was no change between baseline and delay periods ( i . e . , delay is 100% of baseline ) .", "Spearman rank correlation was used to determine the relationship between neural activity ( timescale; theta; high-frequency; χ ) change and working memory accuracy across participants ( Figure 4D and Figure 4—figure supplement 1 ) .", "Since electrode coverage in the MNI-iEEG dataset is sparse and nonuniform across participants ( Figure 2—figure supplement 1 ) , simply averaging across parcels within individuals to estimate an average cortical timescale per participant confounds the effect of age with the spatial effect of cortical hierarchy .", "Therefore , we instead first normalize each parcel by its max value across all participants before averaging within participants , excluding those with fewer than 10 valid parcels ( 71 of 106 subjects remaining; results hold for a range of threshold values; Figure 4—figure supplement 2B ) .", "Spearman rank correlation was used to compute the association between age and average cortical timescale .", "Each cortical parcel had a variable number of participants with valid timescale estimates above the consistency threshold , so we compute Spearman correlation between age and timescale for each parcel , but including only those with at least five participants ( 114 of 180 parcels , result holds for a range of threshold values; Figure 4—figure supplement 2C ) .", "Spatial effect of age-timescale variation is plotted in Figure 4F , where parcels that did not meet the threshold criteria are grayed out .", "Mean age–timescale correlation from individual parcels was significantly negative under one-sample t-test .", "All data analyzed in this manuscript are from open data sources .", "All code used for all analyses and plots are publicly available on GitHub at https://github . com/rdgao/field-echos ( Gao , 2020 ) and https://github . com/rudyvdbrink/surface_projection ( van den Brink , 2020 ) .", "See Tables 1 and 2 for details ." ] ]
[ "Complex cognitive functions such as working memory and decision-making require information maintenance over seconds to years , from transient sensory stimuli to long-term contextual cues .", "While theoretical accounts predict the emergence of a corresponding hierarchy of neuronal timescales , direct electrophysiological evidence across the human cortex is lacking .", "Here , we infer neuronal timescales from invasive intracranial recordings .", "Timescales increase along the principal sensorimotor-to-association axis across the entire human cortex , and scale with single-unit timescales within macaques .", "Cortex-wide transcriptomic analysis shows direct alignment between timescales and expression of excitation- and inhibition-related genes , as well as genes specific to voltage-gated transmembrane ion transporters .", "Finally , neuronal timescales are functionally dynamic: prefrontal cortex timescales expand during working memory maintenance and predict individual performance , while cortex-wide timescales compress with aging .", "Thus , neuronal timescales follow cytoarchitectonic gradients across the human cortex and are relevant for cognition in both short and long terms , bridging microcircuit physiology with macroscale dynamics and behavior ." ]
[ "The human brain can both quickly react to a fleeting sight , like a changing traffic light , and slowly integrate complex information to form a long-term plan .", "To mirror these requirements , how long a neuron can be activated for – its ‘timescale’ – varies greatly between cells .", "A range of timescales has been identified in animal brains , by measuring single neurons at a few different locations .", "However , a comprehensive study of this property in humans has been hindered by technical and ethical concerns .", "Without this knowledge , it is difficult to understand the factors that may shape different timescales , and how these can change in response to environmental demands .", "To investigate this question , Gao et al . used a new computational method to analyse publicly available datasets and calculate neuronal timescales across the human brain .", "The data were produced using a technique called invasive electrocorticography , where electrodes placed directly on the brain record the total activity of many neurons .", "This allowed Gao et al . to examine the relationship between timescales and brain anatomy , gene expression , and cognition .", "The analysis revealed a continuous gradient of neuronal timescales between areas that require neurons to react quickly and those relying on long-term activity .", "‘Under the hood’ , these timescales were associated with a number of biological processes , such as the activity of genes that shape the nature of the connections between neurons and the amount of proteins that let different charged particles in and out of cells .", "In addition , the timescales could be flexible: they could lengthen when areas specialised in working memory were actively maintaining information , or shorten with age across many areas of the brain .", "Ultimately , the technique and findings reported by Gao et al . could have useful applications in the clinic , using neuronal timescale to better understand brain disorders and pinpoint their underlying causes ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia
elife-03896-v2
[ [ "There is significant empirical support for the idea , proposed by Ramón Y Cajal more than a century ago ( Cajal , 1894 ) , that long-term memories are expressed in the brain , in part , by changes in synaptic connectivity .", "A corollary of this idea , accepted by many , if not most , modern neuroscientists , is that memories are maintained by persistent molecular and cellular alterations in synaptic structures themselves ( Bailey and Kandel , 2008; Kandel et al . , 2014 ) .", "Here , we have tested the idea that long-term memory ( LTM ) is stored at synapses using the marine mollusk Aplysia californica .", "The relatively simple behavior and nervous system of this model invertebrate organism offer several major advantages for understanding memory at the level of modifications of individual synapses ( Kandel , 2001 ) .", "A form of learning in Aplysia whose cellular and molecular substrates are particularly well understood is sensitization of the gill- and siphon-withdrawal reflex ( Carew et al . , 1971; Brunelli et al . , 1976; Antonov et al . , 1999; Kandel , 2001; Glanzman , 2010 ) .", "Sensitization of the withdrawal reflex exhibits a long-term ( ≥24 hr ) form ( Pinsker et al . , 1973 ) due , in part , to long-term facilitation ( LTF ) of the monosynaptic connection between the sensory and motor neurons that mediate the reflex ( Frost et al . , 1985 ) .", "Importantly , the monosynaptic sensorimotor connection can be reconstituted in dissociated cell culture , and LTF of the in vitro synapse can be induced by training with pulses of serotonin ( 5HT ) , the monoaminergic neurotransmitter that mediates sensitization in Aplysia ( Brunelli et al . , 1976; Glanzman et al . , 1989; Marinesco and Carew , 2002 ) .", "Cellular and molecular analyses of this form of long-term synaptic plasticity have provided major mechanistic insights into long-term memory in Aplysia ( Goelet et al . , 1986; Dash et al . , 1990; Bartsch et al . , 1995; Martin et al . , 1997 ) , insights that have generalized to learning and memory in other organisms , including mammals ( Yin et al . , 1994 , 1995; Frey and Morris , 1997; Kogan et al . , 1997; Josselyn et al . , 2001 ) .", "Accordingly , we used the in vitro sensorimotor synapse in initial experiments to determine whether LTM is stored at synapses .", "Current evidence supports the idea that LTM can be modified or even eliminated under certain circumstances .", "One of these goes under the rubric of reconsolidation blockade .", "Here , a stimulus is delivered to an animal that serves to reactivate the LTM for a previous learning experience .", "If , immediately after delivery of this reminder , the animal is treated with an inhibitor of protein synthesis ( Nader et al . , 2000 ) , or subjected to electroconvulsive shock ( Misanin et al . , 1968 ) , the LTM will be apparently eliminated .", "Based on this evidence , it has been proposed that the reminder stimulus returns the LTM to a labile state in which new protein synthesis is required to reconsolidate the memory; and that inhibition of protein synthesis during this period of reconsolidation can erase the original memory ( Nader and Hardt , 2009 ) ( but see Lattal and Abel , 2004 ) .", "Another manipulation that can apparently erase LTM permanently is inhibition of the constitutively active catalytic fragment of the atypical protein kinase Cζ; the ongoing activity of this catalytic fragment , named PKMζ , appears to be required for the maintenance of several forms of LTM in mammals ( Sacktor , 2011 ) ; inhibition of PKMζ , in the absence of a reminder , has been reported to abolish consolidated memory ( but see Lee et al . , 2013; Volk et al . , 2013 ) .", "We , and others , have shown that the synaptic memory for LTF of in vitro sensorimotor connections can be apparently eliminated when sensorimotor cocultures are treated with a protein synthesis inhibitor following reminder training ( Cai et al . , 2012; Lee et al . , 2012; Hu and Schacher , 2014 ) .", "In addition , inhibiting the activity of PKM Apl III , the constitutively active fragment of the Aplysia atypical protein kinase C ( PKC Apl III ) ( Villareal et al . , 2009; Cai et al . , 2011; Bougie et al . , 2012 ) , also erases consolidated LTF ( Cai et al . , 2011 ) .", "LTF is mediated partly by the growth of new sensory neuron varicosities ( Glanzman et al . , 1990; Bailey and Kandel , 2008 ) .", "Here we investigated whether blocking the reconsolidation of the memory for LTF , or inhibiting PKM Apl III , altered this long-term change in presynaptic structure .", "We found that the synaptic growth induced by LTF training was reversed by these two memory-disrupting manipulations; however , although the overall number of presynaptic varicosities reverted to the original , pretraining level , the resultant morphological pattern of sensorimotor synapses differed significantly from the original one .", "These results imply that the persistence of memory does not require the stability of particular synaptic connections .", "We provide additional support for this idea with data from behavioral experiments in which we show that LTM can be reinstated in intact Aplysia following its apparent disappearance due to reconsolidation blockade or PKM inhibition .", "Because these two antimnemonic manipulations not only disrupt the behavioral expression of LTM , but also eliminate the synaptic changes—both electrophysiological ( Cai et al . , 2011 , 2012; Lee et al . , 2012 ) and morphological ( present data ) —closely associated with LTM in Aplysia ( Bailey and Chen , 1983 , 1988; Frost et al . , 1985; Glanzman et al . , 1990 ) , our results challenge the idea that the synapse is a cellular site for long-term memory storage in Aplysia .", "In other behavioral experiments we show that both the disruption of LTM through inhibition of PKM Apl III and LTM induction require epigenetic changes .", "These results point to the nucleus of neurons as the potential locus of the engram in Aplysia ." ], [ "In electrophysiological experiments involving 5HT-induced LTF of sensorimotor synapses in dissociated cell culture , we previously showed that a ‘reminder’ stimulus—a single , 5-min pulse of 5HT—could trigger the apparent reconsolidation of synaptic memory , as indicated by the vulnerability of consolidated LTF to disruption by administration of a protein synthesis inhibitor ( anisomycin ) following the reminder .", "Specifically , treatment with a single pulse of 5HT and anisomycin 24 hr or more after the induction of LTF reversed the synaptic facilitation ( Cai et al . , 2012; see also; Lee et al . , 2012; Hu and Schacher , 2014 ) .", "Here we asked whether the blockade of the reconsolidation of LTF also reverses the synaptic growth that underlies LTF ( Glanzman et al . , 1990 ) .", "Sensory neurons ( SNs ) of established sensorimotor cocultures were labeled with dextran fluorescein , and motor neurons ( MNs ) with dextran rhodamine , via pressure injection; the neurons were then imaged using laser scanning confocal fluorescence microscopy , and the presynaptic varicosities in contact with a postsynaptic structure ( either a motor neurite or the postsynaptic soma ) were quantified ( Glanzman et al . , 1990 ) ( Figure 1A , B ) .", "After initial imaging some cocultures received five spaced 5-min pulses of serotonin ( 5X5HT training , 100 µM ) .", "24 hr later the cocultures were reimaged and the varicosities requantified .", "As previously reported ( Glanzman et al . , 1990 ) , the 5X5HT training produced a significant increase in the number of presynaptic varicosities contacting the MN ( Figure 1C ) .", "After reimaging , two groups of the trained cocultures received a single 5-min pulse of 5HT ( 1X5HT , 100 µM ) to reactivate the synaptic memory induced by 5X5HT training ( Cai et al . , 2012 ) .", "Immediately following the reminder pulse of 5HT , one group ( 5X5HT-1X5HT-Aniso group ) received anisomycin ( 10 µM ) treatment for 2 hr .", "Two other groups of 5X5HT-trained cocultures did not get the reminder pulse of 5HT; one of these groups ( 5X5HT-Aniso ) received the anisomycin treatment , whereas the other group ( 5X5HT ) received vehicle solution instead .", "A final group of cocultures ( Controls ) did not receive either 5HT or anisomycin; instead , the Controls were treated with vehicle solution at the experimental times that the 5X5HT training and anisomycin treatment were administered to other groups .", "At 48 hr after the original imaging session all of the cocultures were imaged and the varicosities once more quantified . 10 . 7554/eLife . 03896 . 003Figure 1 . Blockade of memory reconsolidation reverses 5HT-induced synaptic growth .", "( A ) Experimental protocol .", "The vertical blue bars represent pulses of 5HT , and the horizontal red bar represents anisomycin/vehicle treatment .", "A reminder pulse of 5HT ( single blue bar ) was delivered to the 5X5HT-1X5HT-Aniso cocultures prior to the anisomycin .", "( B ) Sample confocal micrographs of a Control coculture .", "Blue arrowheads , presynaptic varicosities present at 0 hr; red arrowheads , new varicosities that appeared during 0–24 hr; and yellow arrowheads , varicosities formed during the 24–48 hr period .", "Scale bar , 20 μm .", "( C ) Mean normalized varicosity number at 24 hr and 48 hr in the Control ( n = 21 ) , 5X5HT ( n = 21 ) , 5X5HT-Aniso ( n = 19 ) , and 5X5HT-1X5HT-Aniso ( n = 26 ) groups .", "The number of varicosities measured at 24 hr and 48 hr was normalized to the number present at 0 hr ( Note that differences in the data for the 5X5HT and 5X5HT-1X5HT groups were not significant , and two groups have been grouped together in the graph . The separate data for the 5X5HT-1X5HT group can be found in Figure 1—figure supplement 1 ) .", "A repeated-measures ANOVA indicated that there was a significant group × time interaction ( F[3 , 83] = 9 . 5 , p < 0 . 0001 ) .", "Planned comparisons using one-way ANOVAs indicated that the group differences at 24 and 48 hr were highly significant ( F[3 , 83] = 8 . 6 for 24 hr and 12 . 5 for 48 hr; p < 0 . 0001 for the results of each ANOVA ) .", "SNK posthoc tests on the 24 hr data showed that the mean normalized varicosity number in the 5X5HT group ( 192 . 8 ± 12 . 7% ) , 5X5HT-Aniso group ( 180 . 9 ± 11 . 6% ) , and 5X5HT-1X5HT-Aniso group ( 179 . 5 ± 14 . 6% ) was each significantly greater than that in the Control group ( 110 . 3 ± 8 . 5%; p < 0 . 001 for all comparisons ) .", "The increase in varicosity number persisted to 48 hr in the 5X5HT group ( 197 . 9 ± 12 . 2% , p < 0 . 001 ) and 5X5HT-Aniso group ( 191 . 6 ± 13 . 2% , p < 0 . 001 ) , but not in the 5X5HT-1X5HT-Aniso group ( 124 . 4 ± 10 . 9% ) , when compared to the Control group ( 108 . 9 ± 15 . 2% ) .", "The difference between the 5X5HT-Aniso and 5X5HT-1X5HT-Aniso groups was highly significant ( p < 0 . 001 ) .", "Asterisks indicate a significant difference for comparisons with the Control group; plus signs indicate a significant difference for comparisons with the 5X5HT-1X5HT-Aniso group .", "Here and in subsequent figures one symbol indicates p < 0 . 05; two symbols , p < 0 . 01; three symbols , p < 0 . 001 .", "Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 00310 . 7554/eLife . 03896 . 004Figure 1—figure supplement 1 . Graphs presenting the normalized mean varicosity numbers for the three 5HT-trained groups not subjected to reconsolidation blockade . The mean normalized varicosity number at 24 hr was 192 . 7 ± 15 . 0% in the 5X5HT group ( n = 18 ) , 193 . 3 ± 17 . 6% in the 5X5HT-1X5HT group ( n = 3 ) , and 180 . 9 ± 11 . 6% in the 5X5HT-Aniso group ( n = 19 ) .", "At 48 hr the mean normalized varicosity number was 192 . 9 ± 13 . 7% in the 5X5HT group , 228 . 3 ± 17 . 4% in the 5X5HT-1X5HT group , and 191 . 6 ± 13 . 2% in the 5X5HT-Aniso group .", "A repeated-measures ANOVA indicated that neither the interaction ( F[2 , 37] = 0 . 8 , p = 0 . 45 ) , nor the overall effects of group ( F[2 , 37] = 0 . 3 , p = 0 . 73 ) , was significant . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 004 The overall increase in the number of presynaptic varicosities induced by 5X5HT training on Day 1 persisted through the third imaging session in the 5X5HT and 5X5HT-Aniso groups of cocultures ( Figure 1C ) .", "( Notice that because there were no significant differences between the 5X5HT and 5X5HT-1X5HT groups , these two groups have been combined into a single group [5X5HT] in Figure 1C . However , the data for the 5X5HT-1X5HT group are presented separately in the graph in Figure 1—figure supplement 1 ) .", "The reminder pulse of 5HT coupled with inhibition of protein synthesis caused the number of varicosities to revert to the pretraining ( 0 hr ) value in the 5X5HT-1X5HT-Aniso group .", "This structural result parallels the electrophysiological results previously reported for Aplysia sensorimotor cocultures ( Cai et al . , 2012; Lee et al . , 2012; Hu and Schacher , 2014 ) , and provides additional support for the notion that 1X5HT reactivated the synaptic memory induced by the 5X5HT training .", "Besides quantifying changes in overall varicosity number , we tracked the fate of each SN varicosity in every coculture over the course of the experiments .", "The varicosities were put into one of three categories: ‘original’ varicosities , that is , the varicosities present at 0 hr; ‘5HT-induced’ varicosities , the varicosities that appeared during the 24 hr after 5HT treatment ( this category pertained only to the groups given 5X5HT training ) ; and ‘new’ varicosities , varicosities formed during the 24–48 hr period .", "For the analysis of varicosity fate the three groups of cocultures that received the 5X5HT training without reconsolidation blockade—that is , the 5X5HT , 5X5HT-1X5HT and 5X5HT-Aniso groups—were consolidated into a single group labeled ‘5HT-No reconsolidation/No blockade’ in Figure 2 .", "Inspection of the fates of individual varicosities in this group yielded surprising results .", "First , many of the 5HT-induced SN varicosities in the 5HT-No reconsolidation/No blockade group did not persist until the final imaging session ( Figures 2A and 3A ) ; instead , there was significant retraction of the 5HT-induced varicosities between 24 and 48 hr in this group .", "Second , there was also retraction of the original varicosities during this period ( Figures 2B and 3A ) .", "Varicosities in the 5X5HT-1X5HT-Aniso group—the group of trained cocultures subjected to reconsolidation blockade—exhibited a similar pattern of retraction of 5HT-induced and original varicosities , but the amount of retraction was significantly greater than that observed in the 5HT-No reconsolidation/No blockade cocultures ( Figures 2A , B and 3B ) .", "A third unexpected finding was the substantial growth of new varicosities in all trained groups between 24 and 48 hr; the amount of the growth was significantly greater , however , in the 5-HT-No reconsolidation/No blockade cocultures than in the 5X5HT-1X5HT-Aniso group ( Figure 2C ) .", "Thus , the 5X5HT trained cocultures subjected to reconsolidation blockade exhibited significantly more retraction and significantly less growth of varicosities during the 24–48 hr period than did the other trained cocultures .", "Interestingly , whereas there was greater retraction of 5HT-induced varicosities than of original varicosities between 24–48 hr in the 5HT-No reconsolidation/No blockade group , the retraction of 5HT-induced varicosities and original varicosities did not differ in the 5X5HT-1X5HT-Aniso group ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 03896 . 005Figure 2 . Effect of reconsolidation blockade on the fates of individual varicosities . For varicosities in the 5HT-induced and original categories , the varicosity count in a given category for each coculture at 48 hr was normalized to the number of varicosities in the same category in that coculture at 24 hr .", "For the varicosities formed >24 hr , the varicosity count for each coculture at 48 hr was normalized to the total number of varicosities in that coculture at 24 hr . ( A ) The mean normalized decrease in the number of 5HT-induced varicosities ( varicosities that appeared within 24 hr after 5X5HT training ) at 48 hr was 67 . 9 ± 4 . 2% in the 5X5HT-1X5HT-Aniso group , which was greater than that in the 5HT-No reconsolidation/No blockade group ( 47 . 7 ± 3 . 7% , p < 0 . 001 , two-tailed t test ) .", "( B ) Retraction of original varicosities in the 5X5HT-1X5HT-Aniso group ( 56 . 4 ± 6 . 1% ) was significantly greater than in the 5HT-No reconsolidation/No blockade group ( 32 . 3 ± 5 . 1% , p < 0 . 01 , two-tailed t test ) .", "( C ) Reversal of the morphological changes induced by 5X5HT training also involved inhibition of synaptic growth from 24–48 hr .", "There was significantly less growth of new varicosities during this period in the 5X5HT-1X5HT-Aniso group ( 36 . 8 ± 5 . 2% ) than in the 5HT-No reconsolidation/No blockade group ( 52 . 9 ± 4 . 2% , p < 0 . 05 , two-tailed t test ) .", "Blue bars , 5HT-No reconsolidation/No blockade; red bars , 5X5HT-1X5HT-Aniso group .", "Asterisks indicate significant differences between the 5HT-No reconsolidation/No blockade and 5X5HT-1X5HT-Aniso groups .", "Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 00510 . 7554/eLife . 03896 . 006Figure 2—figure supplement 1 . Comparison of retraction of original and 5HT-induced varicosities between 24–48 hr in the reconsolidation blockade experiments .", "( A ) The percent retraction of 5HT-induced varicosities ( 47 . 7 ± 3 . 7% ) was significantly greater than that of the original varicosities ( 32 . 3 ± 5 . 1% , p < 0 . 02 , two-tailed t test ) in the 5HT-No reconsolidation/No blockade group .", "( B ) The percent retraction of the 5HT-induced varicosities ( 67 . 9 ± 4 . 2% ) did not differ significantly from that of original varicosities ( 56 . 4 ± 6 . 1% ) in the 5X5HT-1X5HT-Aniso group . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 00610 . 7554/eLife . 03896 . 007Figure 3 . Confocal fluorescence micrographs illustrating the structural effects of 5X5HT training , reconsolidation blockade , and chelerythrine treatment .", "( A ) Coculture that received 5X5HT training alone .", "( B ) Coculture that received the 5X5HT training plus 1X5HT and anisomycin treatment immediately after the 24 hr imaging session .", "( C ) Coculture that received the 5X5HT training plus chelerythrine treatment immediately after the 24 hr imaging session .", "Blue arrowheads , varicosities present at 0 hr; red arrowheads , varicosities formed during 0–24 hr; and yellow arrowheads , varicosities formed during 24–48 hr .", "Scale bars , 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 007 The substantial morphological changes in the 5HT-No reconsolidation/No blockade cocultures between 24 and 48 hr were surprising , because the overall number of varicosities remained stable in these cocultures during this period ( see the data for the 5X5HT and 5X5HT-Aniso groups in Figure 1C ) .", "Moreover , as demonstrated in our earlier electrophysiological investigations of LTF , the amplitude of the sensorimotor EPSP was also stable between 24 and 48 hr after 5X5HT training ( Cai et al . , 2011 ) .", "Our morphological data reveal the operation of a heretofore unrecognized homeostatic mechanism that adjusts the number of SN varicosities—and , presumably , active synaptic sites ( Schacher et al . , 1990; Kim et al . , 2003 ) —for sensorimotor connections according to their learning-related experience , but that seems unconcerned with the identities of the individual varicosities/synaptic sites .", "In the 5HT-No reconsolidation/No blockade cocultures the significant retraction of SN varicosities during 24–48 hr after 5X5HT training was compensated for by significant growth of new varicosities , so that the overall number remained constant ( Figures 1C and 2 ) .", "By contrast , the number of varicosities was reset to the original ( 0 hr ) value in the 5X5HT-1X5T-Aniso group; this resetting involved increased retraction of both 5HT-induced and original varicosities , and decreased growth of new varicosities between 24 and 48 hr .", "Consequently , the morphological state of the cocultures in the 5X5HT-1X5T-Aniso group at 48 hr differed significantly from that at 0 hr with respect to the identities of individual SN varicosities .", "Next we examined the effect of inhibiting PKM on SN varicosity number .", "PKM Apl III , a homolog of mammalian PKMζ ( Sacktor , 2011 ) , is formed from the atypical Aplysia protein kinase C ( PKC Apl III ) ( Bougie et al . , 2009 ) by calpain-dependent cleavage; furthermore , this cleavage is induced by prolonged treatment with 5HT ( Bougie et al . , 2012 ) .", "In previous behavioral and electrophysiological investigations we found that inhibition of PKM Apl III , either with the pseudosubstrate sequence of the regulatory domain of atypical PKC ( ZIP ) or with chelerythrine , a PKC inhibitor selective for PKM at low concentrations ( Ling et al . , 2002; Villareal et al . , 2009 ) , abolished both consolidated long-term sensitization and LTF ( Cai et al . , 2011 ) .", "To determine whether inhibition of PKM Apl III reverses the structural growth that mediates long-term sensitization ( Bailey and Chen , 1983 , 1988 ) and LTF ( Glanzman et al . , 1990 ) , we gave some cocultures ( 5X5HT-Chel group ) 5X5HT training followed 24 hr later by treatment with chelerythrine ( Figure 4A ) .", "Another group of cocultures ( 5X5HT group ) received the 5X5HT training alone .", "Finally , a third group ( Controls ) received the vehicle solution instead of either 5HT or chelerythrine .", "The SNs and MNs of all of the cocultures were labeled with fluorescent dye as before and then imaged on Day 1 ( 0 hr ) of the experiment .", "Immediately afterwards cocultures in the 5X5HT and 5X5HT-Chel groups were given 5X5HT training .", "All cocultures were imaged for a second time at 24 hr , after which cocultures in the 5X5HT-Chel group received chelerythrine ( 10 µM , 1 hr ) , while the other two groups received the vehicle solution .", "( The 5X5HT-Chel group did not receive 1X5HT prior to chelerythrine treatment . )", "The cocultures were imaged for a final time at 48 hr . 10 . 7554/eLife . 03896 . 009Figure 4 . Inhibition of PKM also reverses 5HT-induced synaptic growth .", "( A ) Experimental protocol .", "The vertical blue bars represent pulses of 5HT; the horizontal red bar represents the period of chelerythrine/vehicle treatment .", "The 5X5HT and Control groups received vehicle treatment during this time .", "( B ) Mean SN varicosity number measured at 24 hr and 48 hr for the Control ( n = 14 ) , 5X5HT ( n = 21 ) and 5X5HT-Chel ( n = 14 ) .", "The number of varicosities measured at 24 hr and 48 hr was normalized to the number of varicosities at 0 hr .", "A repeated-measures ANOVA indicated that there was a significant group × time interaction ( F[2 , 46] = 7 . 5 , p < 0 . 001 ) .", "Planned comparisons showed that the group differences were significant for both the 24 and 48 hr measurements ( 24 hr , F[2 , 46] = 3 . 7 , p < 0 . 04; 48 hr , F[2 , 46] = 12 . 8 , p < 0 . 0001 ) .", "The mean normalized varicosity number at 24 hr was 171 . 2 ± 14 . 0% in the 5X5HT group , 166 . 9 ± 22 . 8% in the 5X5HT-Chel group , and 114 . 0 ± 8 . 9% in the Control group .", "Posthoc comparisons indicated that the 5X5HT training produced a significant increase in varicosity number in the 5X5HT and 5X5HT-Chel groups at 24 hr ( p < 0 . 05 for both comparisons with the Control group ) .", "The mean normalized varicosity number at 48 hr in the 5X5HT group ( 197 . 9 ± 18 . 7% ) was greater than that in the Control group ( 104 . 4 ± 7 . 2% , p < 0 . 001 ) , but the 5X5HT-Chel group mean ( 116 . 1 ± 9 . 5% ) was not significantly different from the Control mean .", "The difference between the mean varicosity numbers for the 5X5HT and 5X5HT-Chel groups was highly significant ( p < 0 . 001 ) .", "Asterisks , comparison between 5X5HT and Control groups; plus signs , comparison between 5X5HT and 5X5HT-Chel groups .", "Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 009 As before , the 5X5HT training produced a significant net increase in the number of presynaptic varicosities in the two trained groups at 24 hr ( Figure 4B ) .", "This net increase persisted in the 5X5HT group , but was reversed in 5X5HT-Chel group at 48 hr .", "Monitoring of the fate of individual varicosities in the 5X5HT group revealed the same pattern of structural growth and retraction observed previously in the 5X5HT-trained cocultures not subjected to reconsolidation blockade .", "Specifically , some varicosities induced by the 5X5HT training were lost between 24 and 48 hr , as were some of the original varicosities; this loss , however , was compensated for by the growth of new varicosities; consequently , the overall varicosity number remained stably elevated during the 24–48 hr period ( Figures 3C and 5 ) .", "The varicosities in the 5X5HT-Chel group exhibited significantly greater retraction and significantly less growth between 24 and 48 hr than those in the 5X5HT group , resulting in an overall loss of varicosities .", "Thus , the number of varicosities in the 5X5HT-Chel group at 48 hr was returned to the value at 0 hr .", "This result indicates that inhibiting PKM Apl III , like reconsolidation blockade ( Figure 1C ) , engages a homeostatic mechanism that resets the presynaptic varicosities in sensorimotor cocultures to the number present prior to training .", "Another similarity between the morphological effects of reconsolidation blockade and inhibition of PKM was that there was equal retraction of 5HT-induced and original varicosities in the 5X5HT-Chel cocultures during 24–48 hr , whereas , as before , the retraction of 5HT-induced varicosities was greater in the 5X5HT cocultures during this period ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 03896 . 010Figure 5 . Effect of inhibition of PKM on varicosity fate . For the 5HT-induced and original varicosities , the number of varicosities in each category for each coculture at 48 hr was normalized to the number in the same category in that coculture at 24 hr .", "For the varicosities formed >24 hr , the number of varicosities for each coculture at 48 hr was normalized to the total number of varicosities in the coculture at 24 hr . ( A ) The mean normalized decrease in the number of 5HT-induced varicosities at 48 hr was 53 . 4 ± 5 . 8% in the 5X5HT group and 68 . 0 ± 5 . 8% in the 5X5HT-Chel group ( p < 0 . 05 , one tailed t test ) .", "( B ) The mean normalized decrease in the number of original varicosities at 48 hr was 23 . 5 ± 4 . 7% in the 5X5HT group and 63 . 4 ± 9 . 3% in the 5X5HT-Chel group ( p < 0 . 001 , two-tailed t test ) .", "( C ) More presynaptic varicosities were formed during 24–48 hr in the 5X5HT group ( 63 . 9 ± 9 . 0% ) than in the 5X5HT-Chel group ( 43 . 2 ± 5 . 8% , p < 0 . 05 , one-tailed t test ) .", "Blue bars , 5X5HT group; red bars , 5X5HT-Chel group .", "Asterisks , comparison between 5X5HT and 5X5HT-Chel groups .", "Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 01010 . 7554/eLife . 03896 . 011Figure 5—figure supplement 1 . Comparison of retraction of original and 5HT-induced varicosities between 24–48 hr in the chelerythrine treatment experiments .", "( A ) The difference in the percent retraction between 5X5HT-induced ( 53 . 4 ± 5 . 8% ) and original ( 23 . 5 ± 4 . 7% ) varicosities was highly significant in the 5X5HT group ( p < 0 . 001 , two-tailed t test ) .", "( B ) There was no significant difference in the percent retraction between 5HT-induced ( 68 . 0 ± 5 . 8% ) and original ( 63 . 4 ± 9 . 3% ) varicosities in the 5X5HT-Chel group . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 011 Inspection of the synaptic structure of the Control cocultures revealed a complex underlying pattern of structural change similar to that observed in the reconsolidation experiment .", "There was significant growth and retraction of SN varicosities , both original and new , over the 48 hr of the experiments ( Figures 6 and 1B ) .", "This structural dynamism contrasted with the constancy of overall varicosity number in these cocultures ( Figures 1C and 4B ) , as well as with the stability of the sensorimotor EPSP amplitude observed in Control cocultures over similar time periods in our earlier studies ( Cai et al . , 2011 , 2012 ) . 10 . 7554/eLife . 03896 . 008Figure 6 . Changes in presynaptic varicosities in Control cocultures .", "( A ) The normalized increase in the number of varicosities was 54 . 9 ± 6 . 5% during the 0–24 hr period and 41 . 5 ± 7 . 2% during the 24–48 hr period ( green bars ) .", "( B ) The mean normalized number of original varicosities ( blue bar ) that retracted during the 0–24 hr period was 43 . 2 ± 4 . 4% .", "During the 24–48 hr period 15 . 5 ± 2 . 0% of the original varicosities ( those present at 0 hr , blue segment ) , and 30 . 2 ± 5 . 1% of the varicosities that formed during 0–24 hr period ( red segment ) , retracted; thus , the mean total normalized retraction of varicosities from 24–48 hr was 45 . 7% .", "Note that the numbers of newly formed varicosities during the 0–24 and 24–48 hr periods in each coculture were normalized to the total number of varicosities in that coculture measured at 0 hr .", "The percentage of retracted varicosities was obtained by normalizing the number of varicosities that disappeared during the 0–24 and 24–48 hr periods for a given coculture to the total number of varicosities in that coculture at 0 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 008 The present morphological results challenge the notion that the persistence of sensitization memory in Aplysia depends on the persistence of particular facilitated synapses .", "To further investigate this idea , we tested whether the LTM for behavioral sensitization can be reinstated in Aplysia following reconsolidation blockade and inhibition of PKM , two treatments previously shown to eliminate LTF , the synaptic basis of long-term sensitization ( Cai et al . , 2011 , 2012; Lee et al . , 2012; Hu and Schacher , 2014 ) .", "Long-term sensitization ( LTS ) of the siphon-withdrawal reflex ( SWR ) was induced in intact Aplysia using five bouts of tails shocks ( 5XTrained ) .", "Brief reminder training ( one bout of tail shocks , 1XTrained ) was applied at 48 hr after the original training to trigger reconsolidation of the LTM for sensitization ( Cai et al . , 2012 ) .", "Immediately following the reminder training an intrahemocoel injection of anisomycin was administered to some animals ( Figure 7A ) .", "As previously reported ( Cai et al . , 2012; Lee et al . , 2012 ) , this treatment eliminated LTS , assessed here at 72 hr ( Figure 7B ) .", "Afterwards , some of the animals received three additional bouts of tail shocks ( 3XTrained ) .", "Importantly , the three additional bouts of sensitization training did not induce LTM in naïve animals ( Control-Veh-3XTrained; Figure 7B ) .", "However , the three bouts of training completely restored LTM following its disruption by reconsolidation blockade ( Figure 7B ) . 10 . 7554/eLife . 03896 . 012Figure 7 . Reinstatement of LTS after its elimination by reconsolidation blockade in Aplysia .", "( A ) Experimental protocol .", "The timing of the pretests , training , posttests , and drug/vehicle injections is shown relative to the end of the last training session .", "The time of the intrahemocoel injection of either anisomycin or vehicle is indicated by the red arrow .", "Animals in the 5XTrained-1XTrained-Aniso and 5XTrained-1XTrained-Aniso-3XTrained groups received a reminder episode of sensitization training ( one bout of tail shocks ) immediately after the 48 hr posttest ( black bar ) and prior to the injection of anisomycin , whereas the animals in the 5XTrained-Aniso group did not .", "After the 72 hr posttest animals in the Control-Veh-3XTrained and 5XTrained-1X5HT-Aniso-3XTrained groups received truncated sensitization training ( three bouts of tail shocks ) .", "( B ) The mean duration of the SWR measured at 48 hr , 72 hr and 96 hr for the Control-Veh-3XTrained ( n = 12 ) , 5XTrained-Aniso ( n = 4 ) , 5XTrained-1XTrained-Aniso ( n = 7 ) , and 5XTrained-1XTrained-Aniso-3XTrained ( n = 11 ) groups .", "A repeated-measures ANOVA indicated that there was a significant group × time interaction ( F[9 , 90] = 51 . 0 , p < 0 . 0001 ) .", "Subsequent planned comparisons indicated that the overall differences among the four groups were highly significant on all of the posttests ( 48 hr , F[3 , 30] = 137 . 2 , p < 0 . 0001; 72 hr , F[3 , 30] = 50 . 5 , p < 0 . 0001; and 96 hr , F[3 , 30] = 43 . 8 , p < 0 . 0001 ) .", "SNK posthoc tests on the 48 hr data indicated that the initial sensitization training produced significant LTS in the 5XTrained-Aniso ( 53 . 0 ± 7 . 0 s ) , 5XTrained-1XTrained-Aniso ( 59 . 9 ± 0 . 2 s ) , and 5XTrained-1XTrained-Aniso-3XTrained ( 53 . 7 ± 2 . 9 s ) groups compared to the Control-Veh-3XTrained group ( 2 . 5 ± 0 . 9 s , p < 0 . 001 for each test ) .", "The responses of the trained groups did not differ significantly at 48 hr after sensitization training .", "However , the mean duration of the SWR in the 5XTrained-Aniso group ( 53 . 8 ± 6 . 3 s ) remained prolonged at 72 hr , and was significantly longer than that in the 5XTrained-1XTrained-Aniso group ( 6 . 4 ± 1 . 9 s ) as well as in the 5XTrained-1XTrained-Aniso-3XTrained group ( 4 . 8 ± 3 . 2 s , p < 0 . 001 for both comparisons ) .", "LTS was restored by the three additional tail shocks applied after the 72 hr posttest .", "The mean duration of the SWR in the 5XTrained-1XTrained-Aniso-3XTrained group at 96 hr was 56 . 7 ± 2 . 7 s , which was significantly longer than that for the 5XTrained-1XTrained-Aniso group ( 10 . 4 ± 3 . 9 s ) at 96 hr ( p < 0 . 001 ) .", "The SWR of the 5XTrained-Aniso group at 96 hr ( 53 . 8 ± 3 . 8 s ) was also significantly longer than that in the 5XTrained-1XTrained-Aniso group ( p < 0 . 001 ) .", "Asterisks , comparisons of the Control-Veh-3XTrained group with the 5XTrained-Aniso group , the 5XTrained-1XTrained-Aniso group , and 5XTrained-1XTrained-Aniso-3XTrained group at 48 hr; comparison of the Control-Veh-3XTrained group with the 5XTrained-Aniso group at 72 hr; and comparisons of the Control-Veh-3XTrained group with the 5XTrained-Aniso group and the 5XTrained-1XTrained-Aniso-3XTrained group at 96 hr .", "Plus signs , comparisons of the 5XTrained-1XTrained-Aniso group with the 5XTrained-Aniso group and the 5XTrained-1XTrained-Aniso-3XTrained group at 96 hr .", "Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 012 To test whether LTS could be reinstated after its apparent erasure by inhibition of PKM Apl III , animals were again initially trained using five bouts of tail shocks .", "24 hr after the original sensitization training the animals received an intrahemocoel injection of chelerythrine ( Figure 8A ) .", "Animals treated with chelerythrine exhibited no LTS at 48 hr ( Figure 8B ) , confirming our previous finding ( Cai et al . , 2011 ) .", "The animals that received the original sensitization training were then given three additional bouts of tail shocks ( 5XTrained-Chel-3XTrained group ) , as were control animals that had not received the original sensitization training ( Control-Veh-3XTrained group ) .", "The modest additional training reinstated LTS following its apparent erasure by chelerythrine , but did not produce LTS in the control animals ( Figure 8B ) . 10 . 7554/eLife . 03896 . 013Figure 8 . Reinstatement of LTS following its erasure by PKM inhibition .", "( A ) Experimental protocol .", "The timing of the pretests , training , posttests , and drug/vehicle injections is shown relative to the end of the last training session .", "The time of the intrahemocoel injection of either chelerythrine or vehicle is indicated by the red arrow .", "After the 48 hr posttest animals in both groups received three additional bouts of tail shocks .", "( B ) Mean duration of the SWR measured at 24 hr , 48 hr and 72 hr for the Control-Veh-3XTrained ( n = 9 ) and 5XTrained-Chel-3XTrained ( n = 9 ) groups .", "A repeated-measures ANOVA indicated that there was a significant interaction effect between group and time ( F[3 , 14] = 25 . 4 . p < 0 . 0001 ) .", "A planned comparison indicated that the mean duration of the SWR at 24 hr in the 5XTrained-Chel-3XTrained group ( 44 . 7 ± 5 . 6 s ) was significantly longer than that in the Control-Veh-3XTrained group ( 1 . 1 ± 0 . 1 s , F[1 , 16] = 60 . 2 , p < 0 . 001 ) .", "The mean duration of the SWR at 48 hr ( 4 . 1 ± 1 . 4 s ) in the 5XTrained-Chel-3XTrained group was not significantly longer than that in the Control-Veh-3XTrained group ( 2 . 6 ± 0 . 8 s ) , as indicated by a planned comparison ( F[1 , 16] = 0 . 95 , p = 0 . 34 ) .", "The SWR in the 5XTrained-Chel-3XTrained group at 72 hr was 48 . 7 ± 5 . 0 s , which was significantly longer than that in the Control-Veh-3XTrained group ( 4 . 6 ± 1 . 2 s; planned comparison , F[1 , 16] = 73 . 1 , p < 0 . 001 ) .", "Asterisks , comparison between 5XTrained-Chel-3XTrained and Control-Veh-3XTrained groups .", "Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 013 The above behavioral results indicate that LTM in Aplysia can persist despite elimination of its behavioral expression by reconsolidation blockade and inhibition of PKM Apl III .", "Moreover , because these two antimnemonic treatments also eliminate LTF ( Cai et al . , 2011 , 2012; Lee et al . , 2012; Hu and Schacher , 2014 ) ( and Figures 1C and 4B ) , the results imply that some component of LTM , perhaps a priming process , may persist in the absence of synaptic alterations .", "Possibly , LTM , or the primer for LTM , resides in the nuclei of neurons within the SWR circuitry , encoded as epigenetic changes ( Levenson and Sweatt , 2005; Rahn et al . , 2013; Graff and Tsai , 2013a ) .", "To investigate this possibility , we tested whether chelerythrine's apparent disruption of LTM involves alterations of chromatin structure .", "For this test we used the histone deacetylase ( HDAC ) inhibitor trichostatin A ( TSA ) ( Graff and Tsai , 2013b ) .", "Aplysia were given five bouts of tail shocks , and the strength of the SWR was tested 24 hr later ( Figure 9A ) .", "Immediately after this test the sensitization-trained animals received an intrahemocoel injection of the vehicle solution alone or TSA; 10–15 min later some of the animals treated with TSA were given an injection of chelerythrine ( 5XTrained-TSA-Chel group ) .", "( Untrained control animals received an injection of the vehicle solution after the 24 hr test . )", "In addition , another group of trained animals were given the chelerythrine without a prior injection of TSA ( 5XTrained-Chel group ) .", "All animals were retested at 48 hr .", "TSA treatment blocked the disruption of LTS by chelerythrine ( Figure 9B ) .", "The injection of TSA 24 hr after training by itself did not alter LTS ( 5XTrained-TSA group ) .", "These results indicate that one of chelerythrine's antimnemonic actions is to reverse histone acetylation induced by LTS training . 10 . 7554/eLife . 03896 . 014Figure 9 . Epigenetic regulation of LTM in Aplysia .", "( A ) Experimental protocol .", "The times of the pretests , training , posttests , and drug/vehicle injections are shown relative to the end of the last training session .", "The times of the trichostatin A ( TSA ) /vehicle and chelerythrine/vehicle injections are indicated by blue arrow and red arrow respectively .", "( B ) TSA treatment blocks erasure of LTS by chelerythrine .", "The graph presents the mean duration of the SWR measured at 24 hr and 48 hr in the Control-Veh ( n = 9 ) , 5XTrained-Veh ( n = 5 ) , 5XTrained-TSA ( n = 6 ) , 5XTrained-Chel ( n = 5 ) , and 5XTrained-TSA-Chel ( n = 6 ) groups .", "A repeated-measures ANOVA showed a significant group × time interaction ( F ( 8 , 52 ) = 53 . 2 , p < 0 . 0001 ) .", "Subsequent planned comparisons indicated that the group differences for the 24 and 48 hr posttests were highly significant ( 24 hr , F[4 , 26] = 45 . 5 , p < 0 . 0001; 48 hr , F[4 , 26] = 94 . 4 , p < 0 . 0001 ) .", "For the 24 hr posttest , SNK posthoc tests indicated that the training produced significant sensitization in all four trained groups ( 5XTrained-Veh , 54 . 0 ± 6 . 0 s; 5XTrained-TSA , 49 . 0 ± 7 . 6 s; 5XTrained-Chel , 59 . 6 ± 0 . 4 s; and 5XTrained-TSA-Chel , 57 . 7 ± 2 . 3 s ) compared to the Control-Veh group ( 1 . 6 ± 0 . 6 s , p < 0 . 001 for all tests ) .", "Comparisons of the four trained groups showed that their responses did not differ significantly on the 24 hr posttest .", "However , the responses of 5XTrained-Veh ( 49 . 0 ± 6 . 9 s ) , 5XTrained-TSA ( 56 . 0 ± 4 . 0 s ) , and 5XTrained-TSA-Chel ( 59 . 8 ± 1 . 7 s ) on the 48 hr posttest were significantly more enhanced than those for both the 5XTrained-Chel ( 2 . 8 ± 1 . 8 s ) and Control-Veh ( 1 . 7 ± 0 . 5 s ) groups ( p < 0 . 001 for all tests ) .", "Thus , chelerythrine treatment reversed LTS , and TSA treatment blocked chelerythrine's reversal of LTS .", "There were no significant differences among 5XTrained-Veh , 5XTrained-TSA and 5XTrained-TSA-Chel groups , nor between 5XTrained-Chel and Control-Veh groups , at 48 hr .", "Asterisks , comparisons of the Control-Veh group with the 5XTrained-Veh group , the 5XTrained-TSA group , the 5XTrained-Chel group , and the 5XTrained-TSA-Chel group at 24 hr; and of the Control-Veh group with the 5XTrained-Veh group , the 5XTrained-TSA group , and the 5XTrained-TSA-Chel group at 48 hr .", "Plus signs , comparisons of the 5XTrained-Chel group with the 5XTrained-Veh group , the 5XTrained-TSA group , and the 5XTrained-TSA-Chel group at 48 hr . ( C ) Experimental protocols .", "The times of the pretests , training , posttests , and drug/vehicle injections are shown relative to the end of the last training session .", "The intrahemocoel injection of either TSA or vehicle is indicated by the red arrow .", "Note that TSA was injected into animals prior to the sensitization training .", "( D ) Inhibiting histone deacetylation facilitated the induction of LTS in Aplysia .", "The mean duration of the SWR in the 3XTrained-TSA group ( n = 4 ) at 24 hr was 59 . 8 ± 0 . 3 s , whereas it was only 2 . 0 ± 1 . 0 s in the 3XTrained-Veh group ( n = 4; p < 0 . 001 , unpaired t test ) .", "Asterisks , comparison between 3XTrained-TSA and 3XTrained-Veh groups; Error bars represent ±SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03896 . 014 Previous work has shown that LTF of in vitro Aplysia sensorimotor connections depends on alterations of chromatin structure .", "In particular , 1X5HT , which normally induces only short-term facilitation , was found to induce LTF when applied with TSA ( Guan et al . , 2002 ) .", "In intact Aplysia three bouts of tail shocks are insufficient by themselves to induce LTS ( Control-Veh-3XTrained data , Figures 7 and 8 ) .", "We tested whether three bouts of shocks could induce LTS if delivered in the presence of an HDAC inhibitor .", "Animals were given three bouts of shocks 15 min after an intrahemocoel injection of either vehicle solution or TSA ( Figure 9C ) .", "24 hr later the animals that received three bouts of shocks after an injection of TSA exhibited LTS , whereas animals that received the shocks after an injection of vehicle did not ( Figure 9D ) .", "Thus , HDAC inhibition facilitates the induction of LTM in Aplysia ." ], [ "The present morphological results , together with those of our previous behavioral and electrophysiological investigations ( Cai et al . , 2011 , 2012 ) , suggest that the persistence of sensitization-related LTM in Aplysia does not require the persistence of the synaptic connections generated during learning .", "Rather , LTM appears to be regulated by a homeostatic mechanism that specifies the net synaptic strength according to experience .", "Following 5X5HT stimulation , we observed that the number of presynaptic varicosities in the sensorimotor cocultures increased to a new overall value , and that this value was maintained despite the appearance and disappearance of individual varicosities .", "Furthermore , blockade of memory reconsolidation , or inhibition of PKM , reset the number of varicosities to the original , non-facilitated value .", "Remarkably , this resetting did not result from the straightforward retraction of the varicosities that appeared by 24 hr after 5X5HT stimulation , varicosities whose growth coincides with LTF ( Glanzman et al . , 1990 ) ; rather , the resetting was produced by a complex reorganization that involved retraction of both 5HT-induced and original varicosities , as well as growth of new , additional varicosities .", "Thus , for any given sensorimotor connection , the homeostatic regulatory mechanism appeared indifferent to the identities of the particular presynaptic varicosities and , presumably , active zones ( Schacher et al . , 1990 ) , that mediated baseline synaptic transmission and synaptic facilitation .", "Our data argue that synapses are not ‘tagged’ with respect to memory storage , at least for a connection involving a single SN and a single MN; the data may therefore reflect a fundamental distinction between the cellular and molecular processes of LTM storage and those of LTM induction in Aplysia , in which synaptic tagging plays a critical role ( Martin et al . , 1997 ) .", "The data also suggest that synaptic change is an expression mechanism , rather than a storage mechanism , for LTM in Aplysia ( although see below ) .", "Importantly , neither reconsolidation blockade nor inhibition of PKM caused the varicosity number to drop below the starting value .", "( Notice that the same is true for the amplitude of the EPSP in our earlier synaptic physiological studies , which used the same training regimens [Cai et al . , 2011 , 2012] . )", "This result provides additional evidence for the operation of a homeostatic regulatory mechanism , and suggests that the homeostatic mechanism toggles between two all-or-none states , a facilitated , sensitization-related state and a nonfacilitated one .", "( There must also be a third state , synaptic depression , in which the strength of the sensorimotor connection is reduced below the nonfacilitated level [Montarolo et al . , 1988] . )", "Our results are reminiscent of those from studies in the Drosophila neuromuscular junction and the mammalian CNS , which also indicate that synaptic plasticity is regulated by homeostatic mechanisms ( Turrigiano , 2007 ) .", "The present conclusions appear to conflict with those from morphological investigations of LTM in mice in which dendrites have been chronically imaged in the intact , living brain .", "Several such studies have reported stability of some dendritic spines for periods of weeks-to-months after training ( Xu et al . , 2009; Yang et al . , 2009; Liston et al . , 2013 ) .", "These results , as well as evidence that spines in the adult mammalian cortex exhibit little turnover ( Grutzendler et al . , 2002 ) , have led some to argue that stable spines provide a physical basis for the lifelong maintenance of memories ( Bhatt et al . , 2009 ) .", "But other studies of the mouse brain using very similar in vivo imaging techniques have reported a high degree of spine instability in the adult cerebral cortex ( Trachtenberg et al . , 2002; Holtmaat et al . , 2005 ) ; the contradiction between these two sets of opposed findings remains unresolved .", "Furthermore , even in those studies where spines of adult cortical neurons were reported to be highly stable , there was significant instability of both pre-existing and new spines within the first 2–4 days after a training protocol ( Xu et al . , 2009; Yang et al . , 2009 ) , and we monitored SN varicosities for only 48 hr after training with 5HT .", "Therefore , the present results are not necessarily inconsistent with those from in vivo imaging studies in the mouse brain .", "We found that LTS could be fully reinstated following its disruption by the same antimnemonic treatments previously shown to eliminate LTF ( Cai et al . , 2011 , 2012; Lee et al . , 2012; Hu and Schacher , 2014 ) ( Figures 7 and 8 ) .", "This finding is consistent with two possible explanations .", "First , the LTM for sensitization may be intact in animals following reconsolidation blockade and inhibition of PKM , despite the elimination of both behavioral enhancement and synaptic growth .", "According to this scheme , synapses serve merely to express LTM , they are not sites of LTM storage .", "The reinstatement of LTM expression , then , involves restoring the appropriate number of synapses between the SNs and MNs , as determined by an experience-registering homeostatic mechanism .", "The second possible explanation incorporates the notion of an as-yet-unidentified priming mechanism; here , reconsolidation blockade and inhibition of PKM , can erase the stored sensitization memory through the reversal of pre- and postsynaptic structures induced by the long-term training , but the antimnemonic treatments do not eliminate the primer .", "The primer does not constitute LTM , but is required for its reconstitution via new synaptic growth .", "The priming signal might interact with fresh facilitatory input to the withdrawal circuit , due to the additional ( 3× ) tail shocks , to upregulate the number of synaptic contacts; the appropriate number of contacts could be determined by the homeostatic process , and involve signals from existing and new synapses .", "In a previous study we attempted to reinstate LTS following its disruption by inhibition of PKM Apl III but failed ( Cai et al . , 2011 ) ( see Figure 3B of the earlier study ) .", "Interestingly , we used only a single bout of tail shocks in that attempt .", "In light of the present results ( Figures 7 and 8 ) , our previous failure indicates that one bout of tails shocks is insufficient to fully recruit the signaling pathways that reinstate LTS ( Abel et al . , 1998; Sossin , 2008; Mayford et al . , 2012; Zhang et al . , 2012 ) .", "In future work we will seek to discover how the molecular signals evoked by three bouts of tail shocks differ from those evoked by a single bout .", "The data from our behavioral experiments involving the use of an HDAC inhibitor ( Figure 9 ) implicate epigenetic modifications ( Levenson and Sweatt 2005; Rahn et al . , 2013; Graff and Tsai 2013a ) —either in the SN or MN or , most likely , both—in the storage mechanism for LTM in Aplysia .", "Data from an in vitro study of LTF have also implicated histone acetylation in LTM in Aplysia ( Guan et al . , 2002 ) .", "How might HDAC inhibition block chelerythrine's apparent disruption of LTM ?", "Possibly , TSA , by inhibiting histone deacetylase , activates the transcription of genes that promote LTM; the activation of these genes , in turn , may compensate for a disruptive action of chelerythrine on LTM .", "Findings by others are consistent with this idea .", "Thus , treatment with an HDAC inhibitor rescues cognitive deficits and learning-related synaptic plasticity in mice that are heterozygous for a mutant form of CREB binding protein ( CBP ) ( Alarcon et al . , 2004 ) .", "In addition , it has been found that phosphorylation of CBP by the atypical PKCζ is required for the histone acetylation that promotes the differentiation of developing neural precursors into neurons , astrocytes , and oligodendrocytes; mutant mice haploinsufficient for CBP exhibit abnormal development of the cerebral cortex ( Wang et al . , 2010 ) .", "Furthermore , chelerythrine , by inhibiting the phosphorylation of CBP by PKCζ , blocks the differentiation of cultured precursor cells into astrocytes , and oligodendrocytes ( Wang et al . , 2010 ) .", "A recent study in Aplysia has found that reducing the activity of CBP in SNs through intracellular injection of CBP small interfering RNA ( siRNA ) impaired the induction of LTF in sensorimotor cocultures ( Liu et al . , 2013 ) .", "Interestingly , this deficit in LTF induction could be rescued through the use of a 5HT training protocol designed to maximize the phosphorylation and synthesis of CCAAT/enhancer-binding protein ( C/EBP ) .", "Taken together , these studies suggest that chelerythrine can disrupt epigenetic processes , possibly involving histone acetylation stimulated by CBP , that promote the induction and maintenance of LTM .", "Moreover , because HDAC inhibition can block chelerythrine's deleterious effects on memory , as well as promote the induction of LTM , it is intriguing to speculate that these effects might be due to enhancement of a CBP-dependent pathway .", "But the above explanation for why TSA blocks chelerythrine's effect on consolidated LTM raises a critical question .", "Our data suggest that consolidated LTM persists after chelerythrine treatment ( as well as reconsolidation blockade ) , possibly as nuclear changes .", "If so , then the molecular basis for the persistence of LTM is unlikely to be histone acetylation , because an apparent action of chelerythrine is to reverse histone acetylation .", "Another prominent epigenetic mechanism known to play a role in LTM is DNA methylation ( Day and Sweatt , 2010 ) .", "Although early evidence indicated that learning-induced DNA methylation in the hippocampus was transient and readily reversible ( Miller and Sweatt , 2007 ) , a more recent study has reported that contextual fear conditioning in rats induces DNA methylation of the gene for calcineurin in cortical neurons that persists for at least a month ( Miller et al . , 2010 ) .", "Furthermore , Sweatt and colleagues have shown in a rat model of childhood maltreatment that early trauma can produce changes in the DNA methylation of the gene for brain-derived neurotrophic factor ( BDNF ) in the cortex , changes that persist into adulthood ( Roth et al . , 2009 ) .", "Thus , DNA methylation may constitute an epigenetic mechanism for the lifelong storage of memory ( Day and Sweatt , 2010 ) .", "Interestingly , 5HT has recently been reported to induce DNA methylation of the promoter of the transcriptional repressor of memory CREB2 , thereby facilitating the induction of LTF ( Rajasethupathy et al . , 2012 ) .", "Finally , another potential candidate for a nuclear mechanism of LTM storage is suggested by a recent study by Suberbielle et al . ( 2013 ) that found , remarkably , that learning causes DNA double-strand breaks ( DSBs ) in neurons in the brains of control mice; thus , chromatin remodeling subsequent to DNA DSBs may also encode LTM .", "Could a nonsynaptic storage mechanism based on nuclear changes mediate the maintenance of associative memories , particularly those induced in complex neural circuits in the mammalian brain , where a given neuron may have 1000s or 10s of 1000s of synaptic partners ?", "An obvious difficulty confronting any hypothetical nuclear storage mechanism in the mammalian brain is how the appropriate number of connections can be maintained in a synapse-specific manner after learning has occurred .", "For example , if a synaptic contact that has undergone Hebbian long-term potentiation ( Bliss and Collingridge , 1993 ) as a consequence of associative learning retracts , how could a nuclear storage mechanism restrict the growth of a replacement contact to the correct pair of pre- and postsynaptic neurons ?", "Possibly , there are nonsynaptic ways for neurons to communicate that ensure specificity of associative synaptic plasticity in the face of the significant lability of synaptic structure documented here .", "There are , of course , somal , non-nuclear , mechanisms of memory storage or priming that could account for the apparent persistence of memory following synaptic erasure .", "One such mechanism would be the persistent activity of a kinase ( Hegde et al . , 1993; Bougie et al . , 2012 ) , or persistent inhibition of a phosphatase ( Sharma et al . , 2003 ) , in the cell body of the SN or MN or in both cell bodies .", "Another somal mechanism consistent with our data is ubiquitination of one or more somal proteins ( Hegde et al . , 1997; Chain et al . , 1999 ) ; up-regulation of a ubiquitin-proteasome pathway could degrade proteins that inhibit the storage of LTM , or that block the priming of LTM .", "The present data necessitate a reappraisal of the mnemonic consequences of blockade of memory reconsolidation and inhibition of PKM .", "It has been previously argued that these manipulations can erase consolidated memory ( Nader and Hardt , 2009; Sacktor , 2011; Glanzman , 2013 ) ( although see Lattal and Abel , 2004 ) .", "But our results indicate that the effect of reconsolidation blockade and PKM inhibition is not to delete LTM but , rather , to impair its expression .", "In other words , the antimnemonic actions of these manipulations may result from their ability to at least partially reverse the cellular and molecular changes , including synaptic growth , that mediate the expression , rather than the storage , of LTM .", "Possibly , neither reconsolidation blockade nor PKM inhibition can reverse the nuclear remodeling—involving epigenetic modifications , particularly DNA methylation ( Day and Sweatt , 2010; Graff et al . , 2011; Rahn et al . , 2013 ) , as well as , perhaps , chromatin remodeling subsequent to DNA DSBs ( Suberbielle et al . , 2013 ) —that represents the stored memory trace .", "Alternatively , these antimnemonic manipulations might indeed disrupt the stored memory trace , but leave intact some memory-priming signal .", "Likely candidates for a residual priming signal include the persistent repression of transcription of the CREB repressor , CREB2 ( Bartsch et al . , 1995; Upadhya et al . , 2004; Liu et al . , 2011 ) , and persistent degradation of the regulatory subunit of protein kinase A ( PKA ) ( Chain et al . , 1999 ) .", "Because chelerythrine's disruption of LTM in Aplysia is blocked by TSA , chelerythrine's antimnemonic actions must involve histone deacetylation .", "Previous speculation regarding how chelerythrine and the zeta inhibitory peptide ( ZIP ) disrupt memory maintenance in both Aplysia and rats has focused on the ability of these pharmacological agents to inhibit the phosphorylation of proteins by atypical PKM ( Cai et al . , 2011; Sacktor , 2011 ) ( Box 1 ) .", "The present results imply that chelerythrine and ZIP may interfere with epigenetic processes induced by atypical PKM , as well as inhibit protein phosphorylation by atypical PKM .", "A prior study reported that , following inhibition of PKMζ activity in the insular cortex of rats by the peptide inhibitor ZIP , conditioned taste aversion could not be reinstated by an application of the unconditioned stimulus ( UCS , intraperitoneal injection of lithium chloride ) ( Shema et al . , 2007 ) .", "Our experience ( Cai et al . , 2011 , and the present results ) suggests that the failure to reinstate conditioned taste aversion in this study was not because LTM had been extinguished , but because a single application of the UCS was too weak to reverse the disruptive effects of ZIP on mechanisms of LTM expression , some of which may involve chromatin remodeling ( Figure 7A , B ) .", "In summary , we have found that LTM , or a primer for LTM , can persist following reconsolidation blockade and inhibition of PKM .", "Furthermore , the residual memory/primer must be independent of synaptic plasticity , because it persists following elimination of the synaptic changes induced during learning .", "Our results indicate that consolidated memories may be far more refractory to modification or elimination than generally supposed .", "If confirmed and extended to mammals , the present results would have important implications for treating disorders of LTM , such as posttraumatic stress disorder ( PTSD ) ." ], [ "The sensorimotor cocultures consisted of one pleural SN and one small siphon ( LFS-type ) MN .", "Adult abdominal ganglia and pleural ganglia were removed from 60–100 g Aplysia and then incubated in protease ( 10 mg/ml Dispase II [Roche Applied Science , Indianapolis , IN] in Leibowitz-15 [L-15 , Sigma , St Louis , MO] ) for 2 hr at 35°C before desheathing .", "The appropriate amounts of salts were added to the L15 to yield the following concentrations in mM: 400 NaCl , 11 CaCl2 , 10 KCl , 27 MgSO4 , 27 MgCl2 , 2 NaHCO3 .", "Additionally , the L15 was supplemented with penicillin ( 50 unit/ml ) , streptomycin ( 50 µg/ml ) , dextrose ( 6 mg/ml ) and glutamine ( 0 . 1 mg/ml ) .", "After desheathing , SNs and MNs were individually dissociated from ganglia and paired in cell culture .", "The culture medium contained 50% Aplysia hemolymph and 50% L-15 .", "The cultures were maintained at 18°C in an incubator for 3 day before the start of the experiments .", "The SNs and MNs were labeled with the intracellular dyes dextran fluorescein and dextran rhodamine B ( Molecular Probes , Eugene , OR ) , respectively , on Day 3/4 in culture .", "The dyes were dissolved in 0 . 2 M KCl with 0 . 25% fast green ( final concentration of 10 mg/ml ) and then microinjected into cells via brief pressure pulses ( 6–12 MΩ resistance electrodes , 10–20 psi for 2–5 pulses [40 ms] ) .", "The fluorescent images of the labeled cocultures were acquired with a LSM Pascal ( Zeiss , Thornwood , NY ) confocal microscope during three imaging sessions: immediately prior to , and 24 hr and 48 hr after , 5X5HT training ( or at the equivalent times for Control cocultures ) .", "The images were taken using a 20× , 0 . 5 NA objective .", "The total number of varicosities was counted for each sensory neuron from the confocal images .", "All image analyses were performed using Axiovision 4 . 8 . 2 ( Zeiss , Thornwood , NY ) .", "The counter was blind to the experimental conditions of the imaged cocultures .", "SN varicosities that were in clear contact with , or overlapping , postsynaptic structures ( either the MN soma , major neurite , or fine processes ) were counted .", "The majority of the SN varicosities contacted either the MN soma or its initial segment .", "Only those varicosities having a punctate shape—those in which an oval-shaped body could be distinguished by the narrowing of the neurite on either side—and a measured area of 10 µm2 or greater were counted .", "If a large fluorescent varicosity comprised several visible punctate varicosities in the image , those small varicosities were counted individually; if not , the structure was treated as a single varicosity .", "Three categories of varicosities are tracked and quantified: original varicosities , that is the varicosities present at 0 hr; varicosities formed during 0–24 hr; and varicosities formed during 24–48 hr .", "Immediately after the first imaging session , some of the cocultures were given 5HT training .", "5HT was prepared fresh daily as a 10 mM stock solution in artificial sea water ( ASW ) and then diluted to the final concentration of 100 µM in the perfusion medium immediately before the first application .", "The perfusion solution contained 50% L15 and 50% ASW .", "The 5HT training consisted of five 5 min pulses of 5HT .", "After each 5 min pulse , the 5HT was rapidly washed out with normal perfusion medium for 15 min .", "The Control cocultures were treated with the perfusion solution alone .", "Following 5HT or control treatment , the perfusion medium was replaced with culture medium and the cocultures were returned to the 18°C incubator .", "A stock solution of anisomycin ( Sigma , St Louis , MO ) was made by dissolving the protein synthesis inhibitor in dimethyl sulfoxide ( DMSO ) to a concentration of 40 mM for use in the reconsolidation experiments .", "After the second imaging session ( 24 hr after 5HT training or the equivalent time in Control experiments ) the anisomycin stock solution was diluted to a concentration of 10 µM in perfusion solution and applied to cocultures for 2 hr .", "Immediately prior to the anisomycin treatment , some cocultures were given one 5-min pulse of 5HT ( 100 µM ) as the reminder stimulus .", "The 5HT-containing perfusion medium was washed out with normal perfusion medium in the coclutures that received the reminder .", "To inhibit PKM Apl III chelerythrine ( EMD Biosciences , San Diego , CA ) was dissolved in dH2O to a concentration of 10 mM to make a stock solution .", "After the second imaging session 24 hr after 5HT training , the chelerythrine stock solution was diluted to a concentration of 10 µM in perfusion solution and applied to cocultures for 1 hr .", "Following the anisomycin or chelerythrine treatment , the drug was rapidly washed out of the coculture dishes with normal perfusion medium; afterwards the perfusion medium was replaced with culture medium and the cocultures were returned to the incubator .", "Adult A . californica ( 80–120 g ) were obtained from a local supplier ( Alacrity Marine Biological , Redondo Beach , CA , USA ) .", "Animals were housed in a 50 gal aquarium filled with cooled ( 12–14°C ) , aerated seawater ( Catalina Water Company , Long Beach , CA , USA ) .", "The behavioral training and testing methods were similar to those previously described ( Fulton et al . , 2008; Cai et al . , 2011 , 2012 ) .", "Three pretests were performed at once per 10 min , beginning 25 min before the start of training .", "Full sensitization training consisted of five bouts of electrical shocks delivered to the tail at 20-min intervals .", "During each bout , the animal received three trains of shocks spaced 2 s apart .", "Each train was 1 s in duration; the shocks ( 10-ms pulse duration , 40 Hz , 120 V ) were delivered via a Grass stimulator ( S88 , Astro-Med , West Warwick , RI ) connected to platinum wires implanted in the tail .", "After training the animals were given posttests as indicated in the figures .", "A stock solution of 40 mM anisomycin or 10 mM chelerythrine was prepared as for the cell imaging experiments ( above ) .", "Anisomycin was then diluted in ASW to a concentration of 20 mM ( 50% DMSO ) .", "200 μl per 100 g of body weight of anisomycin was injected into the animals .", "Injections of the same amount of vehicle solution ( DMSO in ASW ) were made in Control experiments .", "The final concentrations of anisomycin in the animal were approximately 40 μM .", "The final concentration of DMSO in the hemocoel was ∼0 . 1% .", "A volume of 200 μl per 100 g of body weight of chelerythrine was injected into the animals .", "Trichostatin A ( TSA ) ( Sigma , St Louis , MO ) was dissolved in DMSO to a concentration of 10 mM to make a stock solution .", "To inhibit the histone deacetylase , a volume of 100 μl per 100 g of body weight of TSA was injected into the animals .", "The specific times at which the intrahemocoel injections were made are indicated in the relevant figures .", "All statistical tests were performed using SPSS 22 . 0 ( IBM , Armonk , NY ) .", "Parametric tests were used for all statistical analyses .", "For each coculture the number of varicosities counted at 24 hr and 48 hr after 5HT training were normalized as described in the figure legends .", "The normalized data were expressed as means ±SEM .", "For the analysis of the data involving monitoring of synapses/behavior over time , the overall data were first assessed with repeated-measures two-way analyses of variance ( ANOVA ) .", "If the repeated-measures ANOVA indicated a significant interaction , one-way ANOVAs were performed on the separate test times , followed by Student-Newman-Keuls ( SNK ) posthoc tests for pairwise comparisons .", "An unpaired Student's t-test was used to determine the statistical significance of the differences when there were only two groups in the data set ( data in Figures 2 , 5 , 6 and 9D ) .", "All reported levels of significance represent two-tailed values unless otherwise indicated ." ] ]
[ "Long-term memory ( LTM ) is believed to be stored in the brain as changes in synaptic connections .", "Here , we show that LTM storage and synaptic change can be dissociated .", "Cocultures of Aplysia sensory and motor neurons were trained with spaced pulses of serotonin , which induces long-term facilitation .", "Serotonin ( 5HT ) triggered growth of new presynaptic varicosities , a synaptic mechanism of long-term sensitization .", "Following 5HT training , two antimnemonic treatments—reconsolidation blockade and inhibition of PKM—caused the number of presynaptic varicosities to revert to the original , pretraining value .", "Surprisingly , the final synaptic structure was not achieved by targeted retraction of the 5HT-induced varicosities but , rather , by an apparently arbitrary retraction of both 5HT-induced and original synapses .", "In addition , we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the same antimnemonic treatments that erase learning-related synaptic growth .", "These results challenge the idea that stable synapses store long-term memories ." ]
[ "Cells called neurons allow information to travel quickly around the body so that we can rapidly respond to any changes that we sense in our environment .", "This includes non-conscious reactions , such as the knee-jerk reflex in humans .", "Reflexes and other behaviors can be influenced by long-term memory , and it is thought that long-term memory is stored by changes in the synapses that connect neurons to each other .", "The reflexes of a sea slug known as Aplysia are often used to study memory because it has a simple nervous system in which individual sensory neurons ( which detect changes ) only form synapses with single motor neurons ( which control muscles ) .", "Chen et al . have now studied whether long-term memory is actually stored in these synapses .", "Sensory neurons and motor neurons removed from Aplysia were grown together in Petri dishes and allowed to form synapses .", "Next , the cells were treated with the hormone serotonin , which promotes long-term memory by , in part , causing the neurons to grow more synapses .", "Afterwards , the cells were given treatments that disrupted long-term memory and also reversed the synaptic growth caused by serotonin .", "However , it was not only new synapses that retracted: some synapses that had existed before the serotonin treatment were also lost .", "This apparently random loss of synapses suggests that the memory was not stored in specific synapses .", "Moreover , long-term memory could be restored after these treatments , which supports that idea that memory does not depend on synapses between the neurons being maintained .", "This work offers hope that it might be possible to develop treatments that help to restore long-term memory in people suffering from Alzheimer's disease and other conditions that affect long-term memory ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Kinesin-1 regulates dendrite microtubule polarity in Caenorhabditis elegans
elife-00133-v1
[ [ "Neurons are highly polarized cells that usually elaborate two sets of morphologically and functionally distinct processes: a single long axon that sends out information to its targets , and multiple shorter dendrites that receive synaptic input from the environment or other neurons .", "Among the many differences between axons and dendrites , one distinct feature is that they have different microtubule ( MT ) organization .", "MTs , composed of strands of tubulin polymers , are dynamic structures with two molecularly and functionally distinct ends: a plus-end that favors polymerization and supports net growth and a minus-end that favors depolymerization ( Howard and Hyman , 2003 ) .", "Since MTs in neurites provide the platform for intracellular transport , and molecular motors such as kinesins and dyneins migrate toward either the plus- or minus-ends , MT polarity might play an important role in determining the directionality of transport events .", "One way to measure MT polarity in vivo is to perform time-lapse imaging studies using the EB family members of the TIPs ( Tip Interacting Proteins ) , which bind transiently to the growing plus-ends of MTs ( Baas and Lin , 2011 ) .", "Based on the behavior of EB1 in neurites , it was cohesively concluded that MTs are uniformly polarized with their plus-ends oriented toward the distal tip in axons from worm to rodents .", "In the dendrites , MTs are mixed in the proximal region and uniformly plus-end-out in distal parts in cultured vertebrate neurons , whereas MTs exhibit a mixed orientation with minus-ends predominantly facing the distal dendrite in worm and fly ( Baas et al . , 1987; Baas et al . , 1988; Stepanova et al . , 2003; Rolls et al . , 2007; Stone et al . , 2008; Kollins et al . , 2009; Kapitein and Hoogenraad , 2011; Maniar et al . , 2012 ) .", "Unlike in nonneuronal cells , where most MTs extend out from the microtubule organization center ( MTOC ) that harbors the minus-ends , the majority of neuronal MTs have ‘free-floating' ends , raising the question of whether these microtubules are locally polymerized or transported from the cell body ( Baas and Lin , 2011 ) .", "Based on direct observation of short MT strand movement in cultured neurons , Baas and colleagues proposed a model in which the MT-based motors dynein and several mitotic kinesins directly slide MT strands into the axon and dendrite ( Baas , 1999 ) .", "More recently , Jan and colleagues showed that dynein was required for the uniform plus-end-out MT organization in Drosophila sensory neuron axons ( Zheng et al . , 2008 ) .", "In dynein mutants , about 30% of the axonal MTs exhibit a minus-end-out orientation , whereas the dendritic MT polarity remains intact .", "Another recent study showed that the axon-localized MT-binding protein UNC-33/CRMP plays an important role in orienting dynamic MTs in both axons and dendrites in Caenorhabditis elegans neurons ( Maniar et al . , 2012 ) .", "In unc-33 mutants , both the axon and dendrite exhibit polarity defects .", "However , how predominant minus-end-out MT polarity is established in the dendrite remains largely unknown .", "The signature pattern of MT polarity provides structural characteristics for axons and dendrites .", "In addition , the polarity pattern of MTs likely instructs the transport of motor-based polarized cargo and distribution of asymmetrical cellular material in the neuron .", "Thus , MT polarity might play an instrumental role in the establishment of neuronal polarity ( Baas , 2002; Hoogenraad and Bradke , 2009 ) .", "Here , we show that kinesin-1 is critical in generating the characteristic minus-end-out MT organization of the dendrite in vivo .", "Kinesin-1 ( previously called kinesin heavy chain or KHC ) is a plus-end-directed motor and has been reported to transport various cargoes including mitochondria , synaptic vesicles , and mRNAs ( Vale , 2003; Hirokawa and Takemura , 2005 ) .", "Like other kinesin family members , kinesin-1 contains an N-terminal motor domain , a coiled-coil region for dimerization , and a C-terminal tail domain for cargo binding and regulation ( Vale , 2003; Adio et al . , 2006 ) .", "It has been showed that the kinesin-1 tail domain directly binds to MTs and mediates MT sliding in vitro ( Navone et al . , 1992; Andrews et al . , 1993; Jolly et al . , 2010; Seeger and Rice , 2010 ) .", "In unc-116 ( kinesin-1/kinesin heavy chain ) mutants , we find that dendritic MT polarity is completely reversed and adopts an axonal-like plus-end-out organization .", "The consequences of this polarity reversal in the dendrite include ectopic accumulation of synaptic vesicles ( SVs ) and active zone proteins , and loss of dendritic enrichment of dendritic proteins .", "These results demonstrate that the proper polarized MT organization is essential for conferring neurite identity because it specifies the compartmental distribution of vital axonal and dendritic constituents such as SVs and neurotransmitter receptors in vivo .", "We also provide evidence that kinesin-1 is able to cross-link anti-parallel MTs in vitro .", "Combined with the structure–function analyses , we propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain ." ], [ "In order to understand the molecular mechanisms and physiological importance of differential neuronal MT organization , we studied MT organization in two bipolar neurons in C . elegans .", "The ventrally localized DA9 cell body elaborates two functionally distinct processes .", "An axon migrates to the dorsal nerve cord , where it extends anteriorly and forms presynaptic specializations , and a dendrite extends anteriorly along the ventral nerve cord ( Figure 1A; White et al . , 1976; Sym et al . , 1999; Klassen and Shen , 2007 ) .", "MT motors fused to a fluorescence tag have previously been used as indicators for MT polarity ( Clark et al . , 1997; Stone et al . , 2008 ) .", "To examine MT polarity in the DA9 neuron , we expressed fluorescent proteins fused with retrograde and anterograde motors , which should accumulate at the minus- and plus-ends of MTs , respectively .", "We found that YFP-tagged UNC-104/Kinesin3 exhibits dim diffuse fluorescence along the axon with dramatic accumulation at the distal tip , but is completely absent from the dendrite ( Figure 1B ) .", "Conversely , DHC-1/dynein heavy chain fused with GFP shows weak staining along the dendrite with accumulation at the distal tip of the dendrite ( Figure 1C ) .", "In addition , GFP fused to the slow Ncd family minus-end kinesin , KLP-16 , exhibits a graded fluorescence signal from proximal to distal dendrite with accumulation at the distal tip of the dendrite ( Figure 1D ) .", "The specific localization of the tagged KLP-16 motor is dependent on its motor activity , as motor-dead versions are largely diffuse through the DA9 processes ( Figure 1—figure supplement 1 ) .", "These localization data are consistent with previous observations made in mammalian and Drosophila neurons , which suggest that MTs are uniformly plus-end-out in the axon and predominantly minus-end-out in the dendrite . 10 . 7554/eLife . 00133 . 003Figure 1 . UNC-116 ( kinesin-1 ) is required for the minus-end-out MT polarity in the DA9 dendrite .", "( A ) Schematic diagram of the morphology of the DA9 neuron .", "Asterisk denotes DA9 or PHC cell body ( throughout all images ) ; D: dorsal; V: ventral; A: anterior; P: posterior .", "( B ) and ( E ) Localization of UNC-104::YFP in a representative wild-type ( B ) or unc-116 worm ( E ) .", "Note that UNC-104::YFP is enriched in the axonal tip ( denoted by an arrowhead throughout ) in wild-type , while it is enriched at the tips of both the axon and dendrite ( dendritic tip is marked by an arrow , dashed black box and shown in higher magnification micrographs throughout ) in unc-116 animals .", "( C ) and ( F ) Localization of DHC-1::GFP in a representative wild-type ( C ) or unc-116 worm ( F ) .", "( D ) and ( G ) Localization of KLP-16::YFP in a representative wild-type ( D1 ) or unc-116 worm ( G1 ) .", "The dendrite is shown in higher magnification for a wild-type ( D2 ) or unc-116 animals ( G2 ) .", "( H ) – ( J ) Quantification of fractions of worms with qualitative defects in UNC-104::YFP ( H ) , DHC-1::GFP ( I ) , and KLP-16::YFP ( J ) localization in DA9 dendrite ( n > 50 for each genotype ) .", "( K ) Schematic diagram of the morphology of the PHC neuron .", "( L ) and ( M ) Localization of KLP-16::YFP in a representative wild-type ( L ) or unc-116 worm ( M ) .", "( N ) Quantification of fractions of worms with qualitative defects in KLP-16::YFPlocalization in the PHC dendrite ( n > 50 for each genotype ) .", "The scale bar represents 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 00310 . 7554/eLife . 00133 . 004Figure 1—figure supplement 1 . Kinesin motor activity is required for the enrichment of KLP-16::YFP in dendrite .", "( A ) Domain structure of KLP-16 .", "From N to C terminus , tail domain is in green , coiled-coil region is in red , and motor domain is in blue .", "The numbers of amino acids are on top .", "( B ) Amino acid sequence alignment of KLP-16 motor domain with KLP-15 , NCD , and Kar3 proteins .", "Identical residues are labeled in blue and similar residues are labeled in orange .", "Functional regions within the motor domain are underlined .", "( C ) A representative image of KLP-16 ( KTH/AAA ) ::GFP expressed in DA9 neuron .", "The scale bar represents 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 00410 . 7554/eLife . 00133 . 005Figure 1—figure supplement 2 . DA9 dendrite MT polarity is altered in unc-116 mutants .", "( A ) – ( C ) Representative images showing KLP-16::GFP localization in wild-type ( A ) , unc-116 ( wy270 ) ( B ) , and unc-116 ( rh24 sb79 ) animals ( C ) .", "Asterisk indicates cell body; arrowhead indicates the tip of DA9 dendrite .", "The scale bar represents 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 00510 . 7554/eLife . 00133 . 006Figure 1—figure supplement 3 . UNC-116 acts cell autonomously in DA9 neuron .", "( A ) – ( C ) Confocal micrographs of 10 wild-type ( A ) , unc-116 ( e2310 ) ( B ) , and unc-116;Ex[Pmig13::unc-116] DA9 ventral axon , cell body and dendrite , straightened and aligned along their A-P axis . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 006 To extend these findings to other neurons , we used similar markers to examine MT polarity in the PHC sensory neurons .", "The two PHC neurons are bipolar with posteriorly oriented dendrites and anteriorly guided axons ( Figure 1K ) .", "Consistent with the expression pattern in DA9 , the plus-end kinesin UNC-104::YFP is enriched at the axonal tip of PHC , whereas the minus-end kinesin KLP-16::YFP decorates the dendrite and shows accumulation in the tip of dendrite ( Figure 1L and data not shown ) .", "The above data provide information regarding the overall steady-state polarity of MTs in neurites .", "To examine the polarity of dynamic MTs in the DA9 and PHC neurons , we fluorescently tagged the microtubule plus-end-tracking protein EBP-2/EB1 .", "We observed characteristic ‘comet' like movements of EBP-2::GFP in the dendrite and axon of both cell types by time-lapse analysis .", "The majority of the EBP-2::GFP comets in the axon move away from soma , whereas the majority of comets in the dendrite move toward the soma ( Figure 2A–D and Videos 1–3 ) .", "Consistent with published results ( Maniar et al . , 2012 ) , these data show that axonal MTs are predominantly plus-end-out and dendritic MTs are largely minus-end-out in wild-type C . elegans neurons . 10 . 7554/eLife . 00133 . 007Figure 2 . Dendrite MTs are plus-end out in the unc-116 ( e2310 ) mutant .", "( A ) and ( B ) Representative kymographs of moving EBP-2::GFP puncta in the PHC neuron dendrite of wild-type ( A ) and unc-116 animals ( B ) .", "The cell body is to the left in both panels .", "Time runs top to bottom; arrowheads mark retrogradely moving puncta; arrows mark anterogradely moving puncta .", "( C ) and ( D ) Bar graphs of the fraction of anterograde and retrograde movements in PHC ( C ) and DA9 neurons ( D ) .", "MT , microtubule; numbers within each column denote the number of puncta counted in the corresponding categories; ***p<0 . 001 , χ2 test . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 00710 . 7554/eLife . 00133 . 008Video 1 . Movement of EBP-2::GFP puncta in the ventral axon of wild-type DA9 neuron .", "Cell body is to the left .", "Displayed 10× speed . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 00810 . 7554/eLife . 00133 . 009Video 2 . Movement of EBP-2::GFP puncta in the dendite of wild-type DA9 neuron .", "Cell body is to the left .", "Displayed 10× speed . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 00910 . 7554/eLife . 00133 . 010Video 3 . Movement of EBP-2::GFP puncta in the dendrite of wild-type PHC neuron .", "Cell body is to the left .", "Displayed 10× speed . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 010 To understand how the minus-end-out MTs are assembled and maintained in the dendrite , we examined the localization of our MT markers in the mitotic kinesin mutant zen-4/kinesin-6 , which is required for mobilizing MTs in neuronal processes in vitro ( Baas et al . , 2006 ) .", "To our surprise , we found no obvious changes in the distribution of MT polarity markers in these mutants .", "Instead , we found that the DA9 dendritic MT organization is altered in mutants of a related kinesin , unc-116/kinesin-1 .", "In unc-116 ( e2310 ) mutants , UNC-104::YFP also accumulates in the tip of dendrite in addition to its normal localization at the axonal tip ( Figure 1E ) .", "Furthermore , the fluorescence signals of DHC-1::GFP and KLP-16::YFP no longer accumulate at the dendrite tip; instead , fluorescence is visible only at the dendritic segment nearest the cell body ( Figure 1F , G , M ) .", "These changes in the distribution of MT polarity markers suggest that dendritic MT organization has changed to an axon-like plus-end-out pattern in the unc-116 mutants .", "Consistently , in the same mutant dynamic MTs in the dendrites of both DA9 and PHC neurons almost completely change their behaviors , adopting a predominantly plus-end-out movement ( Figure 2A–C and Videos 4 and 5 ) .", "These data further indicate that the unc-116/kinesin-1 mutation causes MT polarity in dendrites to become axon-like , but with no effect on MT orientation in the axon . 10 . 7554/eLife . 00133 . 011Video 4 . Movement of EBP-2::GFP puncta in the ventral axon of unc-116 ( e2310 ) DA9 neuron .", "Cell body is to the left .", "Displayed 10× speed . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 01110 . 7554/eLife . 00133 . 012Video 5 . Movement of EBP-2::GFP puncta in the dendrite of unc-116 ( e2310 ) DA9 neuron .", "Cell body is to the left .", "Displayed 10× speed . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 012 UNC-116 encodes the sole C . elegans ortholog of kinesin-1 ( previously called kinesin heavy chain or KHC ) ( Siddiqui , 2002 ) .", "Since complete loss of unc-116 leads to embryonic lethality and unc-116 ( e2310 ) is viable , the e2310 allele likely represents a partial loss-of-function mutant .", "Two additional partial loss-of-function alleles of unc-116 , wy270 and rh24sb79 , show similar mislocalization of MT motors in the DA9 dendrite , suggesting that this phenotype is indeed due to loss of the protein activity ( Figure 1—figure supplement 2 ) .", "Consistent with this notion , cell autonomous expression of wild-type UNC-116 in unc-116 ( e2310 ) mutants rescued the dendritic distribution phenotype of KLP-16 , indicating that UNC-116 functions in the DA9 neuron ( Figure 1—figure supplement 3 ) .", "Taken together , these data support the idea that UNC-116's function is required for establishing the minus-end-out MT organization in the dendrite .", "What are the consequences of MT polarity reversal in the DA9 dendrite ?", "We first compared the development of the DA9 dendrite in wild-type and unc-116 animals .", "In wild-type animals , DA9 axonal outgrowth and neuromuscular junction formation occur embryonically , whereas most dendrite outgrowth takes place postembryonically starting from the L1 stage ( Teichmann and Shen , 2011 ) .", "Upon reaching adulthood , the DA9 axon and dendrite achieve stereotyped lengths .", "In the unc-116 ( e2310 ) mutants , the sequence and directionality of axonal and dendrite outgrowth as well as the length of the axon are indistinguishable from that of the wild-type strain .", "The length of the DA9 dendrite , however , is dramatically longer compared with wild-type animals at every stage of development ( Figure 3—figure supplement 1 ) .", "To determine whether MT-dependent vesicle transport is affected by unc-116 ( e2310 ) , we examined localization of synaptic vesicle ( SV ) markers .", "The UNC-104/Imac/kinesin3 motor traffics SVs to presynaptic specializations ( Hall and Hedgecock , 1991 ) , and synaptic vesicle proteins like RAB-3 exclusively accumulate in the axon of wild-type animals ( Figure 3A ) .", "In unc-104 ( e1265 ) mutants , SV markers are completely absent from the axon , accumulating instead in the cell body and dendrite due to the activity of dynein ( Figure 3C; Ou et al . , 2010 ) .", "Interestingly , we found that in unc-116 ( e2310 ) mutants , both the axon and dendrite contain a similar number of vesicle clusters ( Figure 3E and Figure 3—figure supplement 2 ) .", "If this ectopic accumulation of SVs in the dendrite is due to the switch of MT polarity to axon-like plus-end-out arrangement ( rather than to the activity of dynein ) , then the accumulation of SVs in the DA9 dendrite should depend on the UNC-104 kinesin motor .", "Indeed , in unc-104 ( e1265 ) ;unc-116 ( e2310 ) double mutants , SVs are largely absent from both processes and trapped in the cell body .", "These results argue that UNC-116 is not directly involved in SV transport in the DA9 neuron; rather , it creates the minus-end-out MT polarity of the dendrite .", "As a result of this aberrant MT polarity , the plus-end motor UNC-104 promotes locomotion of cargoes into the dendrite .", "If our model is correct , the unc-116 mutations should not only affect RAB-3 but also other synaptic vesicle proteins and active zone proteins .", "Indeed , the active zone protein SYD-2/liprin and other SV markers like SNG-1/synaptogyrin show similar ectopic localization to DA9 dendrites in the unc-116 ( e2310 ) mutants , suggesting that the changes in MT polarity affect the entire presynaptic structure ( Figure 3—figure supplement 2 ) . 10 . 7554/eLife . 00133 . 013Figure 3 . Dendrite exhibits axonal-like properties in unc-116 mutants .", "( A ) – ( D ) Distribution of GFP::RAB-3 puncta ( left panels ) and represented schematic diagrams ( right panels ) in wild-type ( A ) , unc-116 ( B ) , unc-104 ( C ) , and unc-116;unc-104 mutant animals ( D ) .", "( E ) Average percentage of GFP::RAB-3 fluorescence intensity in the dendrite ( n = 20 ) .", "( F ) Quantification of GFP::RAB-1 fluorescence intensity in the dendrite ( n = 20 ) .", "***p<0 . 0001 .", "Student's t-test .", "The scale bar represents 10 μm .", "Asterisk denotes DA9 cell body . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 01310 . 7554/eLife . 00133 . 014Figure 3—figure supplement 1 . DA9 dendrite is longer in unc-116 mutants .", "( A ) and ( B ) Micrographs of representative wild-type ( A ) and unc-116 ( e2310 ) ( B ) adults expressing cytoplasmic mCherry in DA9 .", "( C ) Average length of DA9 dendrite in 20 wild-type and unc-116 ( e2310 ) adults .", "The scale bar represents 10 μm .", "Bracket indicates dendrite; error bars indicate standard deviations .", "***p<0 . 0001 .", "χ2 test . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 01410 . 7554/eLife . 00133 . 015Figure 3—figure supplement 2 . Presynaptic proteins are mis-localized in unc-116 mutant .", "( A ) and ( B ) Distribution of synaptic vesicle marker GFP::RAB-3 and an active zone marker mCherry::SYD-2 in wild-type and unc-116 ( e2310 ) mutant .", "The scale bar represents 10 μm .", "The high magnifications are shown in the right panels . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 015 To determine whether unc-116 and dendritic MT polarity are critical for localization of dendritic proteins , we analyzed the distribution of several dendritically targeted proteins .", "An acetylcholine receptor ( ACR-2 ) , a receptor tyrosine kinase ( CAM-1 ) , and a gap junction protein ( FBN-1 ) are all reported to be dendritically enriched in DA9 ( Sieburth et al . , 2005; Barbagallo et al . , 2010 ) .", "In wild-type DA9 neurons , fluorescently tagged ACR-2 , CAM-1 , and FBN-1 all localize to the dendrite , cell body , and the initial part of the ventral axon .", "In unc-116 ( e2310 ) animals , however , these dendritic proteins accumulate within the cell body and largely disappear from the dendrite , suggesting that unc-116 is required for dendritic localization of postsynaptic proteins ( Figure 4 ) .", "Taken together , these data suggest that the minus-end-out MT organization in the dendrite is instructive for dendritic cargo transport by molecular motors .", "It discourages the plus-end motor , UNC-104 , and encourage the minus-end motor , dynein , from entering and accumulating in the dendrite .", "This polarity mechanism is thus essential for setting up the polarized distribution of vital axonal and dendritic constituents such as SVs and neurotransmitter receptors . 10 . 7554/eLife . 00133 . 016Figure 4 . DA9 dendrite fails to accumulate dendritic proteins in unc-116 mutants .", "( A ) and ( D ) Distribution of the postsynaptic neurotransmitter receptor ACR-2::GFP in wild-type ( A ) and unc-116 ( D ) animals .", "Bracket indicates dendrite region .", "( B ) and ( E ) Localization of CAM-1::YFP in a representative wild-type ( B ) or unc-116 worm ( E ) .", "( C ) and ( F ) Localization of FBN-1::YFP in a representative wild-type ( C ) or unc-116 worm ( F ) .", "( G ) Quantification of CAM-1::YFP fluorescence intensity in the dendrite ( n = 20 ) .", "***p<0 . 0001 .", "Student's t-test .", "The scale bar represents 10 μm .", "Bracket indicates dendrite .", "Asterisk denotes DA9 cell body . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 016 The canonical function of kinesin-1 is to transport certain cargos , such as mitochondria , along neuronal MTs .", "Like kinesin-1 , UNC-116 is composed of an N-terminal motor domain , a putative coiled-coil dimerization stalk region , and a C-terminal tail domain that binds to kinesin light chains ( KLCs ) , adaptors , and cargos ( Vale , 2003 ) .", "Interestingly , several recent biochemical and cell biology experiments have shown that part of the C-terminal tail domain of kinesin-1 binds directly to MTs ( Navone et al . , 1992; Dietrich et al . , 2008; Seeger and Rice , 2010 ) .", "It was also demonstrated that kinesin-1 could cross-link and glide MTs using in vitro assays ( Andrews et al . , 1993; Palacios and St Johnston , 2002; Yamada et al . , 2010 ) .", "To understand how UNC-116 regulates dendrite MT polarity , we considered two possible models in which UNC-116/kinesin-1 directly transport the minus-end-out MTs into dendrites or transport the plus-end-out MTs out of dendrites via interaction of its C-terminal MT-binding domain with the cargo MTs .", "In either scenario , it requires kinesin-1 selectively cross-link anti-parallel MTs .", "We first tested it using in vitro MTs bundling assay .", "When purified tubulins were polymerized into MTs , the MTs were found as isolated microtubules with the appearance of smooth surface under the electron microscope ( Figure 5A ) .", "Addition of purified kinesin-1 complex from bovine brain to the MTs causes the dispersed MTs to adopt a more ‘rough' surface due to the binding of kinesin-1 to MTs ( Figure 5B ) .", "When both of kinesin-1 and mNUDC were added to the MTs , we found that MTs were bundled ( Figure 5C ) .", "The MT bundles are superficially similar to those reconstructed from flagellar axonemal dyneins and MTs ( Ueno et al . , 2008 ) .", "No such bundles were observed in control samples containing just taxol-stabilized MTs or in mixtures of MTs with kinesin-1 only ( Figure 5B ) .", "Neighboring MTs in the bundles has an interval of ∼80 nm .", "Considering kinesin-1 has a long flexible stalk ( maximum ∼80 nm; Hirokawa et al . , 1989 ) and mNUDC is an adapter protein , it is conceivable that the cross-bridges represent a complex of kinesin-1 and mNUDC .", "Furthermore , we determined the polarity of the cross-linked MTs from their Moiré patterns of protofilaments ( Chretien et al . , 1996 ) , and determined orientation of 13 pairs of MTs cross-linked by kinesin-1 .", "Nine pairs ( 69% ) of microtubule bundles were anti-parallel with a 95% confidence interval ( CI ) of 39% and 91% by F-test for binominal distribution ( Figure 5D-D2 ) .", "The lower limit of CI exceeds 50% when a significant level is 13 . 4% using one-tailed F-tests .", "Therefore , kinesin-1-mNUDC complex tends to cross-link anti-parallel microtubule bundles . 10 . 7554/eLife . 00133 . 017Figure 5 . Purified kinesin-1 complex bundles anti-parallel MTs in vitro .", "( A ) – ( C ) Negatively stained EM images of ( A ) MTs alone , ( B ) kinesin-1-MTs , and ( C ) kinesin-1-NudC-MTs .", "Two enlarged images are in right row .", "Scar bar is 100 nm from ( A–C ) , and 30 nm in the enlarged images .", "Note that long spanned MT bundles were observed only in ( C ) when the samples mixed with both Kinesin-1 and NudC .", "Deeply stained cross-bridging molecules are seen between adjacent MTs . Cryo-EM images of microtubules of ( D ) kinesin-NudC microtubles , ( D1 ) microtubules alone and ( D2 ) kinesin-microtubules . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 017 The ability of UNC-116 to slide anti-parallel MTs requires that the motor have two distinct binding sites for MTs .", "Indeed , the C-terminal domain of kinesin-1 has been shown to bind to MTs in addition to the N-terminal motor domain ( Navone et al . , 1992; Seeger and Rice , 2010 ) .", "To further investigate whether the C-terminal domain of UNC-116 can bind to MTs and whether this binding is required for its function in establishing dendritic MTs , we first examined the amino acid sequence of the C-terminal region of UNC-116 .", "Sequence alignment of UNC-116 with its corresponding fly , rat , and human homologs showed that the MT-binding site is highly conserved ( Figure 6A ) .", "The positively charged residues ( labeled in red ) in this region have been shown to be required for MT-binding in vitro ( Seeger and Rice , 2010 ) .", "Given the high degree of sequence conservation of the MT-binding site , it is likely that its function in binding MTs is also conserved in worms .", "Furthermore , GFP-tagged C-terminal domain of UNC-116 ( 521–863 ) showed colocalization with MTs when expressed in HEK293 cells , suggesting that the C-terminal domain of UNC-116 can indeed bind to MTs ( data not shown ) . 10 . 7554/eLife . 00133 . 018Figure 6 . UNC-116/kinesin-1 orients dendritic MTs .", "( A ) Domain structure of UNC-116 protein with motor domain in blue , dimerization region in red , and C-terminal regulation domain in orange , length of amino acid is shown .", "( B ) Cell-autonomous rescue of KLP-16::YFP localization .", "MD , mutant swapping the amino acids ‘KTH' in P-loop of UNC-116 motor domain with ‘AAA'; MT , mutant swapping the MT-BS in UNC-116 tail region with ‘AAAYAA' .", "n > 50 per group .", "Error bar indicates standard deviation; ***p<0 . 0001 , χ2 test .", "( C ) and ( D ) MTs structure highlighted by GFP::TBA-1 in a representative wild-type ( C ) or unc-116 worm ( D ) .", "Quantification of GFP::TBA-1 fluorescence density ratio between the dendrite and the ventral axon in wild-type and the unc-116 ( e2310 ) animals .", "( E ) Quantification of EBP-2::GFP puncta moving frequency in the dendrite of wild type and the unc-116 ( e2310 ) animals .", "n > 20 per group .", "Error bar indicates standard deviation; **p<0 . 001 , ***p<0 . 0001 , Student's t-test .", "( F ) A schematic model showing kinesin-1 cross-linking and sliding MTs out of the dendrite . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 01810 . 7554/eLife . 00133 . 019Figure 6—figure supplement 1Kinesin light chains are not required for dendrite MT polarity .", "( A ) – ( C ) Distribution of a mitochondria marker , TOM-20::YFP in a representative wild-type ( A ) , unc-116 ( e2310 ) ( B ) , or klc-2 ( km11 ) ( C ) worm .", "( D ) – ( F ) Localization of KLP-16::YFP in a representative wild-type ( D ) , klc-1 ( ok2609 ) ( E ) , or klc-2 ( km11 ) worm ( F ) .", "( G ) Quantification of axonal TOM-20::YFP puncta number in wild-type , unc-116 ( e2310 ) , or klc-2 ( km11 ) worms ( n = 20 ) .", "The scale bar represents 10 μm .", "Error bars indicate standard deviations .", "***p<0 . 0001 .", "χ2 test . DOI: http://dx . doi . org/10 . 7554/eLife . 00133 . 019 Second , we examined the molecular lesions in three unc-116 mutants , all of which show MT polarity defects ( Figure 1—figure supplement 3 ) .", "The molecular lesion of e2310 is a TC5 transposon insertion .", "The transposon was inserted after residue 692 , which truncates the C-terminal MT-binding domain ( Patel et al . , 1993 ) .", "Point mutations are found in the motor domain in wy270 and rh24sb79 alleles , which likely result in partial loss of motor function ( Yang et al . , 2005 and unpublished data ) .", "These results suggest that both motor activity and C-terminal MT-binding might be required for this newly defined function of UNC-116/kinesin-1 .", "To definitively test this , we created a mutant form of UNC-116 in which three point mutations were introduced in the C-terminal MT-binding motif .", "The same mutations diminish the binding of kinesin-1 C-terminal domain to MTs ( Seeger and Rice , 2010 ) .", "Therefore , this mutant form ( UNC-116MT ) might specifically lack its ability to bind to MTs with the C-terminal motif .", "We tested whether this form can still rescue the dendrite polarity phenotypes as well as phenotypes related to the canonical transport function of UNC-116 .", "To assess the canonical organelle transport function of UNC-116 , we examined localization of mitochondria in DA9 .", "In wild-type animals , a mitochondrial marker TOM-20::YFP localizes to discrete puncta in both the axon and dendrite of DA9 ( Figure 6—figure supplement 1 ) .", "In unc-116 ( e2310 ) mutants , the TOM-20 signal is completely absent from both processes and only found in the cell body ( Figure 6—figure supplement 1 ) , suggesting that UNC-116 is required for transporting mitochondria to both axon and dendrite .", "DA9 expression of UNC-116MT efficiently rescues the TOM-20 localization defect in the axon , suggesting that this mutant form of UNC-116 can support the organelle transporting function .", "Interestingly , but the same transgene showed much less rescue of the MT polarity defects in the dendrite , as assayed by KLP-16 localization ( Figure 6B ) .", "This result is consistent with the notion that the C-terminal MT-binding motif of UNC-116 is specifically required for the establishing dendritic MT polarity .", "In addition , UNC-116MT efficiently rescues the abnormally long dendrites in the unc-116 ( e2310 ) mutant , suggesting that this length phenotype is independent of the MT polarity .", "To further explore the difference between the MT-polarity-related function of UNC-116 and its canonical vesicular and organelle transport function , we studied the kinesin light chains , KLC-1 and KLC-2 .", "Consistent with previous reports , KLC-2 is required for axonal localization of TOM-20 , suggesting that the KLCs are required for the organelle transport function of UNC-116 ( Figure 6—figure supplement 1 ) .", "Interestingly , neither klc-1 or klc-2 single mutants nor klc-1; klc-2 double mutants showed any defects in MT polarity markers ( Figure 6—figure supplement 1 ) , suggesting that they are dispensable for the MT polarity function of UNC-116 .", "Indeed , the MT-gliding activity of kinesin-1 in nonpolarized Drosophila S2 cells is also independent of KLCs , suggestive of a similar mode of action in nonneuronal cells ( Jolly et al . , 2010 ) .", "Taken together , our structure–function analyses have revealed a novel function of UNC-116 in regulating dendritic MT polarity .", "This function requires both motor activity and C-terminal MT binding , and can be separated from the canonical vesicular and organelle trafficking function of UNC-116 both at the level of cargo-binding domains as well as the dependency on kinesin light chains .", "How does UNC-116/kinesin-1 promote minus-end-out MTs configuration in the dendrite ?", "Our genetic data so far are consistent with two models .", "In the first model , UNC-116/kinesin-1 directly slides minus-end-out MTs into dendrites .", "Alternatively , UNC-116 might slide plus-end-out MTs out of dendrite and consequently increase the percentage of the minus-end-out MTs in dendrites .", "In order to differentiate these two models , we analyzed the MTs concentration in the dendrite and axon .", "We reasoned that if UNC-116/kinesin-1 slides minus-end-out MTs into dendrite , there would be less MTs in the dendrite of mutant animals .", "If UNC-116/kinesin-1 slides plus-end-out MTs out of dendrite , there would be more MTs in the dendrite of mutant .", "We visualized the MTs structure by expressing GFP::TBA-1/ α-tubulin in the DA9 neuron .", "In wild-type animals , axon has higher expression of GFP::TBA-1 , with a dendrite/ventral axon intensity ratio around 0 . 6 .", "In unc-116 ( e2310 ) animals , axon expression of GFP::TBA-1 remains largely unchanged , whereas expression in the dendrite increases , with a dendrite/axon intensity ratio around 1 . 2 , suggestive of excessive MTs in the unc-116 mutant dendrites ( Figure 6C–E ) .", "Another measure of the number of MTs is the frequency of EBP-2::GFP comets , which directly reflects the number of dynamic plus-ends .", "We found that EBP-2::GFP comets in the unc-116 mutant DA9 dendrite is three times more frequent comparing to wild type , further indicating there are more MTs in unc-116 ( e2310 ) dendrite ( Figures 2C , 6F ) .", "Collectively , these data favor the model that UNC-116/kinesin-1 maintains minus-end-out MT polarity in dendrite through selectively sliding plus-end-out MTs out of dendrite ( Figure 6G ) ." ], [ "We report a new function of kinesin-1 to promote the minus-end-out MT organization in the dendrite .", "Previous studies in neuronal cell cultures showed that intracellular transport of short MTs occurs robustly in neurites and can be driven by mitotic kinesins ( Baas et al . , 2006 ) .", "While we did not find evidence that mitotic kinesins are required for DA9 MT organization , our data provide in vivo evidence that transport of MTs by another kinesin , kinesin-1 , in the dendrite is critical for establishing or maintaining MT polarity .", "An interesting study in Drosophila sensory neurons showed that the minus-end motor dynein is required for the uniform plus-end-out MT organization in the axon but dispensable for dendritic MT organization ( Zheng et al . , 2008 ) .", "A recent study in Drosophila neuron showed that another motor kinesin-2 complex is required to steer MTs growth in the dendrite branch point to maintain the uniform minus-end-out MT polarity in the dendrite ( Mattie et al . , 2010 ) .", "Since kinesin-1 is highly conserved throughout evolution , we anticipate our findings to be a starting point for more sophisticated in vitro or in vivo analyses of MT polarity in neurons .", "If UNC-116/kinesin-1 slides plus-end-out MTs out of dendrites , one immediate question is how this process is restricted to dendrites .", "Maniar et al . ( 2012 ) recently showed that the MT-associated axon initial segment ( AIS ) -enriched protein UNC-33/CRMP is important for MT organization in both the axon and dendrite in worm sensory neurons .", "The AIS has been reported to play important roles in neuronal polarity ( Hedstrom et al . , 2008; Sobotzik et al . , 2009 ) .", "It is conceivable that AIS proteins provide critical regulatory roles for motor-based MT sliding by restricting the direction of MT transport .", "Interestingly , the AIS in vertebrate neurons is known to serve as a diffusion barrier for both transmembrane and cytosolic proteins .", "How it regulates MT transport will be an interesting question for future studies .", "The compartmentalization of neurons is achieved by several cell biological mechanisms including directed intracellular trafficking , local sequestration , diffusion barriers , and transcytosis ( Kennedy and Ehlers , 2006 ) .", "Previous studies have also reported the existence of ‘smart motors' that are capable of distinguishing axonal and dendritic MTs ( Saito et al . , 1997; Marszalek et al . , 1999; Guillaud et al . , 2003; Kapitein et al . , 2010 ) .", "The existing literature raises the question of what functional role is played by MT polarity , an important question that cannot be answered without a polarity-altering tool .", "The unc-116 mutants specifically affect MT polarity in dendrites , and therefore serve as useful reagents to dissect the importance of dendritic MT polarity .", "Our data strongly support the notion that the minus-end-out MTs in dendrites not only allow for the accumulation of dendritic proteins but are also critical for the exclusion of axonal components such as synaptic vesicles and active zone proteins .", "Furthermore , MT polarity appears to affect the length of the dendrite , suggesting that both the structural and transport functions of MTs depend on their polarity .", "Interestingly , the direction and timing of dendrite outgrowth are not affected in unc-116 mutants , suggesting that MT polarity does not play a critical role in dendrite outgrowth , a process in which the actin cytoskeleton is known to play an important role ( Gao et al . , 1999 ) .", "Together , these data argue that polarized MT tracks play instructive roles in formation of axonal and dendritic identities , as well as in the compartmentalization of axonal and dendritic constituents such as SVs , active zone proteins , and neurotransmitter receptors ." ], [ "Worms were raised on NGM plates at 20°C using OP50 Escherichia coli as a food source .", "N2 Bristol was utilized as the wild-type reference strain .", "The following mutant strains were obtained through the Caenorhabditics Genetics Center: FF41 unc-116 ( e2310 ) III , RB1975 klc-1 ( OK2609 ) IV , and CB1265 unc-104 ( e1265 ) II .", "TV6687 unc-116 ( rh24 sb79 ) III was a kind gift from Dr . McNally at University of California at Davis , TV3706 unc-116 ( wy270 ) III , carrying a single L129 to F mutation in motor domain , ( EL , unpublished data ) was previously isolated from our laboratory .", "Expression clones were made in the pSM vector , a derivative of pPD49 . 26 ( A Fire , unpublished data ) with extra cloning sites ( S McCarroll and CI Bargmann , unpublished data ) .", "The plasmids and transgenic strains ( 1–50 ng/μl ) were generated using standard techniques and coinjected with markers Podr1::RFP or GFP ( 40 ng/μl ) : wyEx3892 [Pitr1::dhc-1::GFP] , wyEx 2559 [Pitr1:: unc-104::YFP] , wyEx3128 [Pitr1::klp-16::YFP] , wyIs674 ( Pitr1::klp-16::YFP ) , wyIs309 ( Pmig13::klp-16::GFP ) , wyEx4974 [Pida-1::unc-104::GFP] , wyEx4980 [Pida-1::klp-16::GFP] , wyEx4978 [Pida-1::GFP::rab-3] , wyEx4828 [Pdes-2::ebp-2::GFP] , wyIs349 ( Pmig13::loxP-ebp-2::GFP ) , wyIs85 ( Pitr1::GFP::rab-3 ) , wyEx2505 [Pitr1::syd-2::GFP , Pitr1::mCherry::rab-3] , wyIs386 ( Pitr1::acr-2::GFP ) , wyEx1902 [Pitr1::mCherry] , wyEx403 [Pitr1::cam-1::YFP] , wyEx2396 [Pitr1::fbn-1;;YFP] , and wyEx2709 [Pitr1::tom-20::YFP] .", "Detailed subcloning information will be provided on request .", "Images of fluorescently tagged fusion proteins were captured in live C . elegans using a Plan-Apochromat 63×/1 . 4 objective on a Zeiss LSM510 confocal microscope system .", "Visual inspections and some quantification were done using a Zeiss Axioplan 2 microscope with Chroma HQ filter sets for GFP , YFP , and RFP ( 63×/1 . 4NA objective ) .", "Worms were immobilized using 10 mM levamisole ( Sigma , St Louis , MO ) and oriented anterior to the left and dorsal up .", "Dynamic imaging was performed on an inverted Zeiss Axio Observer Z1 microscope equipped with the highly sensitive QuantEM:512SC camera .", "A Plan-Apochromat 63×/1 . 4 objective was used for acquisition .", "L4 worms were cultured at 22°C for imaging .", "They were mounted onto 2% agarose pads and anesthetized with 6 mM levamisol ( Sigma ) for no longer than 20min .", "All videos were acquired over 25 s with 8 frames per second .", "Videos were then analyzed using the ImageJ software .", "Tubulin was purified from porcine brain and polymerized to microtubules by a previously described method , then stabilized by taxol ( Sloboda and Rosenbaum , 1982 ) .", "Conventional kinesin ( kinesin-1 ) was also purified from porcine brain as described before ( Wagner et al . , 1991 ) .", "Recombinant protein for mNUDC was generated using pGEX-4T expression vector ( GE Healthcare Bio-sciences ) .", "Protein purification was performed using Glutathione Sepharose 4B ( GE Healthcare Bio-sciences ) based on the manufacturer's recommendation .", "To remove GST-tag , we treated recombinant protein with Thrombin ( Merck ) , followed by absorption of thrombin by Benzamidine Sepharose 6B ( GE Healthcare Bio-sciences ) .", "Taxol-stabilized MTs ( 25 μg/ml ) with/without kinesin-1 ( 85 μg/ml ) , NudC ( 50 μg/ml ) , and 2 mM AMP-PNP were mixed in a test tube according to the condition then sit for 10 min .", "The kinesin-1 and NudC were mixed at a molar ratio of 1:5 .", "The mixed solution of 5 μl was loaded on a hydrophilized carbon grid ( 75/300 mesh; VECO , Cu ) to sit for 60 s in a humid chamber .", "Unabsorbed protein was rinsed by BRB80 buffer ( 80 mM Pipes-KOH , 2 mM MgSO4 , 1 mM EGTA , pH 6 . 8 ) containing with 10 μM taxol .", "After the excess solution was blotted with a filter paper , the specimen was stained with an equal volume of 2% uranyl acetate for 40 s .", "The specimens were observed with a transmission electron microscope , Tecnai Spirit ( FEI Co . ) .", "The microscope was operated at 120 kV .", "The magnification for overviewed images was ×21 , 000 and the nominal magnification was ×67 , 000 .", "The defocusing values were usually −1 to −1 . 5 μm .", "The images were recorded using CCD ( 2k × 2k , Eagle1k CCD; FEI ) at 30 μm/pixel corresponding to 0 . 47 nm .", "The electron dose per single image was estimated as 10 e/Å2 .", "To define the polarity of the MTs , electron cryo-microscopy was used .", "First frozen-hydrated specimens were prepared as follows: a 10-μl drop of purified MT/kinesin-1/NudC complex with 2 mM AMP-PNP was mounted on a holey carbon grid ( Quantifoil: Mo R2/2 , Germany ) and blotted with filter paper to remove excess solution and make a thin aqueous layer .", "The grid was then plunged into ethane slush at −185°C to create a thin layer of vitreous ice .", "The frozen-hydrated specimens were examined at liquid nitrogen temperature using a cryo-holder ( CT3500; Oxford Instruments , United Kingdom ) with an EF-2000 microscope ( Hitachi High-Technologies , Japan ) operated at 200 kV .", "The electron micrographs were recorded using a 2 k × 2 k CCD camera developed by TVIPS ( Yasunaga and Wakabayashi , 2008 ) with an defocusing value of −3 to −4 μm .", "The electron dose per single image was estimated as approximately ∼1400 e/Å2 .", "The defocusing values of all obtained images were estimated by ‘ctfDisplay' .", "The images were then corrected by phase flipping after the phase contrast transfer functions ( CTF ) were determined from the defocusing values .", "Corrected images were processed by the band-pass filter ( 1/100 nm to 1/10 nm ) , and so their background noise was reduced .", "The polarity of microtubules was determined by checking their Moiré patterns .", "Most of the image processing was performed by Eos ( Yasunaga and Wakabayashi , 1996 ) ." ] ]
[ "In neurons , microtubules ( MTs ) span the length of both axons and dendrites , and the molecular motors use these intracellular ‘highways' to transport diverse cargo to the appropriate subcellular locations .", "Whereas axonal MTs are organized such that the plus-end is oriented out from the cell body , dendrites exhibit a mixed MTs polarity containing both minus-end-out and plus-end-out MTs .", "The molecular mechanisms underlying this differential organization , as well as its functional significance , are unknown .", "Here , we show that kinesin-1 is critical in establishing the characteristic minus-end-out MT organization of the dendrite in vivo .", "In unc-116 ( kinesin-1/kinesin heavy chain ) mutants , the dendritic MTs adopt an axonal-like plus-end-out organization .", "Kinesin-1 protein is able to cross-link anti-paralleled MTs in vitro .", "We propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain ." ]
[ "Neurons , or nerve cells , are excitable cells that transmit information using electrical and chemical signals .", "Nerve cells are generally composed of a cell body , multiple dendrites , and a single axon .", "The dendrites are responsible for receiving inputs and for transferring these signals to the cell body , whereas the axon carries signals away from the cell body and relays them to other cells .", "Like all cells , nerve cells have a cytoskeleton made up of microtubules , which help to determine cellular shape and which act as ‘highways' for intracellular transport .", "Microtubules are long hollow fibers composed of alternating α- and β-tubulin proteins: each microtubule has a ‘plus'-end , where the β subunits are exposed , and a ‘minus'-end , where the α subunits are exposed .", "Nerve cells are highly polarized: within the axon , the microtubules are uniformly oriented with their plus-ends pointing outward , whereas in dendrites , there are many microtubules with their minus-ends pointing outward .", "This arrangement is conserved across the animal kingdom , but the mechanisms that establish it are largely unknown .", "Yan et al . use the model organism Caenorhabditis elegans ( the nematode worm ) to conduct a detailed in vivo analysis of dendritic microtubule organization .", "They find that a motor protein called kinesin-1 is critical for generating the characteristic minus-end-out pattern in dendrites: when the gene that codes for this protein is knocked out , the dendrites in microtubules undergo a dramatic polarity shift and adopt the plus-end-out organization that is typical of axons .", "The mutant dendrites also show other axon-like features: for example , they lack many of the proteins that are usually found in dendrites .", "Based on these and other data , Yan et al . propose that kinesin-1 determines microtubule polarity in dendrites by moving plus-end-out microtubules out of dendrites .", "These first attempts to explain , at the molecular level , how dendritic microtubule polarity is achieved in vivo could lead to new insights into the structure and function of the neuronal cytoskeleton ." ]
2013
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "immunology and inflammation" ]
CD14 release induced by P2X7 receptor restricts inflammation and increases survival during sepsis
elife-60849-v2
[ [ "Purinergic signaling controls many different processes during infection and inflammation ( Eltzschig et al . , 2012 ) and the P2X7 receptor is one of the key purinergic receptors in modulating the macrophage functions that orchestrate the inflammatory response ( Di Virgilio et al . , 2017 ) .", "The P2X7 receptor in LPS-primed macrophages activates the nucleotide-binding domain and the leucine-rich repeat receptor pyrin domain containing 3 ( NLRP3 ) inflammasome , which in turn leads to the release of pro-inflammatory cytokines from the interleukin ( IL ) −1 family , such as IL-1β ( Di Virgilio et al . , 2017 ) .", "However , the P2X7 receptor can also block NLRP3 if it is activated before the LPS priming of the macrophages ( Martínez-García et al . , 2019 ) .", "This means that the P2X7 receptor can cause different pro- or anti-inflammatory responses depending when it is activated .", "In macrophages , the P2X7 receptor also induces the release of extracellular vesicles that contain IL-1β and MHCII ( MacKenzie et al . , 2001; Pellegatti et al . , 2008; Qu et al . , 2009; Qu et al . , 2007 ) ; however , apart from these proteins , the cargo of P2X7 receptor-induced extracellular vesicles , remains largely unknown .", "A large fraction of IL-1β and other cytosolic proteins are also released via pyroptosis , a type of cell death dependent on inflammasome activation and the formation of large plasma membrane pores by gasdermin D ( Broz et al . , 2020 ) .", "The secretome of the P2X7 receptor in macrophages includes many soluble proteins released by pyroptosis and some prototypic proteins of extracellular vesicles , such as annexin A1 ( de Torre-Minguela et al . , 2016 ) .", "One of the plasma membrane-associated proteins identified as part of the P2X7 receptor secretome is CD14 , a well-known myeloid cell marker ( de Torre-Minguela et al . , 2016; Setoguchi et al . , 1989 ) .", "CD14 is a glycosylphosphatidylinositol ( GPI ) -anchored membrane protein important for transferring LPS to Toll-like receptor ( TLR ) 4 and for controlling TLR4 translocation to endosomes that activate TRAM-TRIF-dependent pathways ( Zanoni and Granucci , 2013 ) .", "Therefore , CD14 is important for ensuring that TLR4 responds optimally to LPS and that macrophages produce pro-inflammatory cytokines .", "CD14 could be released from the cells by the action of proteinase ( Wu et al . , 2019 ) ; however , there are also different release mechanisms independent of proteinase that are not known .", "The pool of extracellular CD14 in vivo , known as soluble CD14 , is detected in different fluids during infection and particularly during sepsis , a life-threating condition resulting from exacerbated inflammation in response to infection ( Barratt-Due et al . , 2017; Bas et al . , 2004 ) .", "Extracellular CD14 during microbial infection is important for host defense , in particular for bacterial clearance , but little is known about the release of CD14 in vivo ( Qin et al . , 2017; Knapp et al . , 2006; Sahay et al . , 2018; Wieland et al . , 2005 ) .", "Despite the fact that the P2X7 receptor induces the release of CD14 ( de Torre-Minguela et al . , 2016 ) , it is not known if P2X7 contributes to the extracellular pool of CD14 during infection or what its role is in defending the host during sepsis .", "In this study , we found for first time that CD14 is a cargo of extracellular vesicles released by P2X7 receptor activation and functionally that the lack of cellular CD14 compromises the production of macrophage pro-inflammatory cytokines .", "Additionally , we also found that during sepsis there is a decrease in the extracellular pool of CD14 in P2rx7−/− mice , which results in high bacterial dissemination and a decreased mice survival and reveals that the P2X7 receptor is important for maintaining an optimum level of CD14 and thus ensuring survival of sepsis ." ], [ "In a previous study , we identified CD14 as a specific component of the P2X7 receptor secretome in macrophages ( de Torre-Minguela et al . , 2016 ) .", "After ATP stimulation of LPS-primed macrophages , we detected the appearance of extracellular CD14 in the 100K pellet together with the tetraspanin CD9 , a well-known extracellular vesicle marker ( Figure 1a ) .", "This pellet contained extracellular vesicles with an average size of 167 . 4 nm ( Figure 1—figure supplement 1a ) .", "Most of the P2X7 receptor secretome examined , with the exception of IL-1β , was present in the soluble fraction and not associated with the 100K pellet ( Figure 1—figure supplement 1b ) .", "The presence of CD14 in extracellular vesicles obtained from macrophage supernatants after P2X7 receptor stimulation was also determined by using a protocol for extracellular vesicle isolation based in polymer-precipitation ( Figure 1b ) .", "The treatment of whole cellular supernatants with Triton X100 before vesicle isolation resulted in a loss of CD14 from the 100K pellet fraction that was detected in the soluble fraction ( 100K supernatant ) ( Figure 1c ) , suggesting CD14 is a cargo of the vesicles .", "The CD14 in the 100K pellet was detected just after 5 min of ATP stimulation ( Figure 1—figure supplement 1c ) and was independent of the macrophage activation polarity , as it was detected from M1 and M2 macrophages ( Figure 1d ) .", "However the number of extracellular vesicles released in LPS-primed macrophages ( M1 ) after P2X7 receptor stimulation was significantly higher than those released from IL-4 treated ( M2 ) or resting macrophages ( Figure 1e and Figure 1—figure supplement 2a ) .", "Interestingly , the release of CD14 induced by the P2X7 receptor in extracellular vesicles was highly dependent on the NLRP3 inflammasome , but not on caspase-1 ( Figure 1f ) .", "Then , we found that the amount of extracellular vesicles released after activation of the P2X7 receptor in Nlrp3−/− and Casp1/11−/− macrophages was smaller when compared with wild-type macrophages ( Figure 1g and Figure 1—figure supplement 2b ) .", "The morphology of Nlrp3−/−-derived extracellular vesicles was similar to that of resting or IL-4-primed wild-type macrophages ( Figure 1—figure supplement 2a , b ) .", "In contrast , Casp1/11−/−-derived extracellular vesicles were similar in morphology to LPS-primed wild-type macrophages ( Figure 1—figure supplement 2a , b ) , indicating that a specific pool of extracellular vesicle dependent on LPS-priming and NLRP3 could be enriched in CD14 and explain the differences of released CD14 found among Nlrp3−/− and Casp1/11−/− macrophages .", "The release of CD14 observed in ATP-treated macrophages resulted in a significant decrease in cell surface CD14 ( Figure 1h ) , thus suggesting that the P2X7 receptor could induce a decrease in CD14-dependent signaling in macrophages as well as being a source of extracellular CD14 .", "CD14 is a co-receptor of TLR4 important for LPS signaling ( Zanoni and Granucci , 2013 ) , so we investigated whether CD14 released after P2X7 receptor activation affect the signaling of LPS in macrophages .", "Treatment of macrophages with extracellular ATP and then subjected to LPS activation resulted in a decrease in LPS-induced expression and a secretion of the pro-inflammatory cytokines IL-6 and TNF-α ( Figure 2a , b ) .", "This effect was abrogated when the specific P2X7 receptor antagonist A438079 was used or when macrophages were isolated from P2rx7−/− mice ( Figure 2a , b ) , thus suggesting that the P2X7 receptor was affecting LPS signaling in macrophages .", "LPS is not able to activate TLR4 in the absence of CD14 at the cell membrane ( Pizzuto et al . , 2018 ) .", "By contrast , lipopolysaccharides with smaller hydrophilic moiety , like monophosphoryl lipid A ( MPLA ) , signals independently of CD14 ( Maeshima and Fernandez , 2013 ) .", "In order to investigate the role of P2X7 receptor-induced CD14 release in the reduction of LPS signaling , we tested whether also MPLA signaling was impaired by P2X7 receptor activation .", "When macrophages were stimulated with MPLA , the production of IL-6 and TNF-α was not affected when P2X7 receptor was activated ( Figure 2c , d ) .", "This effect was similar when CD14 was blocked with a specific antibody; when this was done , it decreased the production of both cytokines after LPS was added to stimulate the macrophages but not when MPLA was used ( Figure 2—figure supplement 1a , b ) .", "Our group has recently reported that in macrophages , activating the P2X7 receptor before LPS-priming also inhibits NLRP3 inflammasome , a phenomena mediated by P2X7 receptor-mediated mitochondrial damage ( Martínez-García et al . , 2019 ) .", "To assess the possible involvement of CD14 release in NLRP3 inhibition , we first measured Il1b gene expression and found that , similarly to Il6 and Tnfa , ATP treatment decreased the expression of Il1b when LPS was used to activate the cells , but not when MPLA was used ( Figure 2e ) , suggesting that the decrease of CD14 on cell membrane affects the priming by LPS .", "However , the release of IL-1β induced by nigericin decreased when macrophages were incubated with ATP before LPS- or MPLA-priming ( Figure 2f ) , which suggests that Il1b production , but not NLRP3 activation , was affected by the release of CD14 induced by the activation of the P2X7 receptor .", "The extracellular pool of CD14 increases during infection and sepsis ( Bas et al . , 2004 ) , and here we confirm that septic patients presented elevated levels of CD14 in the plasma when compared to non-septic volunteers ( Figure 3a and Supplementary file 1-Table 1 ) .", "Similarly , the presence of P2X7 receptor in monocytes was also elevated in septic individuals ( Figure 3a ) , in accordance to previous studies ( Martínez-García et al . , 2019 ) .", "To study if P2X7 receptor is important in maintaining the extracellular pool of CD14 during infection , we performed the cecal ligation and puncture ( CLP ) procedure in wild-type and P2rx7−/− mice and we found that the lack of P2X7 receptor expression resulted in reduced levels of cell-free CD14 in both serum and peritoneal lavage ( Figure 3b ) .", "Similarly , administration of the specific P2X7 receptor-antagonist A438079 to wild-type mice subjected to CLP resulted in a reduction in CD14 in the peritoneal lavage and a mild reduction in serum ( Figure 3c ) .", "This result could be probably due to the site of A438079 injection , which was i . p . , and to its short half-life , which hampers its ability to reach the blood serum ( McGaraughty et al . , 2007 ) .", "Furthermore , the increase in CD14 detected in the peritoneal lavage was mainly associated with the extracellular vesicle pool ( Figure 3d ) , and the presence of CD14 in extracellular vesicles decreased in P2X7 receptor deficient mice ( Figure 3d ) .", "These data suggest that during infection , the P2X7 receptor contribute to the presence of extracellular CD14 in extracellular vesicles .", "In order to determine if the P2X7 receptor-dependent release of CD14 during sepsis was impairing LPS-signaling in vivo , we measured cytokines in the serum of P2X7 deficient mice subjected to CLP .", "In line with our in vitro data , levels of IL-6 appeared higher in the P2rx7−/− mice after CLP compared to wild-type ( Figure 4a ) .", "This was also confirmed for other cytokines , chemokines and acute phase proteins measured in the serum of P2rx7−/− mice as well as in wild-type mice treated with A438079 ( Figure 4b , c ) , which suggests that P2X7 receptor is important for the downregulation of cytokines during sepsis .", "In order to elucidate if extracellular CD14 released by P2X7 has a role during sepsis , we next analyzed bacterial dissemination , as it is known that extracellular CD14 contributes to the clearance of invading bacteria and that the P2X7 receptor is important for controlling bacterial content during sepsis ( Csóka et al . , 2015; Lévêque et al . , 2017 ) .", "As expected , the bacterial burden increased in serum , peritoneal cavity and liver in P2rx7−/− compared to wild-type mice after CLP ( Figure 5a ) .", "Similarly , wild-type mice treated with the P2X7 receptor-antagonist A438079 also presented an increase in bacterial load ( Figure 5b ) .", "Administration of recombinant CD14 to P2rx7−/− mice before the CLP procedure resulted in a significant reduction in bacterial load in the serum , peritoneal lavage and liver ( Figure 5c ) .", "Cytokine and chemokine levels were reduced in the serum of P2rx7−/− mice after CLP and treatment with recombinant CD14 ( Figure 5d , e ) .", "These results suggest that extracellular CD14 is an important element in the P2X7 receptor secretome to control bacterial dissemination and cytokine production during sepsis .", "We and others have found that P2rx7−/− mice present higher mortality during sepsis ( Csóka et al . , 2015; Martínez-García et al . , 2019 ) , and here we also confirm that treating wild-type mice with the pharmacological P2X7 receptor-antagonist A438079 before CLP also increases the likelihood of mortality ( Figure 6a ) .", "In line , CLP resulted in higher decrease of mice body weight and poor well-being score on the supervision protocol in P2rx7−/− mice and in wild-type mice treated with A438079 ( Figure 6—figure supplement 1a , b ) .", "To test if the deficiency in extracellular CD14 in P2rx7−/− mice during sepsis would be detrimental for survival , we treated P2rx7−/− mice with recombinant CD14 before CLP and found that mice survival was significantly increased ( Figure 6b ) , as well as weight loss was preserved ( Figure 6—figure supplement 1a ) .", "This is in agreement with the reduction in the bacterial load induced by recombinant CD14 treatment ( Figure 5c ) .", "We next evaluated organ damage as a direct cause of sepsis mortality and we found that the liver of wild-type mice displayed an unstructured parenchyma and ballooning hepatocytes after CLP ( Figure 6c and Supplementary file 1-Table 2 ) , which was aggravated in P2rx7−/− mice or wild-type mice treated with A437089 and which further presented prominent steatosis and dying hepatocytes ( Figure 6c ) .", "Spleen and lung damage induced by CLP in P2rx7−/− mice or wild-type mice treated with A438079 were also exacerbated ( Figure 6—figure supplements 1c and 2 and Supplementary file 1-Table 2 ) .", "The spleen exhibited a severe depletion of white pulp with the presence of apoptotic bodies and congestion of red pulp ( Figure 6—figure supplements 1c and 2 ) , whereas the lungs showed a marked leukocyte infiltration and intra-alveolar capillary hemorrhages with alveolar thickening ( Figure 6—figure supplements 1c and 2 ) .", "Treating P2rx7−/− mice with recombinant CD14 before CLP resulted in less pronounced damage in liver , spleen , and lung ( Figure 6d and Figure 6—figure supplement 3 and Supplementary file 1-Table 2 ) .", "Altogether , our results demonstrate that the P2X7 receptor controls the release of CD14 in extracellular vesicles , impairing LPS signaling in myeloid cells and controlling bacteria and cytokine production during sepsis , thus reducing tissue damage and improving survival ." ], [ "Our study shows for first time that the cellular release of CD14 induced by the P2X7 receptor has two functional effects on the innate immune system:", "( i ) it decreases CD14-dependent pro-inflammatory signaling in macrophages and", "( ii ) it decrease bacterial dissemination , improving survival during sepsis .", "In macrophages , the activation of the P2X7 receptor controls many different responses , including the activation of the NLRP3 inflammasome or the unconventional release of different cellular proteins ( de Torre-Minguela et al . , 2016; Di Virgilio et al . , 2017 ) .", "The release mechanism for the secretome associated with P2X7 receptor activation is not characterized , but some are proteins released mainly by inflammasome-dependent pyroptosis ( de Torre-Minguela et al . , 2016 ) .", "In this study , we describe that the release of CD14 correlates with the extracellular vesicle fraction , together with the tetraspanin CD9 , rather than with the caspase-1 dependent pyroptotic soluble fraction .", "In fact , the release of CD14 was independent on caspase-1 activity .", "Due to the heterogeneity of extracellular vesicle populations released from cells and the fact that the P2X7 receptor has been associated with the release of different extracellular vesicles such as microvesicles and exosomes ( Kowal et al . , 2016; MacKenzie et al . , 2001; Qu et al . , 2009 ) , our data supports that CD14 could be mainly a component of exosomes because CD14 largely appears associated with the high-speed pellet containing ‘small’ vesicles of ~160 nm .", "However , we could not rule out CD14 containing extracellular vesicles and exosomes may originate in the plasma membrane because there is a net reduction in plasma-membrane-associated CD14 and the presence of CD9 or IL-1β in this fraction has been correlated with both exosomes and plasma membrane-derived ‘small’ vesicles ( Kowal et al . , 2016; MacKenzie et al . , 2001; Qu et al . , 2007 ) .", "Furthermore , the inflammasome deficiency does not affect the release of CD14 , but it does decrease the amount of ‘small’ extracellular vesicles after P2X7 receptor activation , being this finding in accordance with a previous study demonstrating that NLRP3 impairs the release of exosomes from P2X7 receptor-activated dendritic cells ( Qu et al . , 2009 ) .", "Therefore , it remains difficult to determine the exact nature of the extracellular vesicles containing CD14 .", "The release of CD14 occurred 5 min after stimulation in resting macrophages after brief activation of the P2X7 receptor .", "Under these circumstances , the NLRP3 inflammasome is not primed and therefore is not activated , thus protecting the cells from pyroptotic cell death ( Broz et al . , 2020 ) .", "However , it has been recently described that prolonged P2X7 receptor activation would lead to apoptosis in resting macrophages ( Bidula et al . , 2019 ) .", "The brief P2X7 receptor activation in resting macrophages with millimolar concentrations of ATP used in this study did not compromise cell viability and may resemble physiological conditions where ectonucleotidases provoke a fast ATP degradation in the extracellular milieu ( Eltzschig et al . , 2012 ) .", "Under these conditions , there is a reduction in the subsequent production of pro-inflammatory cytokines after a smooth LPS activation that requires CD14 to signal via the TLR4-MD2 complex ( Pizzuto et al . , 2018; Ryu et al . , 2017 ) .", "This suggests that the release of CD14 from macrophages impairs CD14 signaling , and probably also the translocation of TLR4 complex to endosomes , thus impairing TRAM-TRIF-dependent pathways ( Zanoni and Granucci , 2013 ) .", "However , cytokine production was not affected when macrophages were treated with MPLA after P2X7 receptor activation , because MLPA does not require CD14 to signal ( Jiang et al . , 2005; Maeshima and Fernandez , 2013 ) .", "The reduction in cytokine production upon LPS-priming induced by initial P2X7 receptor activation in macrophages is additional to the effect we have described on the inflammasome activation ( Martínez-García et al . , 2019 ) , because NLRP3 activation was affected when macrophages were primed using both LPS and MLPA .", "All these suggest that brief P2X7 receptor activation before LPS priming has a widespread inhibitory effect on the pro-inflammatory functions of the macrophage , which includes reduced CD14 signaling and NLRP3 inflammasome activation .", "However , it should be noted that the stimulation of P2X7 receptor after LPS priming enhance the release of pro-inflammatory cytokines ( de Torre-Minguela et al . , 2016; Solle et al . , 2001 ) .", "When P2X7 receptor is absent or pharmacologically blocked , the reduced levels of circulating CD14 during sepsis is accompanied by an increase of cytokine release .", "Lower levels of cytokines were restored by the addition of recombinant CD14 .", "This strongly suggests that the release of CD14 induced by P2X7 receptor during sepsis reduces the induction of cytokine by bacterial LPS , being in line with the fact that a reduced amount of CD14 at the plasma membrane impairs LPS and other PAMPs signaling ( Akashi-Takamura and Miyake , 2008; Baumann et al . , 2010; Weber et al . , 2012 ) and circulating CD14 binds LPS and impair its signaling from plasma membrane receptors ( Kitchens and Thompson , 2005 ) .", "CD14 release has been described during infection due to proteinase-dependent shedding; however , there is also a proteinase-independent CD14 release that is less well understood ( Wu et al . , 2019 ) .", "Our study demonstrates that the release of extracellular vesicles induced by P2X7 receptor activation is a pathway contributing to the extracellular pool of CD14 .", "During sepsis , cell-free CD14 is present in serum and other body fluids and has been proposed as a marker for septic patients ( Bas et al . , 2004; Zhang et al . , 2015 ) ; in fact , the presence of CD14 in the blood has been validated by different studies as a valuable prognostic capacity to predict mortality ( Behnes et al . , 2014 ) .", "CD14 increases in plasma during the first 24 hr after sepsis initiation and remains elevated at least during the first 8 days , conferring an exceptional long-term prognostic value over acute phase proteins or IL-6 that quickly decrease after 3–8 days of sepsis ( Behnes et al . , 2014; Martínez-García et al . , 2019 ) .", "This is similar to the CLP model presented in this study , where while serum IL-6 concentration remains constant between 1 and 2 days of sepsis initiation , CD14 increased .", "Extracellular CD14 is required for host defense and in particular for bacterial clearance ( Qin et al . , 2017; Knapp et al . , 2006; Sahay et al . , 2018; Wieland et al . , 2005 ) , being necessary for phagocytosis of bacteria ( Grunwald et al . , 1996; Lingnau et al . , 2007; Schiff et al . , 1997 ) .", "Likewise , we found that P2X7 receptor deficiency or its pharmacological inhibition reduces CD14 in peritoneal lavage and serum when mice are subjected to a sepsis model .", "In these circumstances , there is an increase in bacterial dissemination that was controlled by the exogenous reconstitution of extracellular CD14 .", "The decreased levels of CD14 in the infection foci of our model , the peritoneum of P2X7-deficient mice , could be then the cause of the increased to bacterial dissemination from peritoneum and infection of distant tissues and organs , thus compromising animal viability .", "This is in agreement with a previous study that found the P2X7 receptor to be important for bacterial clearance during sepsis ( Csóka et al . , 2015 ) .", "The dissemination of bacteria in the blood during sepsis exacerbates the immune response and leads to life-threatening complications , such as organ failure and ultimately death ( Barratt-Due et al . , 2017 ) .", "P2rx7-deficient mice present aggravated damage to different organs and premature deaths during sepsis and the administration of recombinant CD14 restores survival in the P2rx7−/−genotype mice .", "This effect is similar to wild-type mice , where the administration of CD14 increases survival ( Haziot et al . , 1995 ) .", "Therefore , P2X7 receptor-dependent release of CD14 seems to have a role in bacterial infection restraint , and while we studied CD14 release from macrophages , there are also reports indicating that non-hematopoietic cells such as epithelial or endothelial cells that also express the P2X7 receptor could release CD14 , thus also influencing innate immune functions during infection ( Jersmann , 2005 ) and in turn restoring homeostatic conditions after sepsis ( Zanoni and Granucci , 2013 ) .", "In conclusion , we have identified the release of CD14 by extracellular vesicles as part of the previously identified P2X7 receptor secretome of macrophages .", "The release of CD14 induced by the P2X7 receptor affects CD14 signaling in macrophages because the activation by smooth LPS was affected and fewer pro-inflammatory cytokines were produced .", "During sepsis , the elevation of CD14 levels in the serum and peritoneal lavage , also depended on the P2X7 receptor , were important in controlling cytokine secretion , restricting bacterial dissemination and organ damage , increasing overall survival .", "Therefore , circulating CD14 is not only a marker for sepsis but also an important component of the host’s innate immune system because the P2X7 receptor releases it in a regulated manner in order to control infection and increase survival during sepsis ." ], [ "All experimental protocols for animal handling were refined and approved by the Animal Health Service of the General Directorate of Fishing and Farming of the Council of Murcia ( Servicio de Sanidad Animal , Dirección General de Ganadería y Pesca , Consejería de Agricultura y Agua Región de Murcia , reference A1320140201 ) .", "C57BL/6 mice ( WT , wild-type , RRID:IMSR_JAX:000664 ) and P2X7 receptor-deficient mice in C57BL/6 background ( P2rx7−/−; RRID:IMSR_JAX:005576 ) ( Solle et al . , 2001 ) were obtained from the Jackson Laboratories .", "NLRP3-deficient ( Nlrp3−/− ) ( Martinon et al . , 2006 ) and Caspase-1/11 deficient ( Casp1/11−/− ) ( Kuida et al . , 1995 ) in C57BL/6 background were a generous gift from I . Coullin .", "For all experiments , mice between 8 and 10 weeks of age were used .", "Mice were bred in specific pathogen-free conditions with a 12:12 hr light-dark cycle and used in accordance with the Hospital Clínico Universitario Virgen Arrixaca animal experimentation guidelines , and Spanish national ( Royal Decree 1201/2005 and Law 32/2007 ) and EU ( 86/609/EEC and 2010/63/EU ) legislation .", "The cecal ligation and puncture ( CLP ) -induced sepsis procedure was performed in wild-type and P2rx7−/− mice as previously described ( Rittirsch et al . , 2009 ) .", "Briefly , a laparotomy was performed to isolate the cecum of mice anesthetized with isoflurane ( 3–5% for induction and 1 . 5–2% for maintenance and oxygen flow to 1 L/min ) .", "Approximately 2/3 of the cecum was ligated with a 6–0 silk suture and punctured twice through-and-through with a 21 gauge needle .", "The abdominal wall and incision were closed with 6–0 silk suture .", "Sham operated animals underwent laparotomy without ligation or puncture of the cecum .", "Buprenorphine ( 0 . 3 mg/kg ) was administered intraperitoneally ( i . p . ) at the time of surgery and mice were monitored continuously until recovery from anesthesia .", "Twenty-four or 48 hr after the procedure , the animals were euthanized by CO2 inhalation and peritoneal lavages and blood and tissue samples were collected .", "In some experiments , P2rx7−/− mice received an i . p . injection of human recombinant CD14 ( 10 µg/g , Peprotech , RRID:AB_2877062 ) or vehicle ( sterile physiologic saline ) 30 min prior to the CLP procedure .", "In some experiments , wild-type mice were injected with A438079 ( 100 µM/kg , i . p . ) or vehicle 1 hr prior to the CLP procedure .", "Blood samples were obtained by thoracic aorta and were centrifuged at 12 , 500g for 10 min .", "The recovered serum was stored at −80°C until further use .", "For collecting peritoneal lavage , the abdominal wall was exposed by opening the skin; 4 ml of sterile saline were injected into the peritoneal cavity via a 25 gauge needle .", "The abdomen was gently massaged for 1 min , and the peritoneal fluid was recovered through the needle and centrifuged at 433 g for 10 min to obtain a cell-free peritoneal lavage .", "The supernatant was stored at −80°C until further analysis .", "For tissue harvesting , the abdominal wall was exposed , the organs were removed using scissors and forceps , and they were fixed and paraffin-embedded or stored at −80°C for future analysis .", "Fresh liver samples were homogenized mechanically in sterile physiologic saline .", "Serum , peritoneal lavages , and tissue samples were diluted serially in sterile physiological saline and 100 μl of each dilution was plated in Luria-Bertani agar and cultured on agar plates at 37°C .", "After 24 hr of incubation , the number of bacterial colonies ( CFU ) was counted in the various dilutions and only used the dilutions where separate colonies were obtained .", "Bacterial load was calculated by multiplying CFUs to the corresponding dilution and divided by the volume inoculated to obtain the expressed CFU/ml of serum or peritoneal exudates or CFU/g of liver .", "Liver , spleen , and lung tissues were fixed in 4% p-formaldehyde ( PFA , Sigma ) for 24 hr , processed , paraffin-embedded , and sections stained with hematoxylin and eosin using standard methods to evaluate damage .", "Slides were examined using a Zeiss Axio Scope AX10 microscope with an AxioCamICC3 ( Carl Zeiss ) .", "Bone-marrow-derived macrophages ( BMDMs ) were obtained from wild-type , P2rx7−/− , Nlrp3−/− and Casp1/11−/− mice by differentiating bone marrow cells for 7 days in DMEM ( Lonza ) supplemented with 25% of L929 medium , 15% fetal calf serum ( FCS , Life Technologies ) , 100 U/ml penicillin/streptomycin ( Lonza ) , and 1% L-glutamine ( Lonza ) as described elsewhere ( Barberà-Cremades et al . , 2012 ) .", "Cells were primed with ultrapure E . coli LPS serotype O55:B5 ( 10 ng/ml , Invivogen ) or recombinant mouse IL-4 ( 20 ng/ml , BD Pharmingen , RRID:AB_2868873 ) for 4 hr .", "Cells were then washed three times with physiological buffer before and then stimulated for 20 min with ATP ( 3 mM , Sigma-Aldrich ) in E-total buffer ( 147mMNaCl , 10 mM HEPES , 13 mM glucose , 2 mM CaCl2 , 1 mM MgCl2 , and 2mMKCl , pH 7 . 4 ) .", "In other cases , cells were pretreated with ATP ( 3 mM ) in the presence or absence of the specific P2X7 receptor antagonist A438079 ( Tocris , 10–20 μM ) in E-total buffer and then washed and stimulated with LPS or 1 μg/ml ofS .", "Minnesota Monophosphoryl Lipid A ( MPLA , Invivogen ) for 4 hr .", "Then cells were treated with 10 μM of nigericin sodium salt ( Sigma-Aldrich ) for 30 min in E-total .", "In some experiments BMDMs were incubated with 20 μg/ml of the blocking antibody anti-CD14 clone M14-23 ( Biolegend ) before LPS or MPLA were added .", "Supernatants were collected and clarified at 14 , 000 g for 30 s at 4°C to remove floating cells and stored at −80°C until cytokine determination .", "Cells were lysed immediately in lysis buffer ( 50 mM Tris-HCl pH 8 . 0 , 150 mM NaCl , 2% Triton X-100 ) supplemented with 100 μl/ml of protease inhibitor mixture ( Sigma ) for 30 min on ice and then cell debris was removed by centrifugation at 16 , 000 g for 15 min at 4°C .", "Extracellular vesicles were purified as previously described ( Théry et al . , 2006 ) , diagram shown in Figure 1—figure supplement 2c .", "Briefly , differentiated BMDMs in 150 mm2 plates were washed with PBS and incubated 24 hr in medium with extracellular vesicle-depleted FBS .", "The cells were primed with 10 ng/mL LPS , 20 ng/ml IL-4 or complete cell culture media alone for 4 hr at 37°C , then washed three times with E-total buffer and incubated in the same buffer with ATP 3 mM for 20 min .", "The collected medium was immediately transferred into a tube containing Protease inhibitor mix ( Sigma ) on ice , and then followed by sequential centrifugation at 4°C for 20 min at 2000 g , ( Sigma 3-18KS , rotor 11180 and 13190 ) , 30 min at 10 , 000 g , and 1 hr at 100 , 000 g ( Beckman Ultracentrifuge Optima L-80 XP , SW40 rotor ) .", "The supernatant of this last step was stored at −80°C .", "The pellet from 100 , 000 g was washed in 10 ml of PBS and centrifuged again for 1 hr at 100 , 000 g .", "Finally , extracellular vesicle fraction was collected in the pellet with 50 ml of PBS and stored at −80°C until use .", "ExoQuick precipitation was carried out following the manufacturer’s instructions ( System Biosciences ) , diagram shown in Figure 1—figure supplement 2d .", "Briefly , 800 µl of cell culture supernatant or 2 ml or peritoneal lavage was diluted to 5 ml in PBS and mixed with 1 ml of ExoQuick-TC solution by inverting the tube several times .", "The sample was incubated overnight at 4°C then centrifuged twice at 3000 g for 10 min to isolate extracellular vesicles .", "Later , extracellular vesicles were centrifuged at 1 , 000 g for 30 s in order to purify them .", "Cells lysates , total cell-free supernatants , extracellular vesicle fraction , and extracellular vesicle-free supernatants were resolved in 4–12% precast Criterion polyacrylamide gels ( Biorad ) and transferred to nitrocelulose membranes ( Biorad ) by electroblotting as it is described in de Torre-Minguela et al . , 2016 .", "Cell-free and extracellular-free supernatants were precipitated overnight at −20°C with 6 vol of cold acetone .", "Membranes were probed with different antibodies: anti-CD14 rat monoclonal ( rmC5-3 , BD Pharmingen , RRID:AB_395020 ) , anti-CD9 rabbit monoclonal ( EPR2949 , ab92726 , Abcam , RRID:AB_10561589 ) , anti-MMR rat monoclonal ( MR5D3 , Acris Antibodies , RRID:AB_1611247 ) , anti-Cystatin B rat monoclonal ( Clone #227818 , R and D , RRID:AB_2086095 ) , anti-Cathepsin B rat monoclonal ( Clone #173317 , R and D , RRID:AB_2086935 ) , or anti-Peptidyl-prolyl cis-trans isomerase A rabbit polyclonal ( ab41684 , Abcam , RRID:AB_879768 ) .", "After ultracentrifugation , 100K pellet was analyzed with an NS300 nanoparticle tracking analysis ( NTA ) instrument , ( NanoSight Technology ) to determine the vesicle size distributions and concentrations .", "Data was analyzed with NTA 3 . 1 software ( RRID:SCR_014239 ) .", "Electron microscopy analysis was performed as previously described ( Théry et al . , 2006 ) on pellets of purified extracellular vesicle loaded on form var-carbon-coated grids and fixed in 2% PFA .", "Grids were observed at 80 kV with a JEM-1011 Transmission Electron Microscope ( JEOL Company ) .", "EV were counted for each preparation in five different random fields of TEM pictures taken at 25 , 000x .", "The number of EV was then normalized to the number of cells obtained in each treatment .", "For membrane CD14 flow cytometry , BMDMs seeded in 24-well plates were washed and incubated for 30 min at 37°C in E-total buffer supplemented with or without 5 mM of ATP , in presence or absence of P2X7 receptor antagonist A438079 ( 10 μM ) .", "To stain surface CD14 , cells were washed and incubated with mouse seroblock FcR ( BD biosciences ) and then stained with anti-mouse CD14 ( clone rmC5-3; 553738; BD biosciences; RRID:AB_395020 ) for 30 min at 4°C .", "Cells were washed again and incubated with secondary Alexa Fluor 647 goat anti-rat IgG ( H+L ) ( A21247; Invitrogen , RRID:AB_141778 ) for an additional 30 min at 4°C .", "Finally , cells were washed and fixed with 4% PFA in PBS and then scrapped and aliquoted in flow cytometry tubes .", "For human P2X7 flow cytometry , monocytes were determined from peripheral blood mononuclear cells from non-septic and septic patients by CD3− CD14+ selection , and P2X7 receptor surface expression was determined using the monoclonal anti-P2X7 L4 clone ( Buell et al . , 1998; Martínez-García et al . , 2019 ) .", "All samples were subjected to flow cytometry analysis using a BD FACSCanto flow cytometer ( BD ) and FACSDiva software ( BD , RRID:SCR_001456 ) by gating for BMDM cells based on FSC versus SSC parameters .", "BMDMs , plated in 96-well plates , were stimulated as described above .", "Total RNA extraction was performed using the RNAqueous Micro Kit ( Invitrogen ) , followed by reverse transcription using iScript cDNA Synthesis ( Bio-Rad ) with oligo-dT .", "The mix SYBR Premix ExTaq ( Takara ) was used for quantitative PCR in iCycler My iQ thermocycler ( Bio-Rad ) .", "Specific primers were purchased from Sigma ( KiCq Start SYBR Green Primers ) .", "Only a single product was seen on melting curve analysis , and for each primer set , the efficiency was >95% .", "For the relative expression of mouse Il6 , Tnfa , and Il1b , their Ct was normalized to the housekeeping gene Gapdh using the 2-ΔCt method .", "The samples and data from patients included in this study were provided by the Biobanco en Red de la Región de Murcia ( PT13/0010/0018 ) , which is integrated into the Spanish National Biobanks Network ( B . 000859 ) and approved by the clinical ethics committee of the Clinical University Hospital Virgen de la Arrixaca ( reference numbers PI13/00174 , 2019-9-4-HCUVA , 2019-12-15-HCUVA and 2019-12-14-HCUVA ) .", "All study procedures were conducted in accordance with the declaration of Helsinki .", "Whole peripheral blood samples were collected after receiving written informed consent from intraabdominal sepsis patients ( n = 9 , Supplementary file 1-Table 1 ) at the Surgical Critical Unit from the Clinical University Hospital Virgen de la Arrixaca .", "The blood samples were obtained from septic individuals within 24 hr of the diagnosis of sepsis .", "The inclusion criteria for septic patients were patients diagnosed with intra-abdominal origin sepsis confirmed by exploratory laparotomy , with at least two diagnostic criteria for sepsis ( fever or hypothermia; heart rate >90 beats per minute; tachypnea , leukocytosis , or leukopenia ) and multiple organ dysfunction defined as physiological dysfunction in two or more organs or organ systems ( Singer et al . , 2016 ) .", "We also recruited non-septic volunteers and after they had signed their informed consent agreement whole peripheral blood samples were collected ( n = 10 ) .", "Sera was isolated and stored at −80°C until use .", "Individual culture cell-free supernatants were collected and clarified by centrifugation .", "The concentration of IL-6 ( RRID:AB_2877063 ) , TNF-α ( RRID:AB_2877064 ) , IL-1β ( RRID:AB_2574946 ) , and CD14 ( RRID:AB_2877065 ) was tested by ELISA following the manufacturer’s instructions ( R and D Systems and Thermo Fisher ) .", "Mice serum and peritoneal lavages were collected and the concentration of IL-6 and CD14 was also tested by ELISA ( R and D Systems ) .", "Results were read in a Synergy Mx ( BioTek ) plate reader .", "Multiplexing in mice serum for MCSF , CRP , RAGE , Resistin , VEGF , MIP-1α , MIP-1β , MIP-2 , CCL2 , CCL5 , IL-1α , IL-5 and IL-10was performed using the Luminex color-coded superparamagnetic beads array from R and D Systems following the manufacturer indications , and the results were analyzed in a Bio-Rad Bio-Plex analyzer .", "Statistical analyses were performed using GraphPad Prism 7 ( Graph-Pad Software , Inc , RRID:SCR_002798 ) .", "For two-group comparisons , the Mann-Whitney test was used and Kaplan-Meier survival curves were plotted and the log-rank test was undertaken to determine the statistical significance .", "The χ2 test was used to determine whether there was a significant difference between different clinical variables among groups of septic patients , except for age , where a one-way ANOVA test was used .", "For mouse in vivo data and before statistical analysis , possible outliers were identified with the robust regression followed by outlier identification method with Q = 1% and were eliminated from the analysis and representation .", "All data are shown as mean values and error bars represent standard error from the number of independent assays indicated in the figure legend .", "p Value is indicated as *p<0 . 05; **p<0 . 01; ***p<0 . 001; ****p<0 . 0001; p>0 . 05 not significant ( ns ) ." ] ]
[ "P2X7 receptor activation induces the release of different cellular proteins , such as CD14 , a glycosylphosphatidylinositol ( GPI ) -anchored protein to the plasma membrane important for LPS signaling via TLR4 .", "Circulating CD14 has been found at elevated levels in sepsis , but the exact mechanism of CD14 release in sepsis has not been established .", "Here , we show for first time that P2X7 receptor induces the release of CD14 in extracellular vesicles , resulting in a net reduction in macrophage plasma membrane CD14 that functionally affects LPS , but not monophosphoryl lipid A , pro-inflammatory cytokine production .", "Also , we found that during a murine model of sepsis , P2X7 receptor activity is important for maintaining elevated levels of CD14 in biological fluids and a decrease in its activity results in higher bacterial load and exacerbated organ damage , ultimately leading to premature deaths .", "Our data reveal that P2X7 is a key receptor for helping to clear sepsis because it maintains elevated concentrations of circulating CD14 during infection ." ]
[ "When the immune system detects an infection , it often launches an inflammatory response to fight off the disease .", "This defense mechanism is activated by a cascade of signaling molecules that can aggravate inflammation , causing it to damage the body’s own tissues and organs .", "This life-threatening reaction is referred to as sepsis , and kills around 11 million people each year .", "New approaches are therefore needed to help alleviate the damage caused by this condition .", "The inflammatory response is often triggered by proteins called receptors , which sit on the surface of immune cells .", "When these receptors are activated , they induce cells to secrete proteins that travel around the body and activate immune cells that can eliminate the infection .", "In 2016 , a group of researchers showed that a receptor called P2X7 stimulates the release of a signaling molecule called CD14 .", "Patients with sepsis often have elevated amounts of CD14 in their bloodstream .", "Yet , it remained unclear what causes this rise in CD14 and what role this molecule plays in the development of sepsis .", "Now , Alarcón-Vila et al . – including some of the researchers involved in the 2016 study – have investigated the role of P2X7 in mice undergoing sepsis .", "This was done by puncturing the mice’s intestines , causing bacteria to leak out and initiate an over-active immune response .", "Alarcón-Vila et al . found that mice lacking the P2X7 receptor had less CD14 and struggled to eliminate the bacterial infection from their system .", "This increase in bacteria caused excessive damage to the mice’s organs , ultimately leading to premature death .", "These findings suggest that P2X7 plays an important role in preventing the onset of sepsis by helping maintain high levels of CD14 following infection .", "This result could help to identify new therapies that reduce the mortality rates of septic infections ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "chromosomes and gene expression", "genetics and genomics" ]
Dynamics of genomic innovation in the unicellular ancestry of animals
elife-26036-v3
[ [ "The transition from a unicellular organism to the first multicellular animal , more than 600 million years ago ( Budd and Jensen , 2017; dos Reis et al . , 2015 ) , marks one of the most radical evolutionary innovations within the eukaryotes .", "Although multicellularity has independently evolved multiple times in the eukaryotic lineage , the highest levels of organismal complexity , body plan diversity and developmental regulation are found in the Metazoa ( Grosberg and Strathmann , 2007 ) .", "Key advances in the study of animal origins have been made by comparing the genomes of early branching metazoa , such as cnidarians , ctenophores or sponges ( Putnam et al . , 2007; Srivastava et al . , 2010a; Moroz et al . , 2014; Srivastava et al . , 2008; Fortunato et al . , 2014 ) , with their closest unicellular relatives in the Holozoa clade , such as the choanoflagellates Monosiga brevicollis and Salpingoeca rosetta ( King et al . , 2008; Fairclough et al . , 2013 ) , and the filasterean Capsaspora owczarzaki ( Suga et al . , 2013 ) ( Figure 1 ) .", "By focusing on the transition , it is possible to determine which genomic innovations occurred at the origin of metazoa , and whether it required the invention of novel genes or structural features . 10 . 7554/eLife . 26036 . 003Figure 1 . Evolutionary framework and genome statistics of the study .", "( A ) Schematic phylogenetic tree of eukaryotes , with a focus on the Holozoa .", "The adjacent table summarizes genome assembly/annotation statistics .", "Data sources: red asterisks denote Teretosporea genomes reported here; double asterisks denote organisms sequenced for this study; † previously sequenced genomes ( King et al . , 2008; Fairclough et al . , 2013; Suga et al . , 2013 ) ; ‡ organisms for which transcriptomic data exists but no genome is available ( Torruella et al . , 2015 ) .", "( B ) Overview of the phenotypic traits of each group of unicellular Holozoa , focusing on their multicellular-like characteristics .", "For further details , see ( Torruella et al . , 2015; Mendoza et al . , 2002; Marshall et al . , 2008; Glockling et al . , 2013 ) .", "Figure 1—source data 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 00310 . 7554/eLife . 26036 . 004Figure 1—source data 1 . Table of genome structure statistics , from the data-set of eukaryotic genomes used in the study . Includes genome size and portion of the genome covered by genes , exons , introns and intergenic regions .", "Used in Figures 1 , 3 and 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 00410 . 7554/eLife . 26036 . 005Figure 1—source data 2 . List of genome and transcriptome assemblies and annotations , including abbreviations , taxonomic classification and data sources . Used in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 00510 . 7554/eLife . 26036 . 006Figure 1—figure supplement 1 . Comparisons of gene length of one-to-one orthologs from pair-wise comparisons of all 10 unicellular Holozoa . Dots around the diagonal lines indicate that orthologs from both organisms have identical lengths .", "Note that Abeoforma and Pirum have abundant incomplete orthologous sequences . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 006 We now know that the animal ancestor was already a genomically complex organism , with a rich complement of genes encoding proteins related to a multicellular lifestyle .", "These include transcription factors , extracellular matrix components and intricate signaling pathways that were previously considered animal-specific , but were already poised to be co-opted for multicellularity when animals emerged ( Fairclough et al . , 2013; Suga et al . , 2013; Richter and King , 2013; Manning et al . , 2008; Suga et al . , 2012; de Mendoza et al . , 2013; Sebé-Pedrós et al . , 2017 ) .", "Suggestively , detailed analyses of the transcriptomic and proteomic regulatory dynamics of Capsaspora and Salpingoeca showed that these genes are frequently implicated in the transition to life stages reminiscent of multicellularity – aggregative in Capsaspora ( Sebé-Pedrós et al . , 2013 , Sebé-Pedrós et al . , 2016a ) , and clonal colonies in Salpingoeca ( Fairclough et al . , 2013 ) .", "Furthermore , the genome architectures of extant Metazoa are , in many aspects , markedly different from most other eukaryotes: they have larger genomes ( Elliott and Gregory , 2015a ) , containing more ( Csuros et al . , 2011 ) and longer introns ( Elliott and Gregory , 2015a ) that can sustain alternative splicing-rich transcriptomes ( McGuire et al . , 2008; Irimia and Roy , 2014 ) , have richer complements of repetitive sequences such as transposable elements ( Elliott and Gregory , 2015b ) and are structured in ancient patterns of gene linkage associated with transcriptional co-regulation ( Irimia et al . , 2012; Simakov et al . , 2013 ) – e . g . , the Homeobox clusters ( Ferrier , 2016 ) .", "The relationship between these patterns of genome evolution and multicellularity is , however , unclear: these traits are not exclusive of animals ( cf .", "( Curtis et al . , 2012; Shoguchi et al . , 2013; Michael , 2014; de Mendoza et al . , 2015; French-Italian Public Consortium for Grapevine Genome Characterization et al . , 2007 ) ) ; the existence of secondarily reduced genomes in animals ( smaller , gene-compact , less repetitive ) in animals blurs their link with organismal complexity ( Simakov and Kawashima , 2017; Seo et al . , 2001; Petrov et al . , 1996 ) ; and non-adaptive scenarios can explain the emergence of genomic complexities as a consequence drift-enhancing population-genetic environments ( Lynch and Conery , 2003; Lynch , 2002 , Lynch , 2007 ) .", "Establishing the timeline of genome architecture evolution in the ancestry of Metazoa is thus essential to understand to which extent genomic complexity is linked to multicellularity .", "Overall , gene content has been extensively studied in the unicellular ancestry of animals , but less attention has been devoted to the evolution of genome architecture in this period – covering features such as the repetitive content , intron creation and synteny conservation ( although cf .", "( King et al . , 2008; Irimia et al . , 2012 ) ) .", "This bias is partly due to the multi-million year gap separating animals from their unicellular relatives and the limited genome sampling of unicellular holozoans .", "We now know several examples of the effects of such limitations .", "For instance , our view of the transcription factor repertoire of the animal ancestor was confounded by the gene losses of Monosiga , which only became evident when Capsaspora genome was analyzed ( Sebé-Pedrós et al . , 2011 ) ; and the same happened with the ancestral animal diversity of cadherin and integrin adhesion systems before genomes from choanoflagellates and Capsaspora were analyzed ( Nichols et al . , 2012; Sebé-Pedrós et al . , 2010 ) .", "Therefore , comparative genomics studies are highly sensitive to taxonomic biases , meaning that rare genomic changes can remain elusive , and more frequent events can manifest saturated evolutionary signals .", "To overcome these limitations , we analyze the genomes of the third lineage of close unicellular relatives of animals , the Teretosporea , composed of Ichthyosporea and Corallochytrium limacisporum ( Torruella et al . , 2015 ) .", "As the earliest-branching holozoan clade , Teretosporea are in a key phylogenetic position to complement our current view of premetazoan evolution .", "Interestingly , they display a developmental mode that radically differs from choanoflagellates and filastereans: many ichthyosporeans have a multinucleate coenocytic stage ( Mendoza et al . , 2002; Marshall et al . , 2008 ) , and Corallochytrium develops colonies by binary , palintomic , cell division ( Raghukumar , 1987 ) .", "In both cases , completion of the life cycle frequently involves release of propagules that restart the clonal proliferation ( Mendoza et al . , 2002; Marshall et al . , 2008 ) .", "In addition , the ichthyosporean Creolimax fragrantissima exhibits many features reminiscent of animals , such as transcriptional regulation of cell type differentiation or synchronized nuclei division during its development ( de Mendoza et al . , 2015; Suga and Ruiz-Trillo , 2013 ) .", "Here , we present the complete genomes of four newly sequenced organisms: Corallochytrium limacisporum and the ichthyosporeans Chromosphaera perkinsii ( gen . nov . , sp . nov . ) , Pirum gemmata and Abeoforma whisleri .", "These are added to the already available Creolimax fragrantissima , Ichthyophonus hoferi and Sphaeroforma arctica ( de Mendoza et al . , 2015; Torruella et al . , 2015 ) ( Ichthyosporea ) , and to the afore-mentioned Salpingoeca rosetta , Monosiga brevicollis ( choanoflagellates ) and Capsaspora owczarzaki ( Filasterea ) , totaling 10 unicellular holozoan genomes ( Figure 1 ) .", "Our aim is to provide new insights into the evolutionary dynamics of the genome in the ancestral unicellular lineage leading to animals , at two broad levels: gene family origin and diversification , and conservation of genome architectural features .", "We address the origin of the large and intron-rich animal genomes , changes in gene linkage ( microsynteny ) , and ancient patterns of gene family diversification .", "The leitmotiv of these analyses is to identify and date genomic novelties along the ancestry of Metazoa , aiming to understand the foundations of the transition to multicellularity .", "The emerging picture from this comparative study is one of punctuated , differently-timed bursts of innovation in genome content and structure , occurring in the unicellular ancestry of animals ." ], [ "We obtained the complete nuclear genome sequences of Corallochytrium limacisporum and the ichthyosporeans Chromosphaera perkinsii , Pirum gemmata and Abeoforma whisleri .", "For all these taxa , we sequenced genomic DNA from axenic cultures using Illumina paired-end and mate-pair reads , which were assembled using Spades ( Nurk et al . , 2013 ) .", "Gene annotation was performed using a combination of de novo gene predictions and transcriptomic evidence derived from RNA sequencing experiments ( see Methods ) .", "Of the four genomes presented here , Corallochytrium ( 24 . 1 Mb ) and Chromosphaera ( 34 . 6 Mb ) have the highest completeness and contiguity ( Figure 1 ) .", "Specifically , Corallochytrium has 7535 genes and 83 . 4% of the BUSCO paneukaryotic gene set ( a proxy to genome completeness ( Simão et al . , 2015 ) ) , and 75% of the assembly length is covered by 86 scaffolds ( L75 statistic ) .", "Chromosphaera has 12 , 463 annotated genes comprising 85 . 5% of the BUSCO set , and its L75 statistic is 187 scaffolds .", "In contrast , Abeoforma and Pirum have larger genome assemblies ( 101 . 9 and 84 . 4 Mb ) , but these are fragmented ( L75 = 25 , 133 and 25 , 440 scaffolds ) and incomplete ( 11 . 9% and 17 . 0% of BUSCO ) .", "These lower contiguities are reflected in their partial gene predictions ( Figure 1A , Figure 1—figure supplement 1 ) , which consequently hindered the detection of BUSCO orthologs .", "Overall , together with Capsaspora , the two choanoflagellates and three already available ichthyosporeans , our expanded dataset now comprises 10 genomes from all unicellular Holozoa lineages – eight more than in previous genome analyses ( Fairclough et al . , 2013; Suga et al . , 2013 ) .", "To have a robust phylogenetic framework for our comparative analyses , we investigated the phylogenetic relationships between holozoans with a phylogenomic analysis based on the dataset developed in Torruella et al . ( 2015 ) .", "We classified the newly identified Chromosphaera perkinsii ( gen . nov . , sp . nov . ) as a member of Ichthyosporea , in the order Dermocystida , as it clusters with Sphaerothecum destruens in our phylogenomic analysis ( Figure 2; BS = 100% , BPP = 1 ) .", "Therefore , Chromosphaera , isolated from shallow marine sediments in Hawai'i , is the first described putatively free-living dermocystid Ichthyosporea .", "Indeed , all described dermocystids are strict vertebrate parasites , whereas ichthyophonids are typical animal commensals or parasites ( although free-living species have been described and some have been identified in environmental surveys of marine microbial eukaryotic diversity ) ( del Campo and Ruiz-Trillo , 2013; Glockling et al . , 2013 ) . 10 . 7554/eLife . 26036 . 007Figure 2 . Phylogenomic tree of Unikonta/Amorphea . Phylogenomic analysis of the BVD57 taxa matrix .", "Tree topology is the consensus of two Markov chain Monte Carlo chains run for 1231 generations , saving every 20 trees and after a burn-in of 32% .", "Statistical supports are indicated at each node:", "( i ) non-parametric maximum likelihood ultrafast-bootstrap ( UFBS ) values obtained from 1000 replicates using IQ-TREE and the LG + R7+C60 model;", "( ii ) Bayesian posterior probabilities ( BPP ) under the LG+Γ7 + CAT model as implemented in Phylobayes .", "Nodes with maximum support values ( BPP = 1 and UFBS = 100 ) are indicated by a black bullet .", "See Figure 2—figure supplement 1 for raw trees with complete statistical supports .", "Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 00710 . 7554/eLife . 26036 . 008Figure 2—source data 1 . BVD57 phylogenomic dataset ( Torruella et al . , 2015 ) including 87 unaligned protein domains ( with PFAM accession number ) per species . Used in Fig . 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 00810 . 7554/eLife . 26036 . 009Figure 2—figure supplement 1 . Phylogenomic analysis of the BVD57 matrix using ( A ) IQ-TREE maximum likelihood and the LG + R7+C60 model ( supports are SH-like approximate likelihood ratio test/UFBS , respectively ) ; ( B ) IQ-TREE maximum likelihood and the LG + R7+PMSF model ( fast CAT approximation; non-parametric bootstrap supports ) ; and ( C ) Phylobayes Bayesian inference under the LG+Γ7 + CAT model ( BPP supports ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 009 Our analysis confirms our previous results with regards to the phylogenetic relationships within Holozoa: the Teretosporea , comprising Ichthyosporea and the small free-living osmotroph Corallochytrium ( Raghukumar , 1987 ) , are a sister-group to all the other holozoans ( filastereans , choanoflagellates and animals ) with improved statistical support ( Figure 2; BS = 93% , BPP = 0 . 85 ) .", "The monophyly of Teretosporea rejects alternative scenarios such as the ‘Filasporea’ hypothesis ( a grouping of Filasterea + Ichthyosporea ) ( Ruiz-Trillo et al . , 2008; Liu et al . , 2009 ) or the status of Corallochytrium as an independent opisthokont lineage .", "The Monosiga genome paper by King et al . ( 2008 ) revealed that much of the innovation in gene content seen in the transition to multicellularity is rooted in pervasive 'tinkering' with preexisting gene families , notably by rearrangements of protein domains .", "This mechanism , combined with gene duplication , allows for a functional diversification of gene families by tuning the interactions with other components of the cell—its substrate specificities , sub-cellular localization or partnerships with other proteins within larger complexes .", "Albeit protein domain rearrangements are not uncommon in eukaryotes ( Basu et al . , 2008 , Basu et al . , 2009; Leonard and Richards , 2012 ) , this process is specifically credited with the diversification of many gene families involved in complex signaling and/or multicellular integrated lifestyle in Metazoa ( Suga et al . , 2012; Simakov and Kawashima , 2017; Sebé-Pedrós et al . , 2010; Tordai et al . , 2005; Ekman et al . , 2007; Hynes , 2012; Deshmukh et al . , 2010; Grau-Bové et al . , 2015 ) .", "Here , we present a comprehensive study of gene diversification in Holozoa , using our taxon-rich genomic dataset to reconstruct its effect in the animal ancestry .", "We thus performed a comparative analysis of protein domain architectures across eukaryotes , using the rates of domain rearrangement ( or shuffling ) as a proxy for gene family diversification .", "We compared the phylogenetic distribution of protein domain co-occurrences across species and gene families ( using a dataset comprising 26 , 377 gene families or clusters of orthologs derived from 40 eukaryotic species ( see Methods ) .", "We inferred rates of domain rearrangement at ancestral nodes of the eukaryotic tree using a probabilistic birth-and-death model ( Csűrös and Miklós , 2006 ) to reconstruct the content of specific protein domain architectures in ancestral genomes ( available as Figure 7 ) .", "In our approach , pairs of domains can create novel combinations ( 'gain' ) that diversify existing gene families , or dissociate domains ( 'loss' ) , which results in decreased diversity of multi-domain proteins . 10 . 7554/eLife . 26036 . 026Figure 7 . Evolution of protein domain architectures .", "( A ) Protein domain combination gain and loss per lineage , including extant genomes and ancestral reconstructed nodes .", "Diameter and color of circles denote the number of different domain combinations ( in different gene families ) in that node of the tree .", "Bolder edges mark the line of descent between the LCAs of Opisthokonta and Bilateria , which was generally dominated by gains of protein domain combinations ( see text ) .", "Red and green bars represent the inferred number of gains and losses , respectively .", "( B ) Gain/loss ratio of protein domain diversity in selected ancestors , including animals ( upper chart; from Metazoa to Unikonta/Amorphea ) and unicellular holozoans ( lower ) .", "Heatmap to the right represents the log-ratio value of the diversification rate for selected sub-sets of functionally-related protein domains relevant to multicellularity: green indicates higher-than-average diversification; pink less; white asterisks indicate two-fold or more increases or decreases ( all comparisons relative to the whole set of protein domains ) .", "Source Data Figure 7—source data 1 , 2 , 3 and 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 02610 . 7554/eLife . 26036 . 027Figure 7—source data 1 . Rates of gain and loss of protein domain pairs within a given orthogroup for extant and ancestral eukaryotes , calculated for a phylogenetic birth-and-death probabilistic model that accounts for gains , losses and duplications ( Csurös , 2010 ) .", "Used in Figures 7 , 8 and 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 02710 . 7554/eLife . 26036 . 028Figure 7—source data 2 . Reconstruction of the evolutionary histories of protein domain pairs gains within orthogroups , using a phylogenetic birth-and-death probabilistic model that accounts for gains , losses and duplications ( Csurös , 2010 ) .", "Used in Figures 7 , 8 , 9 and 10 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 02810 . 7554/eLife . 26036 . 029Figure 7—source data 3 . Reconstruction of the evolutionary histories of individual protein domains , using Dollo parsimony and accounting for gains and losses ( Csurös , 2010 ) .", "Used in Figures 7 , 8 , 9 and 10 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 02910 . 7554/eLife . 26036 . 030Figure 7—source data 4 . Rates of gain and loss of orthogroups for extant and ancestral eukaryotes , using a phylogenetic birth-and-death probabilistic model that accounts for gains , losses and duplications . Used in Figures 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 03010 . 7554/eLife . 26036 . 031Figure 7—figure supplement 1 . Gains and losses of individual protein domains across eukaryotes .", "( A ) Ancestral reconstruction of gains ( green ) and losses ( red ) of protein domains per lineage , based on Dollo parsimony .", "Note that , in contrast with the evolution of protein domain combinations here most nodes are dominated by losses .", "The most notable exceptions are the Metazoa and Opisthokonta LCAs ( dominated by gains ) and its unicellular ancestors from Holozoa to Choanoflagellata + Metazoa LCAs ( in which gains and losses are in a relative stasis ) .", "Figure 7—source data 3 .", "( B ) Log-ratio of gains-to-losses for single protein domains ( brown bars ) and protein domain pairs ( blue bars ) , based on the respective ancestral reconstructions .", "Positive values thus mean that gains are greater than losses .", "Loss of single protein domains dominates in most nodes , but gains in protein domain combinations can nevertheless outweigh losses in many ancestors ( e . g . , Holozoa or Metazoa LCAs ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26036 . 031" ], [ "We have identified continued process of gene innovation in terms of protein domain architectures in the animal ancestry , peaking at the LCA of Holozoa .", "This burst of diversification , enriched in TFs and ECM-related domains ( Figure 7B ) , set the foundations of the animal-like gene tool-kits of unicellular holozoans that have been reported in previous studies of gene family evolution regarding signaling pathways ( Suga et al . , 2012; Grau-Bové et al . , 2015 , 2013 ) , cell adhesion systems ( de Mendoza et al . , 2015; Nichols et al . , 2012; Sebé-Pedrós et al . , 2010 ) and transcription factors , often involved in developmental processes ( de Mendoza et al . , 2013; Sebé-Pedrós et al . , 2011 ) .", "The expansion of protein diversity in early holozoans provided fertile ground for the frequent co-option of ancestral genes for multicellular functions in Metazoa ( Richter and King , 2013 ) .", "Overall , our probabilistic reconstruction of the genome content of unicellular animal ancestors ( available as Figure 7—source data 7 ) provides a useful framework for targeted analysis of gene evolution and protein domain architecture evolution .", "As case-in-point examples of our approach , we have established the premetazoan origin of the transcription factors LIM Homeobox ( present in Ichthyopsorea and Capsapsora ) and p300/CBP-like ( all unicellular Holozoa ) ( Figure 9C–E ) , and canonical Type IV collagens , a key element of the animal ECM ( Hynes , 2012 ) ( present in the filasterean amoeba Ministeria vibrans ) .", "We have also investigated the time of origin of intron-rich genomes in Holozoa .", "We detect three independent episodes of massive intron gain: ( 1 ) at the root of Metazoa , ( 2 ) the shared LCA between Metazoa and Choanoflagellata , and ( 3 ) the root of ichthyophonid Ichthyosporea ( Creolimax , Sphaeroforma and Ichthyophonus ) .", "Furthermore , since the early origin of introns in the earliest eukaryotes ( Irimia and Roy , 2014 ) , the ancestry of both animals and ichthyophonids maintained a state of high intron density .", "The evolutionary implications of this circumstance , however , remain an open question .", "First , the independent intron gain episodes of animals and unicellular holozoans are mirrored by two different modes of alternative splicing dominating in each clade: animal transcriptomes are rich in isoform-producing exon skipping ( McGuire et al . , 2008; Irimia and Roy , 2014 ) , whereas most of the alternatively spliced transcripts of Capsaspora ( Sebé-Pedrós et al . , 2013 ) and Creolimax ( de Mendoza et al . , 2015 ) originate by intron retention and are thus more similar to the putative ancestral eukaryote than to Metazoa ( Irimia and Roy , 2014 ) .", "Second , we here show that the holozoan LCAs that underwent intron invasions ( Ichthyophonida , Metazoa and Metazoa + Choanoflagellata ) all possessed the essential NMD machinery and a rich complement of assisting splicing factors ( Figure 5C ) .", "Thus , they were in principle able to reduce the costs imposed by slightly deleterious intron invasions , as predicted by the mutational-hazard hypothesis ( Lynch and Conery , 2003; Lynch , 2002 , Lynch , 2006 ) .", "And third , the protracted state of high intron density in the ancestry of Metazoa and Ichthyophonida could have contributed to maintaining high levels of transcriptome variability that could in turn be co-opted for potentially adaptive , regulated AS events ( Irimia and Roy , 2014; Koonin et al . , 2013 ) .", "However , we cannot at present elucidate the relative importance of adaptation and population-genetic effects in the holozoans’ intron gain episodes: further transcriptomic analyses of unicellular holozoans are required to confirm that intron retention is their ancestrally prevalent AS mode ( Sebé-Pedrós et al . , 2013; Irimia and Roy , 2014; de Mendoza et al . , 2015 ) ; and the scant data on unicellular holozoans’ population genetics hampers the interpretation of genome architecture evolution under the light of the mutational-hazard hypothesis ( Lynch and Conery , 2003; Lynch , 2002 ) .", "We also addressed the evolution of genome size across holozoans .", "The emergence of larger genomes in Metazoa cannot be explained solely by intron gain and gene family expansion ( Elliott and Gregory , 2015a ) .", "Unfortunately , other factors such as the contribution of TE invasions ( Figure 3B ) or the extension of intron sites are not possible to date at the holozoan-wide evolutionary scale due to the lack of conserved signals .", "A possible way out of the conundrum is to study the conserved functions in the non-coding parts of the genome .", "For example , the compact genome of Capsaspora ( median intergenic regions: 373 bp ) has intragenic cis-regulatory elements key to its temporal regulation of cell differentiation ( Sebé-Pedrós et al . , 2016b ) , but the putative regulatory functions in the larger intergenic regions of Creolimax , Sphaeroforma and Salpingoeca ( median intergenic 900–1200 bp ) remain uncharacterized .", "It is tantalizing to note that ( 1 ) Creolimax and Salpingoeca exhibit temporal differentiation of cell types ( Fairclough et al . , 2013; de Mendoza et al . , 2015 ) , and ( 2 ) their intergenic median sizes are in line with those of Amphimedon ( 885 bp ) ( Figure 1—source data 1 ) , a demosponge with bilaterian-like promoters and enhancers , including distal regulation ( Gaiti et al . , 2017a , Gaiti et al . , 2017b ) .", "However , the ancestral gene linkages conserved across Metazoa , frequently due to common cis-regulation ( Irimia et al . , 2012 ) , appear to be animal innovations absent in unicellular holozoans ( Figure 3—figure supplement 1 ) .", "We thus propose that homologous regulatory regions would be rarely conserved between animals and unicellular holozoans; and only common types of regulatory elements could be expected , e . g . distal enhancers or developmental promoters .", "Overall , our results show that extant holozoan genomes have been shaped by both differential retention of ancestral states and secondary innovations , for the multiple genomic traits analyzed here , namely genome size , intron density , synteny conservation , protein domain diversity and gene content ( reviewed in ( Richter and King , 2013 ) ) .", "We can thus conclude that the genomes of unicellular premetazoans were shaped by independent evolutionary pressures on different traits , as has been seen in Metazoa ( Simakov and Kawashima , 2017 ) .", "Our findings can help to delimit the implicit trade-offs of choosing a unicellular model organism for functional and comparative studies with Metazoa , taking into account the loss of animal-like genomic traits relevant to different analyses .", "For example , phylogenetic distances between orthologous genes are shorter between some ichthyosporeans and animals than between choanoflagellates and animals ( Figure 3D ) , yet choanoflagellates are more similar to the animal ancestor in terms of intron structure ( Figure 6A ) and have lower rates of protein domain diversity loss ( Figure 7B ) .", "Interestingly , Capsaspora emerges as a well-suited model with a slow pace of genomic change attested for multiple traits: intron evolution , coding sequence conservation , gene order and ( possibly ) genome size .", "Its remarkable microsynteny conservation with Corallochytrium and Chromosphaera indicates the existence of ancestral holozoan gene linkages that have been disrupted , and rewired , in extant choanoflagellates , ichthyosporeans and animals ( Figure 3C ) .", "However , Capsaspora's lack of close sister groups hampers comparative studies of faster-evolving genomic features , be it the regulatory circuitry ( Sebé-Pedrós et al . , 2016b ) , or co-option of genetic tool-kits for its unique aggregative development ( Sebé-Pedrós et al . , 2013 ) .", "The new genomes from Ichthyosporea and Corallochytrium analyzed here provide novel insights into the reconstruction of premetazoan genomes .", "The Teretosporea clade has a deeper sampling than other unicellular holozoans and exhibit a mixture of slow- and fast-evolving genomic traits , which provides novel insights into the independence of genomic characters during premetazoan evolution .", "For example , Ichthyophonus tends to retain the ancestral intron/exon structure ( Figure 6A ) and is relatively similar to animals in terms of coding sequence conservation ( Figure 3D ) , but it harbors a secondarily expanded genome with disrupted gene linkage ( Figure 3A , C ) .", "Another example is Corallochytrium and Chromosphaera , both with massive simplifications of intron content ( Figure 5A ) , but higher synteny conservation with the inferred ancestral Holozoa ( Figure 3C ) .", "Also , the diversity of protein domain combinations of Chromosphaera is the highest among ichthyosporeans ( in line with values of animals and holozoan ancestors; Figure 7A ) and phylogenetic distances to animal orthologs are comparatively low ( Figure 3D ) .", "These studies of genome history in holozoans are key to our interpretation of functional genomics analyses .", "For example , Creolimax and Sphaeroforma are close species with a broadly conserved life cycle ( Glockling et al . , 2013 ) , and they could therefore be an apt model to test hypotheses of cell type evolution in Holozoa – for example , whether new cell types emerge as lineage-specific transcriptomic specializations , as proposed by ( de Mendoza et al . , 2015 ) .", "This investigation would benefit from taking into account their high microsynteny when analyzing co-regulated gene modules , while considering that Sphaeroforma’s multiple TE invasions could blur the conservation of non-coding regulatory elements in the intergenic regions ( Figure 3A–C ) .", "Our analysis of ten unicellular holozoans has uncovered the timing of genome evolution in the ancestry of Metazoa , at both the architectural and gene content levels .", "In particular , we have observed that holozoan genomes evolved under temporally uncoupled dynamics for synteny reorganization , intron gains , TE propagation , coding sequence conservation and gene family diversification .", "Some of these traits have independent effects in extant holozoans , e . g . , different episodes of intron gain or genome expansion in ichthyosporeans and animals .", "Yet , other traits exhibit conserved dynamics across the unicellularity/multicellularity divide: the diversification of ECM and TF gene families—including molecular tool-kits essential for multicellularity—extends back to the LCA of Holozoa; and the high intron densities in premetazoans suggest a continued state of transcriptome variability , co-optable for regulation or protein innovation , in the unicellular prehistory of Metazoa .", "Overall , our timeline of holozoan genome evolution offers a framework to investigate when and how premetazoan genomic elements—gene tool-kits , linkages and structure , and the non-coding sequences that harbor epigenomic regulatory elements—were functionally co-opted in multicellular animals ." ], [ "Corallochytrium limacisporum , Abeoforma whisleri and Pirum gemmata were grown in axenic culture in marine broth medium ( Difco 2216 ) at 18°C ( Abeoforma and Pirum ) or 23°C ( Corallochytrium ) .", "Chromosphaera was grown in axenic culture at 18°C in YM medium ( containing 3 g yeast extract , 3 g malt extract , 5 g bacto peptone , 10 g dextrose , 14 . 5 g Difco agar , and 25 g sodium chloride , per liter of distilled water ) .", "DNA-seq data was produced for Pirum , Abeoforma , Chromosphaera and Corallochytrium , by sequencing paired-end ( PE ) and Nextera mate-pair ( MP ) libraries .", "DNA extractions were performed from confluent axenic cultures , grown in three flasks of 25 ml for 5 days .", "DNA was extracted using a standard protocol by which cells were lysed in the extraction buffer composed of Tris-HCL , 50 mM EDTA , 500 mM NaCl and 10 mM ß-mercaptoethanol .", "DNA was purified with phenol:chloroform:isoamyl alcohol ( 25:24:1 ) and treated with of Rnase A ( Sigma Aldrich , Saint Louis , MO , USA ) .", "For each library , the read numbers , lengths and insert/fragment sizes were as follows: Pirum , PE 125 bp ( 250·106 reads , 250 bp insert size ) , MP 50 bp ( 108·106 reads , 6 kb fragment size ) ; Abeoforma , PE 100 bp ( 73·106 reads , 600 bp insert size ) , MP 100 bp ( 41·106 reads , 6 kb fragment size ) ; Chromosphaera , PE 125 bp ( 143·106 reads , 250 bp insert size ) , MP 50 bp ( 114·106 reads , 5 kb fragment size ) ; and Corallochytrium , PE 100 bp ( 150·106 reads , 420 bp insert size ) , MP 100 bp ( 47·106 reads , 3 kb fragment size ) .", "All PE and MP libraries were prepared and sequenced at the CRG Genomics Unit ( Barcelona ) , using Illumina HiSeq 2000 and the Trueseq Sequencing Kit v3 ( Abeoforma and Corallochytrium ) or v4 ( Pirum and Chromosphaera ) .", "The only exception was Corallochytrium PE libraries , which were sequenced at the Earlham Institute Genomics Unit ( Norwich , UK ) using Illumina MiSeq and the Trueseq protocol v2 .", "Genome sequencing data has been deposited in NCBI SRA under the BioProject accession PRJNA360047 .", "RNA-seq data was produced for Chromosphaera and Abeoforma .", "RNA extractions were performed from confluent axenic cultures grown in three 25 ml flasks for 5 days .", "RNA was extracted using Trizol reagent ( Life Technologies , Carlsbad , CA , USA ) with a further step of Dnase I ( Roche ) to avoid contamination by genomic DNA , then purified using RNeasy columns ( Qiagen ) .", "We sequenced PE libraries of 125 bp with an insert size of 250 bp , yielding 168·106 reads for Chromosphaera and 178·106 for Abeooforma; which were constructed using the Trueseq Sequencing Kit v4 ( Illumina , San Diego , CA ) .", "The libraries were sequenced in one lane of an Illumina HiSeq 2000 at the CRG genomics unit ( Barcelona ) .", "All transcriptome sequencing data has been deposited in NCBI SRA using the BioProject accession PRJNA360056 .", "Genomic PE and MP libraries were quality-checked using FastQC v0 . 11 . 2 ( Andrews , 2014 ) and trimmed accordingly with Trimmomatic v0 . 33 ( Bolger et al . , 2014 ) to remove remnant adapter sequences ( ad hoc ) and the low-quality 5' read ends ( sliding window = 4 and requiring a minimum Phred quality = 30 ) .", "A minimum length equal to the original read length was required .", "During the quality-trimming process , libraries of unpaired forward reads were kept as single-end reads ( SE ) .", "After trimming , the read survival rate for each DNA library was as follows: Pirum , PE 30 . 2% , MP 91 . 2%; Abeoforma , PE 75 . 5% , MP 31 . 0%; Chromosphaera , PE 81 . 1% , MP 89 . 9%; and Corallochytrium , PE 94 . 7% , MP 73 . 1% .", "Genome assemblies were performed using Spades v3 . 6 . 2 ( Nurk et al . , 2013 ) with the BayesHammer error correction algorithm ( Nikolenko et al . , 2013 ) .", "For each organism , PE data were analyzed using Kmergenie ( Chikhi and Medvedev , 2014 ) to determine the optimal k-mer length for the assembly process , which was used in the Spades assembly in combination with smaller and larger values , including the maximum possible odd length below the maximum read length after trimming .", "The optimized assemble parameters for each genome were as follows: Pirum , max .", "read length = 125 , k = 55 , 123; Abeoforma , max .", "read length = 100 , k = 47 , 91; Chromosphaera , max .", "read length = 125 , k = 91 , 121; Corallochytrium , max .", "read length = 100 , k = 41 , 63 , 91 .", "In the cases of Corallochytrium and Chromosphaera genomes , Spades was run in careful mode , taking into account PE , SE and MP data in the same run .", "In the cases of the highly repetitive Abeoforma and Pirum genomes , an initial Spades assembly of PE and SE libraries was combined with MP libraries using the Platanus v1 . 2 . 1 scaffolding module ( Kajitani et al . , 2014 ) .", "Each assembly was later processed using the GapCloser module from SOAPdenovo assembler with PE data , in order to extend the scaffolded contigs by shortening N stretches ( Luo et al . , 2012 ) .", "Genome assembly statistics ( genome size , N50 , L75 ) were calculated using Quast v2 . 3 ( Gurevich et al . , 2013 ) , and completeness was assessed using the BUSCO v1 . 1 ( Simão et al . , 2015 ) database of universal eukaryotic genes , based on the predicted transcripts .", "Genome feature annotations were produced for Corallochytrium , Chromosphaera , Abeoforma , Pirum and Ichthyophonus .", "We used evidence-based gene finders ( relying on transcript/peptide mapping: Augustus v3 . 1 ( Keller et al . , 2011 ) and PASA v2 . 0 . 2 [Haas et al . , 2003 , 2008] ) , plus complementary ab initio predictors ( based on hidden Markov models for gene structure: GeneMark-ES v4 . 21 ( Lomsadze et al . , 2005 ) and SNAP [Korf , 2004] ) .", "These results were combined to produce a consolidated gene annotation using Evidence Modeler v1 . 1 . 1 ( Haas et al . , 2008 ) .", "SNAP and GeneMark-ES annotations were iterated for three times on the final genome assemblies , using the output of each step as a training set for the next one ( the first SNAP prediction was done using the standard minimal HMM; GeneMark-ES was omitted for Abeoforma and Pirum due to its highly fragmented gene bodies , which impaired intron delimitation ) .", "Transcriptome assemblies were produced to support PASA and Augustus gene predictions .", "RNA-seq PE libraries were assembled using genome-guided Trinity v2 . 0 . 6 and STAR v2 . 5 ( for Corallochytrium , Chromosphaera and Ichthyophonus ) or de novo Trinity ( Pirum and Abeoforma , assemblies from ( Torruella et al . , 2015; Grabherr et al . , 2011; Dobin and Gingeras , 2015 ) .", "In the case of the Corallochytrium , Chromosphaera and Ichthyophonus genome-guided assemblies , quality control was performed as indicated above for the genomic libraries , using the RNA-seq data generated for this study ( Chromosphaera ) or in ( Torruella et al . , 2015 ) ( Ichthyophonus accession: PRJNA264423; Corallochytrium accession: PRJNA262632 ) .", "A minimum k-mer coverage = 2 was used in all Trinity assemblies .", "Transcriptome assemblies were annotated with Transdecoder using Pfam release 29 protein domain database , in order to obtain mRNA and translated peptides .", "Next , PASA annotations were obtained from assembled transcripts , mapped to the genome using GMAP and BLAT v35 ( Kent , 2002; Wu et al . , 2016 ) .", "Only high quality mapping was accepted , with a minimum of 95% identity and 75% transcript coverage .", "We then trained Augustus independently , using protein and mRNA predictions ( mapped to the genome with Scipio 1 . 4 ( Keller et al . , 2008 ) , BLAT and GMAP ) , followed by an optimization round of the species-specific parameters .", "After the training , an Augustus prediction was performed using the optimized parameters .", "Finally , all annotations were consolidated using Evidence Modeler .", "In this step , gene models from PASA and Augustus were given higher relative weights than ab initio-predicted models ( 10 and 5 times more reliability , respectively ) .", "We used an improved version of the dataset published by Torruella et al . ( Torruella et al . , 2015 ) , adding nine single-copy protein domains to the previous version ( which included 78 alignments ) according to the methodology developed in ( Torruella et al . , 2012 ) .", "Since Abeoforma and Pirum genome assemblies were fragmented and contained partial gene models , we used transcriptome assemblies from ( Torruella et al . , 2015 ) instead .", "We compiled a 57-taxa dataset of Unikonta/Amorphea species ( hereby termed BVD57 taxa matrix; including Holozoa , Holomycota , Breviatea , Apusomonadida and Amoebozoa; 24 , 021 amino acid positions ) .", "This dataset represents a ~ 10% increase in the number of aligned positions , compared to the original S70 dataset from ( Torruella et al . , 2015 ) .", "We used the BVD57 dataset to build ML phylogenetic trees using IQ-TREE v1 . 5 . 1 ( Nguyen et al . , 2015 ) , under the LG model with a 7-categories free-rate distribution , and a frequency mixture model with 60 frequency component profiles based on CAT ( LG + R7+C60 ) ( Quang et al . , 2008 ) .", "LG + R7 was selected as the best-fitting model according to the IQ-TREE TESTNEW algorithm as per the Bayesian information criterion ( BIC ) , and the C60 CAT approximation was added because of its higher rate of true topology inference ( Quang et al . , 2008 ) .", "Statistical supports were drawn from 1000 ultrafast bootstrap values with a 0 . 99 minimum correlation as convergence criterion ( Minh et al . , 2013 ) and 1000 replicates of the SH-like approximate likelihood ratio test ( Guindon et al . , 2010 ) , for all models stated above .", "Furthermore , 500 non-parametric bootstrap replicates were computed for the LG + R7+PMSF CAT approximation ( as this was the only CAT approximation for which non-parametric bootstraps could be calculated in a feasible computation time ) .", "We then used the same alignment to build a Bayesian inference tree with Phylobayes MPI v1 . 5 ( Lartillot et al . , 2013 ) , using the LG exchange rate matrix with a 7-categories gamma distribution and the non-parametric CAT model ( Lartillot and Philippe , 2004 ) ( LG+Γ7 + CAT ) .", "A Γ7 distribution was considered to be the closest approximation to the free-rates R7 distribution of the IQ-TREE ML analysis ( as free-rates distributions are not implemented in Phylobayes ) .", "We removed constant sites to reduce computation time .", "We ran two independent chains for 1231 generations until convergence was achieved ( maximum discrepancy <0 . 1 ) with a burn-in value of 32% ( 381 trees ) .", "The adequate burn-in value was selected by sequentially increasing the number of burn-in trees , until we achieved ( 1 ) a minimum value of the maximum discrepancy statistic , and ( 2 ) the highest possible effective size for the log-likelihood parameter .", "The bpcomp analysis of the sampled trees yielded a maximum discrepancy = 0 . 095 and a mean discrepancy = 0 . 001 .", "The tracecomp parameter analysis gave an effective size for the log-likelihood parameter = 37; and the minimum effective size = 11 ( for the alpha statistic ) .", "Our comparative genomics analyses are based on a dataset of 42 complete eukaryotic genomes , with a focus on unicellular and multicellular Holozoa , and using relevant outgroups from the Holomycota , Apusomonadida , Amoebozoa , Viridiplantae , Stramenopila , Alveolata , Rhizaria and Excavata groups .", "The complete list of species , abbreviations and data sources is available as Figure 2—source data 1 .", "Since ancestral state reconstruction requires the assumption of an explicit species tree , we classified the 42 genomes in our dataset according to a consensus of phylogenomic studies ( Torruella et al . , 2015; Derelle et al . , 2015; He et al . , 2014 ) and our own results .", "We remained agnostic about the internal topology of SAR ( Burki et al . , 2016 ) , Fungi ( Torruella et al . , 2015 ) , the contentious hypotheses for the root of eukaryotes ( namely , ‘Opimoda-Diphoda’ or ‘Excavata-first’ ) ( Derelle et al . , 2015; He et al . , 2014 ) and the earliest-branching animal group ( Porifera or Ctenophora ) ( Whelan et al . , 2015; Simion et al . , 2017 ) .", "All these cases were recorded in our species tree as polytomic branchings .", "We inferred two different ortholog datasets using the predicted proteins from the afore-mentioned genomes , using Orthofinder v0 . 4 . 0 with a MCL inflation = 2 . 1 ( Emms and Kelly , 2015 ) .", "The first database included 40 eukaryotic species ( excluding the low-quality gene models of Pirum and Abeoforma ) , whose genes were classified in 162 , 559 clusters of orthologs , 26 , 377 of which contained >1 gene ( henceforth , ‘orthocluster’ ) .", "The second database included all available unicellular holozoan genomes ( i . e . , six ichthyosporeans , two choanoflagellates , Corallochytrium and Capsaspora ) and yielded 58 , 516 orthoclusters , 11 , 925 of which contained >1 gene .", "Repetitive regions were annotated in Holozoa genomes using RepeatMasker open-4 . 0 . 5 ( Smit et al . , 2015 ) and annotations from the 20150807 release of GIRI RepBase database ( Bao et al . , 2015 ) .", "We used the Eukaryota-specific database , with either the slow high-sensitivity search mode ( unicellular holozoans ) or the default search mode ( metazoans ) ; and stored the genome coordinates of TEs , low complexity repeats , tRNA genes , simple repeats and satellite regions .", "Internal similarity of each genome's TE complements was analyzed with blastn self-alignments of all TEs ( considering a minimum 70% identity and 80 bp alignment length ) , and the distribution of percentage identity values was plotted using R . We used the frequency of collinear ortholog pairs as a proxy to estimate microsynteny across holozoans .", "Specifically , we retrieved all sets of single-copy orthologs for each pairwise species comparison within our set of 10 unicellular holozoan genomes .", "We then defined collinear gene pairs for each species pairs if the same two orthologs were adjacent in both genomes ( irrespective of individual gene orientation to account for possible local inversions , as in ( Putnam et al . , 2007 ) ) .", "To account for spurious conservation of gene order , we assigned random positions to each gene using the bedtools v2 . 24 . 0 shuffle utility ( Quinlan and Hall , 2010 ) in 100 independent rounds , for which the number of spurious conserved syntenic pairs was recorded .", "Then , we calculated the gene synteny ratio r of each species pair i-j as follows:rij= ( cij−sijNij ) ( cmax−smaxNmax ) where c denotes the number of syntenic orthologs between i and j; s is the number of spurious syntenic orthologs averaged over 100 random replicates; and N is the number of comparable ortholog pairs between i and", "j . Values are normalized to the 0–1 interval using the maximum values of the dataset as a reference ,", "i . e .", "Sphaeroforma and Creolimax .", "A heatmap representing the degree of similarity in pairwise species comparisons was produced using the synteny ratio ( R gplots library ( Warnes et al . , 2016 ) ) .", "Species were clustered according to their mean synteny .", "The same analysis was performed using the database of 40 eukaryotic genomes , which excluded Abeoforma and Pirum .", "In this case , the maximum values used as a reference were the Nematostella-Aiptasia pair .", "For specific selected species comparisons , syntenic pairs were plotted onto the genome scaffolds using Circos v0 . 67 ( Krzywinski et al . , 2009 ) .", "From our ortholog database using 40 eukaryotic genomes ( excluding Pirum and Abeoforma , which had lower-quality gene annotations due to their fragmented assemblies ) , we selected 143 orthoclusters present in all unicellular holozoans , plus Amphimedon queenslandica , Trichoplax adhaerens , Homo sapiens and Nematostella vectensis ( as representative animal genomes ) .", "We aligned each group of orthologs using MAFFT G-INS-i ( Katoh and Standley , 2013 ) , trimmed the alignments using trimAL automated algorithm ( Capella-Gutiérrez et al . , 2009 ) , and inferred maximum likelihood trees for each ortholog group using RAxML v8 . 2 . 0 ( Stamatakis , 2014 ) and the LG amino acid substitution model .", "Then , for each tree , we recorded all pairwise phylogenetic distances between species as measured by substitutions per alignment position using the cophenetic module of the ape v3 . 5 R library ( Paradis et al . , 2004; Core Team , 2015 ) .", "We retrieved distances between each unicellular holozoan ortholog and , separately , Amphimedon , Trichoplax , Homo and Nematostella orthologs .", "For each inter-species comparison , we tested the significance of differences in phylogenetic distances between unicellular holozoans , using the non-parametric Wilcoxon rank sum test from the R stats library ( Core Team , 2015 ) .", "Intron content of a subset of 40 eukaryotic genomes ( excluding Abeoforma and Pirum , which had lower-quality gene annotations due to their fragmented assemblies ) was analyzed using a set of single-copy orthologous genes , and used to reconstruct ancestral states as described by Csűrös et al . ( Csuros et al . , 2011; Csurös et al . , 2007 , Csurös et al . , 2008 ) .", "We then selected orthocluster present as single-copies in 80% of our species dataset , allowing for paralog genes to occur in just one species per group ( if that was the case , the best-scoring copy in BLAST alignments was kept ) .", "This yielded a group of 342 nearly paneukaryotic genes , whose protein translations were then aligned using MAFFT v7 . 245 G-INS-i algorithm ( Katoh and Standley , 2013 ) and annotated with their intron coordinates ( retrieved from their respective genome annotations ) .", "With this information , we reconstructed the ancestral states of each intron using the Malin implementation of the probabilistic model of intron evolution developed by Csűrös et al . ( Csűrös and Miklós , 2006; Csurös , 2008 ) , starting from the standard null model , running 1000 optimization rounds ( likelihood convergence threshold = 0 . 001 ) and assuming a consensus eukaryotic phylogeny ( see Generation of a species tree for comparative analyses ) .", "Conserved intron sites ( defined as unambiguously aligned in 80% of the orthologs , maximum of 10% of gap positions ) were used to calculate the rates of intron loss and gain for each node of the tree .", "These rates were used to calculate a table of intron sites with a certain probability of presence , gain or loss at every node of the tree ( which , when summed , give the number of introns that are present , gained or lost at that node ( Csűrös and Miklós , 2006 ) ) .", "We computed 100 bootstrap replicates in Malin to assess uncertainty about inferred rate parameters and evolutionary history .", "In particular , we calculated the variance-to-mean ratio of the inferred number of introns in each ancestor with 100 bootstrap replicates ( with values higher than one indicating more dispersed results and less reliable inferences ) .", "For each node i , we calculate the percentage of introns gained ( pG , i ) or lost ( pL , i ) as a percentage of the total number of introns at that node .", "Then , the gain/loss ratio of a node , ri , was calculated as follows:pG , i > pL , i→ri=log10 ( pG , ipL , i ) pL , i < pL . i→ri=log10 ( ( pG , ipL , i ) −1 ) ×−1 We represented the presence and absence of intron sites at each lineage ( extant and ancestral ) , and the number of introns shared between species ( only extant ) , using heatmaps ( R gplots library ( Warnes et al . , 2016 ) ) .", "Inter-species distances were calculated using the pairwise counts of shared introns and the Spearman correlation algorithm , which was used to perform Ward hierarchical clustering as implemented in R stats library ( Core Team , 2015 ) .", "We used the same algorithms to calculate distances of intron presence probability profiles , and subsequent clustering .", "For Capsaspora , the phylostratigraphy of intron sites was combined with the nucleosome-free sites identified by ATAC-seq analysis in ( Sebé-Pedrós et al . , 2016b ) , which were assumed to be putative regulatory sites .", "Then , we compared phylostrata distribution ( 'ancestral' versus 'recent' Capsaspora-specific sites ) for introns with and without regulatory sites , using a Fisher's exact test: 74 recent introns and 465 ancestral introns lacked putative regulatory sites ( ≥50% ATAC site overlap with the intron sequence , calculated using bedtools v2 . 24 . 0 intersect utility ( Quinlan and Hall , 2010 ) ) , while 3 and 22 recent and ancestral introns had regulatory sites .", "Protein domain architectures of the 40 eukaryotic species subset ( excluding Abeoforma and Pirum , which had lower-quality gene annotations due to their fragmented assemblies ) were computed using Pfamscan and the 29th release of the Pfam database ( Punta et al . , 2012 ) .", "For each protein , the domain architecture was decomposed into all possible directed binary domain pairs ( ignoring repeated consecutive domains;", "i . e .", "from protein A-B-B-C , the pairs A-B , A-C and B-C were built ) , and linked to its presence in its corresponding orthocluster ( see Generation of a species tree and ortholog datasets for comparative analyses section ) .", "The final output was a numerical profile of species distribution for each combination of domain pairs in orthoclusters ( considering that a cluster can contain more than one pair , and a pair can be present in more than one cluster , and thence the number of occurrences is recorded ) .", "The numerical profile was analyzed using the general phylogenetic birth-and-death model developed by Csűrös and Miklós ( Csűrös and Miklós , 2006 ) as implemented in Count ( Csurös , 2010 ) .", "This allows the comparative analysis and ancestral reconstruction of discretized quantitative properties of genomes , assuming a specific species tree ( see Comparative analysis of intron content ) .", "We used a gain-loss-duplication model with unconstrained gain/loss and duplication/loss ratios in all lineages , assuming a Poisson distribution of orthocluster size at the LECA ( root ) and no rate variation categories .", "In this context , 'gain' was defined as the acquisition of a new pairwise domain combination in an orthocluster; a 'duplication' as the propagation of the combination ( by gene duplication or convergent domain rearrangements ) ; and 'loss' as pair dissociation .", "Starting from the standard null model , we ran 100 optimization rounds ( convergence threshold = 0 . 1 ) .", "To analyze the modularity of the protein domain networks ( and subnetworks ) for each genome , we 1 ) calculated the community structure of each network using Louvain iterative clustering to obtain communities of domain pairs ( undirected graphs ) , and 2 ) calculated the global network modularity according to these communities .", "The modularity parameter measures the fraction within-community edges minus the expected value obtained from a network with the same communities but random vertex connections ( Newman and Girvan , 2004 ) .", "A maximum value of 1 indicates a strong community structure , while a minimum value of 0 indicates that within-community edges are as frequent as expected in a random network .", "For these analyses we used the relevant algorithms from the igraph R library v1 . 0 . 1 ( Core Team , 2015; Csárdi and Nepusz , 2006 ) .", "Function-oriented domain subnetworks were obtained by retrieving orthologous groups that contained relevant domains , which were obtained from previous studies ( transcription factors from ( de Mendoza et al . , 2013; Weirauch and Hughes , 2011 ) , signaling domains from ( Richter and King , 2013 ) , ECM-related domains from ( Richter and King , 2013; Sebé-Pedrós et al . , 2010; Hynes , 2012 ) , ubiquitination from ( Grau-Bové et al . , 2015 ) ) and pfam2go annotations ( for the subsets mentioned above , and also for protein-binding domains ) ( Mitchell et al . , 2015 ) .", "Monotonic statistical dependence between modularity and the number of domains per community was tested using Spearman's rank correlation coefficient ( ρs ) for all network or subnetwork ( for original and simulated data ) .", "We mapped the presence of individual protein domains across our dataset of 40 eukaryotic species ( excluding Abeoforma and Pirum ) , as predicted by Pfamscan and the 29th release of the Pfam database ( Punta et al . , 2012 ) .", "Using this numerical profile of domain presence in extant genomes , we computed the gains and losses at ancestral nodes using the Dollo parsimony algorithm as implemented in Count ( Csurös , 2010 ) .", "Genome sequencing and assembly data from Corallochytrium , Abeoforma , Pirum and Chromosphaera has been deposited in NCBI using the BioProject accession PRJNA360047 .", "Transcriptome sequencing data from Abeoforma and Chromosphaera has been deposited in NCBI using the BioProject accession PRJNA360056 ." ] ]
[ "Which genomic innovations underpinned the origin of multicellular animals is still an open debate .", "Here , we investigate this question by reconstructing the genome architecture and gene family diversity of ancestral premetazoans , aiming to date the emergence of animal-like traits .", "Our comparative analysis involves genomes from animals and their closest unicellular relatives ( the Holozoa ) , including four new genomes: three Ichthyosporea and Corallochytrium limacisporum .", "Here , we show that the earliest animals were shaped by dynamic changes in genome architecture before the emergence of multicellularity: an early burst of gene diversity in the ancestor of Holozoa , enriched in transcription factors and cell adhesion machinery , was followed by multiple and differently-timed episodes of synteny disruption , intron gain and genome expansions .", "Thus , the foundations of animal genome architecture were laid before the origin of complex multicellularity – highlighting the necessity of a unicellular perspective to understand early animal evolution ." ]
[ "Hundreds of millions of years ago , some single-celled organisms gained the ability to work together and form multicellular organisms .", "This transition was a major step in evolution and took place at separate times in several parts of the tree of life , including in animals , plants , fungi and algae .", "Animals are some of the most complex organisms on Earth .", "Their single-celled ancestors were also quite genetically complex themselves and their genomes ( the complete set of the organism’s DNA ) already contained many genes that now coordinate the activity of the cells in a multicellular organism .", "The genome of an animal typically has certain features: it is large , diverse and contains many segments ( called introns ) that are not genes .", "By seeing if the single-celled relatives of animals share these traits , it is possible to learn more about when specific genetic features first evolved , and whether they are linked to the origin of animals .", "Now , Grau-Bové et al . have studied the genomes of several of the animal kingdom’s closest single-celled relatives using a technique called whole genome sequencing .", "This revealed that there was a period of rapid genetic change in the single-celled ancestors of animals during which their genes became much more diverse .", "Another ‘explosion’ of diversity happened after animals had evolved .", "Furthermore , the overall amount of the genomic content inside cells and the number of introns found in the genome rapidly increased in separate , independent events in both animals and their single-celled ancestors .", "Future research is needed to investigate whether other multicellular life forms – such as plants , fungi and algae – originated in the same way as animal life .", "Understanding how the genetic material of animals evolved also helps us to understand the genetic structures that affect our health .", "For example , genes that coordinate the behavior of cells ( and so are important for multicellular organisms ) also play a role in cancer , where cells break free of this regulation to divide uncontrollably ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology" ]
The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport
elife-02419-v2
[ [ "Cilia and flagella ( interchangeable terms ) are microtubule-based organelles surrounded by a specialized plasma membrane .", "These organelles are found protruding at the surface of a wide range of eukaryotic cells where they exert several roles including cellular motility , sensory reception and developmental signaling ( Fliegauf et al . , 2007; Drummond , 2012 ) .", "Although cilia and flagella can differ by their functions , they share the same basic structure and are mainly assembled by an evolutionary conserved process called intraflagellar transport ( IFT ) ( Kozminski et al . , 1993; Rosenbaum and Witman , 2002 ) .", "During IFT , large protein complexes called IFT trains are moved from the base to the tip of the flagellum by kinesin-2 ( anterograde IFT ) ( Kozminski et al . , 1995 ) and back towards the base by cytoplasmic dynein-1b/2 ( retrograde IFT ) ( Pazour et al . , 1998 ) .", "The IFT particles are composed of at least 20 different proteins ( Cole et al . , 1998; Follit et al . , 2009; Taschner et al . , 2011 ) and can be divided in two different complexes known as complex A ( made of at least 6 proteins ) and B ( made of at least 14 proteins ) that are needed for retrograde and anterograde IFT respectively .", "Inactivation of IFT-B proteins or of the kinesin motor results in the failure to construct the flagellum whereas IFT-A proteins and the IFT dynein contribute to retrograde transport .", "Nevertheless , little is known about how the two processes are interconnected , as the interaction between the IFT complexes and their molecular motors is still poorly understood .", "Structurally , IFT proteins contain several protein–protein interaction domains such as WD40 , tetratricopeptide repeats , β-propeller or coiled-coil domains ( Cole , 2003 ) .", "However , two IFT proteins belonging to the IFT complex B do not share those features .", "Instead , they exhibit sequence homologies with the family of Rab-like small G proteins , which can function to regulate vesicle movement along actin and tubulin networks ( Stenmark , 2009 ) .", "This suggests that they could be involved in IFT regulation .", "The first one , IFT22 ( or Rab-like 5 ) traffics inside the sensory neurons of Caenorhabditis elegans , where its disruption does not affect cilia assembly but produces an intriguing extended lifespan phenotype ( Schafer et al . , 2006 ) .", "IFT22 also undergoes IFT in the flagellum of the protist Trypanosoma brucei but surprisingly knocking down the protein resulted in severe flagellar assembly defects , typical of inhibition of retrograde transport ( Adhiambo et al . , 2009 ) .", "In Chlamydomonas , IFT22 regulates the availability of IFT proteins inside the cell ( Silva et al . , 2012 ) .", "Despite being homologous to the Rab-like proteins , IFT22 lacks the G4 domain needed for recognition of the guanine base ( Paduch et al . , 2001 ) and GTP binding and hydrolysis has not been demonstrated to date .", "Therefore , IFT22 might have diverged from its ancestral GTPase function to complete specific roles in C . elegans , T . brucei , and Chlamydomonas .", "The second protein is termed IFT27 ( or Rab-like 4 ) and contrary to IFT22 , it possesses all five RAS-GTPase consensus sequences needed for GTPase activity and GDP binding .", "Structurally , the protein is similar to Rab8 and Rab11 and is capable of GTP hydrolysis ( Bhogaraju et al . , 2011 ) .", "IFT27 is part of the IFT complex B core , where the protein interacts directly with IFT25 ( Lucker et al . , 2005 ) .", "This IFT25/27 sub-complex is associated with the rest of the IFT complex B prior to flagellum entry , suggesting a possible regulatory role during the entry of the IFT machinery ( Wang et al . , 2009 ) .", "In Chlamydomonas , RNAi-mediated knockdown of IFT27 causes flagellar defects ( Qin et al . , 2007 ) , supporting its role during flagellar assembly , although its exact contribution was not investigated .", "In this study , we analyzed the function of IFT27 in T . brucei , the parasite that causes sleeping sickness and that is also an amenable model to study flagellar assembly ( Kohl and Bastin , 2005; Ralston and Hill , 2008 ) as well as the IFT components ( Absalon et al . , 2008b; Adhiambo et al . , 2009; Franklin and Ullu , 2010; Bhogaraju et al . , 2013; Buisson et al . , 2013 ) .", "We found that IFT27 travels by IFT and associates with other IFT-B proteins .", "RNAi knockdown surprisingly produced short flagella filled with IFT-like material and axoneme assembly defects .", "This phenotype is explained by the absence of both the IFT140 protein ( a member of the IFT-A complex ) and the IFT dynein motor from the flagellar compartment .", "Generation of constitutively active and inactive forms of IFT27 produced further insights: while expression of the active version of the protein complements the RNAi phenotype , this was not the case of the inactive version that was unable to penetrate the flagellar compartment .", "Surprisingly , its expression in the absence of endogenous protein led to the formation of short flagella that do not accumulate IFT-like material .", "This inactive version is unable to interact with two other IFT-B proteins , suggesting that IFT27 must be in a GTP-bound state in order to interact with the B-complex and enter the flagellum .", "These results show that IFT27 , an IFT-B protein , performs two separate functions: one in the import of both the IFT-A complex and IFT dynein motors and one in the assembly of the IFT-B complex ." ], [ "The T . brucei IFT27 gene ( TritrypDB Accession number Tb927 . 3 . 5550 ) has a 552 nucleotide-long sequence that encodes a predicted protein of 183 amino acids ( predicted molecular weight of 20 . 64 kDa ) .", "BLAST analyses show that IFT27 shares significant homology ( E value = 2e−27 ) with the Rab-like 4 ( RABL4 ) GTPase found in Homo sapiens and vertebrates .", "IFT27 homologues are present in the genomes of all ciliated organisms except in Drosophila , C . elegans , Giardia , and some ferns and mosses ( van Dam et al . , 2013 ) .", "The predicted trypanosome protein contains all five consensus domains needed for GTP/GDP binding and GTPase activity found in most Rab proteins ( Figure 1 ) , indicating that IFT27 could be a functional small G protein .", "Additionally , all IFT27 sequences lack the prenylation motif ( two cysteins at the C terminal end ) found in Rab proteins suggesting that the protein is not lipid modified and thus unlikely to interact with the cellular membrane . 10 . 7554/eLife . 02419 . 003Figure 1 . Sequence alignment of deduced amino acid sequences of IFT27 homologues and modified IFT27 sequences . Alignment was generated using CLC main workbench; the most conserved residues are shown in black , the less conserved in yellow .", "G1–G5 indicates conserved motifs implicated in nucleotide binding domains and GTPase activity .", "Dashes indicate gaps introduced to optimize the alignment .", "Arrowheads indicate missense mutations created in IFT27RNAiRES .", "Abbreviations and accession numbers are as follows: Hs: Homo sapiens , NP_006851 . 1 , Mm: Mus musculus , NP_080207 . 1 , Dr: Danio rerio , NP_001008588 . 1 , Cr: Chlamydomonas reinhardtii , XP_001689745 . 1 , Tb: Trymanosoma brucei , Tb927 . 3 . 5550 , Tc: Trypanosoma cruzi , AY371275 , Lm: Leishmania major , LmjF . 29 . 0090 , Tt: Tetrahymena thermophila , TTHERM_00298510 . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 003 Two approaches were used to determine the location of IFT27 in T . brucei .", "First , the full-length protein was expressed and used to produce antisera in mice .", "Second , a GFP::IFT27 fusion protein was expressed in procyclic trypanosomes .", "Western blot analyses using the anti-IFT27 antibody showed a single band migrating at a position close to the predicted size of 20 kDa in wild-type cells ( Figure 2A ) .", "In trypanosomes expressing the GFP::IFT27 fusion , the same antibody detected an additional band migrating close to the 50 kDa marker .", "This molecular weight is compatible with the expected mass of the fusion protein ( Figure 2A ) .", "The anti-IFT27 antibody was then used in immunofluorescence assays in combination with DAPI to stain both nuclear and mitochondrial DNA , the latter being an easy marker of basal body positioning in trypanosomes ( Robinson and Gull , 1991 ) .", "In wild-type cells , the anti-IFT27 antibody produced a signal all along the flagellum , starting at the base of the organelle and reaching its distal tip where it was sometimes brighter ( Figure 2B ) .", "The GFP-fusion protein showed a similar localization , with the presence of the protein at the flagellum base and inside the flagellum .", "Co-staining of the GFP-tagged protein and IFT27 showed a clear colocalization inside the flagellum ( Figure 3A ) and the use of live microscopy demonstrated that the fusion protein clearly traffics along the flagellum ( Figure 2C , D; Video 1 ) where typical bidirectional IFT was visualized .", "Similar IFT trafficking was observed for other IFT-related proteins in trypanosomes including GFP::IFT52 ( Absalon et al . , 2008b ) , GFP::IFT22 ( Adhiambo et al . , 2009 ) , and YFP::IFT81 ( Bhogaraju et al . , 2013 ) .", "Kymographs were made to quantify IFT train speed ( Figure 2D ) .", "The mean anterograde velocity was 2 . 5 ± 0 . 68 µm/s ( n = 234 ) and mean retrograde velocity was measured at 3 . 8 ± 1 . 5 µm/s ( n = 269 ) .", "These speed values are similar to those previously reported for GFP::IFT52 ( Buisson et al . , 2013 ) .", "Overall , these results show that IFT27 distribution and movement are similar to the other IFT-B proteins studied to date in T . brucei . 10 . 7554/eLife . 02419 . 004Figure 2 . IFT27 is found inside the trypanosome flagellum where it travels by IFT .", "( A ) Western blot of wild-type trypanosomes and cells expressing GFP::IFT27 probed with the anti-IFT27 polyclonal antibody .", "( B ) Immunofluorescence of wild-type cells fixed in methanol using the anti-IFT27 polyclonal antibody .", "The first panel shows the phase-contrast image merged with DAPI staining and the second one shows the anti-IFT27 staining ( green ) merged with DAPI staining ( blue ) .", "( C ) Still images of a trypanosome expressing GFP::IFT27 ( Video 1 ) .", "The white arrow shows the fluorescent protein pool at the level of the flagellum base and the yellow arrow shows the distal tip of the flagellum .", "Arrowheads indicate the successive position of an anterograde IFT train .", "( D ) Kymograph from Video 1 shows clear IFT traces .", "Anterograde and retrograde events were separated as previously described ( Chenouard et al . , 2010 , Buisson et al . , 2013 ) .", "Scale bars: 5 µm in B and C . In D , horizontal scale bar is 2 µm and vertical scale bar is 2 s . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 00410 . 7554/eLife . 02419 . 005Figure 3 . GFP::IFT27 and IFT27RNAiRES::YFP colocalise with the endogenous IFT27 . ( A ) Trypanosomes expressing GFP::IFT27 were fixed in methanol and stained simultaneously with the anti-IFT27 ( red ) antibody and the anti-GFP antibody ( green ) .", "Top left and right panels show the phase-contrast image merged with DAPI staining and the anti-IFT27 staining respectively .", "Bottom left and right panels show the anti-GFP staining and the merged image .", "Non-induced ( B ) and 3-day induced ( C ) IFT27RNAiRESYFP cells were fixed in methanol and immunofluorescence assays was performed using simultaneously the anti-IFT27 and the anti-GFP antibodies in order to locate the endogenous and the fluorescent version of IFT27 .", "Immunofluorescence assays reveal that both proteins are found within the flagellum .", "Scale bars: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 00510 . 7554/eLife . 02419 . 006Video 1 . IFT27 is found inside the trypanosome flagellum where it travels by IFT . Live procyclic , wild-type T . brucei cell transfected with GFP::IFT27 observed by time-lapse epifluorescence microscopy using a DMI4000 microscope at room temperature .", "Frames were taken every 250 ms for 30 s by an Evolve 512 EMCCD Camera .", "The resulting video was then exported to . AVI format with the ImageJ 1 . 47g13 software . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 006 To evaluate the role of IFT27 , inducible RNA interference ( RNAi ) was used to deplete the expression of the protein in 29–13 procyclic parasites in culture .", "The 29-13 strain expresses the tetracycline repressor and the viral T7 RNA polymerase , which enables conditional expression of double stranded RNA leading to RNAi-mediated gene inhibition ( Wirtz et al . , 1999; Wang et al . , 2000 ) .", "Western blot analysis using the anti-IFT27 polyclonal antibody showed that the IFT27 expression was inhibited after 48 hr of induction ( Figure 4A ) and IFT27RNAi cells showed a clear growth defect upon depletion of the protein ( Figure 4B ) , in line with the essential role of the flagellum for the trypanosome cell cycle ( Kohl et al . , 2003 ) .", "To analyze the impact of IFT27 depletion on flagellar formation , two markers were used: MAb25 ( monoclonal , axoneme-specific ) and L8C4 ( monoclonal , paraflagellar rod-specific ) .", "Immunofluorescence assays results revealed that IFT27RNAi cells induced for 72 hr assemble abnormally short flagella ( Figure 4C bottom panel , arrowhead ) with a mean axonemal length of 4 . 0 ± 6 . 8 µm ( n = 100 ) instead of 20 . 5 ± 2 . 8 µm ( n = 100 ) for non-induced controls ( Figure 4C , top panel ) .", "Furthermore , L8C4 staining unpredictably displayed a very bright or very weak staining in the flagella of induced IFT27RNAi cells , indicating that an excessive or a restrained amount of paraflagellar rod ( PFR ) material per unit length is found in the flagella of these cells ( Figure 4C , bottom panel , arrow ) . 10 . 7554/eLife . 02419 . 007Figure 4 . Silencing of IFT27 disrupts flagellum formation .", "( A ) Western blot showing decrease of IFT27 protein upon RNAi silencing .", "Total protein samples of non-induced and induced cells were prepared after the indicated number of days .", "The membrane was incubated with the anti-IFT27 or the anti-aldolase as a control .", "( B ) Growth curve of non-induced ( blue ) and induced ( red ) IFT27RNAi cells .", "( C ) Non-induced and 3-day induced IFT27RNAi cells were fixed in methanol , stained with the Mab25 antibody to detect the axoneme ( red ) and the L8C4 antibody to detect the PFR ( green ) ( right panels ) then counterstained with DAPI ( left panels ) .", "The arrowhead shows an abnormally short axoneme with an insufficient amount of PFR and the arrow indicates an excessive amount of PFR .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 007 To evaluate whether and how IFT was affected , we first used immunofluorescence assays with three antibodies raised against different IFT-B proteins: IFT22 ( Adhiambo et al . , 2009 ) , IFT172 ( Absalon et al . , 2008b ) , and PIFTC3 ( Franklin and Ullu , 2010 ) .", "IFT-B proteins in non-induced cells showed a uniform distribution along the flagellum ( Figure 5Aa–d ) .", "However , unexpected accumulations of IFT172 and IFT22 were found in the short flagella of induced IFT27RNAi cells ( Figure 5Ae–h , arrows ) and the same results were observed for PIFTC3 , a candidate IFT-B protein ( Franklin and Ullu , 2010 ) ( unpublished data ) .", "In a second approach , the IFT-B core protein IFT52 was expressed as a GFP fusion protein ( Figure 5B ) .", "Whereas in non-induced cells , GFP::IFT52 was localized in the cytoplasm but also concentrated at the base of the flagellum and inside the organelle ( Figure 5Ba–d ) , 72-hr induced cells clearly showed an accumulation of the protein inside their short flagella ( Figure 5Be–h ) .", "Together , these observations demonstrate that at least four members of the IFT-B complex accumulate inside the flagellum of the induced IFT27RNAi cells . 10 . 7554/eLife . 02419 . 008Figure 5 . IFT complex B proteins accumulate in the short flagella of IFT27RNAi cells .", "( A ) IFT27RNAi cells were non-induced ( a to d ) or induced for 3 days ( e to", "h ) , labeled with the anti-IFT172 ( a , c , e and", "g ) or the anti-IFT22 antibody ( b , d , f and", "h ) and counterstained with DAPI ( blue ) .", "Arrows show the accumulation of IFT proteins in the short flagella .", "( B ) Non-induced ( a to d ) and 3-day induced ( e to", "h ) IFT27RNAi GFP-IFT52 cells were treated as above except that the anti-GFP was used .", "Insets in f and h show the axoneme and GFP::IFT52 localization respectively .", "Scale bar: 5 µm .", "( C ) TEM of non-induced ( a–b ) and induced IFT27RNAi cells ( c–d ) .", "Black arrows show the axonemal defects , and the white arrow points to an excessive amount of IFT-like material .", "An enlarged PFR structure ( bracket ) is also visible .", "fp: flagellar pocket , axo: axoneme , bb: basal body .", "Scale bars: 500 nm in a and c , 200 nm in b and d . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 008 The formation of short flagella filled with IFT proteins is typical of inhibition of retrograde IFT ( Pazour et al . , 1999; Blacque et al . , 2006; Absalon et al . , 2008a ) , suggesting that despite being a protein belonging to IFT-B complex , IFT27 could play a role in retrograde IFT .", "To further characterize this unexpected phenotype , transmission electron microscopy analyses were performed .", "Non-induced IFT27RNAi cells looked like normal wild-type trypanosomes ( Figure 5Ca , b ) , whereas induced cells exhibited major defects in the short flagellum .", "First , a significant accumulation of electron-dense material that likely corresponds to excessive IFT material was observed ( Figure 5Cc–d ) .", "In addition , the axoneme was frequently disrupted and sometimes even split into two ( Figure 5C , D ) and an enlarged , often disorganized PFR structure was frequently observed .", "Over 61% of the flagellar sections ( n = 77 ) displayed the combination of the three phenotypes .", "Other phenotypes included the presence of a membrane sleeve ( 18% ) ( Davidge et al . , 2006; Absalon et al . , 2008b ) , vesicles inside the lumen of the flagellar pocket ( 3% ) and a loss of the alignment of the axonemal central pair relative to the PFR ( 14% ) ( Branche et al . , 2006; Gadelha et al . , 2006; Ralston et al . , 2006; Figure 6 ) . 10 . 7554/eLife . 02419 . 009Figure 6 . Additional phenotypes observed in the IFT27RNAi cell line . Transmission electron micrographs of IFT27RNAi cells induced for 3 days displaying different flagellar defects .", "( A ) Membrane-like material in the flagellar pocket lumen and the presence of a flagellum ‘sleeve’ ( arrows ) .", "( B ) Vesicle-like material inside the flagellar pocket ( arrows ) and misorientation of the basal body ( arrowhead ) .", "( C ) Defect in central pair alignment relative to the PFR .", "Scale bars 500 nm in A , 1 µm in B and 200 nm in C . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 009 To explain the surprising IFT retrograde phenotype , we examined the distribution of two elements involved in the retrograde transport machinery: the IFT dynein and IFT140 , a member of the IFT-A complex .", "We generated an IFT27RNAi cell line expressing the IFT dynein heavy chain ( DHC1b ) fused to GFP , and its localization was analyzed by immunofluorescence assays using the Mab25 and the anti-GFP antibodies ( Figure 7 ) .", "In non-induced IFT27RNAi cells , the GFP::DHC1b signal was present around the base of the flagellum and displayed a punctuated staining inside the organelle and in the cytoplasm ( Figure 7a–d ) confirming previous localization experiments performed with the anti-DHC1b antibody ( our unpublished data ) .", "When IFT27RNAi cells were induced for 3 days , DHC1b was not detected inside the short flagella but instead the protein appeared concentrated at the base of the flagellum ( Figure 7e–h ) , suggesting defects in flagellum entry . 10 . 7554/eLife . 02419 . 010Figure 7 . The IFT dynein is not able to access the flagellar compartment in the absence of IFT27 . ( A ) Immunofluorescence of non-induced ( a to d ) and 3-day induced ( e to", "h ) IFT27RNAi GFP::DHC1b cells fixed in methanol , counterstained with DAPI and stained with the Mab25 and anti-GFP antibodies .", "Insets in f and h show the localization of the axoneme ( red ) and the GFP::IFT dynein ( green ) respectively .", "Scale bars: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 010 To determine the localization of the IFT-A complex , the core component IFT140 protein was fused to the Tandem Tomato ( TdT ) fluorescent protein upon endogenous tagging and expressed in IFT27RNAi cells ( Figure 8 ) .", "In vivo observations confirmed that the fusion protein indeed traffics inside the flagellum ( unpublished data ) and its localization was then analyzed by immunofluorescence assays using an anti-DsRed antibody .", "Slides were stained simultaneously with the Mab25 and anti-IFT172 antibodies to localize the axoneme and the IFT-B complex respectively .", "In non-induced cells , the fusion protein is localized inside the flagellum compartment ( Figure 8b ) as for the anti-IFT172 ( Figure 8c ) .", "In 3-day-induced IFT27RNAi cells , TdT::IFT140 is only found concentrated at the base of the organelle and not inside the flagellar compartment ( Figure 8f , h ) .", "In contrast , IFT172 is still aberrantly accumulated inside the organelle ( Figure 8g , h ) as observed previously . 10 . 7554/eLife . 02419 . 011Figure 8 . IFT140 does not accumulate in the short flagella of IFT27RNAi cells . IFT27RNAi cells were non-induced ( a to d ) or induced for 3 days ( e to", "h ) and simultaneously labeled with the anti-DsRed ( red ) , the anti-IFT172 ( green ) antibodies along with Mab25 ( blue ) and counterstained with DAPI .", "Insets show the localization of the axoneme and TdTomato::IFT140 in f and of the axoneme and IFT172 in g .", "In h , the localization of Td::IFT140 , IFT172 and the axoneme are shown .", "The arrowhead indicates the base of the flagellum .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 011 Therefore , both actors of the retrograde transport fail to enter the flagellum in the absence of IFT27 , suggesting that this protein , despite being a member of the IFT-B complex , is a major controller of retrograde transport .", "To demonstrate the specificity of the IFT27 silencing phenotype , an RNAi-resistant version of the gene was synthetized ( GeneBank Accession Number KF147877 ) and expressed into the IFT27RNAi cell line .", "Officially named IFT27RNAi+IFT27RNAiRES this new cell line will be termed IFT27RNAiRES for simplicity .", "This exogenous copy of IFT27 has a C-terminal TY-1 tag ( Bastin et al . , 1996 ) to allow for independent monitoring .", "Upon induction of IFT27 dsRNA expression , transcripts from the endogenous gene should be silenced , whereas the RNAi-resistant version of IFT27 should remain unaffected .", "Western blot analysis of uninduced IFT27RNAiRES cells using the anti-TY1 antibody showed a single band migrating close to the 20-kDa marker , whereas probing the membrane with the anti-IFT27 antibody revealed two bands , the endogenous IFT27 and the slightly larger exogenous IFT27RNAiRES ( the TY1-tagged protein has a predicted molecular weight of 22 . 64 kDa ) ( Figure 9A ) .", "The inhibition of the endogenous IFT27 occurred when RNAi was induced while the exogenous IFT27RNAiRES continued to be expressed , as was evident in Western blots probed with the anti-IFT27 antibody ( Figure 9B , left panel ) .", "Furthermore , IFT27 inhibition can be reversed after removing tetracycline , whereas IFT27RNAiRES expression remained unchanged ( Figure 9B , right panel ) .", "The growth curve of the IFT27RNAiRES cell line showed no defects after RNAi induction ( Figure 9C ) , and immunofluorescence assays using the anti-Ty1 antibody showed a signal all along the flagellum of both non-induced and induced cells , as expected ( Figure 9D ) .", "These results show that the RNAi-resistant version of IFT27 complements gene function upon silencing of the endogenous IFT27 gene copies and demonstrate the specificity of the phenotypes observed in the IFT27RNAi cell line . 10 . 7554/eLife . 02419 . 012Figure 9 . An epitope-tagged RNAi-resistant version of IFT27 complements the phenotype resulting from endogenous IFT27 depletion in the IFT27RNAi cell line .", "( A ) Western blot of total protein samples of wild-type ( WT ) and non-induced IFT27RNAiRES cells .", "The anti-IFT27 antibody was used first and the anti-TY1 antibody was used after membrane stripping .", "The epitope-tagged RNAi-resistant version of IFT27 is indicated by the red asterisk .", "( B ) Western blot of total protein samples from the IFT27RNAiRES cell line prepared from induced cells and from cells deinduced after the indicated number of days .", "The red asterisk shows the presence of IFT27RNAiRES .", "( C ) Growth curves of the non-induced ( blue ) and induced ( red ) IFT27RNAiRES cells .", "( D ) Immunofluorescence of IFT27RNAiRES cells using the anti-TY1 antibody .", "The first panel shows the phase-contrast image merged with DAPI staining and the second shows the BB2 anti Ty-1 antibody staining .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 012 To evaluate the ability of IFT27RNAiRES to participate in IFT , a version fused to YFP at the C-terminus was synthetized and transfected into the IFT27RNAi cell line in order to exclusively track the fluorescent RNAi-resistant version of IFT27 ( Figure 10 ) .", "Immunoblotting of this IFT27RNAiRESYFP cell line using the anti-IFT27 antibody showed reduced expression of endogenous IFT27 upon induction , while expression of IFT27RNAiRES::YFP seems unaffected ( Figure 10A ) .", "Live microscopy analyses revealed a clear localization of the fusion protein in the flagellum and at the base of the organelle regardless of RNAi induction ( Figure 10B ) .", "As expected , the tracks made in the induced IFT27RNAiRESYFP cell line context are more noticeable than in the non-induced one .", "Kymograph analyses of IFT27RNAiRES::YFP ( Figure 10C; Video 2; Figure 3 ) revealed an average anterograde velocity of 2 . 3 ± 0 . 87 µm/s ( n = 161 ) for the non-induced cells and 2 . 0 ± 0 . 53 µm/s ( n = 177 ) for the induced cells .", "Retrograde IFT mean speed was measured at 4 . 4 ± 1 . 3 µm/s ( n = 211 ) for non-induced cells and 4 . 0 ± 1 . 4 µm/s for induced cells ( n = 227 ) , values comparable with GFP::IFT27 ( Figure 2D ) .", "Furthermore , immunofluorescence assays showed a clear colocalization of the endogenous and the recoded fusion protein in the flagellum and at the base of the organelle in both non-induced and induced context ( Figure 3B , C ) .", "Overall , these results demonstrate the specificity of the IFT27 silencing phenotype since the RNAi-resistant version of IFT27 complements the IFT27RNAi phenotype , indicating that IFT27RNAiRES and the YFP-tagged version of the protein are functional . 10 . 7554/eLife . 02419 . 013Figure 10 . IFT27RNAiRES::YFP traffics inside the trypanosome flagellum in the presence and absence of endogenous IFT27 . ( A ) Western blot using the anti-IFT27 showing a decrease of the IFT27 endogenous protein .", "The anti-PFR L13D6 antibody was used as loading control and cells were induced for the indicated number of days .", "( B ) Live microscopy imaging of non-induced ( top panels ) and induced ( bottom panels ) IFT27RNAiRESYFP cells .", "White arrows show the fluorescent protein pool at the base of the flagellum and yellow arrows show the distal tip of the flagellum .", "Arrowheads indicate the successive position of an anterograde IFT train .", "( C ) Kymographs show clear IFT tracks made by IFT27RNAiRES::YFP in non-induced ( left ) and 3 day induced ( right ) cells ( Video 2 ) .", "Scale bars: 5 µm in B . In C , horizontal scale bar is 2 µm and vertical scale bar is 2 s . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 01310 . 7554/eLife . 02419 . 014Video 2 . IFT27RNAiRES::YFP traffics inside the trypanosome flagellum in the presence and the absence of endogenous IFT27 . Observations of live , non-induced and 3-day-induced IFT27RNAiRESYFP cells using time-lapse epifluorescence microscopy using a DMI4000 microscope .", "Acquisitions were made at room temperature and frames were taken every 250 ms for 30 s by an Evolve 512 EMCCD Camera .", "Videos were exported to .", "AVI format with the ImageJ 1 . 47g13 software . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 014 Rab proteins function as molecular switches cycling between a GTP-bound ‘ON’ form and a GDP-bound ‘OFF’ form .", "While multiple effector proteins recognize the GTP-bound form , the GDP-bound form is unable to form such interactions .", "These two conformational changes also control the cellular localization of Rab proteins ( Stenmark , 2009 ) and as a consequence , mutations leaving the proteins in a GTP-locked or GDP-locked state affect the function and localization of this GTPases .", "To evaluate the significance of the GTP/GDP state of IFT27 , two missense mutations were introduced at critical residues ( Figure 1 , GeneBank Accession Numbers KF147878 and KF147879 ) of the epitope-tagged IFT27RNAiRES gene .", "The Q67L mutation replaces the catalytic glutamine at the G3 region found in most small G proteins with a leucine and is predicted to disrupt GTP hydrolysis , leaving the protein in a GTP-bound , active state .", "On the other hand , the mutation on the G1 domain replacing a threonine with an asparagine , T19N , is predicted to abolish GTP binding and should leave the protein in a GDP-bound , inactive state .", "These mutated versions were incorporated into the genome of the IFT27RNAi cell line context and were consequently named IFT27RNAi+IFT27RNAiREST19N and IFT27RNAi+IFT27RNAiRESQ67L respectively .", "For simplicity , we will refer to them as IFT27RNAiREST19N and IFT27RNAiRESQ67L .", "Western blot showed that the amount of endogenous IFT27 decreased after 1 day of induction in both cell lines , leaving only the modified T19N ( GDP-locked ) and Q67L ( GTP-locked ) RNAi-resistant proteins ( Figure 11A , C , stars ) .", "After induction , the growth rate of the IFT27RNAiRESQ67L cell line did not vary ( Figure 11B ) , while the IFT27RNAiREST19N cells showed impaired growth ( Figure 11D ) .", "Therefore , the modified T19N protein produces a phenotype only when the endogenous IFT27 expression is inhibited , suggesting a recessive effect of the T19N mutation .", "Immunofluorescence assays using the monoclonal antibodies Mab25 and L8C4 showed that the IFT27RNAiRESQ67L ( GTP-locked ) cells were undistinguishable from their non-induced control ( Figure 11E ) whereas IFT27RNAiREST19N ( GDP-locked ) cells build unusually short flagella after 3 days of induction ( Figure 11F , arrows ) .", "The flagellar length of the IFT27RNAiRESQ67L cell line showed no significant differences between non-induced ( 16 . 9 ± 1 . 9 µm , n = 100 ) and induced cells ( 16 . 6 ± 2 . 98 µm , n = 100 ) whereas flagellar length of IFT27RNAiREST19N cells dropped to 3 . 7 ± 3 . 1 µm ( n = 79 ) after induction compared to 17 . 6 ± 2 . 1 µm ( n = 100 ) for the non-induced controls .", "However , by carefully analysing both IFT27RNAiREST19N and IFT27RNAiRESQ67L cells in non-induced conditions , we noticed that the length of the flagellum was shorter by about 3 µm compared to cell that are not expressing a mutated form of IFT27 ( Figure 12 ) .", "This interesting result indicates that the expression of a GDP or GTP-locked form of IFT27 has a slightly negative dominant impact on the formation of the trypanosome flagellum .", "Once the endogenous IFT27 is depleted , this effect is exacerbated in the case of the GDP-locked version but not in the case of the GTP-locked version ( Figure 12 ) .", "These observations reveal that the sole expression of the GTP-locked version , although able to sustain flagellum formation , is not neutral . 10 . 7554/eLife . 02419 . 015Figure 11 . Expression of IFT27RNAiREST19N and IFT27RNAiRESQ67L produces different phenotypes .", "( A and C )", "IFT27RNAiRESQ67L ( A ) and IFT27RNAiREST19N cells ( C ) were induced the indicated number of days and western blots were performed using the anti-IFT27 antibody .", "A decrease in the amount of the endogenous protein can be observed while the expression of the modified RNAi-resistant proteins remains constant ( red stars ) .", "( B and D )", "Growth curves of the non-induced and induced IFT27RNAiRESQ67L ( B ) and IFT27RNAiREST19N ( D ) cell lines .", "( E and F )", "Immunofluorescence assays with the IFT27RNAiRESQ67L ( E ) and IFT27RNAiREST19N ( F ) cells .", "After methanol fixation , non-induced and 3 day-induced cells were stained using L8C4 together with Mab25 to detect the PFR and the axoneme respectively .", "Arrows show abnormal flagella .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 01510 . 7554/eLife . 02419 . 016Figure 12 . Expression of the GTP- or GDP-locked form of IFT27 has a slightly dominant-negative impact on the formation of the trypanosome flagellum . Flagellar length comparison of IFT27RNAiRES ( expressing a non-mutant version of IFT27 ) , IFT27RNAiRESQ67L ( GTP-locked ) , and IFT27RNAiREST19N ( GDP-locked ) cells .", "Flagellum length was measured after the indicated hours upon RNAi induction using the axoneme marker Mab25 ( n = 200 for each induction time ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 016 To investigate if the IFT machinery was disrupted in the IFT27RNAiREST19N cell line , immunofluorescence assays using Mab25 and the anti-IFT172 were performed ( Figure 13A ) .", "While the IFT172 staining looked normal in the non-induced context ( Figure 13Aa–d ) , the induced IFT27RNAiREST19N cells did not show signs of IFT B complex accumulation but IFT172 was found concentrated at the base of the organelle ( Figure 13Ae–h ) in a way reminding of the localization of the IFT140 protein in the IFT27RNAi cell line ( Figure 7 ) .", "This is in stark contrast with the IFT27RNAi cell line where the depletion of IFT27 is accompanied by accumulation of IFT172 inside the flagellum ( Figure 13B ) .", "The phenotype obtained in the induced IFT27RNAiREST19N cells is reminiscent to the ones observed upon disruption of the anterograde IFT machinery ( Pazour et al . , 2000; Absalon et al . , 2008b ) and shows that the expression of IFT27RNAiREST19N in the absence of endogenous IFT27 triggers an anterograde IFT phenotype .", "To understand this intriguing phenotype , the flagellar structure of the IFT27RNAiREST19N cell line was analyzed by TEM and scanning electron microscopy .", "TEM observations were not conclusive , as no visible defects in the axoneme structure were detected .", "These results could be explained by the fact that T . brucei assembles the new flagellum while retaining the old one and that only this one , formed prior to the RNAi-mediated silencing of IFT27 , could be observed .", "On the other hand , SEM revealed the presence of abnormally short flagella with no visible dilation ( Figure 13Aj , arrow ) and the presence of a ‘flagellar sleeve’ , a long and thin extension of the flagellum membrane ( Figure 13Aj , arrowheads ) .", "This is very different from the induced IFT27RNAi cells that possess a short , bulky flagellum , in agreement with the presence of an excessive accumulation of material ( Figure 12Bj , arrow ) .", "Since the IFT dynein is found concentrated at the base of flagellum after IFT27 depletion ( Figure 8 ) , we wondered what could be the localization of the motor in the presence of the Q67L and T19N versions of IFT27 .", "This was addressed by immunofluorescence assays using an anti-DHC1b antibody in non-induced and induced IFT27RNAiRESQ67L ( GTP-locked ) or IFT27RNAiREST19N ( GDP-locked ) cells .", "In IFT27RNAiRESQ67L cells , the dynein signal showed no significant difference between the non-induced and induced conditions ( Figure 14 ) suggesting that the motor is functional and participates normally to IFT .", "In contrast , DHC1b remained at the flagellum base in induced cells solely expressing the T19N mutant protein ( Figure 14e–h , arrow ) .", "In conclusion , the GDP-locked version does not sustain the entry of the IFT-B complex in the flagellar compartment , leading to an anterograde phenotype .", "Nevertheless , dynein is still targeted to the base of the flagellum . 10 . 7554/eLife . 02419 . 017Figure 13 . The IFT27RNAiREST19N cell line displays an anterograde phenotype upon induction .", "( A ) Non-induced ( a to d ) and 3-day-induced ( e to", "h ) IFT27RNAiREST19N cells were fixed in methanol , stained with the anti-IFT172 and Mab25 and counterstained with DAPI .", "Insets show the axoneme and IFT172 localization .", "( i and j )", "Scanning electron microscopy of non-induced and induced IFT27RNAiREST19N cells .", "The white arrow shows the short flagellum and the black arrowheads in j indicate the flagellar sleeve .", "( B ) Immunofluorescence of non-induced ( a to d ) and 3-day-induced IFT27RNAi cells using the same conditions .", "Scanning electron microscopy of the non-induced and induced IFT27RNAi cell line .", "The white arrow in j points to the short flagellum with an abnormally large diameter .", "Scale bars: 5 µm from a to h , 1 µm in i and j . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 01710 . 7554/eLife . 02419 . 018Figure 14 . The IFT27RNAiREST19N cell line shows perturbation of IFT dynein entry into the flagellum . Non-induced and cells induced for 3 days of the IFT27RNAiQ67L ( a to d ) and IFT27RNAiREST19N ( e to", "h ) cell lines were fixed in methanol , probed with the anti-DHC1b antibody and counterstained with DAPI .", "Arrows indicate the dynein pool around the base of the flagellum .", "Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 018 To evaluate the ability of the mutated versions of IFT27 to traffic inside the flagellar compartment , the IFT27RNAiREST19N and Q67L point mutations were expressed as fusion proteins with YFP in the IFT27RNAi cell line .", "Immunofluorescence assays using the anti-IFT27 and the anti-GFP antibodies ( Figure 15 ) showed that IFT27RNAiRESQ67L is localized inside the flagellum in non-induced ( Figure 15Aa–d ) and induced ( Figure 15Ae–h ) cells as the unmodified IFT27RNAiRES::YFP protein ( Figure 3 ) .", "In addition , the fluorescent Q67L modified protein traffics inside the flagellum in both conditions ( Figure 15B; Video 3 ) and kymograph analyses demonstrated anterograde and retrograde train speed similar to the unmodified protein ( Figure 15B , Figure 3 ) . 10 . 7554/eLife . 02419 . 019Figure 15 . IFT27 in its inactive form is unable to penetrate the flagellar compartment .", "( A ) IFT27RNAiRESQ67LYFP cells were non-induced ( a to d ) or induced for 3 days ( e to", "h ) , stained with the anti-IFT27 , the anti-GFP antibody and counterstained with DAPI .", "( B ) Live observation of the non-induced and 3-day-induced IFT27RNAiRESQ67LYFP cell line .", "Yellow arrows indicate the flagellar tip , white arrowheads show the fluorescent protein pool at the base of the flagellum and the asterisk indicates the cells used for kymograph analysis .", "( C ) Immunofluorescence assays of non-induced ( a to d ) or 3 day induced ( e to", "h ) IFT27RNAiREST19NYFP cells using the anti-IFT27 and the anti-GFP antibody .", "Scale bar for ( A ) , and ( C ) : 5 µm .", "In ( B ) , horizontal scale bar is 2 µm and vertical scale bar is 2 s .", "( D ) Immunoprecipitates of total proteins by an anti-GFP antibody were separated on 4–15% polyacrylamide gels for the three different cell lines as indicated .", "Corresponding immunoblots with the anti-IFT172 ( middle row ) and the anti-IFT22 ( bottom row ) are shown and lanes labeled as ‘Control’ were precipitations with a rabbit antibody raised against T . brucei aldolase .", "These results were replicated in four independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 01910 . 7554/eLife . 02419 . 020Video 3 . IFT27 in its active form traffics normally in the flagellar compartment . Live , time-lapse epifluorescence microscopy of non-induced and 3-day induced IFT27RNAiRESQ67L::YFP .", "All acquisitions were made at room temperature with frames taken every 250 ms for 30 s .", "Videos were exported to .", "AVI format with the ImageJ 1 . 47g13 software . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 020 Strikingly , the IFT27RNAiREST19N::YFP fusion protein was always found in the cytoplasm in both non-induced and induced situations ( Figure 15C ) .", "No signal could be detected in the flagellum , neither by immunofluorescence assays ( Figure 15C ) nor by live cell imaging ( Video 4 ) in both non-induced and induced cells , suggesting that the modified protein is not able to access the flagellum .", "Since the induced IFT27RNAiREST19N cell line shows no signs of IFT-B accumulation in contrast to the induced IFT27RNAi cell line ( Figure 12 ) , a possible explanation is that IFT27RNAiREST19N disrupts the assembly of the IFT complex B . 10 . 7554/eLife . 02419 . 021Video 4 . IFT27 in its inactive form is unable to penetrate the flagellar compartment . Live , time-lapse epifluorescence microscopy of non-induced IFT27RNAiREST19N::YFP cells as well as 3-day induced ones .", "All acquisitions were made at room temperature with frames taken every 250 ms for 30 s .", "Videos were exported to .", "AVI format with the ImageJ 1 . 47g13 software . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 021 To first confirm the interaction of IFT27 with the IFT complex B of T . brucei , co-immunoprecipitation assays were performed using the anti-GFP antibody .", "The IFT27RNAiRES::YFP and the two mutated proteins were successfully immunoprecipitated in all three cell lines as confirmed by western blot with the anti-IFT27 ( Figure 15D , top panel ) .", "Next , immunoblotting using two antibodies recognizing IFT-B proteins IFT22 and IFT172 showed that the immune complexes formed by IFT27RNAiRES::YFP and IFT27RNAiRESQ67L::YFP contain both proteins ( Figure 15D , left and middle rows ) .", "However , immunoprecipitation of IFT27RNAiREST19N::YFP failed to bring down IFT22 and IFT172 ( Figure 15D , right panel ) .", "These results demonstrate that IFT27RNAiRESQ67L is associated to the IFT-B complex like the non-modified protein , supporting the in vivo results ( Figure 15B ) .", "In contrast , the T19N mutant version of IFT27 is unable to interact with at least two members of the IFT-B complex .", "Overall , these results demonstrate that IFT27 needs to be in a GTP-bound state to interact with the rest of the IFT complex and access the flagellar compartment ." ], [ "Studies in different model organisms have shed light into the structure and nature of the IFT machinery but few data are available about the mechanisms coordinating the entry of the IFT complexes into the flagellum .", "Amongst the possible regulators , several small GTPases have been associated with the flagellum: ARL-13b is involved in the stabilization of ciliary assembly in C . elegans and its mutations are associated with a ciliopathy named Joubert syndrome ( Cantagrel et al . , 2008; Cevik et al . , 2010; Li et al . , 2010 ) .", "The RAN importin system , known to be involved in nucleocytoplasmic transport , is responsible for the entry of the kinesin KIF17 inside the primary cilium ( Dishinger et al . , 2010 ) .", "However , none of these proteins are part of any of the IFT complexes .", "Here , we have shown by immunoprecipitation that IFT27 likely is a constitutive member of the IFT-B complex in T . brucei .", "Previous purification assays of the B complex using C3/DYF13 in T . brucei identified most of the IFT-B predicted proteins but were not able to detect IFT27 , perhaps because of its short molecular weight and the under-representation of small polypeptides inherent to the MS analysis .", "Significantly , IFT20 and IFT25 , the two other predicted IFT-B proteins of short size , were also missing ( Franklin and Ullu , 2010 ) .", "We observed that GFP-tagged IFT27 traffics in the flagellum in a similar way to IFT52 or IFT81 , two well-studied IFT-B members ( Bhogaraju et al . , 2013; Buisson et al . , 2013 ) showing that IFT27 likely associates to the IFT-B complex and participates to the IFT process ( Figure 16A ) .", "These findings are consistent with the fact that Chlamydomonas IFT27 is part of IFT-B complex and traffics inside the flagellum ( Lucker et al . , 2005; Qin et al . , 2007 ) . 10 . 7554/eLife . 02419 . 022Figure 16 . Model of IFT27 function during IFT assembly .", "( A ) The proposed model shows a GTP-bound IFT27 associated with the IFT-B complex prior to the entry into the flagellum .", "After leaving the flagellar compartment , GTP is hydrolyzed , releasing IFT27 and disassembling the IFT-B complex .", "( B ) After RNAi-mediated depletion of IFT27 , the IFT-B complex enters the flagellum but is unable to exit because of the absence of IFT dynein inside the flagellar compartment .", "To explain the anterograde phenotype seen in after expression of the GDP-bound version of IFT27 , the inactive IFT27 could negatively control the entry of the IFT-B complex ( C ) .", "The PFR was not drawn for clarity . DOI: http://dx . doi . org/10 . 7554/eLife . 02419 . 022 Surprisingly for an IFT-B protein , IFT27 turns out to be essential for retrograde transport as its knockdown triggered the formation of short flagella with an unexpected accumulation of IFT-B proteins such as IFT52 , IFT172 , IFT22 , and PIFTC3 .", "By TEM , this accumulated material appeared as electron dense regions spread around short , irregular and often disrupted axonemes whereas the basal body and transition zone appeared normal .", "All these features are reminiscent of a retrograde phenotype , usually caused by a deficit of either the IFT dynein or the IFT-A complex ( Pazour et al . , 1998; Ou et al . , 2007; Absalon et al . , 2008a ) .", "Interestingly , DHC1b and IFT140 , two essential molecules of the dynein and IFT-A complex respectively , are not able to enter the flagellum in the absence of IFT27 ( Figure 16B ) .", "The IFT dynein is the retrograde motor of IFT that brings back IFT trains from the tip to the base of the flagellum , a process in which IFT-A is also involved although its exact contribution remains to be shown .", "Being an IFT-B protein , IFT27 could therefore regulate IFT-A and dynein transport at the physical interface between IFT-A , IFT-B and dynein complexes to ensure coordinate import in the flagellar compartment .", "Supporting that view , some relationships between IFT-B proteins and the IFT dynein motor have been suggested in the literature .", "First , IFT-B and dynein genes are both controlled at the transcriptional level by the transcription factor RFX whereas IFT-A and kinesin II are regulated differently in metazoans ( Thomas et al . , 2010 ) .", "Second , co-immunoprecipitation experiments indicate that DHC1b interacts with the IFT-B protein IFT172 in Chlamydomonas ( Pedersen et al . , 2006 ) .", "In T . brucei , we could not detect a direct interaction between IFT27 and DHC1b or IFT27 and IFT140 by co-immunoprecipitation ( unpublished data ) but the association might be transient , for example taking place only during protein entry in the flagellar compartment .", "It has been shown that IFT27 forms a complex with IFT25 , and both could be involved in flagellum entry regulation ( Wang et al . , 2009 ) .", "In addition , the IFT25/27 structure displays a surface patch able to mediate protein–protein interactions ( Bhogaraju et al . , 2011 ) .", "This IFT25/27 sub-complex could then act as a scaffold mediating the interaction of the IFT-B complex with dynein and IFT-A complexes .", "In Chlamydomonas , the RNAi knockdown of IFT27 produced cells with abnormally short flagella but the nature of the IFT defect ( effect on anterograde or retrograde IFT ) has not been reported ( Qin et al . , 2007 ) .", "In this organism , it is the only IFT protein whose inhibition triggers cell cycle defects .", "However , the specificity of the phenotype has not been proven and an off-target effect cannot be excluded .", "In trypanosomes , the flagellum is essential for cell morphogenesis and cytokinesis and inhibition of all IFT genes studied so far invariably resulted in growth arrest ( Kohl et al . , 2003; Davidge et al . , 2006; Absalon et al . , 2008a; Franklin and Ullu , 2010 ) .", "In the present study , the possibility of this being an off-target effect has been ruled out upon complementation of the IFT27RNAi phenotype by the expression of an RNAi-resistant version of IFT27 that rescued both the observed growth and flagellar phenotype while displaying normal IFT trafficking in non-induced as well as in induced cells .", "IFT27 has all the necessary features to function as a GTPase , although it displays low intrinsic activity during in vitro assays ( Bhogaraju et al . , 2011 ) .", "The orthologue of IFT27 in the related protozoan Trypanosoma cruzi exhibits GTPase activity in vitro ( Ramos et al . , 2005 ) .", "As the T . cruzi protein is 88 . 5% similar to IFT27 and all the key residues are conserved , it is very likely that the T . brucei protein is also able to hydrolyze GTP .", "We have shown that the Q67L version apparently behaves like the unmodified protein even in the complete absence of the endogenous protein .", "Thus , IFT27 likely interacts with other IFT complex B members , controls the entry of the IFT-A complex and DHC1b and enters the flagellum in a GTP-bound state ( Figure 16A ) .", "These findings could be compared with previous results showing that a constitutively active GTPase activity does not affect the localization of various Rab proteins ( Richardson et al . , 1998; Alvarez et al . , 2003; Nachury et al . , 2007 ) .", "Another possible caveat to explain the absence of phenotype in the cells expressing the Q67L version of IFT27 is that the protein is not actually impaired in its GTPase activity so that it undergoes a normal chemical cycle .", "However , this is not likely the case as we noticed that cells expressing both the endogenous and the GTP-locked protein possessed a slightly shorter flagellum compared to cell that are not expressing a mutated form of IFT27 ( Figure 12 ) .", "These observations reveal that the sole expression of the GTP-locked version , although able to sustain flagellum formation , is not neutral .", "Since the constitutively active version of IFT27 is found inside the flagellum and interacts with the rest of the IFT machinery , one could argue about the need of an inactive state .", "However , T . brucei has a complex life cycle in which the length of the flagellum can vary from 3 µm to 30 µm ( Rotureau et al . , 2011 ) .", "Therefore , it will be interesting to see the impact of the GTP/GDP cycle on assembly of flagella of different length during the life cycle of T . brucei .", "The expression of the T19N version , predicted to leave IFT27 in a GDP-bound form , did not cause a dominant negative phenotype .", "However , the modified T19N protein was not found inside the flagellar compartment neither in the presence nor in the absence of endogenous IFT27 and was unable to associate with the IFT-B complex .", "One would then expect that the exclusive expression of IFT27 in its inactive state should phenocopy the IFT27RNAi cell line .", "Strikingly , this was not the case as only very short flagella that do not exhibit accumulation of IFT proteins were built , typical of anterograde inhibition ( Absalon et al . , 2008b ) .", "Several explanations could be proposed to explain this intriguing result .", "First , the inactive IFT27 protein could sequester other components of the IFT-B complex in the cytoplasm and inhibit their transfer to the base of the flagellum .", "However , immunofluorescence assays revealed that IFT172 is still found concentrated at the base of the flagellum but not inside organelle .", "Second , the inactive protein could interfere with assembly or stability of the IFT-B complex in the cytoplasm , a hypothesis supported by the reported decrease in the amount of several IFT proteins in the Chlamydomonas IFT27 RNAi experiments ( Qin et al . , 2007 ) .", "However , this seems unlikely here because the total amount of IFT172 and IFT22 did not decrease when the IFT27T19N protein was exclusively expressed in the IFT27RNAiREST19N cell line ( unpublished data ) .", "Third , the inactive IFT27 could negatively control the entry of the IFT-B complex hence resulting in an anterograde phenotype while the wild-type protein in non-induced conditions would overhaul this effect .", "Curiously , both IFT27 and IFT25 are absent from C . elegans , D . melanogaster , and Giardia intestinalis ( van Dam et al . , 2013 ) .", "This could be the consequence of different organization of the flagellum base , a sub-compartment involved in the regulation of the entry of flagellar components ( Reiter et al . , 2012 ) .", "No obvious basal body structure can be found at the base of C . elegans cilia ( Perkins et al . , 1986; Williams et al . , 2011 ) and in D . melanogaster , the basal body possesses a unique electron-dense ring structure ( Enjolras et al . , 2012 ) .", "The δ- and ε-tubulins , involved in basal body maturation , are also absent from the genomes of these two organisms ( Dutcher , 2001 ) .", "Finally , the basal bodies are deeply rooted in the cytoplasm in Giardia , with long cytoplasmic axoneme segments ( Dawson and House , 2010 ) .", "Therefore , the absence of IFT27 and IFT25 could be related to the non-canonical structure of their flagellum base , implying a different process for the entry of the IFT proteins .", "Alternatively , the requirement for retrograde transport might be different according to the type of cilia , necessitating specific adaptations for IFT-A/IFT dynein trafficking .", "Inhibition of retrograde IFT moderately affects the length of sensory cilia in Drosophila ( reduced by one third in IFT140 or dynein mutants , Lee et al . , 2008 ) or C . elegans ( reduced by 25% in IFT-A mutants , Blacque et al . , 2006 ) in contrast to the severe reduction observed in Chlamydomonas ( Pazour et al . , 1999 ) , trypanosomes ( Kohl et al . , 2003 ) or Leishmania ( Satir and Christensen , 2007 ) .", "In summary , these findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery ." ], [ "All procyclic T . brucei cell lines were derivatives of the strain 427 and grown in SDM79 medium with hemin and 10% fetal calf serum .", "The 29–13 cell line expressing the T7 RNA polymerase and the tetracycline-repressor ( Wirtz et al . , 1999 ) has been described previously , as well as the cell line expressing GFP::IFT52 ( Absalon et al . , 2008b ) .", "For generation of the IFT27RNAi cell line , a 360-nucleotide fragment of IFT27 ( Tb 927 . 3 . 5550 ) was amplified by PCR and cloned in the pZJM vector ( Wang et al . , 2000 ) , allowing tetracycline-inducible expression of dsRNA generating RNAi upon transfection in the 29-13 recipient cell line .", "The dsRNA is expressed from two tetracycline-inducible T7 promoters facing each other in the pZJM vector .", "Primers were selected using the RNAit algorithm to ensure that the fragment lacked significant identity to other genes to avoid cross-RNAi ( Redmond et al . , 2003 ) .", "The fragment was amplified by PCR with the forward primer cgatcgAAGCTTaaacttgcgtcttcaggtgg and the reverse primer gtcatCTCGAGatttgcgagatctttcccct , digested with XhoI and HindIII ( sites underlined ) and ligated into the corresponding sites of the pZJM vector .", "The plasmid was linearized at the unique NotI site in the rDNA intergenic targeting region before transfection ( Wang et al . , 2000 ) .", "GeneCust Europe ( Dudelange , Luxembourg ) conducted the chemical synthesis of the pPCPFReGFPIFT27 plasmid allowing a GFP tagging at the N-terminal of IFT27 .", "The whole IFT27 gene ( 552 nucleotides ) was synthetized , digested with NheI and EcoRV and cloned into the corresponding sites of the pPCPFR vector ( Adhiambo et al . , 2009 ) .", "The resulting plasmid was linearized with NsiI , targeting integration in the intergenic region of PFR2 .", "Generation of the p2845TdTomatoIFT140 plasmid enabling the expression of TdTomato::IFT140 was performed by modifying the p2845IFT140 mCherry plasmid .", "The mCherry gene was removed from p2845IFT140 using XhoI and HindIII while simultaneasouly getting the TdTomato gene from the p2675TdTomatoIFT81 plasmid with the same restriction enzymes .", "Ligation was performed between the digested p2845IFT140 plasmid and the TdTomato insert overnight at 16°C , the resulting plasmid was then transfected into XL-10 Gold Ultracomptent Cells ( Agilent Technologies , France ) to allow amplification .", "Plasmids from 10 different colonies were double digested with XhoI and HindIII to assess the presence of the TdTomato insert .", "For the RNAi-resistant version of IFT27 , a new version of the gene was chemically synthesized modifying the first 411 nucleotides in order to make synonymous mutations , leaving the amino acid sequence unchanged ( GeneBank Accession Number KF147877 ) .", "This IFT27RNAires gene was then cloned into the pPCPFR vector .", "Additional point mutations ( Figure 1 ) made by GeneCust Europe enabled the creation of the RNAi-resistant versions of IFT27 in a GTP ( Q67L ) or GDP ( T19N ) locked state ( GeneBank Accession Numbers KF147878 and KF147879 ) .", "The YFP-tagged version of IFT27RNAires was made by synthetizing and cloning the YFP gene into the pPCPFRIFT27RNAires vector between the NheI and BamHI restriction sites .", "The Q67L and T19N point mutations were then performed to finally obtain the C-terminus-tagged pPCPFRIFT27RNAiresQ67LYFP and pPCPFRIFT27RNAiresT19NYFP plasmids .", "Linearization was carried out with NsiI .", "In all cases , trypanosomes were transfected with the plasmid constructs by Nucleofector technology ( Lonza , Italy ) as described before ( Burkard et al . , 2007 ) .", "Transfectants were grown in media with the appropriate antibiotic concentration and clonal populations were obtained by limited dilution .", "For antibody production , IFT27 was expressed as a recombinant fusion GST protein in Escherichia coli .", "The full length IFT27 coding sequence was amplified by PCR using Platinum Taq Polymerase High Fidelity ( Invitrogen ) and primer pairs gaggaaGGATCCatggtaaacttgcgtcttcagg and cgcaacagCCCGGGttacctcatttggg ( BamHI and XmaI sites underlined ) .", "PCR products were ligated into the pCR2 . 1-TOPO vector ( Invitrogen , Carlsbad , CA ) following the manufacturer's instructions .", "IFT27 sequence was then excised using BamHI and XmaI enzymes and ligated into compatible sites of the pGEX B vector ( GE Healthcare , Piscataway , NJ ) .", "The plasmid was sequenced to confirm correct fusion with GST and transformed in E . coli BL21 .", "Protein expression was triggered by adding 0 . 1 mM IPTG for 3 hr at 37°C and analyzed by SDS-polyacrylamide gel electrophoresis ( PAGE ) followed by Coomassie staining .", "Glutathione transferase ( GST ) -coupled proteins were purified as described previously ( Smith and Johnson , 1988 ) and 20 μg were administrated by four successive subcutaneous injections ( every 3 weeks ) to five BALB/c mice for immunization .", "After bleeding , sera were absorbed against GST .", "Sera from mice immunized with GST alone were used as negative controls .", "For transmission electron microscopy , cells were fixed in culture media overnight at 4°C in 2 . 5% glutaraldehyde–0 . 1 M cacodylate buffer ( pH 7 . 2 ) and postfixed in OsO4 ( 2% ) in the same buffer .", "After serial dehydration samples were embedded in Agar 100 ( Agar Scientific , Ltd . , United Kingdom ) and left to polymerize at 60°C .", "Ultrathin sections ( 50–70 nm thick ) were collected on Formvar-carbon-coated copper grids using a Leica EM UC6 ultramicrotome and stained with uranyl acetate and lead citrate .", "Observations were made on a Tecnai 10 electron microscope ( FEI ) and images were captured with a MegaView II camera and processed with AnalySIS and Adobe Photoshop CS4 ( San Jose , CA ) .", "For scanning electron microscopy , samples were fixed overnight at 4°C in 2 . 5% glutaraldehyde–0 . 1 M cacodylate buffer ( pH 7 . 2 ) and postfixed in 2% OsO4 in the same buffer .", "After serial dehydration , the samples were dried at the critical point and coated with platinum according to standard procedures .", "Observations were made in a JEOL 7600F microscope .", "Cultured parasites were washed twice in SDM79 medium without serum and spread directly onto poly-L-lysine coated slides .", "The slides were air-dried for 10 min , fixed in methanol at −20°C for 30 s and rehydrated for 10 min in PBS .", "For immunodetection , slides were incubated with primary antibodies diluted in PBS with 0 . 1% Bovine Serum Albumine ( BSA ) for 1 hr .", "Three washes of 10 min were performed and the secondary antibody diluted in PBS with 0 . 1% BSA was added to the slides .", "After an incubation of 45 min , slides were washed three times in PBS for 10 min and DAPI ( 2 µg/µl ) was added .", "Slides were mounted using ProLong antifade reagent ( Invitrogen ) .", "Antibodies used were the anti-IFT27 antisera of one mouse diluted 1/800 , the anti-IFT22/RABL5 mouse polyclonal antibody ( Adhiambo et al . , 2009 ) , Mab25 recognizing TbSAXO1 , a protein found all along the trypanosome axoneme ( Pradel et al . , 2006 ) , L8C4 , recognizing PFR2 , a major paraflagellar rod protein ( Kohl et al . , 1999 ) , BB2 recognizing the TY1-epitope ( Bastin et al . , 1996 ) , the anti-IFT172 mouse monoclonal and anti-DHC1b polyclonal antibodies diluted 1/200 and 1/100 respectively ( our unpublished data ) , the Living Colors DsRed Polyclonal Antibody ( Clontech ) diluted 1/1500 and the rabbit polyclonal anti-GFP ( Invitrogen ) diluted 1/500 .", "Subclass-specific secondary antibodies coupled to Alexa 488 and Cy3 ( 1/400; Jackson ImmunoResearch Laboratories , West Grove , PA ) were used for double labeling .", "Sample observation was performed using a DMI4000 microscope equipped with a 100X NA 1 . 4 lens ( Leica , Wetzlar , Germany ) and images captured with a CoolSnap HQ camera ( Roper Scientific , Tucson , AZ ) .", "Pictures were analyzed using ImageJ 1 . 47g13 software ( National Institutes of Health , Bethesda , MD ) and images were merged and superimposed using Adobe Photoshop CS4 .", "For live video microscopy , cells were covered by a coverslip and observed directly with the DMI4000 microscope at room temperature .", "Videos were acquired using an Evolve 512 EMCCD Camera ( Photometrics , Tucson , AZ ) , the Metamorph acquisition software ( Molecular Probes , Sunnyvale , CA ) .", "Trypanosomes were observed and the IFT was recorded at 250 ms per frame during 30 s using the Evolve 512 EMCCD Camera .", "Videos were mounted using iMovie 2011 .", "Kymographs were extracted and analyzed as described previously ( Chenouard et al . , 2010 , Buisson et al . , 2013 ) .", "Cells were washed in PBS and boiled in Laemmli loading buffer before SDS-PAGE separation , loading 20 μg of total cell protein per lane .", "Proteins were transferred overnight at 25V at 4°C to polyvinylidene fluoride membranes , then blocked with 5% skimmed milk in PBS-Tween 0 . 1% ( PBST ) and incubated with primary antibodies diluted in 1% milk and PBST .", "The anti-IFT27 serum was diluted 1/800 , and BB2 was diluted 1/100 .", "To detect GFP and YFP , we used a mouse monoclonal anti-GFP antibody ( Roche ) diluted 1/500 .", "As loading controls , antibodies anti-PFR ( L13D6 ) ( Kohl et al . , 1999 ) diluted 1/50 and anti-aldolase ( a kind gift of Paul Michels , Brussels , Belgium ) diluted 1/1000 were used .", "Three membrane washes were performed with PBST for 5 min .", "Species-specific secondary antibodies coupled to horseradish peroxidase ( GE Healthcare ) were diluted 1/20 , 000 in PBST containing 1% milk and incubated for 1 hr .", "Final detection was carried out by using an enhanced chemoluminescence kit and a high performance chemoluminescence film according to manufacturer's instructions ( Amersham , Piscataway , NJ ) .", "50 ml of IFT27RNAiRESYFP , IFT27RNAiRESQ67LYFP and IFT27RNAiREST19NYFP cell lines were grown until they reached a concentration of 1 . 107 cells/ml .", "Cells were washed and incubated in IP2 buffer ( 0 . 8M NaCl , 20 mM Tris–HCl pH 8 , 20 mM EDTA , 2% Triton X100 , 10 µg/ml leupeptin and pepstatine A ) for 30 min at room temperature .", "Soluble protein extract was centrifuged at 21 , 130×g at 4°C for 30 min and supernatant was collected and incubated for 12 hr at 4°C with either 5 µl of polyclonal rabbit anti-GFP ( Invitrogen ) or 5 µl of anti-aldolase in the control .", "Prior to the immune complex recovery , protein A-Sepharose beads ( GE Healthcare , NJ ) were washed in IP1 buffer ( 0 . 4M NaCl , 10 mM Tris–HCl pH 7 , 4 , 10 mM EDTA , 1% Triton X100 , 1 mg/ml ovalbumine ) .", "Immune complexes were recovered by incubation with pretreated protein A-Sepharose beads for 15 min at 4°C .", "The beads then were washed three times with IP1 buffer , twice with IP2 buffer and once with IP3 buffer ( 0 . 4M NaCl , 10 mM Tris–HCl pH 7 , 4 , 10 mM EDTA ) .", "Interaction of the immune complex with the beads was disrupted by boiling in 50 μl of Laemmli loading buffer and analyzed by SDS-PAGE followed by immunoblotting ." ] ]
[ "The construction of cilia and flagella depends on intraflagellar transport ( IFT ) , the bidirectional movement of two protein complexes ( IFT-A and IFT-B ) driven by specific kinesin and dynein motors .", "IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport .", "Surprisingly , the small GTPase IFT27 , a member of the IFT-B complex , turns out to be essential for retrograde cargo transport in Trypanosoma brucei .", "We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment .", "To get further molecular insight about the role of IFT27 , GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27 .", "The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation .", "These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery ." ]
[ "Long , thin structures called cilia and flagella are found on the surface of many cells , and perform a range of roles , including propelling the cells around or sensing changes in the surrounding environment .", "A process called intraflagellar transport ( IFT for short ) is responsible for flagellum construction in eukaryotic cells .", "Protein complexes called IFT trains carry the building blocks that make up flagella along microtubule ‘tracks’ between the base and the tip of a flagellum .", "IFT trains are made from two different protein complexes called IFT-A and IFT-B , which are dragged by various molecular motors .", "The IFT-B complex is necessary for the train to move towards the tip of the flagellum , and so enables the flagellum to grow .", "The IFT-A protein complex is required to recycle the train back towards the base of the flagellum .", "Huet et al . examined the role that a protein called IFT27 plays in intraflagellar transport .", "IFT27 is part of the IFT-B complex , and so it was thought to only affect how flagella grow .", "However , short flagella still grow when IFT27 is absent , but they are filled with IFT trains that are not able to reverse back from the tip .", "Huet et al . reveal that the IFT-A complex and the molecular motor that is essential for reversing the train are not transported into the flagellum if IFT27 is not present .", "This is therefore an unusual case of an IFT-B protein affecting the IFT-A complex and the transport back to the base .", "IFT27 also affects how the IFT-B complex forms .", "ITF27 can bind to some small molecules , which can switch the protein ‘on’ or ‘off’ .", "Huet et al . found that when IFT27 is switched off it is not transported into flagella , and also cannot bind to some of the other proteins in the IFT-B complex .", "This means that if IFT27 is locked in an inactive state , the IFT-B complex does not form , and a flagellum cannot grow .", "Therefore , activated IFT27 is needed for putting together the IFT train and to ensure its movement in either direction along the microtubule tracks ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "immunology and inflammation" ]
The mucosal adjuvant cyclic di-GMP enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo
elife-06670-v2
[ [ "Most pathogens enter the body via mucosal surfaces .", "Immunization by mucosal routes is more effective at inducing protective immunity against mucosal pathogens than systemic immunization .", "Moreover , mucosal vaccines have the benefits of low cost and ease of administration , which make mucosal vaccines particularly suitable for developing countries and during emergency .", "Currently , only a dozen mucosal vaccines are approved for human use .", "This is largely due to problems with developing safe and effective mucosal adjuvants .", "Cyclic di-GMP ( CDG ) is a promising mucosal vaccine adjuvant candidate ( Ogunniyi et al . , 2008; Hu et al . , 2009; Chen et al . , 2010; Madhun et al . , 2011; Gray et al . , 2012 ) .", "It is ubiquitously found in bacteria , but is absent in higher eukaryotes .", "Yan et al . found that intranasal administration of CDG , along with the pneumococcal Ag PsaA , elicits a comparable Ag-specific Ab response , and reduces bacterial colonization to those mice immunized with cholera toxin and PsaA ( Yan et al . , 2009 ) .", "Cholera toxin is the most potent experimental mucosal adjuvant .", "CDG also exhibits balanced TH1 , TH2 , and TH17 immune responses ( Ebensen et al . , 2007 , 2011; Gray et al . , 2012 ) .", "A recent study found that CDG is a more potent activator of both TH1 and TH2 immune responses than LPS , CpG oligonucleotides ( ODN ) , and aluminum salt based adjuvant in mice ( Gray et al . , 2012 ) .", "Thus , CDG is an excellent mucosal vaccine adjuvant candidate promoting both strong humoral and cellular immune responses .", "The mechanism by which CDG acts as a mucosal adjuvant in vivo is not known ( Chen et al . , 2010 ) .", "We previously showed that MPYS-deficient mice ( Tmem173−/− ) completely lost CDG induced Ag-specific Ab and TH responses ( Blaauboer et al . , 2014 ) .", "MPYS , also known as STING , MITA , and TMEM173 , is a type I IFN stimulator ( Ishikawa and Barber , 2008; Jin et al . , 2008; Zhong et al . , 2008 ) .", "However , we found that type I IFN signaling is not required for the mucosal adjuvant activity of CDG in vivo ( Blaauboer et al . , 2014 ) .", "CDG activates both type I IFN and NF-κB signaling ( McWhirter et al . , 2009 ) .", "While MPYS is required for both CDG induced type I IFN and NF-κB activations ( Jin et al . , 2011a; Sauer et al . , 2011 ) , we found that these two pathways can be uncoupled in dendritic cells ( DCs ) and macrophages ( Blaauboer et al . , 2014 ) .", "Of note , it is still unknown which cell type responds to mucosal adjuvant CDG in vivo .", "In this study , we investigated how CDG promotes its mucosal adjuvant activity in vivo .", "We found that CDG enhances Ag uptake in vivo , and selectively activates pinocytosis-efficient DCs in vivo .", "Furthermore , we demonstrated that these CDG activities depend on the expression of MPYS in DCs in vivo ." ], [ "CDG is a potent mucosal vaccine adjuvant with activity similar to that of cholera toxin , the gold standard of a mucosal vaccine adjuvant ( Yan et al . , 2009 ) .", "The 2′3′-cyclic GMP-AMP ( cGAMP ) is a newly discovered mammalian cyclic dinucleotide that also has mucosal adjuvant activity in vivo ( Skrnjug et al . , 2014 ) .", "Both CDG and 2′3′-cGAMP can bind MPYS in vitro ( Burdette et al . , 2011; Gao et al . , 2013a; Sun et al . , 2013 ) .", "The 2′3′-cGAMP has a much better binding affinity to MPYS than CDG ( Gao et al . , 2013c ) .", "Furthermore , 2′3′-cGAMP induces stronger type I IFN production than CDG does in mammalian cells ( Gao et al . , 2013c ) .", "We , thus , asked if the 2′3′-cGAMP exhibits superior mucosal adjuvant activity to CDG in vivo .", "We intranasally administered BALB/C mice with CDG plus OVA Ag , or 2′3′-cGAMP , plus OVA Ag three times at 2 weeks interval .", "The serum anti-OVA IgG1 , IgG2A , and nasal IgA were quantified .", "Surprisingly , CDG adjuvanted vaccine induced higher Ag-specific IgG1 and IgA production than the 2′3′-cGAMP adjuvanted vaccine ( Figure 1A ) .", "The production of OVA-specific IgG2A was similar in both vaccines ( Figure 1A ) . 10 . 7554/eLife . 06670 . 003Figure 1 . Cyclic di-GMP ( CDG ) is a better mucosal pneumococcal vaccine adjuvant than the mammalian cyclic dinucleotide 2′3′-cyclic GMP-AMP ( cGAMP ) in mice .", "( A ) BALB/c mice were intranasally ( i . n . ) immunized with three doses ( 14 days apart ) of OVA ( 20 μg ) alone or together with 5 μg CDG or 5 μg 2′3′-cGAMP .", "Each group consisted of four mice .", "Sera or nasal washes from the 4 mice in the same group were pooled .", "Blood and nasal washes samples were collected 14 days after the last immunization .", "Anti-OVA IgG1 , IgG2A , and IgA were quantified by ELISA .", "n = 3 . ( B ) Splenocytes from immunized BALB/c mice were stimulated with 50 μg/ml OVA for 4 days in culture .", "Supernatants from the same group were pooled together .", "Cytokines were measured in the supernatant by ELISA .", "n = 3 . ( C ) C57BL/6 mice were immunized with 3 doses of pneumococcal surface protein A ( PspA ) ( 2 μg ) alone or together with 5 μg CDG or 5 μg 2′3′-cGAMP as in A . Blood and nasal washes were collected 14 days after the last immunization .", "Anti-PspA IgG1 , IgG2C , and IgA were measured by ELISA as in A . n = 3 . ( D ) Splenocytes from immunized C57BL/6 mice were stimulated with 5 μg/ml PspA for 4 days in culture .", "Cytokines were measured in the supernatant by ELISA as in B . n = 3 . ( E ) Immunized mice were infected ( i . n . ) with S . pneumoniae ( ∼5 . 0 × 106 c . f . u . ) .", "At 48 hr post infection , lung and spleen bacterial burden were determined .", "n = 2 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 003 As a mucosal adjuvant , CDG generates balanced TH1 , TH2 , and TH17 responses .", "We next performed the ex vivo recall assay in splenocytes from immunized mice , and examined the TH cytokine production .", "Again , CDG adjuvanted vaccine generated better IL-13 , a TH2 cytokine , and IL-17 production than the 2′3′-cGAMP adjuvanted vaccine ( Figure 1B ) .", "The TH1 cytokine , IFNγ , was similarly produced by both cyclic dinucleotides ( Figure 1B ) .", "We then replaced OVA Ag with pneumococcal surface protein A ( PspA ) , a protein Ag extensively tested in various pneumococcal vaccines ( Feldman and Anderson , 2014 ) .", "We also used a different mouse strain , C57BL/6 , to repeat the immunization experiment .", "We found that CDG adjuvanted PspA based pneumococcal vaccine generated higher titers of PspA-specific IgG1 and nasal IgA ( Figure 1C ) .", "Additionally , they had stronger IL-13 ( TH2 ) and IL-17 ( TH17 ) responses in the ex vivo recall assay than the 2′3′-cGAMP adjuvanted pneumococcal vaccine ( Figure 1D ) .", "The IgG2C and IFNγ ( TH1 ) responses were similar between CDG and 2′3′-cGAMP adjuvanted vaccine ( Figure 1C , D ) .", "Last , we examined the protective immunity against pneumococcal infection in CDG plus PspA vs 2′3′-cGAMP plus PspA immunized mice .", "We found that mice immunized with CDG adjuvanted pneumococcal vaccine have a lower bacterial burden in the spleens and lungs than mice immunized with 2′3′-cGAMP adjuvant pneumococcal vaccine ( Figure 1E ) .", "We concluded that , in mice , CDG , as a mucosal adjuvant , generated better Ag-specific Ab production as well as stronger TH responses than the mammalian cyclic dinucleotide 2′3′-cGAMP .", "This translated into better protection against pneumococcal infection in vivo .", "Next , we examined the safety profile of CDG adjuvant .", "At the dose of CDG used in Figure 1 ( 5 μg ) , we saw only very mild neutrophil infiltration in Bronchoalveolar lavage fluid ( BALF ) ( Figure 2A ) and lungs ( Figure 2D ) .", "We also determined lung permeability by serum albumin level in BALF .", "There was no significant difference in samples from saline or CDG treated mice ( Figure 2B ) .", "Last , lung histology also did not reveal any lung damage in CDG treated mice ( Figure 2C ) .", "We concluded that intranasally administered CDG , at the dose used as an effective mucosal adjuvant , did not cause lung injury . 10 . 7554/eLife . 06670 . 004Figure 2 . CDG does not cause lung injury or excess inflammatory responses in vivo .", "( A ) C57BL/6 mice were treated ( i . n . ) with saline or CDG ( 5 μg ) for 20 hr .", "Cells in Bronchoalveolar lavage fluid ( BALF ) were analyzed by FACScan with indicated Abs .", "Live cells were gated .", "n > 3 . ( B ) Serum albumin level in the collected BALF was measured by ELISA ( #GWB-282C17; GenWay ) .", "n = 3 . ( C ) Lung sections from treated mice were fixed and histology was determined by Hematoxylin and eosin stain .", "n = 3 . ( D–G ) Mice were treated as in A . Lung cells were analyzed by FACScan with indicated Abs and quantified .", "Live cells were gated .", "n = 3 . ( H–I ) C57BL/6 and MPYS−/− mice were treated ( i . n . ) with saline or CDG ( 5 μg ) for the indicated time .", "Cytokines were determined in lung homogenates by ELISA .", "n > 3 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 004 Next , we examined CDG induced cellular responses in vivo .", "Besides a mild increase in the number of neutrophils in lung , there was also a ∼twofold increase in Ly6Chi monocytes in the lung after intranasal CDG administration ( Figure 2D ) .", "There were no significant increases in numbers of Mast cells or eosinophils in lungs at the vaccine adjuvant dose of CDG used ( 5 μg ) ( Figure 2D ) .", "There were also no increases in the number of B cells or NK cells ( Figure 2D , E ) .", "Ly6Chi monocytes could differentiate into DCs , mainly CD11B+ myeloid DCs , in situ .", "We did not find any difference in total DC number , or CD103+ , CD11B+ DCs subset numbers in the lungs after CDG treatment ( Figure 2F , G ) .", "CDG induces the production of the proinflammatory cytokines TNFα and IL-1β in vitro ( Karaolis et al . , 2007 ) .", "We confirmed this in vivo ( Figure 2H ) .", "However , we found that CDG also induced potent IL-10 production , an anti-inflammatory cytokine , in vivo ( Figure 2I ) .", "Furthermore , CDG induced strong IL-22 production in vivo ( Figure 2I ) , which is important for lung epithelium repair ( Paget et al . , 2012; Pociask et al . , 2013 ) .", "The balanced production of inflammatory and anti-inflammatory cytokines by CDG likely explains the absence of excess inflammatory responses in vivo .", "While CDG-induced TNFα and IL-22 production were completely dependent on the expression of MPYS , IL-1β , and IL-10 production in vivo were only partially dependent on MPYS ( Figure 2H , I ) .", "This was surprising considering that MPYS was the proposed direct receptor for CDG in mammalian cells .", "We then investigated the cytokine milieu in the lungs after CDG administration in WT and Tmem173−/− mice .", "We first examined the production of type I IFN , the signature cytokine stimulated by MPYS/STING , in the lungs .", "Although we detected low-level background IFNβ production in the lungs , CDG treatment did not increase IFNβ levels above the background ( Figure 3A ) .", "This was consistent with our previous observation that the mucosal adjuvant activity of CDG is type I IFN independent ( Blaauboer et al . , 2014 ) . 10 . 7554/eLife . 06670 . 005Figure 3 . CDG induces a variety of cytokines in lung that is dependent on the expression of MPYS .", "( A–E )", "C57BL/6 and Tmem173−/− mice were treated ( i . n . ) with saline or CDG ( 5 μg ) for the indicated time .", "Cytokines were determined in lung homogenates by ELISA .", "n > 3 . ( F–G ) C57BL/6 mice were treated ( i . n . ) with saline or CDG ( 5 μg ) for 5 hr .", "IL-12-p35 and IFNγ positive dendritic cells ( DCs ) were identified by intracellular cytokine stains and quantified .", "n = 3 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 00510 . 7554/eLife . 06670 . 006Figure 3—figure supplement 1 . IFNλ production is dispensable for the mucosal adjuvant activity of CDG . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 006 Surprisingly , we detected potent type III IFN ( IFN λ ) production in the lungs after intranasal administration of 5 μg CDG ( Figure 3A ) .", "Type III IFN activates similar groups of interferon stimulating genes ( ISGs ) as type I IFN .", "However , their receptors are mainly expressed on lung epithelial cells ( Zhou et al . , 2007 ) .", "Furthermore , neutralizing IFNλ in vivo did not affect the adjuvant activity of CDG ( Figure 3—figure supplement 1 ) .", "We also detected strong CDG induced type II IFN ( IFN γ ) in vivo ( Figure 3B ) .", "Both type II and III IFN production by CDG were absent in MPYS−/− mice ( Figure 3A , B ) .", "We concluded that intranasally administered CDG , at the dose used as an effective mucosal adjuvant , induces potent type II and III IFN , but not type I IFN production in vivo .", "CDG immunization generates TH1 , TH2 , and TH17 responses .", "Type II IFN is a TH1 polarizing cytokine .", "We examined if CDG induced other TH polarizing cytokines in the lungs .", "Indeed , intranasally administered CDG induced TH1 polarizing cytokine IL-12p70 , TH2 polarizing cytokine IL-5 , to a lesser degree IL-4 and IL-13 , and TH17 polarizing cytokines IL-23 , IL-6 , and TGF-β1 ( Figure 3B–D ) .", "Except for IL-6 production , all these CDG induced cytokines were absent in Tmem173−/− mice ( Figure 3B–3D ) .", "Lung epithelial cells generate unique cytokines when activated , and their in vivo roles in modulating immune responses have been appreciated recently ( Hallstrand et al . , 2014 ) .", "We examined lung epithelium-derived cytokines during in vivo CDG activation .", "Indeed , CDG induced potent IL-33 and , to a lesser degree , IL-1α and TSLP production ( Figure 3E ) .", "Distinct from many of the cytokines examined above , these CDG induced lung epithelium cytokines were only partially dependent on the expression of MPYS ( Figure 3E ) .", "Noticeably , all cytokines were detected at both 6 hr and 24 hr post CDG administration ( Figure 2 and Figure 3 ) .", "In fact , we could detect these cytokines as early as 4 hr post CDG administration in vivo .", "The rapid production of these cytokines by CDG in vivo suggested that CDG induced cytokines were a primary response rather than a secondary effect .", "The rapid generation of TH1 , TH2 , and TH17 polarizing cytokines in the lungs from CDG treated mice led us to hypothesize that CDG directly activated pulmonary DCs in vivo that generated TH polarizing cytokines , leading to differentiated T-helper cell responses .", "To test this hypothesis , we performed intracellular cytokine staining in pulmonary DC from CDG treated mice .", "We focused on detecting TH1 promoting DCs as defined by IL-12p35 or IFNγ production .", "Unlike IL-12p40 , IL-12p35 is unique to IL-12p70 .", "We gated MHC IIhiCD11C+ DCs from total lung and looked for IL-12p35+ or IFNγ+ DC ( Figure 3F ) .", "IL-12p35+ DC accounted for ∼0 . 035% of DCs , which amounted to less than 500 of these cells in a lung from a CDG treated mouse ( Figure 3G ) .", "The percentage of IL-12p35+ IFNγ+ DC was ∼0 . 01% ( Figure 3F , G ) .", "As a control , no IL-12p35+ DCs were detected in saline treated mice ( Figure 3F ) .", "Next , we investigated how CDG affects DCs in vivo .", "We used Alexa Fluor 647 conjugated OVA Ag ( OVA-647 ) to examine Ag uptake and DQ-OVA for Ag processing ( Figure 4A , B ) .", "DQ-OVA is a self-quenched conjugate of OVA that exhibits bright , photostable , and pH insensitive green fluorescence upon proteolytic degradation ( DQ-Green ) ( Figure 4A ) .", "Furthermore , when digested fragments of DQ-OVA accumulate in organelles at a high concentration , it forms excimers emitting red fluorescence ( DQ-Red ) ( Figure 4A ) . 10 . 7554/eLife . 06670 . 007Figure 4 . CDG enhances Ag uptake and activates pinocytosis-efficient antigen presenting cells ( APCs ) in vivo .", "( A–B )", "A cartoon showing mechanism of action of DQ-OVA ( A ) and OVA-647 ( B ) .", "( C ) Flow cytometry analysis of lung cells from C57BL/6 mice treated with saline , DQ-OVA ( 20 μg ) + OVA-647 ( 20 μg ) or 5 μg CDG + DQ-OVA ( 20 μg ) + OVA-647 ( 20 μg ) for 20 hr .", "Live cells were gated .", "n > 3 . ( D ) Flow cytometry analysis of lung cells from C57BL/6 mice treated with OVA-647 ( 20 μg ) or 5 μg CDG + OVA-647 ( 20 μg ) for 20 hr .", "Live cells were gated .", "n > 3 . ( E ) Flow cytometry analysis of lung APCs from C57BL/6 mice treated with 5 μg CDG + OVA-647 ( 20 μg ) for 20 hr .", "Live cells were gated .", "n > 3 . ( F ) Histogram of OVA-647 signals from OVA-647+ APCs .", "n > 3 . ( G ) Cell numbers of activated OVA-647+ APCs were quantified .", "n > 3 . Graph present means ± standard error from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 007 We intranasally administered mice with the OVA-647 plus DQ-OVA in the presence or absence , of CDG .", "After 24 hr , we examined OVA-647+ and DQ+ cells in the lung .", "We found that including CDG in the immunization dramatically improved Ag uptake , as indicated by the increased number of OVA-647+ cells in lung ( Figure 4C ) .", "Furthermore , ∼34% of these OVA-647+ cells were DQ+ , which indicated that only a portion of OVA-647+ cells has the ability to process Ag ( Figure 4C ) .", "The DQ+ cells included both DQ-Green and DQ-Red cells ( Figure 4C ) .", "Of note , the CDG induced OVA-647+ cells included both MHC II+ APCs and MHC II− non-APCs ( Figure 4C ) .", "We focused on MHC II+ APCs .", "There are three populations of antigen presenting cells ( APCs ) from WT mice: MHC IIhiCD11C+ ( i . e . , DCs ) , MHC IIlowCD11C+ and MHC IIintCD11C− ( Figure 4D ) .", "Notably , the majority of OVA-647+MHC IIlowCD11C+ cells were OVA-647hi cells , while the majority of OVA-647+MHC IIhiCD11C+ and OVA-647+ MHC IIintCD11C− cells were OVA-647low cells ( Figure 4F ) .", "A previous study established that OVA-647hi cells were generated via receptor-mediated endocytosis while OVA-647low cells were a result of pinocytosis-mediated Ag uptake ( Burgdorf et al . , 2007 ) .", "Thus , CDG predominantly enhanced receptor-mediated endocytosis in MHC IIlowCD11C+ and pinocytosis in MHC IIhiCD11C+ and MHC IIintCD11C− cells .", "CDG treatment activates cells in vitro , which depends on MPYS ( Jin et al . , 2011a; Pociask et al . , 2013 ) .", "We next wanted to know which APCs were activated during intranasal administration of CDG .", "APCs increase CD86 expression during activation .", "In the OVA-647+ MHC IIlowCD11C+ population , there was no increase of the activation marker CD86 ( Figure 4E ) .", "In the remaining two APC populations , MHC IIhiCD11C+ and MHC IIintCD11C− , the OVA-647+ cells had increased CD86 expression ( Figure 4E ) .", "Thus , CDG activates MHC IIhiCD11C+ and MHC IIintCD11C− , but not MHC IIlowCD11C+ APCs in vivo .", "The total numbers of CD86+MHC IIhi activated OVA-647+ cells were similar between MHC IIhiCD11C+ and MHC IIintCD11C− APCs ( Figure 4G ) .", "Of note , while CDG selectively activated different APCs , it did enhance Ag uptake in all three APCs populations in vivo ( Figure 4D ) .", "This suggested that cell activation is not a prerequisite for CDG enhanced Ag uptake in vivo .", "CDG also dramatically increased numbers of DQ+ cells in vivo ( Figure 5A ) .", "As shown in Figure 4C , only a third of OVA-647+ cells were able to process Ag ( DQ+ ) .", "We , thus , focused on DQ+ cells , where Ag was processed .", "Gated on the DQ+ lung cells , we found that the vast majority of DQ+ cells ( ∼94% ) were OVA-647+ cells ( Figure 5A ) .", "Since cells have to take up Ag ( OVA-647+ ) before processing it ( DQ+ ) , the small percentage of DQ+OVA-647− cells ( ∼5% ) could represent cells that lost the OVA-647 signal during the Ag process .", "Alternatively , DQ-OVA signal could be more sensitive than the OVA-647 signal . 10 . 7554/eLife . 06670 . 008Figure 5 . CDG generates mature DCs in vivo .", "( A ) Flow cytometry analysis of lung cells from C57BL/6 mice treated with saline , DQ-OVA ( 20 μg ) + OVA-647 ( 20 μg ) or 5 μg CDG + DQ-OVA ( 20 μg ) + OVA-647 ( 20 μg ) .", "Live cells were gated .", "n > 3 . ( B ) Histogram of DQ-Red and DQ-Green signals from indicated populations .", "n > 3 . ( C ) Flow cytometry analysis of DQ+ lung cells from CDG + DQ-OVA treated ( i . n . ) C57BL/6 mice .", "Live DQ+ cells were gated .", "n > 3 . ( D ) Flow cytometry analysis of lung cells from CDG + DQ-OVA treated ( i . n . ) C57BL/6 mice .", "Live cells were gated .", "n = 3 . ( E ) Flow cytometry analysis of DQ+ lung cells from DQ-OVA or CDG + DQ-OVA treated ( i . n . ) C57BL/6 mice .", "Live were gated .", "n > 3 . ( F ) Flow cytometry analysis of the indicated population from lung cells of CDG + DQ-OVA treated ( i . n . ) C57BL/6 mice .", "Gated on live DQ+ CD80+MHC II+ or live DQ+ CD86+MHC II+ cells .", "n > 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 008 The DQ+OVA+ consisted of two populations: OVA-647hi and OVA-647low cells ( Figure 5A ) .", "OVA-647hi cells were generated via receptor-mediated endocytosis while OVA-647low cells were a result of pinocytosis-mediated Ag uptake ( Burgdorf et al . , 2007 ) .", "The DQ+OVA-647hi cells had a strong DQ-Red signal , indicating that processed Ag concentration was high in these cells ( Figure 5B ) .", "The DQ+OVA-647low cells were DQ-Red negative , though they still processed Ag as they were DQ-Green+ ( Figure 5B ) .", "Thus , the receptor-mediated Ag endocytosis generates DQ-Green+DQ-Red+ cells , while pinocytosis-mediated Ag uptake generates DQ-Green+DQ-Red− cells .", "We found that the DQ+ cells were almost exclusively APCs ( MHC II+ cells ) ( Figure 5C ) .", "This was different from the OVA-647+ cells , which included both APC and non APCs ( Figure 4C ) .", "Furthermore , the vast majority of the DQ+ lung cells ( >90% of DQ+ cells ) were MHC IIlowCD11C+ APC ( Figure 5C ) .", "The MHC IIhiCD11C+ and MHC IIint CD11C− APCs accounted for ∼1% and 2% of DQ+ cells , respectively ( Figure 5C ) .", "The MHC IIlowCD11C+DQ+ cells were Siglec F+ ( Figure 5C ) cells , which should be characterized as alveolar macrophages .", "This suggested that alveolar macrophages are the dominant Ag uptake and processing cells during intranasal CDG administration .", "Indeed , ∼26% of total lung Siglec F+ alveolar macrophages were DQ+ cells ( Figure 5D ) .", "In comparison , only ∼1% of total lung cells were DQ+ cells ( Figure 5A ) .", "We then investigated which DQ+ APCs were activated by CDG in vivo .", "Studies done in OVA-647+ cells revealed that OVA-647+ MHC IIlowCD11C+ cells were not activated ( Figure 4E ) .", "Only OVA-647+MHC IIhiCD11C+ and OVA-647+MHC IIintCD11C− APCs were activated ( Figure 4E ) .", "However , in DQ+ cells , the only CD86+ APC was MHC IIhi DCs ( Figure 5E ) .", "These cells also had increased CD80 expression ( Figure 5E ) .", "CDG predominantly activated OVA-647low APCs ( Figure 4E , F ) , which took up Ag via pinocytosis .", "We found that OVA-647low cells were all DQ-Red− while OVA-647hi cells were all DQ-Red+ ( Figure 5B ) .", "Similarly , the vast majority of MHC IIhiCD86+CD80+DQ+ cells were DQ-Red− cells ( Figure 5F ) .", "We concluded that during intranasal administration of CDG , the only APCs that took up Ag ( OVA-647+ ) , processed Ag ( DQ-Green+ ) and activated ( CD86+CD80+ ) , were MHC IIhi pinocytosis-efficient ( DQ-Red− ) DCs .", "CDG is a 690 Da small molecule with two phosphate groups that cannot directly cross cell membrane ( McWhirter et al . , 2009; Chen et al . , 2010 ) .", "Thus , during intranasal administration , CDG is likely brought into the cytosol by pinocytosis , and stimulates DCs .", "Pulmonary DCs include CD103+DCs and CD11B+DCs .", "By co-administration of DQ-OVA and CDG , we found that CDG enhanced Ag uptake and processing , as indicated by increased numbers of DQ+ cells , in both CD103+ and CD11B+ DCs ( Figure 6A , D ) .", "We did notice that CD103+DCs had a higher percentage of DQ+ cells than the CD11B+ DCs ( Figure 6A , D ) .", "Both DC subsets had DQ-Red+ and DQ-Red− populations ( Figure 6A , D ) . 10 . 7554/eLife . 06670 . 009Figure 6 . CDG activates pinocytosis-efficient CD103+ and CD11B+ DCs in vivo .", "( A and D )", "Flow cytometry analysis of lung cells from C57BL/6 mice treated ( i . n . ) with DQ-OVA ( 20 μg ) or CDG ( 5 μg ) + DQ-OVA ( 20 μg ) for 20 hr .", "Live cells were gated .", "n > 3 . ( B and E ) Flow cytometry analysis of DQ+ DCs from lung of CDG + DQ-OVA treated C57BL/6 mice .", "Cells were gated on live DQ+CD103+MHC II+ or live DQ+CD11B+MHC II+ cells .", "n > 3 . ( C and F ) Histogram of DQ-Green and DQ-Red signals from cell populations in B and E . n > 3 . ( G ) Total cell number of CD86+DQ+ DCs in lung .", "n = 3 . ( H and J ) Flow cytometry analysis of lung draining lymph nodes ( DLN ) from DQ-OVA or CDG + DQ-OVA treated C57BL/6 mice .", "Live cells were gated .", "n = 3 . ( I and K ) Flow cytometry analysis of DQ+ DCs in DLN of CDG + DQ-OVA treated C57BL/6 mice .", "Cells were gated on live DQ+CD103+MHC II+ or live DQ+CD11B+MHC II+ cells .", "n = 3 . ( L–N ) Total cell numbers of DCs , DQ+DCs , and CD86+DQ+DCs in DLN from DQ-OVA or CDG + DQ-OVA treated mice .", "n = 3 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 009 Activated DCs express high MHC II and co-stimulator factor CD86 .", "Furthermore , they migrate to draining lymph nodes ( DLN ) , where they encounter naïve T cells and stimulate diversified T cell responses .", "We first examined the actions of CD103+ DC .", "A significant portion of lung CD103+DQ+ DCs ( ∼35% ) from CDG treated mice were MHC IIhiCD86+ activated DCs ( Figure 6B ) .", "The absolute number of CD103+DQ+CD86+ cells was also recorded ( Figure 6G ) .", "Interestingly , these activated DQ+CD103+CD86+ DCs were all DQ-Red negative cells ( Figure 6B ) .", "In fact , it appeared that all DQ-Red− cells were CD86+ DCs and all DQ-Red+ cells were CD86− ( Figure 6B , C ) .", "We then examined migratory CD103+ DC in lung DLN .", "CDG treatment increased total CD103+ DCs numbers in DLN ( Figure 6H , L ) .", "However , only a very small percentage of the migratory CD103+ DCs ( ∼1 . 8% ) were DQ+ ( Figure 6H , M ) .", "This indicated that a large portion of CD103+ DCs were migratory , likely activated by CDG , but did not take up the DQ-OVA Ag .", "Among those DQ+CD103+ migratory DCs , the vast majority of them were MHC IIhiCD86+ cells ( ∼89% ) ( Figure 6I ) , which indicated that these migratory DQ+CD103+DCs were indeed activated DCs .", "Consistent with the finding in the lungs , all these migratory DQ+CD103+ in DLN were DQ-Red− cells ( Figure 6I ) .", "The number of DQ+ CD86+ migratory CD103DCs was recorded ( Figure 6N ) .", "We next examined the activation of CD11B+ DCs by CDG in vivo .", "Similar to CD103+DCs , we found that ( 1 ) a portion of lung CD11B+DQ+ DCs were MHC IIhiCD86+ activated DCs ( Figure 6E ) ; ( 2 ) these activated DQ+CD11B+CD86+ DCs were all DQ-Red negative cells ( Figure 6E ) ; ( 3 ) all the DQ-Red−DC-Green+CD11B+ DCs were CD86+ ( Figure 6E ) ; ( 4 ) total CD11B+ DCs numbers were increased in DLN ( Figure 6J , L ) ; ( 5 ) only a very small percentage of these CD11B+ DCs ( ∼0 . 8% ) were DQ+ ( Figure 6J , M ) ; ( 6 ) the vast majority of DQ+CD11B+ migratory DC were MHC IIhiCD86+ cells ( ∼82% ) ( Figure 6K , N ) ; ( 7 ) all these migratory DQ+CD11B+ were DQ-Red− cells ( Figure 6K ) .", "Our investigation , so far , revealed that CDG differentially mobilized two major types of Ag-loaded pulmonary DCs: DQ-Green+DQ-Red−CD86+ and DQ-Green+DQ-Red+CD86− DCs .", "DQ-Red− DQ-Green+ cells represented pinocytosis-efficient DCs ( Figure 6A , B ) .", "The fact that these were the only CD86+ and DQ+ migratory DCs found in DLN after CDG treatment suggested that CDG only activated pinocytosis-efficient DCs in vivo .", "It did not matter whether they were CD103+ or CD11B+ DCs ( Figure 6H , J ) .", "In contrast , all the DQ-Green+DQ-Red+ cells were CD86− and non-migratory , suggesting that though CDG enhanced Ag uptake in these cells ( Figure 4 ) , it did not lead to cell activation .", "It further strengthened the notion that activation of these cells is not a prerequisite for CDG enhanced Ag uptake ( Figure 4E ) .", "Mucosal adjuvant activity of CDG requires MPYS in vivo ( Blaauboer et al . , 2014 ) .", "We next asked how MPYS regulated CDG enhanced Ag uptake and processing in vivo .", "Upon co-administration of OVA-647 and CDG , lung cells from Tmem173−/− mice had no increased OVA-647+ cells ( Figure 7A ) . 10 . 7554/eLife . 06670 . 010Figure 7 . MPYS is critical for CDG induced Ag uptake and activation in vivo .", "( A ) Flow cytometry analysis of lung cells from OVA-647 ( 20 μg ) or OVA-647 ( 20 μg ) + CDG ( 5 μg ) treated ( i . n . ) C57BL/6 or Tmem173−/− mice .", "Live cells were gated .", "n > 3 . ( B ) Flow cytometry analysis of lung cells from DQ-OVA ( 20 μg ) or DQ-OVA ( 20 μg ) + CDG ( 5 μg ) treated ( i . n . ) C57BL/6 or Tmem173−/− mice .", "Live cells were gated .", "n > 3 . ( C and D ) Flow cytometry analysis of lung cells from C57BL/6 or Tmem173−/− mice treated with saline , DQ-OVA ( 20 μg ) or 5 μg CDG + DQ-OVA ( 20 μg ) .", "Live cells were gated .", "n > 3 . ( E ) DQ+CD103+DCs and DQ+CD11B+DCs numbers from DQ-OVA or CDG + DQ-OVA treated C57BL/6 and Tmem173−/− mice .", "n = 3 . ( F ) Flow cytometry analysis of DQ+ lung cells from DQ-OVA ( 20 μg ) + CDG ( 5 μg ) treated ( i . n . ) C57BL/6 or Tmem173−/− mice .", "Live DQ+ cells were gated .", "n > 3 . ( G ) One month after the last immunization , CDG/PspA or PspA immunized WT and Tmem173−/− mice were infected ( i . n . ) with S . pneumoniae ( D39 strain , ∼5 . 0 × 106 c . f . u . ) .", "At 48 hr post infection , lung bacterial burden was determined .", "n = 2 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 010 Since DQ-OVA may be more sensitive than OVA-647 in detecting Ag-loaded APCs ( Figure 5A ) , we examined DQ-OVA signals in lung cells from CDG treated Tmem173−/− mice .", "As expected , Tmem173−/− mice had significantly less CDG induced DQ-OVA+ cells than WT mice ( Figure 7B ) .", "Both CD103+DC ( Figure 7C ) and CD11B+ DC ( Figure 7D ) from CDG treated Tmem173−/− mice , had dramatically decreased DQ+ cells ( Figure 7E ) .", "This included both the DQ-Green+DQ-Red+ receptor-mediated endocytosis and DQ-Green+ DQ-Red− pinocytosis cells .", "We concluded that MPYS is critical for CDG induced DC Ag endocytosis and pinocytosis in vivo .", "We next examined activated DQ+ DCs in Tmem173−/− mice .", "As expected , no CD86+CD80+DQ+ MHC IIhi cells can be detected in CDG treated Tmem173−/− mice ( Figure 7F ) .", "This was consistent with the finding that Tmem173−/− mice had a severe defect on CDG induced cytokine production in vivo ( Figure 3 ) .", "CDG is likely brought into cells by pinocytosis in vivo ( Figures 4E , 5F , 6B , E ) and MPYS is critical for CDG enhanced pinocytosis ( Figure 7A ) .", "Thus , the reasons for the lack of overall activation by CDG in Tmem173−/− mice could be twofold .", "On one hand , CDG cannot efficiently get into MPYS-deficient cells by pinocytosis; on the other hand , the small amount of CDG that does get in cannot activate MPYS-deficient cells .", "Next , we examined CDG/PspA vaccine induced protective immunity in the Tmem173−/− mice .", "CDG/PspA immunization significantly lowered the lung bacterial burden in the WT mice ( Figure 7G ) .", "However , the bacterial burden in lungs from CDG/PspA and PspA immunized Tmem173−/− were not significantly different ( Figure 7G ) .", "We concluded that the mucosal pneumococcal vaccine adjuvant activity of CDG requires MPYS .", "Interestingly , PspA immunized Tmem173−/− mice had significantly lower lung bacterial burden than the PspA immunized WT mice ( Figure 7G ) .", "We further found that Tmem173−/− mice , without PspA immunization , are much more resistant to Streptococcus pneumoniae infection than the WT mice ( unpublished data ) .", "Currently , we are dissecting the in vivo mechanism underlying this MPYS-mediated susceptibility to S . pneumoniae infection .", "Our investigation revealed two mechanisms by which CDG promotes its adjuvant activity in vivo: ( 1 ) enhances Ag uptake in vivo; ( 2 ) activates and mobilizes DCs in vivo , specifically , the pinocytosis-efficient DQ-Green+DQ-Red− DCs .", "MPYS expression is required for both actions .", "We then asked whether this MPYS requirement was DC-intrinsic .", "To achieve that , we generated ItgaxCreTmem173Flox/Flox mice ( Figure 8—figure supplement 1 ) .", "Since essentially all DQ+ ( Ag-processing ) cells were CD11C+ ( Figure 5C ) , the ItgaxCreTmem173Flox/Flox mice will eliminate MPYS expression in the vast majority of DQ+ cells except for the CD11C−MHC IIint APCs , which accounts for ∼2% of DQ+ cells ( Figure 5C ) .", "We detected MPYS expression by Flow cytometry intracellular staining .", "We used the same type of cell from Tmem173−/− mice as a negative control and the same type of cell from WT mice as a positive control .", "BALF cells , which are overwhelmingly CD11Chi alveolar macrophages , had dramatically decreased MPYS expression ( >90% ) in ItgaxCreTmem173Flox/Flox mice ( Figure 8—figure supplement 1B ) .", "MPYS expression in spleen B cells ( IgD+ ) or T cells ( CD4+ or CD8+ ) did not change in ItgaxCreTmem173Flox/Flox mice ( Figure 8—figure supplement 1C ) .", "There were two major CD11Chi populations in lung cells: CD11C+MHC IIlow and CD11C+MHC IIhi ( Figure 8—figure supplement 1D ) .", "MPYS expression was dramatically decreased in both populations in ItgaxCreTmem173Flox/Flox mice ( Figure 8—figure supplement 1D ) .", "When we separated the DC population ( CD11C+MHC IIhi ) into CD103+ and CD11B+ DCs , we found that MPYS expression was eliminated in both DCs subsets ( Figure 8—figure supplement 1D ) .", "The MPYS expression was down ∼40% in MHC IIint CD11C− cells from ItgaxCreTmem173Flox/Flox mice ( Figure 8—figure supplement 1D ) .", "NK cells also showed ∼40% decreased MPYS expression in ItgaxCreTmem173Flox/Flox mice ( Figure 8—figure supplement 1E ) .", "We wanted to see if the partial decrease of MPYS expression affected CDG adjuvant activity .", "MPYS heterozygous mice , which had ∼50% decreased MPYS expression ( Figure 8—figure supplement 1F ) , were immunized with CDG and OVA .", "The total OVA-specific IgG production was similar in WT as in the Tmem173+/− mice ( Figure 8—figure supplement 1G ) .", "Thus , the partial decreased MPYS expression ( ∼50% ) does not affect CDG adjuvant activity .", "We then examined CDG generated DQ-OVA+ cells in the lungs of ItgaxCreTmem173Flox/Flox mice .", "The total numbers of DQ+ cells were dramatically decreased in ItgaxCreTmem173Flox/Flox mice ( Figure 8A , B ) .", "The decreased number of DQ+ cells was seen in both CD103+DC ( Figure 8C , D ) and CD11B+ DC ( Figure 8C , E ) from ItgaxCreTmem173Flox/Flox mice .", "The numbers of decreased DQ+ DCs in ItgaxCreTmem173Flox/Flox/flox was comparable to that of Tmem173−/− mice ( Figure 8B ) .", "Thus , CDG induced DC Ag uptake requires MPYS expression in CD11C+ cells . 10 . 7554/eLife . 06670 . 011Figure 8 . CDG induced DC Ag uptake and activation requires MPYS expression in CD11C+ cells .", "( A ) Flow cytometry analysis of lung cells from DQ-OVA ( 20 μg ) or DQ-OVA ( 20 μg ) + CDG ( 5 μg ) treated ( i . n . ) Tmem173Flox/Flox or ItgaxCre Tmem173Flox/Flox mice .", "Live cells were gated .", "n = 3 . ( B ) Total DQ+ lung cells from DQ-OVA or CDG + DQ-OVA treated Tmem173Flox/Flox , ItgaxCre Tmem173Flox/Flox , and Tmem173−/− mice .", "n = 3 . ( C ) DQ+ DCs numbers from DQ-OVA or CDG + DQ-OVA treated Tmem173Flox/Flox , ItgaxCre Tmem173Flox/Flox and Tmem173−/− mice .", "n = 3 . ( D and E ) Flow cytometry analysis of DQ+ DCs from lung of DQ-OVA or CDG + DQ-OVA treated mice Tmem173Flox/Flox , ItgaxCre Tmem173Flox/Flox .", "Live cells were gated .", "n = 3 . ( F–K ) Tmem173Flox/Flox , ItgaxCreTmem173Flox/Flox or Tmem173−/− mice were treated with saline or CDG ( 5 μg ) for 20 hr .", "Indicated cytokines were measured in lung homogenates by ELISA .", "n = 3 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 01110 . 7554/eLife . 06670 . 012Figure 8—figure supplement 1 . Generation of ItgaxCreTmem173Flox/Flox mouse . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 012 Intranasally administered CDG generated a lung cytokine milieu that is dependent on the expression of MPYS ( Figure 3 ) .", "We then examined the cytokine milieu in ItgaxCreTmem173Flox/Flox mice .", "CDG induced TH1 polarizing ( IL-12p70 and IFNγ ) and TH17 polarizing ( IL-23 and IL-6 ) cytokines were significantly decreased in ItgaxCreTmem173Flox/Flox mice ( Figure 8F , H ) .", "Surprisingly , we did not see much of a decrease in the TH2 polarizing cytokines ( IL-5 , IL-13 ) ( Figure 8G ) .", "Thus , MPYS expression in CD11C+ cells is critical for TH1 and TH17 polarizing cytokine production in vivo .", "CDG induced IFN-λ , IL-22 , TNF-α , and IL-1β productions were also dramatically decreased in ItgaxCreTmem173Flox/Flox mice ( Figure 8I , J ) .", "Since the CD11C+MHC IIhi DCs were the only activated CD11C+ cells by CDG in vivo ( Figures 4E , 5E ) , we concluded that DCs expression of MPYS was critical for the generation of TH1 and TH17 polarizing cytokine during intranasal administration of CDG .", "The lung epithelial cytokine TSLP was slightly lower in CDG treated ItgaxCreTmem173Flox/Flox mice than in the Tmem173Flox/Flox mice , but it was not statistically significant ( Figure 8K ) .", "However , the lung epithelial cytokine IL-33 production was significantly lower in CDG treated ItgaxCreTmem173Flox/Flox mice than in the Tmem173Flox/Flox ( Figure 8K ) .", "We favored the idea that there is a crosstalk/communication between lung epithelial cells and CD11C+ cells during CDG induced immune response .", "The ItgaxCreTmem173Flox/Flox mice are defective in CDG induced DCs Ag uptake and activation in vivo .", "To determine if these mice were defective in CDG adjuvanted immune responses , we immunized these mice with CDG plus OVA and measured anti-OVA Ab productions .", "ItgaxCreTmem173Flox/Flox mice exhibited significantly decreased production of anti-OVA IgG1 , IgG2C , and nasal IgA ( Figure 9A ) .", "Noticeably different from the Tmem173−/− mice , where no anti-OVA Ab could be detected , CDG/OVA immunized ItgaxCreTmem173Flox/Flox mice still generated decent amounts of anti-OVA Ab ( Figure 9A ) . 10 . 7554/eLife . 06670 . 013Figure 9 . MPYS expression in CD11C+ cells is required for the optimal mucosal adjuvant activity of CDG .", "( A ) Tmem173Flox/Flox , ItgaxCreTmem173Flox/Flox or Tmem173−/− mice were intranasally administered OVA ( 20 μg ) alone or together with 5 μg CDG as in Figure 1A .", "Anti-OVA IgG1 , IgG2C and IgA were determined by ELISA .", "n = 3 . ( B ) Tmem173Flox/Flox , ItgaxCreTmem173Flox/Flox or Tmem173−/− mice were immunized with PspA ( 2 μg ) alone or together with 5 μg CDG as in Figure 1C .", "Anti-PspA IgG1 , IgG2C , and IgA were measured by ELISA .", "n = 3 . ( C–D ) Splenocytes and lung cells from PspA or CDG + PspA immunized Tmem173Flox/Flox , ItgaxCreTmem173Flox/Flox or Tmem173−/− mice were stimulated with 5 μg/ml PspA for 4 days in culture .", "Cytokines were measured in the supernatant by ELISA as in Figure 1D n = 3 . ( E ) 1 month after the last immunization , CDG/PspA or PspA immunized Tmem173Flox/Flox and ItgaxCreTmem173Flox/Flox mice were infected ( i . n . ) with S . pneumoniae ( D39 strain , ∼5 . 0 × 106 c . f . u . ) .", "At 48 hr post infection , lung bacterial burden were determined .", "n = 2 . Graph present means ± standard error from three independent experiments .", "Significance is represented by an asterisk , where p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 01310 . 7554/eLife . 06670 . 014Figure 9—figure supplement 1 . The impaired CDG response in ItgaxCreTmem173Flox/Flox mice is not due to the over-expression of Cre in CD11C+ cells . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 014 We next immunized ItgaxCreTmem173Flox/Flox mice with pneumococcal vaccine consisting of CDG and PspA and examined their Ab responses .", "Again , ItgaxCreTmem173Flox/Flox mice showed decreased anti-PspA IgG1 , IgG2C , and nasal IgA production in comparison to the immunized WT mice ( Figure 9B ) .", "Similar to the CDG/OVA immunization , ItgaxCreTmem173Flox/Flox mice still had elevated anti-PspA Ab responses compared to the MPYS−/− mice ( Figure 9B ) .", "The CDG/PspA immunized ItgaxCreTmem173Flox/Flox mice also showed dramatically decreased TH1 , TH2 , and TH17 responses in the ex vivo recall assay on splenocytes ( Figure 9C ) .", "Lungs can form Bronchus associated lymphoid tissue after immunization and initiate an adaptive immune response in situ .", "We did the recall assay on the lung cells from immunized mice to examine the local immune responses .", "Similar to the responses in splenocytes , TH1 , TH17 and , to a lesser degree , TH2 responses were decreased in lung cells from ItgaxCreTmem173Flox/Flox mice ( Figure 9D ) .", "Next , we examined the CDG/PspA vaccine induced protective immunity in the ItgaxCreTmem173Flox/Flox mice .", "While CDG/PspA immunization significantly lowered the lung bacterial burden in the Tmem173Flox/Flox mice , it did not alter the bacterial burden from lungs of the ItgaxCreTmem173Flox/Flox mice ( Figure 9E ) .", "We concluded that the mucosal pneumococcal vaccine adjuvant activity of CDG requires MPYS expression in CD11C+ cells .", "Noticeably , unlike the Tmem173−/− mice , PspA immunized Tmem173Flox/Flox and ItgaxCreTmem173Flox/Flox mice had similar lung bacterial burden ( Figure 9E ) .", "The ItgaxCreTmem173Flox/Flox mice also overexpressed the Cre gene in the CD11C+ cells .", "To exclude the possibility that the defect seen in the ItgaxCreTmem173Flox/Flox mice was due to the Cre overexpression , we compared ItgaxCreTmem173Flox/Flox mice with the ItgaxCre-C57BL/6 mice upon intranasal CDG/PspA immunization .", "Similar to the observation in Figure 9 , CDG/PspA immunized ItgaxCreTmem173Flox/Flox mice had a severe defect in anti-PspA Ab production compared to immunized ItgaxCre-C57BL/6 mice ( Figure 9—figure supplement 1A ) .", "Their TH responses in spleen cells were largely non existent , except for IL-5 ( Figure 9—figure supplement 1B ) .", "A similar observation was made in lung recall assay ( Figure 9—figure supplement 1C ) ." ], [ "Our study revealed two novel in vivo mechanisms of action of the mucosal vaccine adjuvant CDG ( Figure 10 ) .", "First , CDG enhances Ag uptake in APCs and non-APCs in vivo .", "Second , CDG activates pinocytosis-efficient cells in vivo .", "CDG has two phosphate groups preventing it from directly passing through the cell membrane .", "The mammalian receptor for CDG , MPYS , is located inside cells .", "Thus , though intranasally administered CDG enhances Ag uptake in all types of cells , only cells that efficiently take up CDG , via pinocytosis , will be activated ( Figure 10A ) . 10 . 7554/eLife . 06670 . 015Figure 10 . In vivo mechanisms of the mucosal vaccine adjuvant CDG .", "( A ) The formula of CDG .", "Red arrows indicate the phosphate groups that prevent CDG from directly crossing the cell membrane .", "( B ) Mechanism I: CDG enhances Ag uptakes in APCs and non-APCs .", "Among OVA647+APCs , only a portion of MHC IIhiCD11C+ ( DCs ) and MHC IIint CD11C− cells up-regulate CD86 expression in vivo .", "They are mainly OVA647low cells , which take up Ag by pinocytosis .", "The activation of these cells generate a cytokine milieu that acts on other cells leading to enhanced Ag uptake ( OVA647hi cells ) but not cell activation .", "CDG also activates lung epithelial cells ( LEC ) , leading to TSLP and IL-33 production .", "But this is only partially dependent on MPYS and is not sufficient to enhance Ag uptake in vivo ( Figure 8 ) .", "( C ) Mechanism II: CDG selectively activates pinocytosis-efficient DCs in vivo .", "After administering DQ-OVA together with CDG , the DQ+MHC IIhiCD11C+ Ag-loading DCs can be separated into two distinct populations: DQ-Green+DQ-Red−CD86+ and DQ-Green+DQ-Red+CD86− .", "DQ-Green+DQ-Red− cells are OVA-647low cells , while DQ-Green+DQ-Red+ cells are OVA-647hi cells .", "Only the DQ-Green+DQ-Red−CD86+ cells migrated to lung DLNs . DOI: http://dx . doi . org/10 . 7554/eLife . 06670 . 015 How does CDG , as a mucosal adjuvant , enhance Ag uptake in vivo ?", "Three observations in this study may shed light on the mechanism .", "First , CDG enhances Ag uptake by both pinocytosis and receptor mediated endocytosis ( Figure 4 ) ; Second , while CDG enhances Ag uptake in all types of cells ( Figure 4 ) , deletion of MPYS in only CD11C+ cells severely impaired that ( Figure 8 ) ; Third , MPYS expression in CD11C+ cells is mainly responsible for the CDG induced cytokine milieu in lungs ( Figure 8 ) .", "We propose that CDG enhances MPYS-dependent Ag uptake in cells directly taking up CDG ( pinocytosis-efficient , OVA-647Low , DQ-Green+DQ-Red− cells ) .", "In cells that do not take up CDG ( OVA-647hi , DQ-Green+DQ-Red+ cells ) , Ag uptake is enhanced by the cytokine milieu generated mainly by CDG activated CD11C+ cells ( Figure 10B ) .", "We favored the hypothesis that intranasally administered CDG directly primed pulmonary DCs , leading to MPYS-dependent production of TH polarizing cytokines in vivo ( Figure 10C ) .", "Two pieces of data support this hypothesis .", "First , we detected IL-12 and IFNγ producing DCs in vivo as early as 5 hr post treatment ( Figure 3F , G ) .", "Second , the ItgaxCreTmem173Flox/Flox mice had dramatically decreased CDG-induced TH1 and TH17 cytokine in vivo ( Figure 8F , H ) .", "There are two CD11C+ populations in the lung: MHC IIhi and MHC IIlow .", "Among Ag positive ( OVA-647+ ) cells , only the MHC IIhiCD11C+ population ( i . e . , DCs ) were activated by CDG in vivo ( Figure 4E ) .", "This suggested that the deletion of MPYS in MHC IIhiCD11C+ cells ( DCs ) was responsible for the impaired TH1 and TH17 polarizing cytokine production in vivo .", "Intriguingly , the production of TH2 polarizing cytokines IL-5 and IL-13 was less dependent on the expression of MPYS in DCs ( Figure 8G ) .", "Indeed , unlike Tmem173−/− mice , ItgaxCreTmem173Flox/Flox mice still have some Ab and TH responses after CDG immunization ( Figure 9 ) .", "Therefore besides DCs , MPYS expression in other cells contributes to the adjuvant activity of CDG in vivo .", "We found that the OVA-647+CD11C−MHC IIint APC was activated by CDG in vivo ( Figure 4E ) .", "The total number of OVA-647+ activated cells in this CD11C−MHC IIint population are comparable to that of the CD11C+ MHC IIhi population ( Figure 4G ) .", "Thus , these CD11C− OVA-647+ MHC IIint CD86+ cells may contribute to the adjuvant activity of CDG in ItgaxCreTmem173Flox/Flox mice .", "How does CDG enhance MPYS-mediated Ag uptake in pinocytosis-efficient cells in vivo ?", "CDG activates MPYS-TBK1-IRF3-Type I IFN signaling in vitro .", "However , intranasally administered CDG did not induce Type I IFN production in vivo .", "Instead , it generates type II , type III IFN , and various cytokines that depend on NF-κB activation .", "We previously showed , in vitro , that CDG induced type I IFN and NF-κB activation can be uncoupled in DCs and macrophages ( Blaauboer et al . , 2014 ) .", "Thus , MPYS is not just a type I IFN stimulator .", "New molecular mechanisms by which CDG enhances MPYS-dependent Ag uptakes as well as activation of Type II , III IFN and NF-κB signaling in pinocytosis-efficient cells in vivo remains to be discovered .", "Tmem173−/− mice still made several cytokines , namely IL-1α , IL-1β , IL-6 , IL-10 , IL-33 , and TSLP , after CDG treatment in vivo .", "This indicates that CDG can activate MPYS-independent signaling in vivo .", "A previous study showed that CDG activated NLRP3 inflammasome independent of STING/MPYS ( Abdul-Sater et al . , 2013 ) .", "CDG also bound to hyperpolarization-activated cyclic nucleotide-gated channel 4 ( HCN4 ) and inhibited cAMP regulated heart rate ( Lolicato et al . , 2014 ) .", "Very recently , it was found that cyclic di-AMP , a similar cyclic dinucleotide for STING/MPYS , induces human monocyte apoptosis independent of STING/MPYS ( Tosolini et al . , 2015 ) .", "Thus , other mammalian receptors for CDG exist .", "We showed that CDG is a superior mucosal pneumococcal vaccine adjuvant than the 2′3′-cGAMP in mice ( Figure 1 ) .", "As a mammalian cyclic dinucleotide , 2′3′-cGAMP can be hydrolyzed by the ecto-nucleotide phosphodiesterase ( ENPP1 ) found in mammalian cells ( Li et al . , 2014 ) .", "On the contrary , as a bacterial cyclic dinucleotide , CDG may be more resistant to hydrolysis when introduced into mammalian cells .", "Further study is needed to determine if CDG is a better human adjuvant than 2′3′-cGAMP .", "The anti-tumor molecules 10-carboxymethyl-9-acridanone ( CMA ) ( Cavlar et al . , 2013 ) and 5 , 6-dimethylxanthenone-4-acetic acid ( DMXAA ) ( Conlon et al . , 2013 ) activate mouse , but not , human MPYS signaling .", "CDG , on the other hand , is functional in human cells as well ( Karaolis et al . , 2007; Yi et al . , 2013 ) .", "In fact , CDG binds to mouse MPYS/STING ( Burdette et al . , 2011 ) and human MPYS/STING ( Ouyang et al . , 2012; Shang et al . , 2012; Shu et al . , 2012; Yin et al . , 2012 ) with similar dissociation constant ( Kd: 2–5 μM ) .", "Nevertheless , we first discovered that human TMEM173 gene displays great heterogeneity ( Jin et al . , 2011b ) .", "We further identified a loss-of-function human TMEM173 variant HAQ ( R71H-G230A-R293Q ) that is carried by ∼20% of Americans ( Jin et al . , 2011b ) .", "In vitro studies demonstrated that many of these human TMEM173 variants are functionally different from the R232 ( wild type ) TMEM173 allele ( Ablasser et al . , 2013; Gao et al . , 2013b; Yi et al . , 2013; Zhang et al . , 2013 ) .", "To develop CDG or other cyclic dinucleotide as a human mucosal vaccine adjuvant , it becomes critical to determine if the adjuvant activity of cyclic dinucleotides is influenced by human TMEM173 variations in vivo .", "In summary , we found that CDG enhances Ag uptake and selectively activates pinocytosis-efficient cells in vivo .", "These qualities should be explored further for the development of CDG as an effective human mucosal vaccine adjuvant ." ], [ "6–12 week old mice were used for all experiments .", "Tmem173−/− mice ( Tmem173<tm1Camb> ) have been described previously ( Jin et al . , 2011a , 2013 ) .", "The ItgaxCreTmem173Flox/Flox mouse was generated as in Figure 4A .", "All mice are on a C57BL/6 background .", "Mice were housed and bred in the Animal Research Facility at Albany Medical College .", "All experiments with mice were performed in accordance to the regulations and approval of Albany Medical College ( Albany , NY ) and the Institutional Animal Care and Use Committee .", "The following reagent was obtained through BEI Resources , NIAID , NIH , Bethesda , MD:S .", "pneumoniae Family 1 , Clade 2 PspA ( UAB055 ) with C-Terminal Histidine Tag , Recombinant from Escherichia coli , NR-33178 .", "Mice were immunized with three doses ( 14 days apart ) of OVA ( 20 μg , cat# vac-efova; Invivogen , San Diego , CA ) or PspA ( 2 μg , BEI Resources ) with , or without , CDG ( 5 μg , cat# vac-cdg; Invivogen ) or 2′3′-cGAMP ( 5 μg , cat# vac-cga23; Invivogen ) .", "Groups of mice ( 4 per group ) were intranasally vaccinated with adjuvanted protein Ag , or Ag alone .", "For intranasal vaccination , animals were anaesthetized using isoflurane in an E-Z Anesthesia system ( Euthanex Corp , Palmer , PA ) .", "Ag , with or without CDG , was administered .", "Sera and nasal washes were collected 14 days after the last immunization .", "The Ag-specific Abs were determined by ELISA .", "The anti-IgG-HRP used was anti-mouse IgG1-HRP ( cat#1070-05; Southern Biotech , Birmingham , AL ) , anti-mouse IgG2C-HRP ( cat#1079-05; Southern Biotech ) , and anti-mouse IgA-HRP ( cat#1040-05; Southern Biotech ) .", "Total anti-OVA IgG2A , IgG1 and IgA were quantified using a mouse anti-OVA IgG2A kit ( cat#3015; Chondrex , Redmond , WA ) , anti-OVA IgG1 kit ( cat#3013; Chondrex , Redmond , WA ) , and anti-OVA IgA kit ( cat#3018; Chondrex , Redmond , WA ) .", "Mice were sacrificed at the indicated time by CO2 asphyxiation and lungs were lavaged with 0 . 8 ml ice-cold PBS .", "The lavage fluid was centrifuged at 2000×g for 1 min .", "Collected cells were analyzed by Flow cytometry .", "Mice were intranasally administered 5 μg CDG ( vaccine grade ) , then sacrificed at the indicated time by CO2 asphyxiation .", "BALF was collected and the lungs were subsequently perfused with cold PBS .", "The harvested lungs were washed in PBS once , then stored in 0 . 7 ml tissue protein extraction reagent ( T-PER ) ( cat#78510; Thermo Scientific , Grand Island , NY ) containing protease inhibitors ( cat#11836153001; Roche , Indianapolis , IN ) at −80°C .", "Later , the lung was thawed on ice and homogenized on ice in the T-PER homogenate buffer in a 2 ml homogenizer .", "Lung homogenates were transferred to a 1 . 5 ml tube and spun at 14 , 000×g for 30 min at 4°C .", "Supernatant was collected and analyzed for cytokine production .", "S . pneumoniae D39 ( serotype 2; ATCC , Manassas , VA ) were grown in Todd-Hewitt broth containing 0 . 5% yeast extract ( THY; BD Biosciences , San Jose , CA ) to an optical density ( OD ) of 0 . 4 ( ∼108 cfu/ml ) .", "Mice were intranasally administered ∼5 × 106 cfu .", "CFUs were confirmed by colony counting of log10 serial dilutions of bacteria cultured overnight on a TSA II with 10% sheep blood agar plate ( cat#221162; BD Bioscience ) .", "Mice were intranasally administered 20 μg DQ-Ovalbumin ( DQ-OVA ) ( D12053; Life Technologies , Grand Island , NY ) or 20 μg Ovalbumin Alexa Fluor 647 ( OVA-647 ) ( O34784; Life Technologies ) with , or without , the adjuvant CDG ( 5 μg , vaccine-grade ) .", "After 20 hr , the lungs were lavaged and perfused with ice-cold PBS .", "Excised lungs were digested in DMEM contain 200 μg/ml DNase I ( 10104159001; Roche ) , 25 μg/ml Liberase TM ( 05401119001; Roche ) at 37°C for 3 hr .", "Red blood cells were then lysed and a single cell suspension was prepared and analyzed by BD LSR II and FACScan flow cytometry .", "The following Abs from Biolegend , San Diego , CA were used in the flow cytometry: CD80 ( 16-10A1 ) , CD86 ( GL1 ) , Ly6C ( HK1 . 4 ) , CD11B ( M1/70 ) , Ly6G ( 1A8 ) , IgD ( 11-26c . 2a ) , CD11C ( N418 ) , FcεRIa ( MAR-1 ) , NK-1 . 1 ( PK136 ) , MHC II ( M5/114 . 15 . 2 ) , CD103 ( 2E7 ) .", "The following Abs from BD Biosciences were used: Siglec F ( E50-2440 ) , c-Kit ( 2B8 ) , and CD68 ( FA-11 ) .", "The intracellular cytokine staining was performed using the Cytofix/Cytoperm kit from BD Biosciences ( cat#555028 ) .", "Briefly , mice were intranasally administered saline or CDG ( 5 μg , vaccine-grade ) .", "The lungs were lavaged , perfused , and harvested at 5 hr post treatment .", "Excised lungs were washed in PBS and digested in DMEM containing 200 μg/ml DNase I ( 10104159001; Roche ) , 25 μg/ml Liberase TM ( 05401119001; Roche ) , and Golgi-plug at 37°C for 6 hr .", "The single lung cell suspension was fixed in Cytofix/perm buffer ( BD Biosciences ) in the dark for 20 min at RT .", "Fixed cells were then washed and kept in Perm/wash buffer at 4°C .", "Golgi-plug was present during every step before fixation .", "The following Abs from eBioscience were used: IL-12p35 ( 4D10P35 ) and IFNγ ( XMG1 . 2 ) .", "Cytokine concentrations were measured using ELISA kits from eBioscience , San Diego , CA according to the manufacturer's instructions .", "The ELISA kits used were IL-1α ( cat#88-5019 ) , IL-1β ( cat#88-7013 ) , IL-4 ( cat#88-7044 ) , IL-5 ( cat#88-7054 ) , IL-6 ( cat#88-7064 ) , IL-10 ( cat#88-7105 ) , IL-12/p70 ( cat#88-7921 ) , IL-13 ( cat#88-7137 ) , IL-17A ( cat#88-7371 ) , IL-22 ( cat#88-7422 ) , IL-23 ( cat#88-7230 ) , IL-33 ( cat#88-7333 ) , TNF-α ( cat#88-7324 ) , TGF-β1 ( cat#88-8350 ) , TSLP ( cat#88-7490 ) , IFN-λ ( cat#88-7284 ) , and IFN-γ ( cat#88-7314 ) .", "The IFNβ ELISA kit was from PBI InterferonSource , Piscataway , NJ ( cat#42410-1 ) .", "All data are expressed as means ±SEM .", "Statistical significance was evaluated using Prism 5 . 0 software to perform a Student's t-test ( unpaired , two tailed ) for comparison between mean values .", "The online supplemental materials include 3 supplemental figures ." ] ]
[ "Effective mucosal adjuvants enhance the magnitude and quality of the vaccine response .", "Cyclic di-GMP ( CDG ) is a promising mucosal vaccine adjuvant .", "However , its in vivo mechanisms are unclear .", "Here , we showed , in mice , that CDG elicits stronger Ab and TH responses than the mammalian 2′3′-cyclic GMP-AMP ( cGAMP ) , and generated better protection against Streptococcus pneumoniae infection than 2′3′-cGAMP adjuvanted vaccine .", "We identified two in vivo mechanisms of CDG .", "First , intranasally administered CDG greatly enhances Ag uptake , including pinocytosis and receptor-mediated endocytosis in vivo .", "The enhancement depends on MPYS ( STING , MITA ) expression in CD11C+ cells .", "Second , we found that CDG selectively activated pinocytosis-efficient-DCs , leading to TH polarizing cytokines IL-12p70 , IFNγ , IL-5 , IL-13 , IL-23 , and IL-6 production in vivo .", "Notably , CDG induces IFNλ , but not IFNβ , in vivo .", "Our study revealed previously unrecognized in vivo functions of MPYS and advanced our understanding of CDG as a mucosal vaccine adjuvant ." ]
[ "The presence of a bacterium , virus , or other pathogen in the body generally triggers a response by the body's immune system .", "As well as trying to destroy the infectious agent , the immune system will also generate ‘memory cells’ that are primed and ready to recognize and help eliminate the pathogen if it is ever re-encountered .", "The parts of the invader that the memory cells recognize are called antigens .", "A vaccine is a biological preparation that improves immunity to a particular disease .", "Vaccines normally contain a dead or weakened version of a pathogen , or its toxins or surface proteins .", "This exposes the immune system to the antigens in a harmless way , and creates memory cells that are able to fight off the harmful pathogen in the future before the individual becomes ill from the infection .", "Substances called adjuvants must also be added to many modern vaccines .", "Adjuvants help to present antigens to immune cells , and by doing so stimulate a stronger and more targeted immune response .", "While many vaccines are currently injected , there is growing interest in developing and improving vaccines that can be inhaled .", "This delivers the vaccine directly to the mucosal surfaces that line the nose and lungs , which is a more effective way to produce immunity against certain bacteria and viruses .", "As these mucosal vaccines are also relatively cheap and easy to apply , they would also be suitable for use in developing countries and during emergencies .", "Current licensed pneumococcal vaccines do not provide strong mucosal protection against the infection .", "As a result , pneumococcal diseases kill more people than all vaccine-preventable diseases combined .", "Developing safe and effective mucosal vaccine adjuvants is key to reducing the impact of pneumococcal diseases .", "Cyclic di-GMP , a molecule found primarily in bacteria , is a powerful mucosal adjuvant .", "However , before it can be widely used in vaccines , it first needs to be known how cyclic di-GMP stimulates the immune system .", "Blaauboer , Mansouri et al . studied the immune response of mice to cyclic di-GMP applied through the nose .", "This revealed two ways that cyclic di-GMP enhances the body's immune response to a vaccine .", "First , cyclic di-GMP improves the uptake of antigens by certain cells exposed to the vaccine , a process that ensures a large number of cells will alert the immune system to the perceived threat .", "Second , Blaauboer , Mansouri et al . explain that cyclic di-GMP selectively activates immune cells known as dendritic cells , which then produce proteins called cytokines that signal to other cells and coordinate the immune response .", "A gene called STING ( stimulator of interferon genes ) controls both cyclic di-GMP induced antigen uptake and the activation of dendritic cells .", "Further research into these processes is now needed to investigate whether cyclic di-GMP is a suitable mucosal pneumococcal vaccine adjuvant for humans ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Decreased brain connectivity in smoking contrasts with increased connectivity in drinking
elife-40765-v2
[ [ "Recent statistics have shown not only the widespread use of cigarettes and alcohol , but also the co-occurrence of these high risk behaviors .", "Although dopamine mechanisms in the basal ganglia have been implicated in addiction there is also considerable evidence for cortical , especially prefrontal cortical , involvement ( Ersche et al . , 2011; Goldstein and Volkow , 2011; Ersche et al . , 2012; Ersche et al . , 2013 ) .", "For example , a voxel-based morphometry ( VBM ) study found that smokers exhibited reduced gray matter volumes and gray matter densities in frontal regions including prefrontal cortex , orbitofrontal cortex , and anterior cingulate gyrus , as compared with non-smokers ( Wang et al . , 2015 ) .", "Resting state functional connectivity differences involving the prefrontal cortex and insula have also been described in smokers ( Fedota and Stein , 2015; Yuan et al . , 2016; Bi et al . , 2017; Sutherland and Stein , 2018 ) .", "A functional magnetic resonance imaging ( fMRI ) study found that increased ventromedial prefrontal cortex ( vmPFC ) and anterior cingulate cortex ( ACC ) activation during ‘relaxing’ trials was correlated with high alcohol cue–induced and stress-induced craving in early recovering alcohol-dependent patients ( Seo et al . , 2013 ) .", "These neuroimaging studies in substance use behaviors suggest that the prefrontal cortex plays a key role in such behaviors .", "The high levels of comorbidity in using cigarettes and alcohol demonstrated by a wealth of epidemiological and genetic data ( Farrell et al . , 2001; Agrawal and Lynskey , 2006 ) makes it important to consider the neurobiological commonalities and differences associated with these two drugs , especially when they are consumed together .", "Thus , King et al . ( 2010 ) found that consumption of a moderately intoxicating oral dose of alcohol increased ratings of the desire to smoke , and moreover that alcohol amplified ventral striatum reactivity to appetitive cues associated with smoking in young individuals who tend to use cigarettes in the context of alcohol intoxication .", "Most studies have used relatively small sample sizes and focused on brain regions rather than brain circuits identified with functional connectivity .", "Further , common and distinct connectivity features underlying the use of different substances such as nicotine and alcohol have not been fully explored with large numbers of participants ( Zhu et al . , 2017; Sutherland and Stein , 2018 ) .", "In this study , we examined whole-brain functional connectivity ( i . e . not limited by particular hypotheses and therefore unbiased ) in groups using nicotine , or alcohol , or both , based on a large sample size ( 831 subjects from the Human Connectome Project ( HCP ) ( and cross-validated by another 1176 subjects from the IMAGEN collaboration as described in the Materials and methods ) with resting-state functional magnetic resonance imaging ( fMRI ) data .", "The aim was to identify shared and distinct patterns of different functional connectivity in smokers and drinkers in a sufficiently large sample to ensure sufficient statistical power to provide robust findings on associations between functional connectivity and smoking and drinking .", "Moreover , by including assessment of smoking and drinking in the same study , we aimed to provide robust evidence on whether there were differences in functional connectivity in smokers and drinkers .", "We also investigated aspects of substance use and impulsive behavior that might relate to the changes in neural connectivity , and that might provide information about the causation and aetiology of substance use ." ], [ "In the HCP participants , 273 links had significantly lower resting state functional connectivity in the smoking group compared to the non-smoking group ( after FDR correction p<0 . 005 ) .", "Table 1 shows the strengths of the top 30 significantly different FC links between smokers and non-smokers .", "Figure 1A shows in the lower triangle matrix that functional connectivity across all FC links of the smoking group relative to the non-smoking group was lower overall ( Figure 1 and Figure 1—figure supplements 1 , 2 , 3 and 4 ) ; and the upper triangle matrix shows which FC links were significantly lower in the smoking group ( p<0 . 005 , FDR corrected ) .", "The links that are lower in the smoking group are shown in Figure 2B .", "Many ( 90 ) of the significantly different links involved the lateral orbitofrontal cortex ( AAL2 areas OFClat and Frontal_Inf_Orb_2 ) and the adjacent inferior frontal gyrus ( pars triangularis BA45 and pars opercularis BA44 ) ( Table 1 ) .", "These areas had lower functional connectivity with areas such as the hippocampus , temporal lobe , supramarginal gyrus , and insula ( Figure 2 ) .", "Figure 2B and Table 1 show that in addition to these lower functional connectivities of the lateral orbitofrontal cortex and related inferior temporal gyrus areas , there was also lower functional connectivity for the middle and superior frontal gyri , mid- and superior temporal gyri , precuneus , hippocampus , and basal ganglia ( caudate , putamen , and pallidum ) .", "Table 1 shows , by contrast with the lateral orbitofrontal cortex , that there were only two significant medial orbitofrontal cortex areas ( OFCmed ) in this set of different functional connectivities in the smoking group .", "In the HCP participants , the top 30 significantly different FC links between the low drinking and high drinking group are shown in Table 1 ( and Figure 3 shows all 214 significantly different FC links after FDR correction with p<0 . 05 ) .", "All the significantly different links are higher in the high level of drinking group ( HA , high amount ) compared to the low level of drinking group ( LA , low amount ) ( except one FC between OFClat_R and Angular_R ) .", "Figure 1B shows in the lower triangle matrix that across all links ( except one ) the functional connectivity of the HA group relative to the LA group was higher overall; and the upper triangle matrix shows the FC links that were significantly higher in the HA drinking group than the LA group ( p<0 . 05 , FDR corrected ) .", "Those links that are higher in the HA group than the LA group are shown in Figure 3 , which show that , in addition to much higher functional connectivity involving the left medial orbitofrontal cortex , there was also higher functional connectivity for the cingulate cortex , supramarginal gyrus , globus pallidus , and pre-/post-central cortex .", "Table 1 shows the top 30 links for the high drinking group , which are all higher compared to the low drinking ( LA ) group .", "Ten of the top 30 links involved higher functional connectivity of the medial orbitofrontal cortex , and 13 higher functional connectivity of the anterior cingulate cortex .", "For the HCP participants , Figure 1C shows the distribution of the t values of all 4371 FC links ( i . e . between all 94 AAL2 areas ) in the drinking and smoking groups respectively .", "Almost all links across the whole brain were increased in the drinking group but decreased in the smoking group .", "Further analysis shows that the t values of the links in the smoking group were significantly negatively correlated with those in the drinking group ( r = −0 . 289 , p<10−10 ) ( Figure 1D ) and this finding was well cross-validated in an independent IMAGEN dataset ( Figure 7C , r = −0 . 442 , p<10−10 ) .", "This finding indicates a complementarity between the functional connectivities in drinkers and smokers .", "That is , if links were high in the smoking group , they tended to be low in the drinking group .", "This provides evidence that a difference in one direction ( lower ) may relate to smoking , and in an opposite direction ( higher ) to drinking .", "We also performed a direct comparison between drinkers and smokers , to ensure that the results were not subject simply to possible baseline differences in the different control groups .", "In this contrast , we found , as predicted , that links involving the lateral orbitofrontal cortex ( Frontal_Inf_Orb_2 and OFClat ) were significantly ( with FDR correction ) positive , indicating that the lateral orbitofrontal cortex links are lower in smokers than drinkers .", "Further , also as predicted , medial orbitofrontal cortex and related areas ( including OFCpost and Olf ) were significantly more positive , indicating that the medial orbitofrontal cortex links are greater in drinkers than smokers , consistent with the other analyses in this investigation .", "Details are provided in the source data of Figure 4 .", "To analyse functional connectivity in those who both smoke and drink , the following comparison between four groups was made , with a common baseline group who neither smoked nor drank .", "Functional connectivity was compared over four groups ( i . e smoking only n = 60 , drinking only n = 219 , and both smoking and drinking n = 143 ) using a common baseline comparison group ( a no smoking and low drinking group n = 198 ) from the HCP dataset .", "Figure 4A shows the distribution of t values of all the links for all AAL2 areas for the three comparisons .", "This shows that the functional connectivity values for the smoking plus drinking group were similar to those for the only smoking group .", "For Figure 4B–D only the significantly different links identified in the HCP dataset as shown in Figures 2 and 3 are considered .", "Figure 4B confirms that all these links are lower in the smoking only group .", "Figure 4C confirms that almost all these links are higher in the drinking only group .", "Figure 4D shows that there are mainly lower links in the group that both smokes and drinks .", "Figure 4—source data 1 shows that for the links that are lower in the smoking group as shown in Table 1 , they are also lower in the smoking only group , and the both smoking and drinking group .", "Figure 4—source data 2 shows that for the links that are higher in the drinking group as shown in Table 1 , they are also higher in the drinking only group , while some are higher and some are lower in the both smoking and drinking group .", "Thus the group that both smokes and drinks shows a combination of the links found to be different in the smoking group , and of those found to be different in the drinking group .", "However , it was noticeable that in the combined smoking and drinking group the difference in functional connectivity from the controls for the increases and decreases ( expressed as a t value ) found were smaller than in the only drinking or only smoking group ( Figure 4 ) .", "In summary , participants who both smoked and drank had a combination of the specifically different links in those who only smoked or only drank , and these links had on average lower functional connectivity .", "An implication is that the differences in functional connectivity in smokers and drinkers described in this paper can , when combined , be related to , and potentially contribute to , both smoking and drinking in the same individual .", "Figure 5A , B shows that for the smoking group , there was a negative correlation between the mean strength of functional connectivity for both the significantly different links ( n = 369 , r = −0 . 102 , p=0 . 031 , permutation test ) and the links for the whole brain ( n = 830 , r = −0 . 080 , p=0 . 026 ) with the frequency of smoking across individuals .", "( This is consistent with the finding that the functional connectivity is lower in smokers . ) .", "It should be noted that all correlation analyses involving identified links were performed only within the smoking ( or high drinking ) group .", "Figure 5C , D shows that for the drinking group , there was a positive correlation between the mean strength of FCs for the links for the whole brain ( n = 782 , r = 0 . 075 , p=0 . 037 ) and a trend also for significantly different links ( n = 471 , r = 0 . 069 , p=0 . 07 , permutation test ) and the amount of drinking across individuals ( drinks per drinking day in the past 12 months ) .", "( This is consistent with the finding that the FCs are higher in drinkers ) .", "Further , we found that this pattern of changes with the amount of use also applied to most of the significant links for both smoking ( Figure 5E ) and drinking ( Figure 5F ) .", "Specifically , 269 out of 273 links associated with smoking showed a negative correlation with the frequency of smoking , and 54 of these 273 identified links were significant ( p<0 . 05 uncorrected ) .", "In addition , 177 out of 214 links associated with drinking showed a positive correlation with the drinks per drinking day , and 38 of these 214 identified links were significant ( p<0 . 05 uncorrected ) .", "We also investigated the relationship between the amount of smoking and drinking , with inhibitory control related to impulsivity ( measured by the delay discounting score ) .", "Figure 6A , B shows that there was a negative correlation between the frequency of smoking and both the delay discounting scores , DDisc_AUC_200 ( n = 830 , r = −0 . 120 , p=6 . 0 × 10−4 ) and DDisc_AUC_40K ( n = 830 , r = −0 . 088 , p=0 . 012 ) .", "There was also a negative correlation between the amount of drinking and both the delay discounting scores , DDisc_AUC_200 ( n = 782 , r = −0 . 122 , p=7 . 0 × 10−4 , Figure 6C ) and DDisc_AUC_40K ( n = 782 , r = −0 . 033 , p=0 . 036 , Figure 6D ) .", "Thus higher impulsivity was associated with more drinking and more smoking in the drinking and smoking groups .", "Furthermore , canonical correlation analysis ( CCA ) revealed a significant model that relates functional connectivity levels to the delay discounting score for the drinking group ( Figure 6G , n = 782 , r = 0 . 575 , p=9 . 9 × 10−4; Figure 6H , r = 0 . 572 , p=0 . 002 ) with the same direction for the smoking group ( Figure 6E , n = 830 , r = 0 . 643 , p=0 . 005; Figure 6F , r = 0 . 610 , p=0 . 185 ) .", "( The links involved in this analysis were all of the links shown in Figure 2B ( for smoking ) and Figure 3B ( for drinking ) , ‘all links’ ( with a different number of links for the two cases ) ) .", "The findings are consistent with decreases of functional connectivity in areas such as the lateral orbitofrontal cortex and the adjacent inferior frontal cortex being associated with greater impulsivity in the smoking group as measured with the delay discounting score .", "For the drinking group , the significant increases in functional connectivity in the medial orbitofrontal cortex found in this group ( Figure 3B ) were associated with greater impulsivity .", "This may indicate that higher functional connectivity of the reward-related medial orbitofrontal cortex areas ( Rolls , 2017; Rolls , 2018b ) can be associated with increased impulsivity , possibly arising because rewards are driving behavior more strongly .", "We further found that these measures of impulsivity had relatively high correlations , with respect to the 68 behavior measures in the HCP , with the smoking and drinking , and their related functional connectivities .", "Using a separate dataset ( IMAGEN ( Schumann et al . , 2010 ) ) we cross-validated the findings , as follows , with details provided in the Supplementary Material .", "The results of this cross-validation for smoking are shown in Figure 7 .", "Figure 7A shows the distribution of t values of the links between all AAL2 areas in the HCP and IMAGEN dataset at age 19 .", "( Age 19 was used because by then some participants were smoking , and some were not . )", "The lower global ( i . e . overall ) functional connectivity found with the HCP dataset was confirmed in the IMAGEN dataset , with significant differences from zero at age 19 for smoking ( t = −55 . 53 , p<10−10 , one sample t-test ) .", "With respect to cross-validation of the specific links identified in the HCP dataset , Figure 7D shows that these links also had a significantly lower distribution of values compared to the comparison control group for the IMAGEN dataset at age 19 ( one sample t-test , t = −13 . 00 , p<10−10 ) .", "For the drinking , the results of the cross-validation are also shown in Figure 7 .", "Figure 7B shows the distribution of t values of the links between all AAL2 areas in the HCP and IMAGEN datasets at age 19 .", "( Age 19 was used because by then some participants were drinking , and some were not . )", "The higher global functional connectivity found with the HCP dataset was confirmed in the IMAGEN dataset , with a significant difference from zero for the IMAGEN dataset at age 19 ( t = 98 . 00 , p<10−10 , one sample t-test ) .", "Figure 7E shows that the significant links for drinking identified in the HCP dataset also had a significantly higher distribution of t values compared to the comparison control group for the IMAGEN dataset at age 19 ( one sample t-test , t = 15 . 32 , p<10−10 ) .", "As described in the Supplementary Material , the comparison group was the low-drinkers .", "To investigate whether the functional connectivity at 14 might be related to whether a participant became a smoker by age 19 , a longitudinal analysis with the same IMAGEN participants was performed .", "The 19 years olds were split into two approximately equal size groups , of smokers vs non-smokers .", "For each link , its value in the 14 years olds was compared between those who were smokers and non-smokers at age 19 with a two sample t test .", "For the overall connectivity , this provided 4371 t values .", "A one-sample t-test was then performed for whether the mean of these t-values was lower than 0 in those who became smokers at 19 .", "That test was significant ( t = −47 . 51 , p<10−10 , Figure 7A ) , providing evidence that the value overall of the links was lower at 14 in those who became smokers at 19 .", "With respect to the significantly different links between smokers and non-smokers identified in the HCP dataset , the test was also significant ( t = −12 . 23 , p<10−10 , Figure 7D ) , providing evidence that the values of the significantly different links found in the HCP dataset were lower at age 14 in the IMAGEN dataset in those who became smokers at 19 .", "An implication is that the low functional connectivities may not simply be produced by smoking , but may instead contribute to the tendency to smoke .", "Similar tests for drinking found analogous effects ( t = 15 . 31 , p<10−10 for the overall links ( Figure 7B ) , and t = 7 . 06 , p<10−10 for the specific links ( Figure 7E ) ) in female , but not in male , IMAGEN participants .", "The analyses of impulsivity with the IMAGEN dataset are considered next .", "First , the impulsivity is significantly positively correlated with the frequency of smoking ( r = 0 . 106 , p=1 . 4 × 10−4 ) and the amount of drinking ( r = 0 . 064 , p=0 . 022 ) at age 19 .", "The results for impulsivity at age 14 are shown in Figure 7F .", "The 14 year olds who will smoke at age 19 ( n = 421 ) have higher impulsivity than those who will not smoke at 19 ( n = 184 ) ( t = −3 . 72 , p=2 . 20 × 10−4 ) .", "The 14 year olds who will drink at age 19 ( n = 527 ) have higher impulsivity than those who will not drink at 19 ( n = 298 ) ( t = −3 . 67 , p=2 . 60 × 10−4 ) .", "Thus higher impulsivity at 14 is associated with who will smoke , or drink , at age 19 ." ], [ "Smokers and drinkers had contrasting patterns of cortical functional connectivity involving the orbitofrontal cortex .", "Smokers relative to non-smokers exhibited reduced functional connectivity involving the lateral orbitofrontal cortex which is implicated in stopping and changing behavior and thus to impulsive-compulsive disorders , and in related areas including the inferior frontal gyrus , superior temporal gyrus , precuneus , and supramarginal gyrus ( Figure 2A ) .", "By contrast , high drinkers relative to low drinkers had higher functional connectivity in networks including the medial orbitofrontal cortex ( bilaterally though more on the left ) which is involved in reward processing , and in related structures including the anterior cingulate cortex , parahippocampal cortex , supramarginal gyrus , insula , and superior temporal gyrus .", "In addition , the mean functional connectivity across the whole brain was lower in smokers than non-smokers .", "In contrast , mean functional connectivity across the whole brain was higher in high drinkers than low drinkers .", "The differences in both the overall and the identified links were correlated with the amount of smoking or drinking .", "For both the smoking and drinking , the differences in overall functional connectivity were correlated with increased impulsivity .", "These findings with the HCP dataset were cross-validated with the IMAGEN dataset .", "In addition , analysis of the IMAGEN dataset showed that for the significantly different links between smokers and non-smokers identified in the HCP dataset , those links were lower at age 14 in the IMAGEN dataset in those who became smokers at 19 ( Figure 7D ) .", "An implication is that the low functional connectivities may not simply be produced by smoking , but may instead contribute to the tendency to smoke .", "The same was found for the global functional connectivity ( Figure 7A ) .", "For drinking , analysis of the IMAGEN dataset showed that for the significantly different links between high drinkers and low drinkers identified in the HCP dataset , those links were higher at age 14 in the IMAGEN dataset in those who became high drinkers at 19 ( Figure 7B , E ) for females , though that effect was not significant in the male participants .", "The low functional connectivity in smokers relative to non-smokers especially involved the lateral orbitofrontal cortex ( Brodmann area 47/12 ) which is implicated in stopping and changing behavior , including when reward is not received ( Kringelbach and Rolls , 2003; Robbins , 2007; Grabenhorst and Rolls , 2011; Rolls , 2014; Rolls , 2016 ) , and when there is an instruction to stop in the stop-signal task ( Deng et al . , 2017 ) .", "This reduced connectivity also involved the inferior frontal gyrus , pars opercularis ( BA 44 ) and pars triangularis ( BA45 ) .", "Further , damage to the lateral orbitofrontal cortex ( Berlin et al . , 2004; Rolls , 2014; Rolls , 2016 ) and to the right inferior frontal gyrus ( Aron et al . , 2014 ) produces impulsive behavior ( measured for example in the stop-signal task ) , where impulsive behavior can be considered as behavior not directly inhibited by non-reward or punishment feedback signals ( Rolls , 2014; Rolls , 2017 ) .", "Consistent with this , lower functional connectivity involving the lateral orbitofrontal cortex in the present study was related to increased impulsiveness as measured by the delay discounting score ( Figure 6 ) .", "An implication is that smokers may smoke in part because of increased impulsiveness and reduced sensitivity to non-reward and punishment associated with reduced functional connectivity of the lateral orbitofrontal cortex and adjacent inferior temporal gyrus .", "Consistently , we found that the delay discounting score ( DDS ) was positively correlated with the strength of many of the lateral orbitofrontal cortex links ( in the HCP dataset ) .", "Thus high FC of the lateral orbitofrontal cortex ( OFC ) is correlated with low impulsivity .", "The increased functional connectivity in drinkers relative to non-drinkers involved the medial orbitofrontal cortex which is involved in reward processing ( Grabenhorst and Rolls , 2011; Rolls , 2014; Rolls , 2017 ) and structures to which it is connected including the anterior cingulate cortex , parahippocampal cortex , supramarginal gyrus , and superior temporal gyrus .", "We found that the delay discounting score ( DDS ) was negatively correlated with the strength of many of the medial orbitofrontal cortex links ( in the HCP dataset ) .", "Thus high FC of the many medial OFC links was correlated with high impulsivity , which correlated in turn with the amount of drinking .", "In rats , steeper delay discounting ( interpreted as greater impulsivity ) is produced by lesions of the lateral OFC , and reduced discounting by lesions of the medial OFC ( Mar et al . , 2011 ) , possibly homologous regions to those in the primate brain , suggesting a similar opponency of control over impulsivity by these prefrontal cortex regions to that found in the present investigation in humans .", "In the short term just after administration , alcohol increases impulsivity and decreases behavioral inhibition ( Smith et al . , 2014 ) ( and increases neurophysiological inhibition in some brain areas ) ( Stephens et al . , 2017 ) .", "The mechanism for the increase in impulsivity produced by alcohol may include reduced activity in the lateral orbitofrontal cortex , which tends to have activations reciprocally related to those of the medial orbitofrontal cortex ( O'Doherty et al . , 2001; Kringelbach and Rolls , 2003; Rolls , 2016; Rolls et al . , 2018a ) .", "The fact that both drinkers and smokers exhibited greater impulsive choice is consistent with a possible causal role for impulsivity in either the initiation of drinking or smoking or else its exacerbation , as well as other behaviors associated with impulsivity , including aggression in the case of alcohol consumption ( Garofalo and Wright , 2017 ) .", "To check the main findings of this investigation , and to control for possible differences in the comparison groups , we directly compared functional connectivity in drinkers and smokers .", "The hypotheses were that functional connectivity involving the lateral orbitofrontal cortex would be smaller in smokers than drinkers; that functional connectivity involving the medial orbitofrontal cortex would be greater in drinkers than in smokers; and that the mean functional connectivity would be higher in drinkers than non-smokers .", "All these hypotheses were confirmed by the direct comparison of drinkers – smokers in the Results section ( see Figure 4 ) .", "Using a separate dataset ( IMAGEN ( Schumann et al . , 2010 ) ) we cross-validated the findings obtained with the HCP dataset .", "In addition , analysis of the IMAGEN dataset showed that there was an association between the low connectivity in smoking at age 14 ( when participants were not smoking ) and whether at age 19 individuals were smoking .", "This association was found for both the global functional connectivities ( Figure 7A , B ) and the specifically different functional connectivities ( Figure 7D , E ) .", "A similar relation was found in drinkers , namely that there was an association between the high functional connectivitities at 14 and wherher an individual would be in the high drinking group at age 19 .", "This was significant for females , but not for males .", "An implication is that the differences in brain functional connectivities identified in this investigation may play a causal role in who will become smokers or drinkers .", "It will be of interest in future to assess further whether the differences in functional connectivity described here in smokers and drinkers cause the smoking or drinking , for example in research on alcohol drinking endophenotypes or smoking endophenotypes ( Ray et al . , 2010; Ducci and Goldman , 2012 ) .", "It is of course also a possibility that drinking may by increasing impulsivity exacerbate the drinking .", "The low functional connectivity that we describe here in smokers in frontal and associated areas may be reflected in the hypofrontality that has previously been described in smokers and has been associated in genome-wide association studies with single-nucleotide polymorphisms ( SNPs ) in the human CHRNA5 gene , encoding the α5 nAcetylcholine receptor subunit , that increase the risks for both smoking and schizophrenia ( Barch et al . , 2001; Hong et al . , 2010; Tobacco and Tobacco and Genetics Consortium , 2010 ) ; Schizophrenia Working Group of the Schizophrenia Working Group of the Psychiatric Genomics Consortium , 2014; Koukouli et al . , 2017 ) .", "So it is possible that smokers self-administer nicotine which acting on this cholinergic receptor may increase cortical neuronal firing , which stabilizes the activity of prefrontal attractor networks , making them more efficient at cognitive control tasks involved in executive function .", "This has been proposed for the smoking in schizophrenia ( Rolls et al . , 2008 ) .", "Thus , we suggest that lower initial whole brain FC in smokers may encourage them to enhance their functional connectivity and hence the functioning of the neural circuitry implicated in cognitive control; smoking can then be seen as a form of ‘self-medication’ which optimizes their behavior .", "For female drinkers there was also an association between high functional connectivity at age 14 and who would be in the high drinking group at age 19 .", "In this case a possible account for the drinking is that it is related to increased functional connectivity or sensitivity of medial orbitofrontal cortex and related reward circuitry , which makes the alcohol more rewarding , and which may also because of the increased sensitivity to reward produce some increase in reward-related impulsivity ( Whelan et al . , 2012 ) .", "The acute effects of the alcohol may themselves reduce behavioral inhibition and increase the drinking , thus exacerbating the drinking ( Whelan et al . , 2014 ) .", "Figures 1D and 7C show a negative correlation between the t values for all the connectivities in the brain for drinkers vs smokers .", "This may be related to the fact that high values of t for the functional connectivity relate to high drinking , and low values t for the functional connectivity relate to smoking .", "Thus the negative correlation reflects whether individuals are more likely to perform an action of either smoking or drinking .", "This reflects the fact that in this dataset those who smoke also are more likely to drink .", "A common underlying factor might be impulsivity .", "We note that although there were overall higher in FC in drinkers , and lower in smokers , to place this in context , FCs were also smaller in males than females in this dataset in smokers and drinkers ( Figure 1—figure supplement 4 ) .", "Accordingly , whilst we would not wish to over-interpret the differences in global mean FC in drinking and smoking , the negative correlation between all FCs in drinking and smoking shown in Figure 1E is very suggestive that there is indeed complementarity of the differences found in drinkers and smokers .", "An interpretation is that smoking and drinking are related to opposite differences in some of many of the links involved .", "Examples from this investigation are that medial orbitofrontal cortex links are decreased in smokers , but increased in drinkers; and vice versa for lateral orbitofrontal cortex links .", "The differences in functional connectivity described in this investigation were not due to any obvious possible confounding factors such as gender , ethnicity , level of education , marijuana use , or basic brain structure ( volume of gray and white matter ) , which were regressed out of the analysis .", "The use of resting state fMRI is useful for this type of investigation as it does not require task performance that might differ between participants , and such studies can for example predict behavior ( Rosenberg et al . , 2016 ) and task performance ( Tavor et al . , 2016 ) in different individuals .", "Structure based morphological comparisons for drinkers and smokers have been performed , but do not necessarily relate closely to what is found with functional investigations , as parts of the brain may have different physiology without much gross anatomical difference .", "Possible limitations of this investigation are that linear analyses were used .", "It is also important to stress that all participants were healthy adults , and that the levels of smoking and drinking exhibited were not directly associated with diagnoses of substance dependence , although they may be relevant to these disorders .", "Thus the effect size of this study involving the normal use of alcohol and cigarettes could be lower than in a case/control study .", "But the design of this study , use of individuals in the general population , enable this investigation to have impact on public health , since the use of substances for most of the smokers and drinkers was within a normal range , and we could identify these changes by the use of two large-scale datasets .", "Although the current study was based on the general population , the findings are in general consistent with previous studies based on clinical populations .", "For example , chronic nicotine exposure associated with lower functional connectivity was reported in a number of studies ( Hong et al . , 2009; Weiland et al . , 2015 ) .", "In addition , many studies report that the orbitofrontal cortex , the key region identified in current study , play an important role in the neurobiological mechanisms of addiction ( Lucantonio et al . , 2012; Hu et al . , 2015; Koob and Volkow , 2016 ) .", "However , the results described here were obtained on populations from the general community , and do not necessarily apply to heavy smokers or drinkers .", "Moreover , we demonstrate with the IMAGEN dataset that the effect size is sufficient that there is a relation between the brain functional connectivities that we identify at age 14 and who will smoke at age 19 .", "This is good evidence that the effects described in this paper are meaningful , in that the functional connectivity at age 14 can be related to what will happen in terms of smoking and drinking five years later .", "Additionally , we have removed several possible confounders that may distinguish the two groups , but it might be useful in future investigations to investigate the relation of depression to smoking and drinking .", "Another possible limitation is that impulsivity was assessed by delay discounting , and it would be of interest to examine the relation to other measures of impulsivity .", "Another possible area in which future research might extend the findings reported here is that we focused on the amount of drinking using the measure ‘drinking amount per drinking day’ , as the relation between the frequency of drinking and the functional connectivity was complex .", "But this enables us to make an interesting point .", "The amount of alcohol consumed on a drinking day , the ‘amount’ measure , may well be related to how rewarding the consumption of the alcohol is .", "Once started on a drinking day , an individual in the High Amount drinking group tends to have more , and this is consistent with the reward value being high .", "That in turn is consistent with the higher functional connectivity of the medial orbitofrontal cortex found in the High Amount drinkers in this investigation , and with the evidence that the medial orbitofrontal cortex contains a representation of reward value ( Rolls , 2014; Rolls , 2017; Rolls , 2018b ) .", "Another possible limitation of the drinking analysis is that the control group was a low drinking group rather than a non-drinker group .", "The reason for this choice was that only 55 participants did not drink any alcohol in the past 12 months , which would have resulted in too small a sample size .", "As shown in Figure 4 , most of the links were lower in the group that both smokes and drinks .", "One explanation is that the effect size for the comparison between non-smokers and regular smokers is higher than the effect size for the comparison between low-drinkers and high drinkers .", "In conclusion , we have related for the first time reduced functional connectivity involving the lateral orbitofrontal cortex and related inferior frontal gyrus to smoking .", "We interpret the smoking as being related to the increased impulsivity and decreased behavioral inhibition .", "In addition , smokers have globally lower functional connectivity .", "A possible interpretation is that smokers may self-administer nicotine in order to increase brain functional connectivity and thereby attention and alertness .", "We also describe for the first time that drinkers have increased functional connectivity of the medial orbitofrontal cortex , a reward area , and globally increased functional connectivity as well as impulsive behavior .", "An interpretation is that there is a role in drinking of increased reward sensitivity implemented in the medial orbitofrontal cortex .", "The increased impulsivity in drinkers may also be related to increased reward processing in the medial orbitofrontal cortex and its connected areas .", "These differences in functional connectivity may play a causal role , as shown by analyses showing that there was an association between smoking or drinking at age 19 and the functional connectivities at age 14 of individuals who at 14 are not smoking or drinking .", "In both smokers and drinkers , the increased impulsivity may exacerbate substance use .", "These findings open the way for further investigations of the extent to which these differences contribute to the substance use , or are caused by it .", "In any case , we note that there may be many factors that influence smoking and drinking , though we have revealed some of the possible underlying factors and brain mechanisms in this investigation ." ], [ "The dataset used for this investigation was selected from the November 2015 public data release from the Human Connectome Project ( HCP , N = 900 ) , WU-Minn Consortium .", "Our sample includes 831 subjects ( ages 22–35 years , 463 females ) scanned on a 3 T Siemens connectome-Skyra scanner .", "The HCP consortium is a public shared large-scale neuroimaging dataset which aims to map macroscopic human brain circuits and their relationship to behavior in a large population of healthy adults ( Van Essen et al . , 2012; Van Essen et al . , 2013 ) and which has been widely used in neuroimaging studies ( Finn et al . , 2015; Smith et al . , 2015; Glasser et al . , 2016 ) .", "The sibships with individual having severe neurodevelopmental disorders , neuropsychiatric disorders or neurologic disorders were excluded , but individuals who are smokers , are overweight , or have a history of heavy drinking or recreational drug use without having experienced severe symptoms were included in HCP consortium .", "The details on the inclusion and exclusion criteria of HCP consortium were provided in the previous studies ( Van Essen et al . , 2012; Van Essen et al . , 2013 ) and the HCP website ( https://www . humanconnectome . org/ ) .", "It should be noted that the HCP dataset was not specifically designed for the study of substance use , but it is useful for investigating the impact of alcohol and cigarette use in a community sample .", "Two resting state fMRI acquisitions on different days were used .", "The four resting-state runs of approximately 15 min each were acquired in separate sessions on two different days , with eyes open with relaxed fixation on a projected bright cross-hair on a dark background .", "The WU-Minn HCP Consortium obtained full informed consent from all participants , and research procedures and ethical guidelines were followed in accordance with the Washington University Institutional Review Boards ( IRB #201204036; Title: ‘Mapping the Human Connectome: Structure , Function , and Heritability’ ) .", "The demographic characteristics of participants are summarized in Table", "2 . More details of subjects are provided in the Supplementary Material and the collection and preprocessing of the data are provided at the HCP website ( http://www . humanconnectome . org/ ) .", "We obtained minimally preprocessed R-fMRI data conducted using the HCP Functional Pipeline v2 . 0 ( Glasser et al . , 2013 ) involving gradient distortion correction , head motion correction , image distortion correction and spatial transformation to the 2×2×2 mm3 Montreal Neurological Institute ( MNI ) space using one step spline resampling from the original functional images followed by then intensity normalization ( Glasser et al . , 2013 ) .", "In this study , these minimally preprocessed images were further analyzed using FSL ( Jenkinson et al . , 2012 ) and AFNI ( Cox , 1996 ) .", "Briefly , first the constant , linear and quadratic trend were removed from these functional images .", "Next , several nuisance signals were regressed from the time course of each voxel using multiple linear regression , including cerebrospinal fluid signal , white matter signal , and Friston’s 24 head motion parameters .", "Then , temporal band-pass filtering ( 0 . 01-0 . 1 Hz ) was performed to reduce the influence of low-frequency drift and the high-frequency physiological noise .", "Finally , 3D spatial smoothing is applied to each volume of the fMRI data using a Gaussian kernel with Full-width at Half Maximum ( FWHM ) equaling to 4 mm .", "The first 50 volumes were discarded to suppress equilibration effects and participants without the full 1200 time points in four resting-state runs were also removed from the following analysis .", "Any data affected by head motion ( mean framewise displacement larger than 0 . 3 mm ) were excluded using the protocol of Power et al . ( 2014 ) .", "The resulting time courses were used for the construction and analysis of the brain network .", "We also compared the findings based on the pipeline described above and elsewhere ( Cheng et al . , 2016 ) with alternative pipelines for data preprocessing including the HCP minimal preprocessing pipeline , and our pipeline but with global signal removal .", "More details on the effect of the preprocessing pipelines are shown in the Supplementary Material ( Figure 1—figure supplements 1 ) .", "After preprocessing , the whole brain ( gray matter ) was parcellated into 94 regions of interest ( ROI ) according to the automated anatomical labeling ( AAL2 ) atlas ( Rolls et al . , 2015 ) ( 47 regions in each hemisphere ) , and the time series were extracted in each ROI by averaging the signals of all voxels within that region .", "The names of the ROIs and their corresponding abbreviations are listed in Supplementary file", "1 . ( This is a standard parcellation scheme , which provides a usable number of different divisions for statistical purposes when differences between regions are being investigated , and includes a useful parcellation of the orbitofrontal cortex . ) .", "For comparison , we also provide the results ( Figure 1—figure supplement 2 ) based on another atlas ( Shen et al . , 2013 ) which has also been validated in resting state fMRI studies ( Finn et al . , 2015; Rosenberg et al . , 2016 ) , although its parcellation of the orbitofrontal cortex is less good than the AAL2 atlas .", "The Pearson cross-correlations between all pairs of regional BOLD signals were calculated for each subject followed by z-transformation to improve normality , and the whole-brain functional connectivity network ( 94 × 94 region-based network with 4371 links ) was constructed .", "Finally , the mean functional connectivity across two scans ( each scan containing left to right and right to left phase encoding directions ) was used for the following analysis to provide a more reliable estimation of functional connectivity .", "The correlation of the functional connectivities was high between the two different scans as shown in the Supplementary Material ( Figure 1—figure supplement 3 ) .", "The following factors were considered in choosing the measures to define the groups .", "The main purpose of the current study was to investigate whether there were differences in functional connectivity that were related to smoking or drinking .", "Accordingly , in the initial analysis , we categorized the participants for the smoking analysis into two groups , one which used significant amounts of cigarettes , and the other using no or low amounts of cigarettes .", "The same was performed for the division into two groups with respect to the amount of alcohol drunk .", "In making the cutoff , we ensured that there were reasonable numbers of participants in the high and the low amount groups .", "After the initial analysis , we wished to investigate the effect of the different amounts of use of cigarettes or alcohol , and therefore used measures of the different amounts of drinking of each participant , and the number of times each participant had smoked , as described in the results section ( Figures 5 and 6 ) .", "Accordingly , to investigate smoking-associated functional connectivity , we divided all participants into three groups according to their smoking history ( SSAGA_TB_Smoking_History ) , that is non-smokers ( 417 participants ) , occasional smokers ( 161 participants ) and regular smokers ( 203 participants ) .", "The non-smoker group contained participants who had never smoked during their lifetime .", "The occasional smoker group contained participants who experimented with cigarettes at least once in their lifetime but never became a regular smoker .", "The regular smoker group included those who smoked at least 100 cigarettes in his or her lifetime and smoked at least one day per week when they were smoking regularly .", "( A score of 3 indicates a regular smoker; of 1 and 2 indicates an occasional smoker , and 0 a non-smoker . )", "In the main analyses described in this paper to investigate whether there was an effect of smoking , two of these groups were used , the non-smoker and regular smoker groups .", "The third group was helpful in further analyses to investigate effects of the amount of smoking ( Figure 5 ) .", "To investigate drinking-associated functional connectivities , we divided all participants into two groups .", "A Low Amount ( LA ) group with 311 participants drank one or fewer drinks per drinking day in the past 12 months .", "A High Amount ( HA ) group with 470 participants drank two or more drinks per drinking day in the past 12 months .", "( The descriptor was SSAGA_Alc_12_Drinks_Per_Day , and this division into two groups was made to help comparison with the initial analysis for smokers , which also used two groups . )", "It should be noted that the ‘amount’ of drinking in the current study refers to the amount of drinks per drinking day ( and not for example to the total number of drinks in the past 12 months ) .", "The rationale for the use of this measure is provided in the Discussion section , and a complementary analysis showed that there was a good correlation between the association pattern of the functional connectivities when the measures were the 'total drinks' and the 'drinks per drinking day' ( r = 0 . 72 , p<1 × 10−10 ) .", "In order to compare the results for smoking and drinking , only participants with information about both drinking and smoking behavior were included .", "The demographic characteristics of the participants in each group are summarized in Table", "2 . In order to investigate the relationship between functional connectivity ( FC ) and smoking , two sample two-tailed t tests were used to test whether smoking was associated with functional connectivity after removing the confounding effects of age , gender , years of education , race , handedness , head motion ( mean framewise displacement , FD ) , BMI , blood pressure ( diastolic and systolic ) , total gray matter volume , total white matter volume , drinking , and marijuana use .", "Considering the purpose of this study described above and the sample size in each sub-group , we used a two sample t-test rather than a 2 × 2 ANOVA .", "Then a Storey's FDR procedure was used to correct for multiple comparisons ( Storey , 2002; Storey and Tibshirani , 2003; Erlikhman and Caplovitz , 2017 ) .", "Storey’s FDR is a modification of FDR , also called positive False Discovery Rate ( pFDR ) by conditioning on one false positive finding having occurred .", "The Storey's FDR method was implemented by the Matlab function mafdr . m with default parameters .", "In the present study FDR correction for the functional connectivity between any pair of AAL2 regions was used , and the results for smoking are presented based on this statistical test with FDR p<0 . 005 .", "We then performed the same statistical analysis on the high drinking and low drinking groups with the FDR correction at p<0 . 05 .", "The effects of any use of the other substance was always removed by regression when we performed the analysis on the use of one of the substances .", "For the effect of the most recent drink/cigarette use , a correlation analysis confirmed that the association pattern was highly consistent in the cases with and without the covariates of the total drinks and total times smoked in the past 7 days ( r = 0 . 957 for smoking and r = 0 . 927 for drinking ) .", "This provides evidence that it is whether one is a smoker or drinker that is relevant to the results described in this paper , and not just the most recent drug use .", "We explored the relationship between the functional connectivity links identified above as different in substance use groups with behavioral measures ( i . e . drinks per drinking day in the past 12 months and the amount of smoking ) .", "Specifically , a partial correlation analysis was used to measure the correlation between the identified functional connectivity and these behavioral measures with removal of the confounding variables of age , gender , years of education , race , handedness , head motion ( mean FD ) , BMI , blood pressure ( diastolic and systolic ) , total gray matter volume , total white matter volume and use of the other substance .", "We also investigated the relationship between the functional connectivity links identified above as different in substance use groups and behavioral scores related to self-regulation and impulsivity .", "We performed a partial correlation of the delay discounting score with the amount of drinking per drinking day and the amount of smoking respectively .", "The score of self-regulation and impulsivity was based on the delay discounting task which measures the undervaluing of rewards delayed in time .", "We used an area-under-the-curve discounting measure ( AUC ) that provides a valid and reliable index of how steeply an individual discounts delayed rewards ( Myerson et al . , 2001 ) .", "Specifically , we calculated the canonical correlation between the area under the curve for discounting of $200 and $40 , 000 ( DDisc_AUC_200 and DDisc_AUC_40K ) with the strength of associated functional connectivities for drinking and smoking respectively , after removing the effects of age , gender , years of education , race , handedness , head motion ( mean FD ) , BMI , blood pressure ( diastolic and systolic ) total gray matter volume , total white matter volume and use of the other two substances .", "We used another separate large longitudinal fMRI dataset ( the IMAGEN dataset ( Schumann et al . , 2010 ) ) which included 1176 participants to test whether the findings based on the HCP dataset could be cross-validated with an independent dataset , which would considerably strengthen the findings .", "This dataset also enabled examination of whether functional connectivity at age 14 when participants neither smoke nor drank was associated with who would become drinkers or smokers at age 19 .", "This potentially thus provides a way to investigate whether differences between individuals that were not caused by smoking and drinking ( as there was little smoking and drinking when the participants were 14 ) might lead to smoking or drinking by the time that the participants were 19 .", "The IMAGEN dataset also enabled investigation of gender differences in the behaviors that may be predictable from brain functional connectivity in this important period of development .", "The details of demographic characteristics of participants from the IMAGEN dataset are presented in Supplementary file", "2 . IMAGEN adopted a longitudinal design to collect genetic , neuroimaging , environmental , and behavioural data in the UK and Europe , starting at age 14 years old with a sample 2087 healthy adolescents , and followed at ages 16 and 19 years .", "The neuroimaging data were acquired at ages 14 and 19 years ( Schumann et al . , 2010 ) .", "The demographic characteristics of participants who included resting state fMRI scans are summarized in Supplementary file", "2 . IMAGEN 19: For the dataset at age 19 , by using a similar criterion to define the sub-groups as in the HCP database , a Low Amount ( LA ) drinking group with 378 participants who drank two or fewer drinks per drinking day , and a High Amount ( HA ) drinking group with 566 participants who drank three or more drinks per drinking day , were identified .", "The small difference in the different definitions for the drinking groups for the HCP and IMAGEN samples is because they used different questionnaires .", "We used the European School Survey Project on Alcohol and Other Drugs ( ESPAD ) to assess the lifetime drinking and smoking in the IMAGEN dataset .", "The measure of drinking in the IMAGEN dataset is as follows: “How many drinks containing alcohol do you have on a typical day when you are drinking ?", "1: 1 or 2 2: 3 or 4 3: 5 or 6 4: 7 to 9 5: 10 or more’ For comparison , the measure of drinking in the HCP dataset is: ‘Drinks consumed per drinking day in past 12 months: 0 , 1 , 2 , 3 , 4 , 5–6 = 5 , 7+ = 6 . ’ In fact , the control group for the HCP and IMAGEN datasets were similar , in that they had not drunk alcohol .", "For the smoking dataset , a smoking group ( regular smoker ) was defined as those who smoked at least 100 cigarettes in his or her lifetime and smoked at least one day per week when they were smoking regularly ( 180 participants ) , and a non-smoking group ( who had never smoked , 295 participants ) .", "IMAGEN 14: Since almost all participants did not take any cigarettes and alcohol at age 14 , it was not suitable to assess the difference between smoking and non-smoking group ( or low drinking and high drinking group ) .", "However , this longitudinal data enabled us to investigate possible causal associations between substance use behaviors and functional connectivities .", "Therefore , for the dataset at age 14 , to investigate possible causal associations between drinking and functional connectivities , we focused only on the subjects who drank two or fewer drinks per drinking day .", "( This was the great majority of participants . )", "We also divided all participants into two groups according to their drinking behavior at age 14 and 19 .", "Specifically , a Future Low Amount ( FLA ) group with 86 participants drank with two or fewer drinks per drinking day at both age 14 and 19 .", "A Future High Amount ( FHA ) group with 91 participants drank with three or more drinks per drinking day at age 19 .", "It should be noted that both of these groups drank two or fewer drinks per drinking day at age 14 .", "Similarly to the above , for the analysis of smoking , we still focused on the participants who never smoked cigarettes at age 14: specifically , on a future non-smoking group with 56 participants who never smoked at both age 14 and 19; and a future regular smoking group with 19 participants who experienced smoking at age 19 .", "Then a two sample two-tailed t-test was used to assess the difference between the ( F ) LA group and ( F ) HA group and the smoking and non-smoking groups in the datasets at both the ages of 14 and 19 after removing the effect of gender , BMI , head motion ( mean FD ) and the use of the another substances use .", "We also investigate the impulsivity of participants at age 14 by using the IMAGEN dataset .", "The impulsivity measure is based on the Substance Use Risk Profile Scale ( Woicik et al . , 2009 ) by using the sum of the following question scores: We used the European School Survey Project on Alcohol and Other Drugs ( ESPAD ) at ages 14 and 19 years to assess life time drinking and smoking .", "The primary question of interest was regarding lifetime alcohol and cigarette use and the questionnaire were as follows ( Schumann et al . , 2010 ) :", "1 . On how many occasions during your lifetime have you smoked cigarettes ?", "2 . How frequently have you smoked cigarettes during the last 30 days ?", "3 . How many drinks containing alcohol do you have on a typical day when you are drinking ?", "4 . On how many occasions over the last 12 months have you had any alcoholic beverage to drink ?", "Data preprocessing was performed using DPARSF ( Chao-Gan and Yu-Feng , 2010 ) ( http:// restfmri . net ) which is a toolbox based on the SPM8 software package .", "The first 10 EPI scans were discarded to suppress equilibration effects .", "The remaining scans of each subject underwent slice timing correction by sinc interpolating volume slices , motion correction for volume to volume displacement , spatial normalization to standard Montreal Neurological Institute ( MNI ) space using affine transformation and nonlinear deformation with a voxel size of 3×3×3mm3 , followed by spatial smoothing ( 6 mm Full Width Half Maximum FWHM ) .", "To remove the sources of spurious correlations present in resting-state BOLD data , all fMRI time-series underwent band-pass temporal filtering ( 0 . 01-0 . 1 Hz ) , nuisance signal removal from the ventricles , and deep white matter , and regressing out any effects of head motion using Friston’s 24 head motion parameters procedure .", "Finally , subjects with the mean framewise displacement ( FD ) of head motion large than 0 . 3 were completely excluded from the analysis as it is likely that such high-level of movement would have had an influence on several volumes ( Power et al . , 2014 ) ." ] ]
[ "In a group of 831 participants from the general population in the Human Connectome Project , smokers exhibited low overall functional connectivity , and more specifically of the lateral orbitofrontal cortex which is associated with non-reward mechanisms , the adjacent inferior frontal gyrus , and the precuneus .", "Participants who drank a high amount had overall increases in resting state functional connectivity , and specific increases in reward-related systems including the medial orbitofrontal cortex and the cingulate cortex .", "Increased impulsivity was found in smokers , associated with decreased functional connectivity of the non-reward-related lateral orbitofrontal cortex; and increased impulsivity was found in high amount drinkers , associated with increased functional connectivity of the reward-related medial orbitofrontal cortex .", "The main findings were cross-validated in an independent longitudinal dataset with 1176 participants , IMAGEN .", "Further , the functional connectivities in 14-year-old non-smokers ( and also in female low-drinkers ) were related to who would smoke or drink at age 19 .", "An implication is that these differences in brain functional connectivities play a role in smoking and drinking , together with other factors ." ]
[ "To understand why people become addicted to alcohol or smoking , it is important to look at how the brains of people who use these substances may be different than those who abstain .", "Many studies show that substance use activates the reward systems in the brain via a chemical called dopamine .", "Changes or differences in parts of the brain that control decision-making and restraint also have been implicated in substance use .", "Functional magnetic resonance imaging ( fMRI ) is one tool scientists can use to explore such differences .", "It can measure how well different parts of the brain are communicating with each other by measuring their activity when a person is at rest .", "The patterns of activity reveal which parts of the brain are working closely together or have high functional connectivity and which parts are less well connected , or have low functional connectivity .", "Cheng , Rolls et al . measured the functional connectivity between different parts of the brain in people who smoke and people who drink alcohol .", "Smokers had a low overall functional connectivity between brain regions .", "Specifically , they had weaker connections involving two brain regions that help people change or stop a behavior , the lateral orbitofrontal cortex and inferior frontal gyrus .", "These differences may make people more impulsive and less able to resist smoking .", "The stimulating effects of nicotine may enhance communication between different parts of the brain , so people also may use it to overcome some underlying communication deficits .", "Those who drink alcohol had high overall functional connectivity .", "Reward-related systems , including the medial orbitofrontal cortex and the cingulate cortex , were especially strongly connected .", "This may make them more sensitive to the rewarding aspects of drinking , or more impulsive .", "To confirm their results , Cheng , Rolls et al . analyzed fMRI data from another study .", "These showed that the characteristic differences in brain connectivity were already present in 14-year olds who would go on to drink or smoke at age 19 .", "This suggests that these functional connectivity differences in the brain make people more likely to smoke or drink ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Distinct cortical codes and temporal dynamics for conscious and unconscious percepts
elife-05652-v2
[ [ "The scientific investigation of consciousness divides into two major branches ( Koch , 2004; Dehaene et al . , 2014 ) .", "The first branch , which studies the ‘state of consciousness’ , focuses on general vigilance or a person's ability to perceive , interact , and communicate with the environment , by examining the regulation of sleep and waking , and their pathological disruption by coma , epilepsy , and sleep disorders ( Laureys et al . , 2004 ) .", "The second branch of inquiry , which studies ‘access to consciousness’ , focuses on the processes that make a specific content subjectively experienced—what differences in brain activity distinguish conscious vs unattended or subliminal stimuli ( Dehaene et al . , 2006 ) .", "To a large extent , both endeavors rely on characterization of the neural activity that underlies the conscious state or conscious perception , and they are traditionally approached in a similar manner ( Dehaene and Changeux , 2011 ) .", "In order to examine the neuronal processes that underlie conscious states , the neuronal activity associated with one state of consciousness ( e . g . , sleep ) is compared to another ( e . g . , awake ) .", "Similarly , in order to extract the neural correlates of conscious perception , threshold-level stimuli are presented to the subject such that they are sometimes consciously perceived and sometimes not .", "Trials are then sorted according to their subjective perception ( ‘Seen’ or ‘Unseen’ ) , and the neurophysiological signatures associated with these categories are contrasted to extract the correlates of consciousness perception .", "Such paradigms have yielded convergent findings ( Koch , 2004; Dehaene and Changeux , 2011; Dehaene et al . , 2014 ) , but they have also been criticized on several grounds ( Aru et al . , 2012 ) .", "First , differences in neural activity may stem from physical differences in the stimuli .", "To control this , many experiments now employ identical stimuli and rely on participants' subjective reports to differentiate seen and unseen conditions .", "Even then , stimuli also differ in their depth of processing: performance is typically much higher for conscious trials ( Lau and Passingham , 2006 ) , and several operations may only be feasible in the conscious state ( Sackur and Dehaene , 2009; De Lange et al . , 2011 ) .", "The main effect of seen vs unseen trials may , therefore , reveal processing differences unrelated to the actual cerebral encoding of a conscious or unconscious stimulus , and reflecting solely , the operations that precede follow or even coincide with conscious access ( Aru et al . , 2012; Pitts et al . , 2012; Frässle et al . , 2014; Pitts et al . , 2014 ) .", "Another limitation is that these paradigms do not isolate the brain mechanisms underlying the conscious representation of a specific content ( Haynes , 2009 ) .", "The broad contrast between brain activity on ‘seen’ vs ‘unseen’ trials may include generic processes of attention , alerting , or reporting that should be carefully kept distinct from the more limited set of processes that represent the current mental content .", "For instance , the late P3 component of event-related potentials , which is a frequent signature of conscious perception ( Dehaene and Changeux , 2011 ) , has been proposed to reflect either a non-specific alerting effect arising from noradrenergic neurons of the locus coeruleus ( Nieuwenhuis et al . , 2005 ) or the activation of generic reporting processes unnecessary to conscious perception itself ( Pitts et al . , 2012 , 2014 ) .", "To resolve this issue , it is necessary to identify which patterns of brain activity , unique to conscious trials , faithfully encode the contents of subjective experience and to separate them from other non-specific brain responses ( Haynes , 2009 ) .", "The presence or absence of additional information is a crucial feature that separates major theories of conscious access .", "Some theories propose that subjective experience emerges from a generic pattern of brain activity without any stimulus-specific change ( Lau and Rosenthal , 2011 ) .", "According to these models , the cognitive representation of a stimulus is built during unconscious perception , and the additional activity that gives rise to consciousness tags it as ‘perceived’ but adds no perceptual or coding benefit .", "Conversely , other models ( Dehaene et al . , 2003 , 2006 ) suggest that conscious perception relies on a massive amplification and cortical broadcasting of stimulus-specific information .", "The Global Neuronal Workspace ( GNW ) model asserts that perceptual stages may unfold identically on conscious and non-conscious trials , but that , when a stimulus gains access to consciousness , stimulus-specific information is amplified and re-encoded in additional areas , including a prefrontal–parietal network .", "This ‘workspace’ maintains the information online and dispatches it to additional processors , thus making the stimulus reportable .", "This view predicts that , ceteris paribus , additional stimulus information should be present on conscious trials .", "Finally , theories that associate conscious perception with posterior visual loops ( Lamme , 2006 ) or with the integration of information into a coherent whole ( Oizumi et al . , 2014 ) are agnostic with respect to this issue: although greater reverberation or integration may cause an amplification of neuronal activity , ( Fahrenfort et al . , 2012 ) it could also , on the contrary , lead to diminished or sparser activity due to the greater predictability of the distributed brain signals being integrated ( Friston , 2005 ) .", "In summary , for both empirical and theoretical reasons , it is essential to study the temporal propagation of conscious vs unconscious information in the brain and to probe the presence of additional stimulus-specific cortical codes on seen trials relative to unseen trials , while excluding any difference in perception and behavior .", "To fulfill this demanding agenda , we used time-resolved MEG and EEG recordings to track the internal representation of a flashed stimulus under visible and invisible conditions .", "This strategy was made possible by advances in multivariate decoding , which can detect stimulus-specific information in brain activity , and evaluate its localization ( Kamitani and Tong , 2005; Haynes and Rees , 2006; Haynes , 2009 ) and its time course ( King and Dehaene , 2014 ) .", "We chose visual location as the decoded parameter of the stimulus , because multiple macroscopic retinotopic maps are present throughout the human brain , including the frontal lobe ( Sereno et al . , 1995; Hagler and Sereno , 2006 ) , and therefore , the full cortical processing stream of this parameter should be more easily decodable than other more microscopic parameters , such as object identity .", "To further address the concern of differences in processing depth , we placed ourselves under ‘blindsight’ conditions by asking subjects to perform a forced-choice location task in which performance was excellent even on subjectively invisible trials .", "Selecting only the correct trials allowed us to compare visible and invisible trials with identical stimuli and responses ( Lamy et al . , 2009 ) ." ], [ "To track the cortical representation of the target location through time , we trained multivariate pattern classifiers separately at every time sample to predict the target location from the recorded brain signals ( see ‘Materials and methods’ ) .", "For each time sample , we extracted classifiers' calibrated posterior probabilities ( Platt , 1999 ) associated with the eight spatial locations .", "We first trained the classifiers on all trials , regardless of participants' responses .", "Figure 2A depicts the classifiers' assigned probability for the correct target location as a function of time .", "Four stages could be distinguished ( this division will be used throughout our analyses ) .", "In a first stage ( 0–115 ms post stimulus ) , classification probability was at chance ( 0 . 1251 ± 0 . 0001; mean ± sem ) , t < 1 .", "From 115 to 162 ms , decoding probability rose above chance and peaked sharply at 147 ms post stimuli ( 0 . 141 ± 0 . 0028 ) , t ( 11 ) = 5 . 9 , p < 0 . 0001 .", "Then came a transition period ( 162–271 ms ) with reduced but still significant decoding ( 0 . 137 ± 0 . 0023 ) , t ( 11 ) = 5 . 48 , p < 0 . 0001 .", "Finally , in a fourth stage ( 271–800 ms ) , decoding probability rose again and remained above chance for about 500 ms ( 0 . 157 ± 0 . 0083 ) , t ( 11 ) = 3 . 8 , p < 0 . 0001 . 10 . 7554/eLife . 05652 . 004Figure 2 . Time course of location information .", "( A ) .", "Average posterior probability of a correct classification of target location , as a function of time .", "Chance = 12 . 5% ( 1/8 ) .", "Decoding confusion matrices are shown at the two decoding peaks .", "( B ) Same data sorted as a function of subjective visibility ( seen/unseen ) and objective localization performance ( correct/incorrect ) .", "The lower part shows the time course of average classifier probability as a function of distance between the decoded and actual target location . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 00410 . 7554/eLife . 05652 . 005Figure 2—figure supplement 1 . Event-Related Fields and potentials for ‘Seen–Correct’ vs ‘Unseen–Correct’ .", "ERF and ERP components time windows were determined according to the global field power ( upper panel ) .", "Two time windows were chosen; on each time window , cluster analysis was performed .", "In the 296–332 ms time window , Magnetometers showed a significant cluster ( p = 0 . 025 ) .", "EEG electrodes exhibited a significant cluster on the 300–600 ms time window p = 0 . 02 . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 00510 . 7554/eLife . 05652 . 006Figure 2—figure supplement 2 . Event-Related Fields and potentials for ‘UnSeen–Correct’ vs ‘Unseen–InCorrect’ .", "ERF and ERP components time windows were determined according to the global field power ( upper panel ) .", "Two time windows were chosen; on each time window , cluster analysis was performed .", "No significant differences were found . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 00610 . 7554/eLife . 05652 . 007Figure 2—figure supplement 3 . Classifying visibility . The black line portrays the mean classification probability of classifier trained to classify ‘Seen–Correct’ and ‘Unseen–Correct’ .", "The lighter line portrays classifier trained on the dataset with shuffled classes .", "In the 270–800 ms time window , mean classification probability ( 0 . 503 ± 0 . 001 ) was slightly higher than chance ( 0 . 5 ) t ( 11 ) = 2 . 45 , p = 0 . 03 .", "Shuffled trials classification probability ( 0 . 498 ± 0 . 001 ) did not differ from chance t ( 11 ) = 2 . 11 , p = 0 . 057 . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 00710 . 7554/eLife . 05652 . 008Figure 2—figure supplement 4 . Controls for eye movements and motor-based decoding .", "( A ) Average posterior probability of a correct classification of target location , across time , separately for ‘Seen–Correct’ and ‘Unseen–Correct’ trials , for classifiers trained on EOG channels only .", "Eye-based decoding is much lower than with the full set of sensors and , crucially , does not differentiate seen and unseen trials .", "Classification rose above chance 256 ms post stimuli and went back to chance 1000 ms post stimuli .", "Importantly , within this time frame , classification for ‘Seen–Correct’ ( 0 . 135 ± 0 . 001 ) and ‘Unseen–Correct’ ( 0 . 134 ± 0 . 001 ) did not differ t ( 11 ) < 1 .", "( B ) Average posterior probability of a correct classification of the manual response on target-absent trials , across time .", "Classifiers were trained on target's location in valid trials and tested on responses in absent trials .", "A flat curve indicates that target location decoding reported in the results section did not rely on motor preparation and execution information . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 008 We next examined how classifier performance varied with the subject's report and performance ( See Figure 2B ) .", "Our behavioral procedure allowed us to differentiate between two levels of processing in the ‘Unseen’ category: ‘Unseen–Correct’ trials where the response contained genuine location information , and ‘Unseen-Incorrect’ in which it did not .", "Remarkably , even in the latter trials , where behavioral evidence did not indicate any processing of stimuli , posterior probabilities for decoding spatial information were higher than chance even in the latest time window ( 0 . 143 ± 0 . 005 ) , t ( 11 ) = 3 . 37 , p < 0 . 01 .", "This result is striking for two reasons; first , it allows us to broaden the concept of blindsight as observed in V1 lesioned patients ( Weiskrantz , 1996 ) and monkeys ( Cowey and Stoerig , 1995 ) or induced in normal subjects ( Lamy et al . , 2009 ) .", "In blindsight , above-chance forced-choice behavior indicates that the observer perceived the stimuli , even though he denies any subjective perception .", "Here we can see that , even when subjects fail to perform the forced-choice task , their brain still perceived and retained the spatial information .", "Although unconscious perception is considered as transient and short-lived ( Rossetti , 1998 ) , here we see that even with the lowest end of perception , spatial information is encoded for up to 800 ms . Nonetheless , the dynamics of classification probability associated with ‘Unseen–Correct’ and ‘Unseen-Incorrect’ were not identical; they shared the same classification probability for the first three stages but diverged in the fourth stage ( 271–800 ms ) where the mean classification probability was higher for ‘Unseen–Correct’ ( 0 . 155 ± 0 . 009 ) than for ‘Unseen–Incorrect’ ( 0 . 143 ± 0 . 005 ) .", "These results indicate , unsurprisingly perhaps , that additional information about location is available in the brain on trials in which subjects responded correctly than in trials in which they did not .", "The main aim of this paper was to track the temporal dynamics of neuronal encoding of consciously and unconsciously perceived spatial information , that is , the differences between ‘Seen–Correct’ and ‘Unseen–Correct’ , which were strictly identical in terms of both stimuli and responses .", "‘Seen–Correct’ and ‘Unseen–Correct’ trials did not differ in classification probability during the first three stages but diverged only during the fourth stage ( see Figure 2B ) .", "Mean classification probability between 272–800 ms was higher for ‘Seen–Correct’ trials ( 0 . 162 ± 0 . 009 ) than for ‘Unseen–Correct’ ( 0 . 155 ± 0 . 009 ) , t ( 11 ) = 2 . 93 , p = 0 . 014 .", "This difference survived correction for correct responses produced by chance ( Lamy et al . , 2009 ) in the ‘Unseen–Correct’ condition .", "Classification was higher for ‘Seen–Correct’ trials ( 0 . 162 ± 0 . 009 ) than for ‘Unseen–CorrectChanceCorrected’ ( 0 . 157 ± 0 . 009 ) , t ( 11 ) = 2 . 93 , p = 0 . 029 ( one-tailed ) .", "Our fourth time window , where significantly improved location decoding was observed on seen compared to unseen trials , corresponds to the timing of N2 and especially P3 event-related potentials , which several past studies using the traditional contrast of ‘Seen’ vs ‘Unseen’ unseen trials have associated to conscious perception ( see Dehaene and Changeux , 2011 for review ) .", "To examine whether our study replicated those findings , we compared the event related potentials ( ERP ) - event related fields ( ERF ) signatures of our critical conditions ( see Figure 2—figure supplement 1 and Figure 2—figure supplement 2 ) .", "We tracked the components by computing Global Field Power and applied cluster analysis across channels .", "While the ‘Unseen–Correct’ vs ‘Unseen-Incorrect’ did not yield any significant difference in evoked responses , the ‘Seen–Correct’ vs ‘Unseen–Correct’ did: MEG magnetometers showed different activities in the N2 time window , while EEG electrodes showed a difference in the P3 time window , with the appropriate topography ( Figure 2—figure supplement 1 ) .", "Thus , these late events again appear as correlates of conscious perception .", "Unsurprisingly , ERP-ERF analysis revealed a slightly different temporal pattern than the dynamics revealed with the multivariate classification , because the former corresponds to a search for generic brain states allowing or not allowing content to access consciousness , while the latter corresponds to a search for brain states encoding a specific consciously perceived content .", "Training a classifier to use those event-related responses in order to separate ‘Seen–Correct’ vs ‘Unseen–Correct’ trials yielded a modest but significant classification success , again based on the late part of the epoch ( see Figure 2—figure supplement 3 ) .", "Our results suggest that , in the present experiment , M/EEG recordings are dominated by content-specific information about location , both on seen and on unseen trials , while it is more difficult to train a generic decoder for seen/unseen trials that cuts across all such contents .", "It also should be noted that this experiment was not optimally designed for such an analysis .", "In order to restrict classifier to using attributes that are related exclusively to visibility and not to stimuli location , we equated the number of trials representing specific stimulus location in the ‘Seen–Correct’ and the ‘Unseen–Correct’ location .", "This reduced the number of trials on which decoder was trained on and affected classification results .", "Because there was a fixed mapping between stimulus location and motor responses ( made using four fingers of each hand ) , the decoding of location could conceptually be based on conscious motor codes .", "To evaluate this possibility , we performed a control analysis in which we applied the decoder to target absent trials in which no stimulus appeared , but subjects still had to produce a response .", "Classification of subjects' responses in these trials was at chance , indicating that the previous decoder focused strictly on stimulus-based location information ( see Figure 2—figure supplement 4 ) .", "Although it remains possible that conscious response planning modulates the late stages of location processing , several aspects of our experiments argue against a contribution of motor codes to our results .", "In terms of experimental design , our main analyses focused on trials in which the decoder was trained to extract the objective location of the target on all trials , errors included .", "Thus , the above-chance extraction of the objective target location on ‘Unseen-Incorrect’ trials is not likely to reflect motor code , nor can the improved classification performance for ‘Seen–Correct’ over ‘Unseen–Correct’ , since those trials involve exactly the same stimuli and responses .", "Another control analysis evaluated the role of microsaccades .", "Although eye movements were carefully removed with principal component analysis ( PCA ) , it could be suggested that residual ocular activity in the MEG/EEG ( MEEG ) signal governed the classification of spatial location .", "Residual eye movement artifacts , if present , should primarily affect the anterior eye channels .", "To test this possibility , we examined the dynamics of classification on electro-oculograms ( EOG ) channels alone .", "Classification revealed a different temporal pattern of classification that was observed with the full MEEG classification: above-chance location decoding started only around 250 ms , remained much lower that with the whole data set ( ∼14% , where chance = 12 . 5% ) , and crucially , did not discriminate between the seen and unseen trials ( see Figure 2—figure supplement 4 ) , suggesting that eye movements did not play a dominant role in the above decoding results .", "Source localization ( see below ) is also incompatible with a single eye movement artifact .", "Our basic analysis indicated that consciously perceived stimuli are coded more reliably than unconscious ones , but the nature of this difference could not be completely appreciated from this analysis .", "‘Seen–Correct’ trials were more numerous than ‘Seen-incorrect’ trials , and this factor alone could perhaps explain why they were more efficiently decoded .", "In this case , if conscious and unconscious codes differ , then training specifically on unconscious trials should revert the pattern and yield better decoding on unseen compared to seen trials .", "The GNW model ( Dehaene et al . , 2003 ) , however , makes a different prediction concerning the asymmetry of decoding on seen and unseen trials .", "Specifically , the model predicts that all the processing stages present on unseen trials should continue to be observed on seen trials , while the converse should not be true: on seen trials , there should be brain activity encoding the perceived stimulus within the GNW and not present on unseen trials .", "We examined these two options by testing for cross-condition generalization: we trained the decoder on a subset of trials ( either the unseen–correct trials , or the seen–correct trials ) and then tested for generalization , either to left-out trials within the same category or to trials belonging to the other category .", "One technical difficulty was that , since the experimental conditions were defined by subjects' responses , the number of trials at each location was no longer balanced within each of these conditions , and this imbalance affected the pre-stimulus bias of the 8-category classifier as well as its capacity to generalize .", "To address this problem , we turned to a simpler binary classifier , which was trained to simply sort the stimuli into left-hemifield vs right-hemifield stimuli .", "Within each subject , we matched the number of trials in these two classes and also selected equal numbers of seen–correct and unseen–correct trials .", "Figure 3 shows that this binary decoder , now operating with a chance level of 50% , still performed above chance with approximately the same time course as the 8-location decoder . 10 . 7554/eLife . 05652 . 009Figure 3 . Asymmetrical cross-condition generalization . A classifier trained in one condition and then tested on new data either from the same condition or the other condition ( e . g . trained on ‘Seen–Correct’ trials and tested on new ‘Seen–Correct’ trials and ‘Unseen–Correct’ trials ) .", "To equalize the number of trials , the classifier was trained to discriminate left- vs right-hemifield targets , hence chance = 50% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 009 The end result demonstrated the predicted asymmetry .", "When trained on ‘Unseen–Correct’ trials , classification generalized identically to ‘Seen–Correct’ trials , but when trained on ‘Seen–Correct’ trials , classification performance dropped when tested on ‘Unseen–Correct’ ( Figure 3 ) .", "This effect was apparent in two time windows , the third time window ( 178–225 ms; average classification probability for ‘Seen–Correct’ = 0 . 526 ± 0 . 0064; for ‘Unseen–Correct’ = 0 . 515 ± 0 . 005; t ( 11 ) = 2 . 8 , p = 0 . 017 ) , and the fourth late time window ( 272–800 ms; ‘Seen–Correct’ = 0 . 575 ± 0 . 017; ‘Unseen–Correct’ = 0 . 547 ± 0 . 016; t ( 11 ) = 2 . 44 , p = 0 . 032 ) .", "These analyses indicate that seen–correct trials contained the same decodable stimulus information as unseen–correct trials , plus additional information unique to conscious trials .", "The GNW model predicts that the additional information associated with conscious representation should be jointly encoded in a network of distributed prefrontal and parietal regions .", "In order to assess the contribution of these brain areas to the encoding of consciously perceived contents , we modeled the brain activity in 68 regions of interest covering the whole cortex ( See ‘Materials and methods’ ) and re-trained our decoders using just the distributed source signals from these regions .", "The results with eight output classes ( Figure 4—figure supplement 1 ) confirmed the existence of two main stages: an early one ( ∼100–200 ms ) where location information was essentially confined to occipital , ventral visual , and lateral and mesial parietal regions , and a later one ( >250 ms ) where it became highly distributed to multiple cortical areas , particularly in prefrontal and anterior cingulate cortex ( Figure 4—figure supplement 1 ) .", "To avoid an unnecessary increase in the number of statistical tests , we confined the subsequent analyses of seen and unseen trials , to four a priori regions of interest covering all areas of the dorsal visual pathway containing retinotopic maps for location ( Sereno et al . , 1995; Hagler and Sereno , 2006 ) : pericalcarine , superior parietal cortex , rostral medial frontal cortex , and superior-lateral frontal cortex .", "We then repeated the generalization across conditions and procedures .", "This region-based decoding revealed the activation of successive spatial codes ( Figure 4 ) : both pericalcarine and superior parietal cortices contained decodable information as early as 115 ms post stimuli ( [0 . 519 ± 0 . 005] , t ( 11 ) = 3 . 82 p = 0 . 0028 and [0 . 516 ± 0 . 003] , t ( 11 ) = 4 . 52 p < 0 . 0001 , respectively ) .", "The two frontal areas revealed a more delayed pattern as the curve associated with superior frontal cortex showed first above-chance decoding only after 194 ms ( 0 . 504 ± 0 . 001 ) , t ( 11 ) = 2 . 33 p = 0 . 0396 and rostral medial frontal as late as 365 ms . 10 . 7554/eLife . 05652 . 010Figure 4 . Source-based decoding and cross-condition generalization . Classifiers were trained as in Figure 3 , but using a restricted subset of cortical sources: pericalcarine ( A ) , superior parietal ( B ) , rostro-medial frontal ( C ) , or superior frontal ( D ) .", "Note , how asymmetrical cross-condition generalization ( right columns , same format as Figure 3 ) successively arises in visual cortex , then superior parietal , and superior frontal regions . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 01010 . 7554/eLife . 05652 . 011Figure 4—figure supplement 1 . Time course of location information for the different cortical sources . Average posterior probability of a correct classification of target location , as a function of time for in 68 regions of interest .", "Chance = 12 . 5% ( 1/8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 011 Only two of the chosen regions , superior frontal and superior parietal cortices , demonstrated the asymmetry between seen and unseen trials obtained with sensor-based decoding .", "In both cases , when trained on activity restricted to the superior frontal regions , a complete generalization obtained when trained on ‘Unseen–Correct’ but when trained on ‘Seen–Correct’ trials , decoding probability dropped for ‘Unseen–Correct’ in the fourth time window .", "Classification probability was significantly higher for ‘Seen–Correct’ ( 0 . 552 ± 0 . 017 ) than for the ‘Unseen–Correct’ trials ( 0 . 541 ± 0 . 017 ) , t ( 11 ) = 2 . 35 , p = 0 . 038 .", "In the superior parietal cortex , when trained on ‘Unseen–Correct’ trials , asymmetry was apparent in all time windows: classifiers generalize completely for ‘Seen–Correct’ trials ( Figure 4 ) but when trained on ‘Seen–Correct’ trials and tested on ‘Unseen–Correct’ , posterior probabilities dropped .", "In the second window classification , probability was significantly higher for ‘Seen–Correct’ ( 0 . 534 ± 0 . 007 ) than for the ‘Unseen–Correct’ trials ( 0 . 525 ± 0 . 006 ) , t ( 11 ) = 2 . 5 , p = 0 . 026 .", "In the third time window , ‘Seen–Correct’ decoding probability was higher than ‘Unseen–Correct’ ( [0 . 537 ± 0 . 008] [0 . 523 ± 0 . 01] , t ( 11 ) = 2 . 3 , p = 0 . 036 ) .", "This pattern was kept in the fourth time window , as ‘Seen–Correct’ decoding probability ( 0 . 559 ± 0 . 011 ) was higher than ‘Unseen–Correct’ decoding probability ( 0 . 545 ± 0 . 01 ) , t ( 11 ) = 3 . 9 , p = 0 . 002 .", "These findings go beyond the distinction of conscious and unconscious processing and allow us to demonstrate a representation of stimulus location outside visual areas .", "Above-chance classification could be achieved in all regions , including superior parietal , rostral middle frontal , and superior frontal regions , supporting the prior finding that these regions contain retinotopic maps ( Sereno et al . , 1995; Hagler and Sereno , 2006 ) .", "Importantly , however , our results show that there is no specific region uniquely dedicated to the encoding of consciously perceived information .", "Instead , a better encoding on seen trials is obtained in the same overall sectors of the superior parietal cortex and superior frontal cortex that also contain location information on unseen–correct trials ( i . e . , blindsight trials ) .", "Again , the key difference is asymmetrical generalization , indicating the presence of superior encoding of spatial information on seen trials , only in dorsal parietal and frontal regions .", "This pattern primarily occurs in the late time window ( beyond 270 ms ) , but it is already present at early stages of processing in the superior parietal cortex .", "According to the GNW model , conscious access corresponds to an amplification and broadcasting of the selected information .", "Conscious processing is , therefore , predicted to consist in a series of information-processing stages , each associated with the settling of brain activity into a temporary metastable state ( stable for a duration of ∼100–300 ms ) , which allows the information to be flexibly transmitted to the appropriate specialized processors ( Dehaene et al . , 2014 ) .", "The series of processing stages may differ as a function of task demands and may include verbal report ( Frässle et al . , 2014 ) , executive control ( Van Gaal et al . , 2010 ) , or meta-cognitive evaluation ( Charles et al . , 2014 ) .", "On unconscious trials , the accumulation of incoming activation would fail to attain the critical threshold level needed to trigger such a discrete series of stages ( ‘failed ignition’ ) , and the unconscious information would , therefore , decay over a period of a few hundreds of milliseconds ( Dehaene and Naccache , 2001 ) .", "To evaluate those predictions concerning the dynamics and transient stability of internal codes , we used the temporal generalization method ( King and Dehaene , 2014 ) .", "This method consists in probing if a classifier trained at a certain time point t can generalize to other time points , t′ .", "If the representation is stable , the classifier should remain efficient even if applied at a different latency .", "If , however , the information is successively re-encoded in a series of different brain systems , then we should see a failure of generalization beyond a certain temporal duration ( i . e . , away from the diagonal where t = t′ ) ( King and Dehaene , 2014 ) .", "We , therefore , quantified the endurance of conscious and unconscious representations by training and testing classifiers on all pairs of time samples ( t , t′ ) .", "To measure a classifier's durability independently of classification efficacy , we defined Classification Endurance ( CE ) , as the number of samples forward and backward in time , for which decoding performance remained above 50% of its level at the original training time ( t = t′ ) —that is , the decoder's half-life time .", "Matrices of temporal generalization ( Figure 5 ) indicated that , up to 272 ms , decoding was narrowly restricted to the diagonal for both seen and unseen trials , suggesting a fast-changing chain of perceptual processes ( King and Dehaene , 2014 ) consistent with a feedforward propagation of unconscious location information .", "Beyond this point and up to ∼800 ms post stimulus , classifiers generalized to a wider neighborhood of latencies , consistent with the entry of information into evidence accumulation systems with longer time constants .", "The generalization pattern was examined using an analysis of variance conducted on CE in the 272–800-ms time window with factors of Visibility ( Seen/Unseen ) , Direction ( Forward/Backward in time ) , and Timeframe ( 5 successive time windows ) ( see Figure 5—figure supplement 1 for statistics ) .", "All main effects and their interactions were significant .", "On average , CE was larger for generalization forward than for generalization backward and also increased in time .", "We were mainly interested in the different generalization patterns of the ‘Seen–Correct’ and ‘Unseen–Correct’ trials .", "Surprisingly , generalization was more extended in time for unseen trials .", "Forward generalization showed a larger CE in unseen trials compared to seen trials ( mean = 382 ms vs 201 ms , p < 0 . 005 ) , and the same pattern was apparent for backward generalization to a lesser extent ( mean = 159 ms vs 78 ms , p < 0 . 05 ) ( see Figure 5—figure supplement 2 for statistics ) .", "Furthermore , in the forward direction , these differences interacted with time .", "Forward CE was constant in seen trials , while for the unseen trials , it was linearly decreasing with time .", "Backward CE did not differ for seen and unseen trials , but increased steadily as a function of time ( see Figure 5—figure supplement 3 for statistics ) . 10 . 7554/eLife . 05652 . 012Figure 5 . Generalization of location decoding over time . 8-location classifiers trained at a specific time were then tested on data from all other time points .", "( A ) Average classification probability as a function of testing time for each training time ( the diagonal , where testing time = training time , gives the curve for classical decoder performance over time ) .", "( B ) Same information plotted as a function of temporal distance from training time ( positive or negative ) , with asterisks indicating the Classification Endurance ( CE ) measure .", "( C ) Tentative model of a sequence of brain activations , which could yield the observed generalization matrices . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 01210 . 7554/eLife . 05652 . 013Figure 5—figure supplement 1 . Analysis of variance ( ANOVA ) on Classification Endurance ( CE ) .", "The table gives the statistics and significance values for the main effects of Visibility ( Seen–Correct vs Unseen–Correct ) , Direction of generalization ( forward , backward ) , and Timeframe ( 5 levels ) , as well as their interactions . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 01310 . 7554/eLife . 05652 . 014Figure 5—figure supplement 2 . ANOVAs on CE with factors of Visibility ( Seen–Correct vs Unseen–Correct ) and Timeframe ( 5 levels ) , separately for forward and backward generalization .", "( A ) Forward generalization ( B ) Backward generalization . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 01410 . 7554/eLife . 05652 . 015Figure 5—figure supplement 3 . Significance Values of the Effect of Timeframe ( 5 levels ) for forward and backward generalization in Seen and unseen trials . DOI: http://dx . doi . org/10 . 7554/eLife . 05652 . 015 Figure 5C shows a possible theoretical interpretation consistent with those results .", "The high level of decoding observed on seen trials , over a long time window of ∼270–800 ms , combined with a low level of off-diagonal temporal generalization beyond a fixed horizon of ∼160 ms , implies that a conscious location is successively encoded by a series of about four distinct stages , each terminating relatively sharply after a processing duration of ∼160 ms . Conversely , the lower decoding level and longer endurance observed for unconsciously perceived stimuli indicate that they do not enter into the same processing chain .", "The square pattern of the temporal generalization matrix on unseen trials ( Figure 5A ) suggests that unconscious information lingers in a single representational state , which decays slowly over time , thus yielding a paradoxically longer endurance than on seen trials in the forward direction .", "All of these findings are consistent with the above predictions as well as with earlier decoding results indicating a chain of additional processing stages unique to conscious trials ( Charles et al . , 2014 ) .", "They suggest that conscious access can be described as the entry of information into a dynamic routing system ( Sackur and Dehaene , 2009; Dehaene et al . , 2014 ) in which information is stabilized , transferred to a processor , exploited in a determined time , and then transferred again to the next stage .", "As described in accumulation-of-evidence models of decision making ( Shadlen and Kiani , 2011 ) , a failure to attain a threshold-level activation at one of these stages cuts the process short , prevents the attainment of a conscious-level representation , and causes the information to linger and decay ." ], [ "Previous experiments exploring the signatures of conscious access have primarily contrasted the overall brain activity evoked by perceived and unperceived stimuli .", "However , this comparison may be contaminated by non-specific attention , alerting , performance , and reporting confounds .", "The quest for brain mechanisms of consciousness implies an isolation of neural populations that encodes the details of subjective experience ( Haynes , 2009 ) .", "As an effort in this direction , we applied multivariate decoding techniques in order to track the neural encoding of a conscious or unconscious representation of stimulus location through time .", "To control over stimulus and performance confounds , we capitalized on the blindsight phenomenon and selectively analyzed a large fraction of trials with identical stimuli and accurate responses , which differed only in subjective reports of seeing or not seeing the stimulus .", "The results revealed that ( 1 ) the location of a briefly flashed stimulus can be accurately decoded from MEG and EEG signals for up to 800 ms , whether or not the stimulus is seen .", "This finding is coherent with the existence of multiple spatial maps in occipital , parietal , and frontal cortex ( Sereno et al . , 1995; Hagler and Sereno , 2006 ) .", "( 2 ) Seen and unseen stimuli are initially encoded identically , but after ∼270 ms , the information is selectively amplified on ‘seen’ trials .", "This observation replicates and extends earlier observations with seen and unseen words , digits and pictures during masking ( Del Cul et al . , 2007; Fisch et al . , 2009 ) , and the attentional blink ( Sergent et al . , 2005 ) .", "( 3 ) Asymmetrical generalization indicates that shared spatial codes are active on unconscious and conscious trials , but that conscious trials also contain additional neural codes for stimulus location that are absent or weaker on unconscious trials .", "Source localization traces those conscious codes to superior parietal and superior frontal cortex .", "While the present method does not allow us to decide whether the difference is quantitative ( the same codes are activated more strongly ) or qualitative ( additional neural codes are recruited ) , they are convergent with the prior observation that , of the many maps for space in the cortex , superior parietal and frontal maps are selectively amplified by attention , while earlier retinotopic maps are activated in an automatic manner ( Saygin and Sereno , 2008 ) .", "( 4 ) The dynamics of cortical coding , as revealed by temporal generalization matrices ( King and Dehaene , 2014 ) , also differs on seen and unseen trials: conscious information undergoes a series of representational transformations , such that each decoder fails to generalize beyond ∼160 ms , while unconscious information , although weaker , generalizes over a longer period .", "The latter finding fits with earlier observations that only conscious stimuli can be passed through a series of discrete information-processing stages ( Sackur and Dehaene , 2009; De Lange et al . , 2011; Charles et al . , 2014 ) .", "The observed characteristic peak-to-end duration of about 160 ms is also compatible with the finding that conscious percepts tend to be locked to an ongoing theta ( ∼4–7 Hz ) or high delta ( ∼1–3 Hz ) rhythm ( Melloni et al . , 2007; Doesburg et al . , 2009; Nakatani et al . , 2014; Sitt et al . , 2014 ) , which is also the frequency range of slow positive event-related potentials , such as the P3 .", "Doesburg et al . found that perceptual switching in a binocular rivalry paradigm corresponds to the emergence of synchronized gamma rhythm embedded in theta envelope ( Doesburg et al . , 2009 ) .", "In an attentional blink paradigm , the synchrony of fast oscillations ( beta and gamma ) with slow activity ( theta and high delta ) increased with practice , in parallel to improved stimulus visibility ( Nakatani et al . , 2014 ) .", "Theta activity was also shown to be higher on visible trials compared to invisible trials in a masking paradigm ( Melloni et al . , 2007 ) and was successfully used to classify conscious vs vegetative patients ( Sitt et al . , 2014 ) .", "It thus seems plausible that conscious information is able to enter into a series of computational stages that are betrayed by a series of theta- or delta-like peaks ( Dehaene and Sigman , 2012 ) .", "Conversely , unconscious information fails to be sequentially dispatched to the successive steps of a serial task and , therefore , remains blocked within a fixed representational state , where it slowly decays .", "Behavioral priming studies confirm that unconscious information lingers at detectable levels for hundreds of milliseconds ( Greenwald et al . , 1996 ) or even seconds ( Soto et al . , 2011 ) .", "Our results are incompatible with theories that postulate identical codes for conscious and unconscious information , which would only be distinguished by second-order metacognitive tags ( Lau and Passingham , 2006 ) .", "They fit with theories that postulate that a conscious episode is distinguished by an amplification of incoming sensory information , possibly through reverberating loops ( Lamme , 2006 ) , and its distributed representation in multiple distinct regions including parietal and prefrontal cortices ( Dehaene and Naccache , 2001; Dehaene et al . , 2006 ) .", "These results are highly convergent with intracranial local-field potentials and single-neuron recordings during binocular rivalry and continuous flash suppression ( Sheinberg and Logothetis , 1997; Kreiman et al . , 2002; Panagiotaropoulos et al . , 2012 ) , which indicate that late neural discharges in higher cortical areas reflect which of two rivaling images are subjectively perceived .", "In the case of object identity , decodable neural activity is found in inferior and anterior temporal cortex ( Sheinberg and Logothetis , 1997; Kreiman et al . , 2002 ) but also in prefrontal cortex ( Panagiotaropoulos et al . , 2012 ) as reported here .", "Thus , this region appears at the confluence of conscious perception of object identity and location , making it a primary candidate for the integrated perception of a unified conscious scene or event ( Oizumi et al . , 2014 ) .", "While numerous previous studies have compared brain activity evoked by ‘seen’ and ‘unseen’ stimuli , the advantage of the present study is to specifically pursue the neural dynamics underlying a specific conscious content ( stimulus location ) .", "Figuratively speaking , one may say that previous studies have extracted the ‘canvas’ on which conscious perception is painted , while our approach is aimed at extracting the ‘painting’ itself .", "Nevertheless , this approach suffers from several limitations .", "One of them is the low level of decodability achieved: single-trial EEG and MEG responses have a low signal-to-noise ratio , which limits our inferences and would not afford , for instance , a reliable brain–computer interface .", "Another limit of our design is that it reduces the rich experience associated with conscious perception to a single feature of a very lean stimulus , presented at threshold , which is the sole focus of attention and which is reported on every trial .", "Whether the present results would generalize to broader real-life situations , especially in the absence of an overt report is an important question for further research .", "Some recent experiments suggest that removing the need to report radically reduces the neurophysiological correlates of conscious perception , including the P3b component of event-related potentials , leaving only a posterior mid-latency component ( Pitts et al . , 2012 , 2014a , 2014b; Frässle et al . , 2014 ) .", "However , many of those experiments involved a dual task , which is known to delay and dilute late ERPs ( Sigman and Dehaene , 2008 ) and to distort conscious access ( Marti et al . , 2010 ) .", "It might be preferable to adopt a ‘passive attentive’ paradigm involving mere attention without report .", "In such an auditory paradigm , late ERPs including the P3b component were again found to contain decodable information about the conscious percept ( Wacongne et al . , 2011; King et al . , 2013 ) .", "A correlation of late neurophysiological activity with perceptibility was also obtained in a variety of neurophysiological studies of human visual perception ( Quiroga et al . , 2008; Kouider et al . , 2013 ) or rodent tactile perception ( Manita et al . , 2015 ) .", "With due caution , we , therefore , hypothesize that the neurophysiological correlates of conscious perception uncovered here might be generalized to other experimental conditions ." ], [ "17 right-handed healthy adults ( 10 men , 19–30 years of age ) with no history of neurological or psychiatric disorders participated the study .", "This study was ethically approved by CPP IDF 7 under the reference CPP 08 021 .", "All participants reported normal or corrected-to-normal visual acuity .", "The data of 5 subjects were excluded due to failure in calibration , and these observers reported more than 85% of the target trials as seen ( even with maximum masking contrast ) .", "The intensity values to which subjects were calibrated ranged from 0 to 227 , mean 131 . 46 ( SE = 19 . 76 ) .", "The target stimulus was a line segment subtending a visual angle of 0 . 33° , tilted by 45° to the left .", "On target-present trials , the target was randomly presented at one of the eight possible locations .", "The potential locations were allocated on the outline of an imaginary circle centered at the fixation point with a radius of 2 . 85° of visual angle .", "Target locations were separated by radial angles of 45° with an offset of 22 . 5° from the meridians .", "The mask comprised crosses , each subtending 0 . 5° of visual angle , always present at all the eight possible locations .", "The fixation cross subtended 0 . 15° of visual angle .", "On target-absent trials , only the mask was displayed .", "All stimuli but the mask were black on white background; mask contrast was determined individually for each subject in a calibration phase .", "Stimuli were generated offline using Matlab ( MathWorks , Natick , MA ) , and their presentation was controlled using the Psychtoolbox package for Matlab ( Brainard , 1997 ) .", "Stimuli were presented with a Panasonic PT D7700E-K video projector ( refresh rate 60 Hz ) to a backprojection screen inside the magnetically shielded at a distance of 1 m from the eyes of the subject .", "Behavioral responses were collected with two 5-button cylindrical fiber optic response pads ( ‘fORP’; Current Designs Inc . , Philadelphia , PA ) .", "At the beginning of each trial , as an alerting signal , the fixation point was magnified twofold for 100 ms and then reverted to its normal size ( fixation was present throughout the trial ) .", "800 ms later , the target was presented for 2 screen refresh cycles ( ∼33 ms ) .", "The mask followed immediately and remained on the screen for 400 ms . A blank screen was presented for at least 1500 ms or until the first response ( retention period ) .", "Then , two consecutive images prompting the second and third responses were presented until response .", "A new trial began 500 ms after the third response ( see Figure 1 ) .", "As noted , three responses were collected on each trial .", "First , participants were required to produce a speeded forced-choice response to the location of the target .", "The second response was a second-order response , reporting whether the participants thought that their first response was correct or incorrect .", "With the third response , participants had to indicate whether they had seen the target or merely guessed its location .", "Because of the procedure's high task demand , the experiment was designed in a way that enabled participants to adapt to it .", "We introduced responses in a gradual way , using a slide that explained the nature of the specific response and a movie that illustrated the corresponding keys .", "The experiment started with a description of the localization task , which was immediately practiced during an eight-trial practice block .", "In this block , the mask contrast was set to zero , so targets were completely visible , and participants had to correctly respond with the designated response-pad buttons .", "A correct response turned the target's color to green and an incorrect response turned it to red .", "Next , participants were introduced to the second-order response and performed another training block with three objectives: first , to continue the gradual adaptation to the task; second , to ensure a fast , automatic response; third , to make sure that subjects complied with the task and caught their occasional errors .", "In the training phase , the target was completely visible once again , and subjects had to report its location and whether they made an error with their first response .", "Average localization performance ( RTs and accuracy ) and average second-order report accuracy were calculated every 5 trials .", "If the participant localized the target correctly in all five trials , or his/hers average RT was above 800 ms , a slide appeared before the next training block encouraging him/her to respond faster .", "On the other hand , if the participant localized the target correctly in less than four trials , the slide prompted him/her to respond slower .", "If participant's second-order report was correct in less than four of the trials , then the slide urged the participant to be more accurate with his/her second-order report .", "Training ended after 16 blocks or if the average performance during the last 4 blocks had reached specific criteria of localization performance between 85% and 95% , average RT below 800 ms and the second-order report correct in more than 90% of trials .", "Following the training , participants were introduced to the subjective visibility response and started a calibration phase .", "The calibration phase was designed to determine the mask contrast that would yield an approximately equal number of trials in which the target stimulus would be seen or not seen ( a 50% detection threshold ) .", "A trial was considered as a ‘Seen’ trial if the participant reported that he/she had seen the target and had correctly localized it , or if he/she reported the target as seen , failed to correctly localize it , but detected the error .", "We used a modified version of the threshold estimation procedure described by Levitt ( Levitt , 1971 ) , changing mask contrast according to participant's ‘Seen’ trials proportion in a block .", "We manipulated contrast by adjusting the pixel intensity values in a unified fashion .", "Initial intensity was set to 230 ( range 0–255 ) .", "The number of trials in each block increased as calibration persisted , so the first two blocks comprised four trials , the third block six trials , the forth eight trials , and from the fifth block on , all blocks comprised ten trials .", "The change in Red Green Blue ( RGB ) values was determined by the proportion of seen trials in a block , and the number of calibration blocks that participant had already completed .", "The maximal possible change after a single block was 80 .", "This kind of change was applied when the proportion of seen trials was either 1 or 0 .", "However , this maximal value decreased as the number of blocks participant has completed increased .", "The stopping rule for the calibration was that RGB change was smaller than 1 . 5 , or subjects had completed 80 trials .", "After mask contrast was determined , participant started the experimental phase where the contrast was fixed .", "The experimental phase was similar to the calibration phase with the following changes: it included 540 trials divided into 6 blocks , 1/9 of these trials were catch trials .", "MEG and EEG data were low-pass filtered at 330 Hz and recorded simultaneously at 1000-Hz sampling rate with a 306-channel Elekta Neuromag MEG system ( Elekta Oy , Helsinki , Finland ) , which comprises 102 triple sensors ( each with two orthogonal planar gradiometers and one magnetometer ) in a helmet-shaped array .", "Four head position indicator coils were placed on the scalp of the subject , and their locations were digitized with respect to anatomical landmarks prior to the MEG recording .", "By briefly energizing the coils , the head position was measured at the beginning of each block .", "EEG signal was recorded from 60 electrodes that were referenced to the nose .", "The ground electrode was on the clavicle bones .", "Horizontal and vertical EOG and electrocardiogram ( ECG ) were also recorded for offline rejection of eye movements and cardiac artifacts .", "Acquired data were processed with the MaxFilter software package ( Elekta Oy ) that implements the Signal Space Separation ( SSS ) method ( Taulu et al . , 2004 ) to suppress ambient magnetic interference .", "Gradiometers and magnetometers with amplitudes continuously exceeding 3000 fT/cm and 3000 fT , respectively , were marked as bad channels and were interpolated by SSS .", "Eye blinks , eye movements , and cardiac activity were detected on the EOG and ECG channels , and to suppress these artifacts , data were averaged with respect to the onset of each artifact separately , and PCA was used to determine the dominant components of these artifacts in the MEG/EEG signals .", "One to three components were removed according to visual inspection .", "Using Fieldtrip software ( Oostenveld et al . , 2011 ) , continuous data were low-pass filtered at 30 Hz and cut to 2 . 5-s epochs starting 500 ms before the stimulus onset .", "Data were downsampled to 64 Hz .", "EEG data from one subject were omitted due to technical problems .", "Thus , data from 12 subjects were analyzed , with one subject missing EEG data .", "To combine data from MEG magnetometer and planar gradiometers as well as from EEG channels , a normalization procedure was first applied; the baseline standard deviation was estimated for each channel using all the trials in the experiment , and then all the samples were divided by this standard deviation to yield a z-score , which was entered in the classification algorithm .", "We used a multivariate classification procedure to characterize the temporal dynamics of information processing when the stimulus was perceived consciously vs unconsciously .", "A distinct classifier was trained for each subject and for each time sample , using the data from all sensors .", "In all analyses , we employed a linear support vector machine ( SVM ) algorithm ( with cost parameter C = 1 ) that was complemented with a continuous output method providing for each sample tested the probability of belonging to each of the possible classes ( i . e . , spatial locations ) ( Platt , 1999 ) .", "Classification was done with the package libsvm ( Chang and Lin , 2011 ) .", "The data were randomly divided into 15 non-overlapping folds; the classifier was trained on fourteenfolds and tested on the one that was left out; this procedure was repeated 15 times for each time sample , so all fifteenfolds were eventually used for testing .", "In the first analysis , the classifier was trained on all the data ( except of the test data ) and eight output classes ( locations ) .", "The general classification probability for the correct location was then split according to participants' subjective reports of visibility and their performance in the forced-choice task .", "The second analysis was aimed at examining cross-condition generalization , that is , whether the features that are used to decode spatial location in one condition of visibility are the same as those used in the other .", "Since the experimental conditions were defined by subjects' responses , the number of trials at each location was no longer balanced within each of these conditions , and this imbalance affected the pre-stimulus bias of the 8-category classifier as well as its capacity to generalize .", "To address this problem , we turned to a simpler binary classifier , which was trained to simply sort the stimuli into left-hemifield vs right-hemifield stimuli .", "Within each subject , we matched the number of trials in these two classes and also selected equal numbers of seen–correct and unseen–correct trials .", "Similarly to the previous analysis , for each perceptual condition , the data were divided into fifteenfolds; the classifier was trained on 14 and tested on the one left out and additionally on the converse perceptual condition .", "A third analysis aimed to find out in which cortical regions the processing of seen and unseen stimuli differed .", "We tested the roles of each region by running decoding analyses separately on sources confined to this region .", "We first parceled the cortex into 68 regions with FreeSurfer ( Desikan–Killiany atlas ) .", "This parcellation is based on cortical curvature patterns that are estimated from individual MRI surface reconstructions .", "While these are large regions , the precision of MEG source reconstruction , combined with the requirements of the decoding approach , does not afford a very small focus .", "Smaller regions generally did not contain enough reconstructed dipoles with distinctive information to support accurate decoding .", "Neuronal current sources underlying single-trial MEG and EEG signals were estimated using linear minimum-norm estimates with fixed cortical source orientations .", "A three-layer volume conductor model based on individual head geometry was used in the forward computations ( MNE-Suite [Gramfort et al . , 2014] Matlab toolbox ) .", "Noise covariance was estimated from the baseline periods of all accepted trials .", "We then ran an individual decoding analysis for each of the 68 regions .", "The estimated current distribution within a ROI at a given latency was fed to the same SVM analyses as for the sensor-level data .", "The results with eight output classes confirmed the existence of early focal and late distributed location-coding stages ( see Figure 4—figure supplement 1 ) .", "To avoid an unnecessary increase in the number of statistical tests , we confined the subsequent analyses of seen and unseen trials , to four a priori regions of interest: pericalcarine , superior parietal cortex , rostral medial frontal cortex , and superior-lateral frontal cortex .", "In the latter case , the decoder outputs were calibrated class probabilities for ‘Seen–Correct’ and ‘Unseen–Correct’ classes after 10-fold cross-validation .", "The fourth type of analysis aimed to test the stability over time .", "As in the first analysis , data from all trials were used to train classifiers at each time point ranging from 200 ms before to 1500 ms after the target and then test generalization to every other time point , as described in ( King and Dehaene , 2014; King et al . , 2014 ) .", "Two additional control analyses were conducted .", "First , we used cross-condition generalization to ensure that the classification performance was not derived from brain activity related to response selection but instead from neural activity related to perceptual processes .", "Therefore , we trained classifiers on the perceptual conditions and tested them on catch trials where the target was absent , but that were labeled according to the location of the participants' responses ( 8 possibilities ) .", "A second analysis was aimed to check whether superior posterior decoding probability for the ‘Seen–Correct’ correct trials rose from eye movements .", "To this aim , we fed the same classifier solely with EOG data , namely horizontal and vertical electro-oculograms ." ] ]
[ "The neural correlates of consciousness are typically sought by comparing the overall brain responses to perceived and unperceived stimuli .", "However , this comparison may be contaminated by non-specific attention , alerting , performance , and reporting confounds .", "Here , we pursue a novel approach , tracking the neuronal coding of consciously and unconsciously perceived contents while keeping behavior identical ( blindsight ) .", "EEG and MEG were recorded while participants reported the spatial location and visibility of a briefly presented target .", "Multivariate pattern analysis demonstrated that considerable information about spatial location traverses the cortex on blindsight trials , but that starting ≈270 ms post-onset , information unique to consciously perceived stimuli , emerges in superior parietal and superior frontal regions .", "Conscious access appears characterized by the entry of the perceived stimulus into a series of additional brain processes , each restricted in time , while the failure of conscious access results in the breaking of this chain and a subsequent slow decay of the lingering unconscious activity ." ]
[ "Our senses constantly receive information from the world around us , but we consciously perceive only a small portion of it .", "Nonetheless , even stimuli that are not consciously perceived are registered in our brain and influence our behavior .", "This is known as unconscious perception .", "Researchers disagree about how brain activity differs during conscious and unconscious perception .", "Some think that both consciously and unconsciously perceived objects are processed in the same way in the brain , but that the brain is more active during conscious perception .", "Others think that different neurons process the information in different types of perception .", "Salti et al . have now investigated this issue .", "While recording participants' brain activity , a line was briefly presented in one of eight different possible locations on a screen .", "The line was masked so it would be consciously perceived in roughly half of the presentations .", "Participants had to report the location of the line and then say whether they had seen it or had merely guessed its location .", "Even when they reported that they were guessing , participants identified the location of the line better than by chance , indicating unconscious perception on ‘guess’ trials .", "This enabled Salti et al . to compare how the brain encodes consciously perceived and unconsciously perceived stimuli .", "Unlike previous studies in which the brain activity associated with ‘seen’ and ‘unseen’ stimuli was compared , Salti et al . used a different approach to extract the neural activity underlying consciousness .", "A classifying algorithm was trained on a subset of the data to recognize from the recorded brain activity where on the screen a line had appeared .", "Applying this algorithm to the remaining data revealed the dynamics of stimulus encoding .", "Consciously and unconsciously perceived stimuli are encoded by the same neural responses for about a quater of a second .", "From this point on , consciously perceived stimuli benefit from a series of additional brain processes , each restricted in time .", "For unconsciously perceived stimuli , this chain of processing breaks and a slow decay of encoding is observed .", "Salti et al . , therefore , conclude that conscious perception is represented differently to unconscious perception in the brain and produces more extensive and structured brain activity .", "Future work will focus on understanding these differences in neural coding and their contribution to the interplay between conscious and unconscious perception ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics" ]
Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals
elife-05421-v2
[ [ "Radiation damage often limits the resolution and accuracy of macromolecular crystal structures ( Garman , 2010; Zeldin et al . , 2013 ) .", "Femtosecond X-ray free electron laser ( XFEL ) pulses enable the possibility of visualizing molecular structures before the onset of radiation damage , and allow the dynamics of chemical processes to be captured ( Solem , 1986; Neutze et al . , 2000 ) .", "Thus , from the first XFEL operation at the Linac Coherent Light Source ( LCLS ) in 2009 , there has been considerable effort dedicated to the development of methods to utilize this rapid succession of bright pulses for macromolecular crystallography , with the aim of obtaining damage-free , chemically accurate structures .", "Most of the structures reported from XFELs to date use a liquid jet to inject small crystals into the beam ( DePonte et al . , 2008; Sierra et al . , 2012; Weierstall et al . , 2014 ) , but diffraction data have also been measured from crystals placed in the beam with a standard goniometer setup ( Cohen et al . , 2014; Hirata et al . , 2014 ) .", "In both cases , the illuminated volume diffracts before suffering damage by a single XFEL pulse .", "Because the crystal is effectively stationary during the 10–50 fs exposure , ‘still’ diffraction patterns are obtained , in contrast to standard diffraction data collection where the sample is rotated through a small angle during the exposure .", "Extracting accurate Bragg peak intensities from XFEL diffraction data is a substantial challenge .", "An XFEL data set comprises ‘still’ diffraction patterns generally containing only partially recorded reflections , typically from randomly oriented crystals .", "The full intensity then has to be estimated from the observed partial intensity observations .", "Most XFEL diffraction data processing approaches reported to date have approximated the full intensity by the so-called “Monte Carlo” method , in which thousands of partial intensity observations of a given reflection are summed and normalized by the number of observations , which assumes that these observations sample the full 3D Bragg volume .", "Because a single diffraction image—in which each observed reflection samples only part of each reflection intensity–contains much less information than a small continuous wedge of diffraction data ( as used in conventional crystallography ) , this method requires a very large number of crystals to ensure convergence of the averaged partial reflection intensities to the full intensity value ( Kirian et al . , 2010 ) .", "Moreover , shot-to-shot differences in pulse intensity and energy spectrum that arise from the self-amplified stimulated emission ( SASE ) process ( Kondratenko and Saldin , 1979; Bonifacio et al . , 1984 ) , along with differences in illuminated crystal volume , mosaicity , and unit-cell dimensions , contribute to intensity variation of the equivalent reflections observed on different images .", "These differences are assumed to be averaged out by the Monte Carlo method ( Hattne et al . , 2014 ) .", "Thus , accurate determination of these parameters for each diffraction image should , in principle , provide more accurate integrated intensities , and converge with fewer measurements .", "Furthermore , it is desirable to assemble a data set from as few diffraction images as possible , since the potential of XFELs has been limited by the very large amounts of sample required for the Monte Carlo method , compounded by severe limitations in the availability of beamtime .", "In the 1970's , the Harrison and Rossmann groups developed ‘post-refinement’ methods ( Rossmann et al . , 1979; Winkler et al . , 1979 ) , in which the parameters that determine the location and volume of the Bragg peaks are ‘post’-refined against a reference set of fully recorded reflections following initial indexing and integration of rotation data .", "Accurate estimation of these parameters , including the unit-cell lengths and angles , crystal orientation , mosaic spread , and beam divergence enables accurate calculation of what fraction of the reflection intensity was recorded on the image , i . e . , its ‘partiality’ , which is then used to correct the measurement to its fully recorded equivalent .", "Applied to virus crystals , for which only a few images can typically be collected before radiation damage becomes significant , post-refinement made it possible to obtain high-quality diffraction data sets collected from many crystals ( Rossmann et al . , 1979; Winkler et al . , 1979 ) .", "The implementation of post-refinement for XFEL diffraction data poses unique challenges .", "Firstly , since XFEL diffraction data generally do not contain fully recorded reflections , the initial scaling and merging of images is difficult .", "Secondly , since the XFEL diffraction images are stills rather than rotation data , different approaches are required for the correction of measurements to determine the full spot equivalent .", "Other schemes for implementing post-refinement of XFEL diffraction data have been described previously , but thus far they have been only applied to simulated XFEL data ( White , 2014 ) , and to pseudo-still images collected using monochromatic synchrotron radiation ( Kabsch , 2014 ) .", "We have developed a new post-refinement procedure specifically designed for diffraction data from still images collected from crystals in random orientations .", "We implemented our method in a new computer program , prime ( post-refinement and merging ) , that post-refines the parameters needed for calculating the partiality of reflections recorded on each still image .", "We describe here our method and demonstrate that post-refinement greatly improves the quality of the diffraction data from XFEL diffraction experiments with crystals of three different proteins .", "We show that our post-refinement procedure allows complete data sets to be extracted from a much smaller number of diffraction images than that necessary when using the Monte Carlo method .", "Thus , this development will help make XFEL crystallography accessible to many challenging problems in biology , including those for which sample quantity is a major limiting factor ." ], [ "Units are arbitrary unless specified in parenthesis .", "Iobs , observed intensity .", "Iref , reference intensity .", "w , weighting term ( inverse variance of the observed intensity ) .", "G , function of linear scale ( G0 ) and resolution-dependent ( B ) factors that scales the different diffraction images to the reference set .", "Eoc , Ewald-offset correction function .", "rh , offset reciprocal-space distance from the center of the reflection to the Ewald sphere ( Å−1 ) .", "rp , radius of the disc of intersection between the reciprocal lattice point and the Ewald sphere ( Å−1 ) .", "rs , radius of the reciprocal lattice point ( Å−1 ) .", "θx , θy , θz , crystal rotation angles ( see Figure 1A; ° ) . 10 . 7554/eLife . 05421 . 003Figure 1 . Geometry of the diffraction experiment and calculation of the Ewald-offset distance , rh .", "( A ) A reciprocal lattice point intersects the Ewald sphere .", "The inset shows the coordinate system used in cctbx . xfel and prime .", "The vector S0 represents the direction of the incident beam ( –z-axis ) and forms the radius of the Ewald sphere of length 1/λ .", "The reciprocal lattice point i is expressed in reciprocal lab coordinates using Equation 5 as represented by the vector xi .", "The Ewald-offset distance , rh , is the difference between the distance from the Ewald-sphere center to the reciprocal lattice point ( length of Si ) and 1/λ .", "The inset shows the definition of the crystal rotation axes; they are applied in the following order: θz , θy , θx .", "( B ) Shown is the volume of a reciprocal lattice point with radius rs .", "The offset rh defines the Ewald-offset correction Eocarea , which is the ratio between the area intersecting the Ewald sphere , Ap , and the area at the center of the volume , As . DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 003 γ0 , parameter for Equation 3 ( Å−1 ) .", "γe , energy spread and unit-cell variation ( see Equation 3; Å−1 ) .", "γx and γy , beam divergence ( see Equation 4; Å−1 ) .", "{uc} , unit-cell dimensions ( a , b , c ( Å ) , α , β , and γ ( ° ) ) .", "Vc , reciprocal-lattice volume correction function ( Å−3 ) .", "xobs and xcalc , observed and predicted spot positions on the detector ( mm ) .", "x , position of the reciprocal lattice point ( Å−1 ) .", "S , displacement vector from the center of the Ewald sphere to x ( Å−1 ) .", "S0 , incident beam vector with length 1/wavelength ( Å−1 ) .", "O , orthogonalization matrix .", "R , rotation matrix .", "fL and fLN , Lorentzian function and its normalized counterpart .", "Γ , full width at half maximum ( FWHM ) of the Lorentzian function .", "Partiality can be modeled by describing the full reflection as a sphere ( Figure 1A ) .", "In a still diffraction pattern , assuming a monochromatic photon source , the observed intensity Iobs , h for Miller index h is a thin slice through a three-dimensional reflection .", "To calculate partiality , we assume that the measurement is an areal ( i . e . , infinitely thin ) sample of the volume ( Figure 1B ) .", "The maximum partial intensity that can be recorded for a given reflection will occur when its center lies exactly on the Ewald sphere .", "By definition , the center of the reflection will be offset from the Ewald sphere by rh , and the corresponding disc will have a radius rp .", "The offset rh is determined by various experimental parameters , including the crystal orientation , unit-cell dimensions , and X-ray photon energy .", "The offset distance is used to calculate the Ewald offset correction , Eocarea , defined as the ratio between the areas defined by rp and rs ( implemented as a smoothed correction function Eoch as defined in ‘Materials and methods’ ) .", "The Ewald-offset corrected intensity is then converted to the full intensity in 3D by applying a volume correction factor , Vc .", "We define the target Tpr for the post-refinement of a partiality and scaling model by: ( 1 ) Tpr= ∑hwh ( Iobs , h−G ( G0 , B ) Eoch ( θx , θy , γ0 , γe , γx , γy , {uc} ) Vc , h−1 × Iref , h ) 2 , which minimizes the difference between the observed reflections Iobs and a scaled and Ewald-offset corrected full intensity ‘reference set’ Iref using a least-squares method .", "The sum is over all observed reflections with Miller indices", "h . In alternate refinement cycles , we also minimize the deviations between predicted ( xcalc ) and observed ( xobs ) spot positions on the detector using a subset of strong spots as has been suggested previously ( Hattne et al . , 2014; Kabsch , 2014 ) : ( 2 ) Txy=∑h ( xobs−xcalc ) 2 .", "Sets of parameters associated with each diffraction image ,", "i . e .", ", G0 , B , θx , θy , γ0 , γe , γx , γy and the unit-cell constants , are iteratively refined in a series of ‘microcycles’ against the current reference set ( Figure 2 ) . 10 . 7554/eLife . 05421 . 004Figure 2 . Post-refinement protocol . The flowchart illustrates the iterative post-refinement protocol , broken up into ‘microcycles’ that refine groups of parameters iteratively ( blue boxes ) , and ‘macrocycles’ .", "At the beginning of first macrocycle , a reference diffraction data set is generated .", "At the end of each macrocycle , the reference diffraction data set is updated .", "Both the micro- and macrocycles terminate either when the refinement converges or when a user-specified maximum number of cycles is reached . DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 004 Procedures for generating the initial reference set Iref ( initial ) are described below .", "After convergence of the microcycles , scaled full intensities are calculated from the observed partial intensities Iobs by multiplication of the inverse of the Ewald-offset correction and the scale factor G , along with the volume correction factor Vc .", "These scaled full reflections are then merged for each unique Miller index , taking into account estimated errors of the observed intensities , σ ( Iobs ) , and propagation of error estimates for the refined parameters .", "This merged and scaled set of full reflections is then used as the new reference set in the next round of post-refinement using the target functions ( Equations 1 and 2 , for details see ‘Materials and methods’ ) .", "These ‘macrocycles’ are repeated until convergence is achieved , after which the merged and scaled set of full intensities is provided to the user .", "The prime program controls post-refinement of specified parameters in a particular microcycle ( Figure 2 ) .", "One can refine all parameters together , or selectively refine groups of parameters iteratively , starting from ( 1 ) a linear scale factor and a B-factor , ( 2 ) crystal orientations , ( 3 ) crystal mosaicity , beam divergence , and spectral dispersion , and ( 4 ) unit-cell dimensions .", "Space-group-specific constraints are used to limit the number of free parameters for the unit-cell refinement .", "A particular microcycle is completed when the target functions converge or when a specified number of iterations is reached; the program then generates the new reference intensity set to replace the current reference set for the next macrocycle .", "Finally , the program exits and outputs the latest merged reflection set either when the macrocycles converge or when a user-specified maximum number of cycles has been reached .", "The starting point for our post-refinement method is a set of indexed and integrated partial intensities , along with their estimated errors , obtained from still images .", "For this study , diffraction data and their estimated errors were obtained from the cctbx . xfel package ( Sauter et al . , 2013; Hattne et al . , 2014 ) , although in principle integrated diffraction data from any other program can be used .", "Observed intensities on the diffraction image were classified as ‘spots’ by the program Spotfinder ( Zhang et al . , 2006 ) , which identifies Bragg spots by considering connected pixels with area and signal height greater than user-defined thresholds .", "By trial and error , we accepted reflections larger than 25 pixels with individual-pixel intensity more than 5 σ over background for myoglobin and hydrogenase ( collected on a Rayonix MX325HE detector with pixel size of 0 . 08 mm and beam diameter [FWHM] of 50 μm ) .", "For thermolysin ( collected on a Cornell-SLAC pixel array detector with pixel size of 0 . 1 mm and beam size of 2 . 25 μm2 ) , where reflections are generally smaller , these values were 1 pixel and 5 σ .", "A full list of parameters is available on the cctbx . xfel wiki ( http://cci . lbl . gov/xfel ) .", "Separate resolution cutoffs for each image were applied by cctbx . xfel , at resolutions where the average I/σ ( I ) fell below 0 . 5 ( Hattne et al . , 2014 ) .", "Prior to post-refinement , the experimentally observed partial intensities need to be corrected by a polarization factor .", "The primary XFEL beam at LCLS is strongly polarized in the horizontal plane , and we calculate the correction factor as a function of the Bragg angle ( θ ) and the angle ϕ between the sample reflection and the laboratory horizontal planes ( Kahn et al . , 1982; see ‘Materials and methods’ ) .", "For a stationary crystal and a monochromatic beam , a Lorentz factor correction is not applicable; the spectral dispersion of the SASE beam ( δE/E ∼ 3 × 10−3 for the data sets studied here ) is accounted for by the γe term ( see ‘Materials and methods’ ) .", "An essential step to initiate post-refinement is the generation of the initial reference set Iref ( initial ) .", "This reference set has to be estimated from the available unmerged and unscaled partial reflection intensities after application of the polarization correction .", "For the results presented here , linear scale factors for each diffraction image were chosen to make the mean intensities of each diffraction image equal .", "Since this procedure can be affected by outliers in the observed intensities , we select a subset of reflections with user-specified resolution range and signal-to-noise ratio ( I/σ ( I ) ) cutoffs .", "From this selection , we calculate the mean intensity on each diffraction image and then scale each image to make the mean intensity of all images equal .", "We correct the scaled observed reflections to their Ewald-offset corrected equivalents using the starting parameters , and then merge the observations , taking into account the experimental σ ( Iobs ) , to generate the initial reference set .", "The initial values for crystal orientation , unit-cell dimensions , crystal-to-detector distance , and spot position on the detector were obtained from the refinement of these parameters by cctbx . xfel .", "The photon energy was that provided by the LCLS endstation system and is not refined .", "Initial values for the parameters of the reflection width model are described in the ‘Materials and methods’ section .", "In order to separately assess the effects of scaling , the Ewald offset correction ( Equation 1 ) , and post-refinement , we refer to three alternative schemes for processing the diffraction data sets: ( 1 ) ‘Averaged merged’ , in which intensities were generated by averaging all observed partial intensities from equivalent reflections without Ewald-offset correction and scaling; ( 2 ) ‘Mean-intensity partiality corrected’ , in which intensities were generated by scaling the reflections to the mean intensity and also applying the Ewald-offset correction determined from the initial parameters obtained from the indexing and integration program , followed by merging; and ( 3 ) ‘Post-refined’ , in which intensities were from the final set of scaled and merged full reflections after the convergence of post-refinement .", "We note that although the ‘averaged merged’ process is similar to the original Monte Carlo method ( Kirian et al . , 2010 ) , the integrated , unmerged partial intensities used in our tests were obtained from the program cctbx . xfel ( Hattne et al . , 2014 ) , which also refines various parameters on an image-by-image basis ( Sauter et al . , 2014 ) .", "We tested our post-refinement method on experimental XFEL diffraction data sets from three different crystallized proteins of known structure: myoglobin , hydrogenase , and thermolysin ( Table 1 ) .", "For quality assessment , we performed molecular replacement ( MR ) with Phaser ( McCoy et al . , 2007 ) using models with selected parts of the known structures omitted , followed by atomic model refinement with phenix . refine ( Afonine et al . , 2012 ) , and inspection of ( mFo-DFc ) omit maps .", "We further used three different metrics: CC1/2 , and the crystallographic Rwork and Rfree of the fully refined atomic model .", "We then compared changes in the three quality metrics between merged XFEL diffraction data sets after scaling , partiality correction , and post-refinement .", "We also investigated the effect of reducing the number of images used by randomly selecting a subset from the full set of diffraction images and repeating the entire post-refinement , merging , MR and refinement processes using this subset . 10 . 7554/eLife . 05421 . 005Table 1 . XFEL diffraction data sets used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 005MyoglobinClostridium pasteurianum hydrogenaseThermolysinSpace groupP6P42212P6122Resolution used ( Å ) 20 . 0–1 . 3545 . 0–1 . 6050 . 0–2 . 10Unit cell dimensions ( Å ) a = b = 90 . 8 , c = 45 . 6a = b = 111 . 2 , c = 103 . 8a = b = 92 . 7 , c = 130 . 5No .", "of unique reflections46 , 55585 , 27319 , 995No .", "of images* indexed75717712 , 692No .", "of images with spots to resolution used307751957Average no .", "of spots on an image ( to resolution used ) 16283640352Energy spectrumSASE†SASE†SASE†DetectorRayonix MX325HERayonix MX325HECSPAD‡Sample delivery methodfixed targetfixed targetElectrospun jet*This is the number of images indexed using cctbx . xfel program , and in the case of thermolysin it is the number of images indexed for one of the two wavelengths .", "†SASE: self-amplified spontaneous emission .", "‡CSPAD: Cornell-SLAC pixel array detector .", "Diffraction data for both myoglobin and hydrogenase were collected from frozen crystals mounted on a standard goniometer setup ( Cohen et al . , 2014 ) , whereas the thermolysin data were collected using an electrospun liquid jet to inject nanocystals into a vacuum chamber ( Sierra et al . , 2012; Bogan , 2013 ) .", "The completeness of each data set was better than 90% at the limiting resolution used in our tests ( Tables 2 , 3 , 4 ) .", "Each diffraction data set involved a different number of images due the differing diffraction quality of the crystals . 10 . 7554/eLife . 05421 . 006Table 2 . Statistics of post-refinement and atomic model refinement for myoglobinDOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 006No .", "images100757Resolutiona ( Å ) 20 . 0–1 . 35 ( 1 . 40–1 . 35 ) 20 . 0–1 . 35 ( 1 . 40–1 . 35 ) Completenessa ( % ) 80 . 0 ( 22 . 2 ) 97 . 7 ( 79 . 8 ) Average no .", "observations per unique hkla4 . 0 ( 1 . 2 ) 25 . 7 ( 2 . 0 ) Averaged-mergedMean-scaled partiality correctedPost-refinedAveraged mergedMean-scaled partiality correctedPost-refinedPost-refinement parametersb Linear scale factor G01 . 00 ( 0 . 00 ) 2 . 79 ( 5 . 02 ) 1 . 00 ( 1 . 04 ) 1 . 00 ( 0 . 00 ) 2 . 19 ( 3 . 83 ) 0 . 89 ( 1 . 07 ) B0 . 0 ( 0 . 0 ) 0 . 0 ( 0 . 0 ) 3 . 2 ( 7 . 8 ) 0 . 0 ( 0 . 0 ) 0 . 0 ( 0 . 0 ) 6 . 2 ( 8 . 3 ) γ0 ( Å−1 ) NA0 . 00135 ( 0 . 00028 ) 0 . 00128 ( 0 . 00022 ) NA0 . 00147 ( 0 . 00042 ) 0 . 00132 ( 0 . 00034 ) γy ( Å−1 ) NA0 . 00 ( 0 . 00 ) 0 . 00007 ( 0 . 00080 ) NA0 . 00 ( 0 . 00 ) 0 . 00007 ( 0 . 00009 ) γx ( Å−1 ) NA0 . 00 ( 0 . 00 ) 0 . 00010 ( 0 . 00011 ) NA0 . 00 ( 0 . 00 ) 0 . 00008 ( 0 . 00010 ) γe ( Å−1 ) NA0 . 00200 ( 0 . 00 ) 0 . 00344 ( 0 . 00266 ) NA0 . 00200 ( 0 . 00 ) 0 . 00423 ( 0 . 00323 ) Unit cell a ( Å ) :90 . 4 ( 0 . 4 ) 90 . 4 ( 0 . 4 ) 90 . 5 ( 0 . 4 ) 90 . 4 ( 0 . 4 ) 90 . 4 ( 0 . 4 ) 90 . 5 ( 0 . 3 ) c ( Å ) 45 . 3 ( 0 . 4 ) 45 . 3 ( 0 . 4 ) 45 . 3 ( 0 . 3 ) 45 . 3 ( 0 . 3 ) 45 . 3 ( 0 . 3 ) 45 . 3 ( 0 . 3 ) Average Tpr Start/EndNANA19 . 39 ( 7 . 68 ) /7 . 17 ( 3 . 38 ) NANA19 . 83 ( 7 . 54 ) /6 . 02 ( 2 . 59 ) Average Txy ( mm2 ) Start/EndNANA169 . 74 ( 132 . 56 ) /132 . 02 ( 104 . 08 ) NANA170 . 66 ( 144 . 52 ) /133 . 42 ( 109 . 58 ) CC1/2 ( % ) 81 . 379 . 686 . 591 . 895 . 798 . 2Molecular replacement scoresc LLG2837 . 5043 . 5291 . 8264 . 8364 . 9320 .", "TFZ10 . 513 . 013 . 413 . 713 . 814 . 0Structure-refinement parameters R ( % ) 39 . 428 . 023 . 521 . 120 . 317 . 8 Rfree ( % ) 42 . 129 . 424 . 823 . 122 . 519 . 7 Bond r . m . s . d . 0 . 0060 . 0060 . 0040 . 0060 . 0060 . 006 Angle r . m . s . d . 1 . 140 . 980 . 791 . 031 . 350 . 86 Ramachandran statistics Favored ( % ) 98 . 098 . 098 . 098 . 098 . 098 . 0 Outliers ( % ) 0 . 00 . 00 . 00 . 00 . 00 . 0aValues in parentheses correspond to highest resolution shell . bPost-refined parameters are shown as the mean value , with the standard deviation in parentheses . cMolecular replacement scores reported by Phaser ( McCoy et al . , 2007 ) : log-likelihood gain ( LLG ) and translation function ( TFZ ) . 10 . 7554/eLife . 05421 . 007Table 3 . Statistics of post-refinement and atomic model refinement for hydrogenaseDOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 007No .", "images100177Resolutiona ( Å ) 45 . 0–1 . 60 ( 1 . 66–1 . 60 ) 45 . 0–1 . 60 ( 1 . 66–1 . 60 ) Completenessa ( % ) 83 . 0 ( 47 . 7 ) 91 . 2 ( 63 . 5 ) Average no .", "observations per unique hkla4 . 4 ( 1 . 7 ) 7 . 13 ( 2 . 3 ) Averaged-mergedPost-refinedAveraged-mergedPost-refinedPost-refinement parametersb Linear scale factor G01 . 00 ( 0 . 00 ) 0 . 56 ( 1 . 27 ) 1 . 00 ( 0 . 00 ) 0 . 53 ( 1 . 22 ) B0 . 0 ( 0 . 0 ) 10 . 0 ( 7 . 0 ) 0 . 0 ( 0 . 0 ) 10 . 5 ( 6 . 9 ) γ0 ( Å−1 ) NA0 . 00132 ( 0 . 00042 ) NA0 . 00126 ( 0 . 00041 ) γy ( Å−1 ) NA0 . 00002 ( 0 . 00004 ) NA0 . 00002 ( 0 . 00004 ) γx ( Å−1 ) NA0 . 00008 ( 0 . 00009 ) NA0 . 00008 ( 0 . 00011 ) γe ( Å−1 ) NA0 . 00269 ( 0 . 00138 ) NA0 . 00288 ( 0 . 00160 ) Unit cell a ( Å ) :110 . 1 ( 0 . 4 ) 110 . 4 ( 0 . 3 ) 110 . 1 ( 0 . 4 ) 110 . 3 ( 0 . 4 ) c ( Å ) 103 . 1 ( 0 . 4 ) 103 . 1 ( 0 . 2 ) 103 . 0 ( 0 . 4 ) 103 . 0 ( 0 . 2 ) Average Tpr Start/EndNA28 . 20 ( 10 . 86 ) /5 . 92 ( 2 . 35 ) NA26 . 47 ( 12 . 70 ) /5 . 22 ( 2 . 72 ) Average Txy ( mm2 ) Start/EndNA623 . 36 ( 314 . 57 ) /381 . 23 ( 198 . 44 ) NA564 . 30 ( 267 . 45 ) /372 . 28 ( 202 . 28 ) CC1/2 ( % ) 62 . 077 . 371 . 784 . 8Molecular replacement scoresc LLG53 , 352 . 9612 . 7229 . 11774 .", "TFZ69 . 275 . 975 . 079 . 0Structure-refinement parameters R ( % ) 33 . 425 . 329 . 122 . 0 Rfree ( % ) 36 . 728 . 931 . 325 . 0 Bond r . m . s . d . 0 . 0060 . 0070 . 0070 . 007 Angle r . m . s . d . 1 . 431 . 501 . 681 . 97 Ramachandran statistics Favored ( % ) 96 . 397 . 097 . 096 . 7 Outliers ( % ) 0 . 00 . 00 . 00 . 0aValues in parentheses correspond to highest resolution shell . bPost-refined parameters are shown as the mean value , with the standard deviation in parentheses . cMolecular replacement scores reported by Phaser ( McCoy et al . , 2007 ) : log-likelihood gain ( LLG ) and translation function ( TFZ ) . 10 . 7554/eLife . 05421 . 008Table 4 . Statistics of post-refinement and atomic model refinement for thermolysinDOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 008No .", "images200012 , 692Resolutiona ( Å ) 50 . 0–2 . 10 ( 2 . 18–2 . 10 ) 50 . 0–2 . 10 ( 2 . 18–2 . 10 ) Completenessa ( % ) 81 . 3 ( 24 . 3 ) 96 . 5 ( 74 . 8 ) Average no .", "observations per unique hkla32 . 8 ( 1 . 2 ) 176 . 6 ( 2 . 4 ) Averaged-mergedPost-refinedAveraged-mergedPost-refinedPost-refinement parametersb Linear scale factor G01 . 00 ( 0 . 00 ) 1 . 65 ( 1 . 66 ) 1 . 00 ( 0 . 00 ) 2 . 26 ( 75 . 12 ) B0 . 0 ( 0 . 0 ) 23 . 0 ( 33 . 8 ) 0 . 0 ( 0 . 0 ) 30 . 1 ( 59 . 8 ) γ0 ( Å−1 ) NA0 . 00052 ( 0 . 00040 ) NA0 . 00051 ( 0 . 00039 ) γy ( Å−1 ) NA0 . 00001 ( 0 . 00003 ) NA0 . 00001 ( 0 . 00003 ) γx ( Å−1 ) NA0 . 00002 ( 0 . 00004 ) NA0 . 00002 ( 0 . 00004 ) γe ( Å−1 ) NA0 . 00110 ( 0 . 00129 ) NA0 . 00103 ( 0 . 00128 ) Unit cell a ( Å ) :92 . 9 ( 0 . 3 ) 92 . 9 ( 0 . 2 ) 92 . 9 ( 0 . 3 ) 92 . 9 ( 0 . 3 ) c ( Å ) 130 . 5 ( 0 . 5 ) 130 . 4 ( 0 . 4 ) 130 . 5 ( 0 . 5 ) 130 . 4 ( 0 . 4 ) Average Tpr Start/EndNA1 . 15 ( 0 . 49 ) /0 . 55 ( 0 . 23 ) NA1 . 15 ( 0 . 52 ) /0 . 28 ( 0 . 13 ) Average Txy ( mm2 ) Start/EndNA168 . 13 ( 117 . 29 ) /167 . 72 ( 106 . 14 ) NA169 . 01 ( 122 . 20 ) /170 . 00 ( 122 . 57 ) CC1/2 ( % ) 77 . 793 . 594 . 398 . 8Molecular replacement scoresc LLG3590 . 4491 . 5477 . 6022 .", "TFZ8 . 99 . 724 . 124 . 6Structure-refinement parameters R ( % ) 25 . 219 . 520 . 718 . 4 Rfree ( % ) 29 . 124 . 023 . 921 . 1 Bond r . m . s . d . 0 . 0040 . 0020 . 0020 . 002 Angle r . m . s . d . 0 . 750 . 580 . 590 . 62 Ramachandran statistics Favored ( % ) 95 . 994 . 695 . 294 . 9 Outliers ( % ) 0 . 00 . 00 . 00 . 0 Zinc peak height Zn ( 1 ) ( σ ) 14 . 016 . 014 . 320 . 9 Zn ( 2 ) ( σ ) 3 . 65 . 17 . 77 . 1 Average peak height for calcium ions ( σ ) 9 . 711 . 314 . 216 . 1aValues in parentheses correspond to highest resolution shell . bPost-refined parameters are shown as the mean value , with the standard deviation in parentheses . cMolecular replacement scores reported by Phaser ( McCoy et al . , 2007 ) : log-likelihood gain ( LLG ) and translation function ( TFZ ) .", "For myoglobin , we used both an XFEL diffraction data set consisting of 757 diffraction images ( Table 1 ) collected by the SSRL-SMB group using a goniometer-mounted fixed-target grid ( Cohen et al . , 2014 ) , and a randomly selected subset of 100 diffraction images .", "The diffraction images were from crystals in random orientations , with a single still image collected from each crystal .", "XFEL diffraction data for Clostridium pasteurianum hydrogenase were measured from eight crystals by the Peters ( University of Montana ) and SSRL-SMB groups using a goniometer-mounted fixed-target grid ( Cohen et al . , 2014 ) .", "This experiment generated 177 diffraction images that could be merged to a completeness of 91% , with more than half of the diffraction images containing reflections to 1 . 6 Å ( each diffraction image typically has approximately 3000 spots ) .", "We also used a randomly selected subset of 100 diffraction images to assess the effect of post-refinement on a smaller number of images .", "The CC1/2 value improved significantly with post-refinement ( Table 3 ) .", "For quality assessment , the Fe-S cluster was omitted from both the molecular replacement search model ( PDB ID 3C8Y ) and subsequent atomic model refinement .", "The omit map densities for the post-refined diffraction data sets using the complete set of 177 diffraction images and the randomly selected subset of 100 diffraction images ( 83% complete ) clearly show the entire Fe-S cluster whereas the densities using the averaged merged data sets are much poorer ( Figure 8A ) .", "Upon atomic model refinement with the Fe-S clusters and water molecules included , the R and Rfree values for both post-refined data sets were significantly better than the averaged merged case ( Figure 8B ) . 10 . 7554/eLife . 05421 . 014Figure 8 . Impact of post-refinement on the hydrogenase diffraction data set .", "( A ) Difference Fourier ( mFo-DFc ) omit maps of one of the four Fe-S clusters ( which were omitted in molecular replacement and atomic model refinement ) for the averaged merged and the post-refined hydrogenase XFEL diffraction data sets consisting of all 177 diffraction images ( Table 1 ) and a randomly selected subset of 100 diffraction images .", "The maps are contoured at 3 σ .", "( B ) Crystallographic R and Rfree values vs resolution after atomic model refinement using the specified diffraction data sets with inclusion of the three Fe-S clusters and water molecules . DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 014 For thermolysin , we tested the entire deposited XFEL diffraction data set consisting of 12 , 692 diffraction images ( Table 1 ) ( Hattne et al . , 2014; the diffraction data are publicly archived in the Coherent X-ray Imaging Data Bank , accession ID 23 , http://cxidb . org ) , as well as a randomly selected subset of 2000 diffraction images .", "In this experiment , the crystal-to-detector distance gave a maximum resolution of 2 . 6 Å at the edge and 2 . 1 Å at the corners of the detector .", "Thus , a large number of diffraction images were required to achieve reasonable completeness of the merged data set for reflections in the 2 . 1—2 . 6 Å resolution range .", "As in the other two cases , post-refinement significantly improved the CC1/2 value ( Table 4 ) .", "For quality assessment , zinc and calcium ions were omitted from the thermolysin molecular replacement search model ( PDB ID: 2TLI ) and subsequent atomic model refinement .", "Post-refinement improved the peak heights of both the zinc and calcium ions ( Table 4 ) .", "The completeness of the merged data sets has a direct impact on the overall quality of the diffraction data set ( CC1/2 ) , quality of the electron density maps and the refined structures ( Tables 2–4 , and Figure 6 ) .", "When completeness is high , adding more images to increase the multiplicity of observations has only a modest impact on the quality of the final refined structures using the post-refined diffraction data .", "For example , when subsets ranging from 2000 to 12 , 000 thermolysin diffraction images ( all subsets 100% complete at 2 . 6 Å ) were post-refined the peak height in the omit map for the larger of the two anomalous sites ( Figure 11C ) , the CC1/2 values , and the R values of the refined structures did not improve significantly when more than 8000 images were used . 10 . 7554/eLife . 05421 . 017Figure 11 . Convergence of structure refinements for the post-refined thermolysin XFEL data set at 2 . 6 Å resolution , using increasing numbers of diffraction images .", "( A ) Average number of observations per unique hkl .", "( B ) CC1/2 for merged subsets using 2000–12 , 000 images ( 100% completeness for all subsets ) .", "( C ) Peak height ( σ ) in the omit map for the largest peak .", "( D ) Rwork and Rfree after refining the thermolysin model without zinc and calcium ions against the corresponding post-refined diffraction data sets . DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 017" ], [ "Diffraction data collection using conventional x-ray sources typically employs the rotation method , in which a single crystal is rotated through a contiguous set of angles , and the diffraction patterns are recorded on a 2-D detector .", "If a full data set can be collected from a single crystal without a prohibitive level of radiation damage , diffraction data processing is a well-established and reliable process .", "In contrast , processing of XFEL diffraction data , which are collected from crystals in random orientations as ‘still’ diffraction images , requires new methods and implementations such as those described here .", "Improved data collection and processing methods , particularly those that can significantly reduce the amount of sample needed to assemble a complete and accurate diffraction data set , are important for making XFELs useful for certain challenging investigations in structural biology .", "We developed a post-refinement method for still diffraction images , such as those obtained at XFELs , and implemented it in new computer program , prime , that applies a least-squares minimization method to refine parameters as defined in our partiality model .", "Other post-refinement methods for XFEL diffraction data have been described recently ( Kabsch , 2014; White , 2014 ) , but our implementation differs from these reports .", "Kabsch uses a partiality model in which an Ewald offset correction is defined as a Gaussian function of angular distance from the Ewald sphere .", "White used the intersecting volume between the reflection and the limiting-energy Ewald spheres defined by the energy spectrum for the partiality calculation , and calculates the initial reference data set by averaging all observations without scaling .", "Neither report describes an application to experimental XFEL diffraction data , so we cannot compare these methods to the results presented here .", "We have demonstrated here that our implementation of post-refinement substantially improves the quality of the diffraction data from three different XFEL experiments .", "Moreover , the resulting structures can be refined to significantly lower Rfree and R values , with electron density maps that reveal novel features more clearly , than those using non-post-refined XFEL data sets .", "A key feature of our method is that the parameters that define the diffracted spot are iteratively refined against the reference set .", "This approach is superior to methods that only consider each diffraction image individually .", "Moreover , our post-refinement procedure allows accurate diffraction data sets to be extracted from a much smaller number of images ( average number of observations ) than that necessary without post-refinement .", "Thus , this development will make XFEL crystallography accessible to many challenging problems in biology for which sample quantity is a major limiting factor .", "At present , it is difficult to assess the relative quality of post-refined XFEL data studied here with conventional rotation data measured at a synchrotron .", "The comparison of myoglobin omit maps ( Figure 7 ) suggests that the SR data are perhaps somewhat better , but more systematic studies will be needed to understand the relative merits of the different data sets .", "We suspect that rotation data would be better due to the ability to directly measure full reflections ( at least by summation of partials ) without modeling partiality , which is still a relatively crude process ( see below ) .", "However , a comparison between still data sets measured at a synchrotron and an XFEL is needed to deconvolute the effect of rotation vs other differences between these sources .", "Our formulation of post-refinement employs the simplifying assumption that reflections are spherical volumes .", "More sophisticated models consider crystal mosaicity to have three components , each with a distinct effect on the reciprocal lattice point ( Juers et al . , 2007; Nave , 1998 , 2014 ) .", "First , the domain size ( the average size of the coherently scattering mosaic blocks ) produces reciprocal lattice points of constant , finite size: small domains produce large-sized spots , while large domains produce small spots , as there is an inverse ( Fourier ) relation between spot size and domain size .", "Second , unit-cell variation among domains produces reflections that are spheres whose radii increase with distance from the origin .", "In cctbx . xfel , mosaicity ( modeled as isotropic parameter ) and effective domain size are taken into account when predicting which reflections are in diffracting position prior to integration ( Sauter et al . , 2014; Sauter , 2015 ) .", "Third , orientational spread among mosaic domains produces spots shaped like spherical caps .", "Each cap subtends a solid angle that depends on the magnitude of the spread .", "In addition , anisotropy in crystal mosaicity is not considered; this would require refining separate parameters along each lattice direction .", "Finally , the rugged energy spectrum that results from the SASE process of the XFEL is not yet considered in our current model .", "These issues will require future investigation ." ], [ "The observed intensity Ih ( i ) for observation i of Miller index h is a thin slice through a three-dimensional reflection .", "To calculate partiality , we assume that the measurement is an infinitely thin , circular sample of a spherical volume ( Figure 1B ) .", "We assume a monochromatic beam as the starting point to define the Ewald offset correction Eocarea .", "The Eocarea of any reflection centered on the Ewald sphere is defined as 1; this position corresponds to the maximum partial intensity that could be measured for the reflection .", "The Eocarea for any other position is defined as a function of the normal distance from the Ewald sphere to the center of the reciprocal lattice point ( the offset distance , rh ) , and of the reciprocal-lattice radius of the spot rs , which is a function of the crystal mosaicity and spectral dispersion ( Figure 1B ) .", "The Eocarea can be described by the ratio of the observed area ( Ap ) with a radius rp to the Ewald-offset corrected area ( As ) with a radius rs ( Figure 1B ) .", "The SASE spectrum emitted by the XFEL is broad and varies from shot-to-shot ( Zhu et al . , 2012 ) .", "To calculate the Ewald sphere , we set the wavelength to be the centroid of the SASE spectrum recorded with each shot .", "For XFEL data measured with a seeded beam ( Amann et al . , 2012 ) , the spectrum is narrow and constant from shot-to-shot , and this single value can be used in this case .", "In order to model spectral dispersion and the possible effects of asymmetric beam divergence , we adapt the rocking curve model described in Winkler et al . ( 1979 ) .", "The four-parameter function used for the rocking curve is rs ( γ0 , γe , γx , γy ) = rs ( θ ) + rs ( α ) , where the first term includes the contribution by spectral dispersion and the second term models beam anisotropy .", "Specifically , ( 3 ) rs ( θ ) =γ0+γe tan θ , where γ0 is a parameter that is initially set to the r . m . s . d . of the Ewald offset calculated for all the reflections on a given image , γe represents the width of the energy spread and the unit-cell variation ( the initial value of γe is calculated from the average energy spread ) , and θ is the Bragg angle .", "The second term is provided by: ( 4 ) rs ( α ) =[ ( γy cos α ) 2+ ( γx sin α ) 2]1/2 , where α is the azimuthal angle going from meridional ( α = 0 ) to equatorial ( α = π/2 ) .", "The values of γy and γx are initially set to 0 .", "The distribution of rh values for the myoglobin case with 757 images after post-refinement is shown in Figure 12 .", "The parameters γe , γy , γx , γ0 are refined within a microcycle ( Figure 2 ) . 10 . 7554/eLife . 05421 . 018Figure 12 . Distribution of the Ewald sphere offset rh . The histogram shows the distribution of rh calculated after post-refinement for myoglobin using 757 diffraction images .", "The number of observations after applying the reflection selection criteria for merging and outlier rejections for this 1 . 35 Å data set is 1 , 136 , 447 ( ∼96% of the total observed reflections ) .", "The standard deviation is 0 . 0016 1/Å or approximately 0 . 12° ( when calculated with the mean of the energy distribution ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 018 The crystal orientation is described in a right-handed coordinate system with the z-axis pointing to the source of the incident beam and the y-axis vertical ( Figure 1A ) .", "We define the crystal orientation by rotations in the order θz , θy , θx about these axes .", "For each Miller index h ( i ) , the reciprocal lattice point vector x ( i ) is obtained by applying orthogonalization and rotation matrixes O and R: ( 5 ) x ( i ) =ROh ( i ) , wherex ( i ) = ( x ( i ) , y ( i ) , z ( i ) ) , h ( i ) = ( h ( i ) , k ( i ) , l ( i ) ) , O= ( a∗b∗ cos γ∗c∗ cos β∗0b∗ sin γ∗c∗ ( cos α∗− cos γ∗ ) /sin γ∗00c∗ cos ( c∗ , c ) ) , R=RθxRθyRθz , where Rθi is the rotation matrix for a rotation around the i-th axis , a∗ , b∗ , c∗ , α∗ , β∗ , γ∗ are the reciprocal unit-cell parameters , and cos ( c∗ , c ) = ( 1+2 cos α∗ cos β∗ cos γ∗−cos2α∗−cos2β∗−cos2γ∗ ) 1/2/sin γ∗ .", "As shown in Figure 1A , the displacement to x ( i ) from the center of the Ewald sphere is given by: ( 6 ) S ( i ) = x ( i ) +S0 , where S0 = ( 0 , 0 , −1/λ ) .", "The offset distance is thus the difference between the length of S ( i ) and the Ewald-sphere radius , ( 7 ) rh= |S ( i ) |−1/λ .", "We introduce a smooth approximation of the area ratio Eocarea ( see ‘Results’ ) in order to circumvent the undefined first derivative when the ratio is zero .", "We use a Lorentzian function ( fL ) to model the radius as function of distance from the Ewald sphere: ( 8 ) fL= 1π12Γ ( rh ) 2+ ( 12Γ ) 2 .", "The function is normalized so that fL ( rh = 0 ) = 1 . 0 when the reciprocal-lattice point is centered on the Ewald sphere , so that ( 9 ) fLn= πΓ2fL .", "We then use the ratio of the observed area ( Ap ) with a radius rp to the Ewald-offset corrected area ( As ) with a radius rs ( Figure 1B ) that corresponds to the full width at half maximum ( FWHM ) , Γ , in the Lorentzian function .", "Using the Lorentzian function to describe the falloff in radius as we move away from the Ewald sphere makes the Eoc function differentiable at rh = rs .", "For the reciprocal lattice volume being bound by a sphere of radius rs centered on the reciprocal lattice point , the intersecting area of the volume is given by: ( 10 ) Ap=πrp2 , whererp= ( rs2−rh2 ) 1/2 .", "The Eoc is then given by the ratio of this intersecting area to the area when this reflection is centered on the Ewald sphere ( As ) , ( 11 ) Eocarea=ApAs=πrp2πrs2=1−rh2rs2 .", "By setting the FWHM of Γ proportional to the radius , rs , at half Eocarea , ( 12 ) Eocarea=1−rh2rs2=0 . 5 , ( 13 ) Γ=rsat 0 . 5 Eocarea=2rh , we arrive at the Ewald-offset correction function ( Figure 13A ) ( 14 ) Eoc=rs22rh2+rs2 . 10 . 7554/eLife . 05421 . 019Figure 13 . The Ewald-offset correction function .", "( A ) Ewald-offset correction Eoc ( Equation 14 ) viewed as a function of the reciprocal-lattice radius ( rs ) and the offset distance ( rh ) .", "( B ) A slice through Eoc at rs = 0 . 003 , comparing Eoc ( Equation 14 ) and Eocarea ( Equation 11 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 019 The use of this Lorentzian approximation to derive the Eoc function vs an actual sphere function , Eocarea , is illustrated in Figure 13B .", "To adjust the observed still intensity to its equivalent at zero offset , we apply the Ewald-offset correction to the observed intensity , ( 15 ) IEoc , h ( i ) =Ih ( i ) Eoch ( i ) Gm , where Ih ( i ) is the observed partial intensity i of Miller index h on image m , Eoch ( i ) is the Ewald-offset correction , and Gm is a scale function for image m .", "We then convert this maximum partial intensity to a full intensity estimate by correcting for the volume of the spot , a factor of 43πrs3πrs2 =43rs: ( 16 ) Ifull , h ( i ) =Vc , h ( i ) IEoc , h ( i ) , whereVc , h ( i ) =43rs , h ( i ) .", "Note that Ifull , h ( i ) will be on an arbitrary scale , and appropriate scaling methods may be applied to place the data on a quasi-absolute scale prior to structure determination and refinement , as is done for conventional rotation data .", "We refine image m by first minimizing the target function: ( 17 ) Tpr=∑h∑iWh ( i ) ( Ih ( i ) −GmEoch ( i ) Vc , h−1 ( i ) Ih ) 2 , where1/Wh ( i ) =σh2 ( i ) , and the scale function Gm comprises a linear scale factor G0 and a B-factor: ( 18 ) Gm=G0 , me−2Bm ( sin θh ( i ) /λm ) 2 .", "We apply a spot position restraint as a second target function in subsequent steps during a microcycle using the x , y positions determined by the spot-finding step of data processing ( Hattne et al . , 2014; Kabsch , 2014 ) .", "( 19 ) Txy=∑h∑i ( xhobs ( i ) −xhcalc ( i ) ) 2 , where xhobs ( i ) and xhcalc ( i ) are the observed and calculated spot centroids , respectively .", "The Levenberg–Marquardt ( LM ) algorithm from the scipy python library ( Oliphant , 2007 ) , which is a combination of the gradient descent and the Gauss–Newton iteration , is used to minimize the target function residuals .", "The refinement of the unit-cell parameters ( a , b , c , α , β , γ ) takes crystal symmetry constraints into account to make the procedure more robust .", "After these iterative refinement cycles are complete , we apply the refined parameters to the reflection intensities of each still , and then merge the same reduced Miller indices ( from all stills ) to obtain the zero-offset still intensities , which are used for the new reference intensity set ( see next section ) .", "At each step in a microcycle , the user can select reflections that are used for post-refinement of a parameter group using the following criteria: resolution range , signal strength ( I/σ ( I ) ) , and the Ewald offset correction value .", "In addition to these selection criteria , deviations from the target unit-cell dimensions ( specified as a fraction of each dimension ) can also be used in the merging step so that only diffraction patterns with acceptable unit-cell dimension values are included in the merged reflection set .", "Each post-refinement parameter group can have its own separate set of reflection selection criteria .", "Starting from the observed intensities , we obtain the full-volume intensity , Ifull , h ( i ) , from IEoc , h ( i ) by first applying the Ewald offset correction ( Equation 15 ) and then the full-intensity correction ( Equation 16 ) .", "Prior to merging equivalent observations , we detect outliers using an iterative rejection scheme , discarding reflections with intensity more or less than a user-specified cutoff ( 3 σ default , where σ is defined as the standard deviation of the distribution of the full reflections Ifull , h ) .", "Finally , in order to obtain the merged reflection set , we calculate 〈Ih〉 from the intensity of reflections with the same reduced Miller indices using the sigma-weighted average: ( 20 ) 〈Ih〉=∑iWh ( i ) I full , h ( i ) ∑iWh ( i ) , whereWh ( i ) =1σ2 ( i ) [I full , h ( i ) ] , and σ ( i ) [I full , h ( i ) ] is derived from the calculation of error: ( 21 ) ( ΔIfull , h ( i ) Ifull , h ( i ) ) 2= ( ΔIh ( i ) Ih ( i ) ) 2+ ( ΔGG ) 2+ ( ΔEocEoc ) 2 .", "Since G is a function of G0 and B , and Eoc is a function of crystal orientation , mosaicity , and unit-cell parameters , the error estimates for G can be further calculated as: ( 22 ) ΔG2= ( ∂G∂G0 ) 2ΔG02+ ( ∂G∂B ) 2ΔB2 , and ΔEoc2 can be calculated similarly by summing all over products of partial derivatives and errors estimated for each parameter in the Eoc function ( square root of the diagonal elements of the covariance matrix ) .", "We use CC1/2 as a quality indicator for the diffraction data sets ( Diederichs and Karplus , 2013 ) .", "We calculate CC1/2 by randomly partitioning all ( partial ) intensity observations of a given reflection into two groups .", "We reject any reflections with fewer than four observations; for all other reflections , we merge the observations in each group using Equation 20 .", "CC1/2 is then calculated as the correlation between these two independently merged diffraction data sets .", "Let ( 23 ) g=1σ ( I−G0e−2B ( sin θ/λ ) 2EocVc−1I ) , for observed partial intensity i of miller index h .", "The XFEL beam is nearly 100% polarized in the horizontal direction .", "The optics at both the LCLS XPP and CXI stations do not introduce additional polarization .", "To account for the polarization of the primary beam , for a given reflection , we consider the angle ϕ between the sample reflection plane formed by the h vector and the -z-axis , and the laboratory horizontal ( Figure 14 ) . 10 . 7554/eLife . 05421 . 020Figure 14 . Geometry of the incident and diffracted beam for polarization correction . The diagram shows a reflection on a plane formed by its reciprocal-space vector and the -z-axis at angle ϕ .", "This reflection is affected by the polarization of the incoming primary beam in both the horizontal", "( x ) and vertical", "( y ) directions . DOI: http://dx . doi . org/10 . 7554/eLife . 05421 . 020 As described in Kahn et al . ( 1982 ) , the beam I0 incident on the sample crystal can be described in terms of two components , one parallel ( σ ) and the other perpendicular ( π ) to the plane of reflection: ( 29 ) I0=Iσ+Iπ .", "Each of these components is affected by the polarization of the primary beam in both the horizontal", "( x ) and vertical", "( y ) directions .", "Using fx and fy as the fractions horizontal and vertical in the laboratory frame ( fx + fy = 1 ) , ( 30 ) Iσ= ( fx cos2 ϕ+fy sin2 ϕ ) I0 , and ( 31 ) Iπ= ( fx sin2 ϕ+fy cos2 ϕ ) I0 , where fx and fy are the polarization fractions in the x and y directions .", "After reflection , only Iσ is attenuated: ( 32 ) I′=I′π+ I′σ= |F|2 ( Iπ+Iσ cos22θ ) .", "By substituting Iσ and Iπ from Equations 30 and 31 in Equation 32 , we arrive at ( 33 ) I′=|F|2[fx ( sin2 ϕ+cos2 ϕ cos22θ ) +fy ( cos2 ϕ+sin2 ϕ cos22θ ) ]I0 , where the bracketed expression is P ( Kahn et al . , 1982 ) .", "To ensure atomic model refinements against the various diffraction data sets were as comparable as possible , we used a standard semi-automated solution and refinement protocol .", "First , we performed molecular replacement phasing with known structures as search models ( PDB ID 3U3E for myoglobin , 3C8Y for hydrogenase , and 2TLI for thermolysin ) with all heteroatoms , water molecules , and ligands removed .", "Molecular replacement was carried out with Phaser ( McCoy et al . , 2007 ) using default settings , with r . m . s . d . set to 0 . 8 .", "The resulting solutions were then refined using phenix . refine ( Afonine et al . , 2012 ) in two cycles .", "In the first cycle , we carried out rigid body refinement , positional ( xyz ) refinement with automatic correction of Asn , Gln and His sidechain orientations , and atomic displacement parameter ( ADP ) refinement .", "We then used the difference density maps for missing ligands and heteroatoms obtained from this cycle to calculate real-space correlation coefficients using phenix . get_cc_mtz_pdb from the PHENIX software suite ( Adams et al . , 2010 ) for myoglobin and thermolysin and the program ‘Map Correlation’ from the CCP4 software ( Winn et al . , 2011 ) for hydrogenase .", "These omit difference density maps are shown in Figures 6 , 7 , 8 , 10 .", "In the second cycle , all ligands and heteroatoms were placed in the difference density maps and combined with the refined structure from the first cycle using Coot ( Emsley et al . , 2010 ) .", "The second cycle employed positional and ADP refinement with target weights optimization and water update was carried out with these complete models .", "The structures were validated by MolProbity ( Chen et al . , 2010 ) .", "Final refinement statistics ( Tables 2 , 3 , 4 ) were analyzed with phenix . polygon ( Urzhumtseva et al . , 2009 ) and found to be within acceptable range for other structures at similar resolutions .", "For the thermolysin structure obtained from anomalous diffraction data ( processed keeping Friedel pairs separate ) , only one cycle of atomic model refinement was carried out .", "All figures were made in PyMOL ( The PyMOL Molecular Graphics System , Version 1 . 5 . 0 . 4 Schrödinger , LLC . ) .", "The computer program , prime , is implemented as a part of the cctbx computational crystallography toolbox ( Grosse-Kunstleve et al . , 2002 ) .", "Download and installation instructions are available on the cctbx website ( http://cctbx . sourceforge . net ) .", "Subsequent to acceptance of this article , a paper was published by Ginn et al . ( 2015 ) describing an alternative method for orientation refinement as compared to the method of Sauter et al . ( 2014 ) , and partiality estimation for each individual image , but without post-refinement ." ] ]
[ "There is considerable potential for X-ray free electron lasers ( XFELs ) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources .", "Prior XFEL studies often involved the collection of thousands to millions of diffraction images , in part due to limitations of data processing methods .", "We implemented a data processing system based on classical post-refinement techniques , adapted to specific properties of XFEL diffraction data .", "When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters , our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps .", "Moreover , the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations .", "These developments will help expand the applicability of XFEL crystallography to challenging biological systems , including cases where sample is limited ." ]
[ "Large biological molecules ( or macromolecules ) have intricate three-dimensional structures .", "X-ray crystallography is a technique that is commonly used to determine these structures and involves directing a beam of X-rays at a crystal that was grown from the macromolecule of interest .", "The macromolecules in the crystal scatter the X-rays to produce a diffraction pattern , and the crystal is rotated to provide further diffraction images .", "It is then possible to work backwards from these images and elucidate the structure of the macromolecule in three dimensions .", "X-ray beams are powerful enough to damage crystals , and scientists are developing new approaches to overcome this problem .", "One recent development uses ‘X-ray free electron lasers’ to circumvent the damage caused to crystals .", "However , early applications of this approach required many crystals and thousands to millions of diffraction patterns to be collected—largely because methods to process the diffraction data were far from optimal .", "Uervirojnangkoorn et al . have now developed a new data-processing procedure that is specifically designed for diffraction data obtained using X-ray free electron lasers .", "This method was applied to diffraction data collected from crystals of three different macromolecules ( which in this case were three different proteins ) .", "For all three , the new method required many fewer diffraction images to determine the structure , and in one case revealed more details about the structure than the existing methods .", "This new method is now expected to allow a wider range of macromolecules to be studied using crystallography with X-ray free electron lasers , including cases where very few crystals are available ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience", "genetics and genomics" ]
Co-expression of xenopsin and rhabdomeric opsin in photoreceptors bearing microvilli and cilia
elife-23435-v2
[ [ "Animal eyes are amongst the best-investigated sensory organs and are subject to studies not only in sensory biology , but also in general molecular , developmental and evolutionary biology .", "Comparative investigations yield insights into the diversity of light-detection mechanisms and their function across animals .", "Further , the integrative data sets on eyes and their cellular components has had considerable impact on the characterization of different kinds of light-detecting cells , and on concepts how to trace their evolution and that of the organs they constitute ( Arendt , 2003; Arendt et al . , 2016; Oakley and Speiser , 2015; Wagner , 2014 ) .", "These studies have dealt with the general question of whether gradual change or rather integration and reshuffling of modules that also exist elsewhere is the main driving force in the evolution of light-detecting cell types .", "Traditionally , photoreceptor cells ( PRCs ) have been characterized mainly by their general electrophysiological response and their fine structure , which reveal clear differences between the two main systems of interest , the eyes of insects and those of vertebrates .", "The PRCs in insects have been shown to enlarge the sensory area by microvilli and to depolarize in response to light , whereas vertebrate eyes are hyperpolarizing and bear a modified cilium ( Eakin , 1963; 1968 , 1979 ) .", "These findings initiated a wealth of comparative studies and resulted in pro-longed debates on whether a few conserved lineages of animal eye PRCs exist or whether both those PRCs andwhole eyes evolved multiple times independently ( Eakin , 1982; von Salvini-Plawen , 1982; Vanfleteren , 1982 ) .", "Subsequent data on molecular physiology and transcriptional regulation of cell specification allowed much more detailed characterization of PRCs , suggesting that two conserved PRC types are indeed the main sensory components of animal cerebral eyes:", "( a ) microvillar depolarizing r-opsin-expressing PRCs , which signal via the Gq-mediated IP3 cascade , and", "( b ) ciliary hyperpolarizing c-opsin-expressing cells , which signal via the Gi/t-mediated cGMP cascade ( Arendt et al . , 2002a , 2004; Fain et al . , 2010; Fernald , 2006; Gehring , 2014 ) .", "This evolution-based classification of PRCs became a sound basis for many kinds of comparative eye research and for ideas about how animal eyes evolved .", "The r-opsin+ type of PRCs constitutes the main visual elements in many protostome cerebral eyes , but they are also present in the ganglion cell layer of vertebrate eyes , where they are involved in the pupillary reflex and the entrainment of the circadian clock ( Hattar et al . , 2003; Koyanagi et al . , 2005; Panda et al . , 2002 ) .", "Ciliary c-opsin+ PRCs on the other hand are predominantly known as the visual PRCs of vertebrate eyes and the frontal eye of cephalochordates ( Vopalensky et al . , 2012 ) , while their role in protostomes is controversially discussed .", "In protostomes , c-opsins were initially reported only in arthropod and annelid deep brain photoreceptors ( Arendt et al . , 2004; Velarde et al . , 2005 ) .", "It was assumed , therefore , that this visual pigment was ancestrally employed by unpigmented brain PRCs that were fulfilling non-visual functions and were only secondarily integrated into the eyes of chordates ( Arendt et al . , 2004; Vopalensky et al . , 2012 ) .", "Reports on the presence of ciliary PRCs and c-opsins in the eyes of protostomes challenged this view and also the concept of gradually evolving ancient PRC types in animal eyes .", "Accordingly , a rather flexible employment of different opsin types by eye photoreceptor cells or frequent gain and loss of photoreceptor cell types in cerebral eyes during the evolution of bilaterian animals were suggested ( Passamaneck et al . , 2011 ) .", "Advances in opsin phylogeny , however , suggest that several protostome sequences , which share characteristic sequence motifs with c-opsins and were initially classified as such , fall into their own group , now termed xenopsins ( Ramirez et al . , 2016 ) .", "In this situation , it is highly desirable to obtain deeper insights into the evolutionary history of c-opsins and xenopsins and their employment in animal photoreceptor cells , especially in those of protostome invertebrates .", "By deeply mining public and new data , we find xenopsins to be present in several taxa of protostome invertebrates .", "Although we did not find even a single case of co-existence of c-opsins and xenopsins in any organism , our phylogenetic and gene structure analyses strongly suggest that xenopsins and c-opsins are two distinct opsin subgroups .", "A new xenopsin from Leptochiton asellus , a member of the basally branching mollusk group of chitons , is found to be co-expressed with r-opsin in PRCs bearing both microvilli and cilia .", "This raises interesting questions about the light transduction and molecular physiology of the cells and their evolutionary origin .", "Recompilation of existing and new data challenges the dichotomous distinction of bilaterian eye PRCs into ciliary c-opsin+ and microvillar r-opsin+ cell types , but nevertheless supports the view that conserved lineages of eye PRCs exist .", "We suggest that a highly plastic PRC type of mixed microvillar/ciliary organization and frequent lineage-specific loss of c-opsins and xenopsins were important players in animal eye evolution ." ], [ "By screening RNA-seq data of Leptochiton asellus , we detected a sequence showing clear opsin characteristics .", "This was substantiated by reciprocal BLAST as well as domain and motif analyses .", "The sequence possesses the Pfam tm7_1 domain , Lys296 ( referring to homologous bovine opsin positions ) , which is predictive for opsin chromophore binding , as well as the NPXXY motif and the tripeptide motif on positon 310–312 crucial for G-protein activation ( Figure 1D ) .", "The latter matches the NKQ motif , which is characteristic for c-opsins but is also present in some sequences of the recently described group of xenopsins ( Ramirez et al . , 2016 ) and a few cnidops .", "Correct placement of the new L . asellus opsin is highly relevant for our study and , according to Ramirez et al . ( 2016 ) , several sequences initially described as c-opsins fall into xenopsins .", "We therefore performed a thorough phylogenetic analysis with a main focus on the relationship of c-opsins and xenopsins .", "For this purpose , we ran both maximum-likelihood and Bayesian analyses to check for congruence of different tree inference algorithms , we chose closely related representatives from amongst rhodopsin-like G-Protein coupled receptors as an outgroup , and we extensively tested standard and data-set specific ( DS-GTR ) evolutionary models for appropriateness .", "In order to increase the taxon sampling of potential c-opsins and xenopsins in protostomes , we mined publicly available genomic and transcriptomic resources and de novo assembled transcriptomes from publicly available raw Illumina RNA-seq data from 9 nemerteans , 10 flatworms , 7 aceoelomorphs and Xenoturbella and from new RNA-seq data from the annelid Owenia fusiformis .", "An initial maximum-likelihood run with RAxML yielded low support for many basal branches .", "Thus , we performed a leaf-stability analysis based on the bootstrap results to identify rogue sequences .", "One sequence of Strongylocentrotus purpuratus ( opsin2 ) and one of the brachiopiod Lingula anatina ( peropsin A ) as well as ctenopsin sequences exhibited leaf-stability values less than 0 . 55 .", "As we were interested in whether ctenopsins are closely allied to c-opsins and xenopsins , only the opsin2 and peropsin A sequences from S . purpuratus and L . anatina were removed from the dataset .", "Subsequent maximum-likelihood and Bayesian runs yielded nearly identical tree topologies .", "In line with the study by Ramirez et al . ( 2016 ) , c-opsins and xenopsins form distinct and highly supported groups ( Figure 2 , Figure 2—figure supplement 1 ) .", "The new opsin from L . asellus groups with high support within xenopsins and is thus termed L . asellus xenopsin .", "In addition to the xenopsins reported by Ramirez et al . ( 2016 ) from mollusks , brachiopods and rotifers , we discovered this opsin type in Owenia fusiformis , a member of the basally branching annelid group of Oweniidae , in several polyclad flatworms and in the triclad flatworm Schmidtea mediterranea .", "By contrast , no previously unidentified c-opsin sequence showed up in any new major animal group .", "Other groups might be devoid of both xenopsins and c-opsins .", "We did not find any c-opsin or xenopsin in the nine screened RNA-seq datasets from acoelomorphs or xenoturbellids .", "Although reasonable numbers of opsins were found in some of those species , reciprocal blast suggests that these proteins are mainly related to r-opsins .", "The situation is more ambiguous in nemerteans .", "From the nine screened RNA-seq datasets , we found higher numbers of opsin sequences only in Lineus longissimus , but no hints that there are c-opsins or xenopsins in any nemertean species .", "The low yield of opsin sequences may reflect the real situation or may be due to low sequencing coverage or the tissue samples taken .", "Further , our data suggest that xenopsins diversified at an early stage of protostome evolution .", "Xenopsin paralogs of several platyhelminth species are distributed across two well-supported xenopsin subgroups , one of which also contains sequences from many other protostome taxa ( Figure 2 , Figure 2—figure supplement 1 ) .", "Notably , the tripeptide motif that is crucial for G-protein activation shows a pattern similar to that found in c-opsins ( NKQ ) within that xenopsin subgroup , which has a broad taxonomic distribution , and in the sequence of Idiosepius paradoxus , while this pattern differs considerably in other xenopsins ( Figure 1—figure supplement 1 ) .", "Ramirez et al . ( 2016 ) reported that xenopsins were secondarily lost in several major animal groups .", "Our data support this view and the same is obviously true for c-opsins .", "Thus , we specifically compared the presence of c-opsins and xenopsins across bilaterian animals .", "Even though genomic resources were screened for many species , stunningly we could not recover a single species possessing both c-opsins and xenopsins .", "This also holds true for higher taxonomic levels , since as sequences of just one of the two opsin groups were found in deuterostomes and in major protostome clades such as mollusks , rotifers , brachiopods , flatworms , arthropods and tardigrades .", "As the only exception , we found xenopsins ( but no c-opsins ) in the annelid Owenia fusiformis , whereas other annelids such as Platynereis dumerilii possess only c-opsins or lack both types as do Capitella teleta or Helobdella robusta ( Figure 2 , Figure 2—figure supplement 1 ) .", "Such a mutually exclusive taxonomic distribution of protein subfamilies is difficult to explain from an evolutionary perspective and raises the question of whether the distinction of xenopsins and c-opsins may be caused by tree inference artifacts .", "Thus we performed gene structure analyses in order to obtain independent information on this matter .", "Position and phase of introns were determined for all those sequences for which genomic information was publicly available and the new sequences for L . asellus opsin and P . dumerilii c-opsin , for which the whole gene sequences cloned from genomic DNA .", "The obtained data were than mapped onto the un-curated protein alignment to identify introns located in homologous sequence positions ( Figure 2 , Figure 2—figure supplement 2 ) .", "This revealed highly conserved and specific exon–intron patterns for many major opsin groups .", "The patterns differ considerably between xenopsins and c-opsins .", "Three introns are highly conserved in position and phase ( Figure 2 , Figure 2—figure supplement", "2 ) and phase ( Figure 2—figure supplement", "3 ) throughout protostome c-opsins , deuterostome encephalopsins , TMT opsins , VA-opsins , parapinopsins and pinopsins , and the vertebrate visual opsins .", "By contrast , all xenopsins share two different intron positions with conserved intron phase ( Figure 2—figure supplements 2 and 3 ) , with the exception of one sequence from Adineta vaga .", "While the second xenopsin intron position is clearly distinct from those of any c-opsin intron , the first xenopsin intron has a position similar to that of the second c-opsin intron .", "In order to investigate whether the minor positional difference between these two c-opsin and xenopsin introns are potentially caused by alignment artifacts rather than independent evolutionary origin , we mapped the gene structures onto several different alignments obtained with MAFFT- E-INS-i , MAFFT-G-INS-I , GLProbs , ProbCons and MUSCLE .", "In all cases , the three conserved c-opsin and two xenopsin introns are recovered .", "The position of the second c-opsin intron is not stable only in the MUSCLE alignment .", "This coincides with the poor quality of the MUSCLE alignment in this region when compared to the other alignment variants .", "The xenopsin and c-opsin intron positions do not overlap in any of the alignments .", "Thus , gene structure provides strong evidence that xenopsins and c-opsins are indeed distinct opsin subgroups , irrespective of their mutually exclusive distribution amongst bilaterian animals .", "Due to the lack of introns in cnidops , the gene structure analyses cannot provide further support for the sistergroup relationship of cnidops and xenopsins .", "Likewise , ctenopsins show a very characteristic gene structure and subdivision into two groups , but no clear similarities to c-opsins or xenopsins exist .", "Notably , Strongylocentrotus purpuratus opsin2 and Lingula anatina peropsin-like A , which were excluded from the current analysis due to low leaf stability , share conserved introns ( Figure 2—figure supplement 2 ) , strongly corroborating the very small but ancient group of bathyopsins reported by Ramirez et al . ( 2016 ) .", "In order to investigate the expression pattern of Las-xenopsin , we performed in-situ hybridization analyses in different larval stages of L . asellus .", "This revealed expression in the posttrochal eye region ( Figures 1B and 3F ) as well as in cells at the very apical ( Figure 1B inset ) and posterior ends of the larva ( Figure 1C ) , an expression pattern strongly resembling that of Las-ropsin ( Vöcking et al . , 2015 ) .", "Double in-situ hybridization unambiguously showed cellular coexpression of Las-xenopsin and Las-ropsin in all PRCs ( Figures 3F and 4A ) .", "The eye PRCs of L . asellus have been shown to be largely of rhabdomeric organization and the r-opsin protein is most likely localized in the microvillar extensions ( Vöcking et al . , 2015 ) .", "The only xenopsin with known expression site is from the brachiopod T . transversa .", "It was originally described as a c-opsin and is expressed in the ciliary PRCs of the larval eyes .", "For this reason , we searched for indicators of cilia development in the PRCs of L . asellus by means of in-situ hybridization , i . e . we looked for the transcription factors Foxj1 and RFX , which are involved in ciliary development in different animals ( Alten et al . , 2012; Choksi et al . , 2014; Dubruille et al . , 2002; Yu et al . , 2008 ) .", "Both are expressed in the PRCs of 7 dpf L . asellus larvae ( Figure 4G , H , Figure 4—figure supplement 1 ) and Las-Foxj1 was also found to be expressed in the prototroch cells and the apical organ of younger larvae ( Figure 4—figure supplement 1 ) .", "As this points towards the formation of cilia by the PRCs , we screened the apical surface of the PRCs for cilia by means of electron microscopy ( Figure 3A–E ) .", "Indeed , we found two cilia in each PRC in addition to the many microvillar extensions ( Figure 3A , C , D ) , corroborating ultrastructural data from other chitons ( Bartolomaeus , 1992; Fischer , 1980; Rosen et al . , 1979 ) .", "The cilia show a 9 × 2 + 2 microtubule pattern ( Figure 3C inset ) .", "Neither the cilia nor the microvillae exceed the cuticle nor exhibit specialized surface extensions ( Figure 3A , C ) , but they are longer than the rudimentary cilia found in surrounding epithelial cells and do not taper ( Figure 3A , C , E ) .", "As we were interested in whether the cells feature a mechanism to transport opsin into cilia , we looked into transporters that are known to be involved in ciliary opsin targeting .", "Although the C-terminal VxPx motif known for cargo binding in vertebrate ( but not protostome ) c-opsin is not conserved in xenopsins ( Figure 4—figure supplement 2 ) , several ortholgos of the general players of ciliary targeting that are also active in ciliary opsin targeting , namely Las-Arf4 , Las-rab8 , Las-FIP3 , Las-RPGR and Las-MyosinVIIa ( Liu et al . , 1999; Wang and Deretic , 2014 , 2015; Wang et al . , 2012 ) , were found to be specifically expressed in the PRCs by double in-situ hybridization with Las-r-opsin ( Figure 4B–F , Figure 4—figure supplements 3–5 ) ." ], [ "In vertebrates , c-opsins drive the important process of vision , as they constitute the visual pigments of the retinal rods and cones and they serve additional functions when expressed in the pineal or in deep brain PRCs .", "Thus , the identification of c-opsins in protostome invertebrates provided the possibility to explore the role of c-opsin+ PRCs in distant relatives and to uncover the evolutionary roots of vertebrate vision .", "For a long time , only very few protostome c-opsins were known and these unambiguously group within the clade of c-opsins .", "This narrowed down the choice of potential study subjects .", "Basically , there were few pteropsins from arthropods and one sequence from the annelid Platynereis dumerilii ( Arendt et al . , 2004; Eriksson et al . , 2013; Velarde et al . , 2005 ) .", "In addition , new protostome sequences , which resemble c-opsins in certain aspects , were not instantly included in broad opsin analyses and this led to weakly supported orthology assignments that suggested a broader distribution of c-opsins in protostome invertebrates .", "Seemingly , this is not the case .", "Initial evidence that the new sequences do not encode c-opsins , but are closer affiliated to cnidops , has been provided by Hering and Mayer ( 2014 ) , though with low support .", "This new group included sequences of hitherto uncharacterized visual pigments from the mollusks Crassostrea gigas and Lottia gigantea , a sequence from the brachiopod T . transversa that originally was described as c-opsin ( Passamaneck et al . , 2011 ) , and a weakly associated sequence from Strongylocentrotus purpuratus ( opsin2 ) that did not group with other major opsin sequence groups in other analyses ( D'Aniello et al . , 2015; Ooka et al . , 2010; Raible et al . , 2006 ) .", "Probably because of the increased taxon sampling and formal tree reconciliation , the recent phylogenetic study of Ramirez et al . ( 2016 ) provides for the first time strong support for a new group of xenopsins , which are distinct from c-opsins .", "We show that irrespective of the remarkable mutually exclusive taxonomic distribution of xenopsins and c-opsins , this distinction does not rely on tree inference artifacts , as can be seen from the specific gene structures of both opsin types .", "Our data reveal a broader occurrence of xenopsins in protostomes than hitherto anticipated , pointing towards a high relevance of this opsin type in animal physiology .", "Accordingly , xenopsins are present not only in mollusks , rotifers and brachiopods as already reported by Ramirez et al . ( 2016 ) , but also in several flatworms and in Oweniidae , the first branch within annelids .", "The situation in nemerteans is not clear because the screened RNA-seq datasets are probably not representative due to the low total number of opsins found .", "In accordance with Ramirez et al . ( 2016 ) , we have no evidence that xenopsins exist in deuterostomes .", "Strongylocentrotus purpuratus opsin2 is the only deuterostome sequence that has ever been suggested to show affinities to sequences now classified as xenopsins ( Hering and Mayer , 2014 ) .", "However , the high similarity of the gene structure to that of Lingula anatina peropsin A strongly supports a position of both sequences in the ancient small group of bathyopsins sensu Ramirez et al . ( 2016 ) , which exclusively comprises only a few echinoderm and brachiopod sequences .", "Nevertheless , the data provide clear evidence that xenopsins were already present in the last common ancestor of deuterostomes and protostomes .", "In the analysis of Ramirez et al . ( 2016 ) and in our study , xenopsins form the sister group to cnidops , and this may very likely reflect the evolutionary history .", "As the basal interrelationships of the major opsin groups were shown to be strongly affected by outgroup composition , taxon sampling , tree inference algorithms and parameters ( compare Cronin and Porter , 2014; Feuda et al . , 2012 , 2014; Hering and Mayer , 2014; Porter et al . , 2012 ) , this hypothesis may , however , need further investigation .", "Unfortunately , gene structure data are not informative due to the lack of introns in cnidops .", "For this reason and in contrast to Ramirez et al . ( 2016 ) , we apply the term xenopsin only to bilaterian sequences and not to cnidops .", "Nevertheless , the assumption that xenopsins emerged within protostomes would require a sister group relationship between xenopsins and another clade of protostome-specific opsins .", "This would need major rearrangements of opsin tree topology and is not supported by gene structure data .", "In the ( unlikely ) scenario that xenopsins do not turn out to be sister to cnidops , it remains to be determined whether xenopsins were already present in the ancestor of Bilateria or emerged in the last common ancestor of protostomes and deuterostomes .", "This depends on the final phylogenetic position of acoelomorphs in relation to Bilateria and on further upcoming sequence data .", "We could not detect any xenopsin or c-opsin in the screened RNA-seq data from seven acoelomorphs and one xenoturbellid .", "According to our data , frequent lineage-specific loss of either xenopsins or c-opsins ( or both ) during animal evolution has to be assumed .", "To date , no explanation can be given as to why the two opsin groups do not co-occur in a single organism .", "The intriguing possibility remains that an underlying causal relation drove the evolution of a large protein family that way .", "This will be an interesting case to study on the molecular physiological and the genomic levels .", "The observed co-expression of xenopsin and r-opsin in the eye PRCs of L . asellus is remarkable from a functional perspective .", "As a general observation , the vast majority of PRCs express only one type of visual opsin .", "In nearly all cases where more than one visual opsin was found in PRCs , the respective opsins belong to the same opsin subgroup and evoke the same phototransduction cascade .", "This has been shown for r-opsins in several arthropods , as for example in the eye PRCs of Limulus polyphemus ( Katti et al . , 2010 ) , fiddler crabs ( Rajkumar et al . , 2010 ) and a butterfly ( Arikawa et al . , 2003 ) , and for c-opsins in for example mice ( Applebury et al . , 2000 ) , guinea pigs ( Parry and Bowmaker , 2016 ) , salamander ( Isayama et al . , 2014 ) and cichlid fishes ( Dalton et al . , 2015 ) .", "In most cases , expansion or tuning of the visual spectrum is the suggested function .", "Co-expression of opsins belonging to different , evolutionary distinct opsin types is reported only very rarely , as in the case of the annelid P . dumerilii , where Go-opsins and r-opsin are employed in eye PRCs ( Gühmann et al . , 2015 ) .", "Here , the main function likewise seems to be the expansion of the visual spectrum of the cells .", "Our findings in L . asellus point towards another direction .", "The L . asellus eye PRCs express all relevant elements of standard r-opsin-mediated GNAQ-dependent IP3 signaling ( Vöcking et al . , 2015 ) .", "By contrast , the NKQ tripeptide motif present in L . asellus xenopsin is known to be involved in the GNAI- and cGMP-based signaling ( Marin et al . , 2000 ) of c-opsins and is similar to motifs in cnidops , some of which were shown to mediate GNAS- and cAMP-based signaling ( Koyanagi et al . , 2008; Liegertová et al . , 2015 ) .", "The eye PRCs might thus be able to integrate different light signals directly .", "To our knowledge , the only case known in which co-expressed opsins evoke competing signaling cascades is in the pineal of the lizard Uta stasburiana ( Su et al . , 2006 ) .", "Functional analysis of the molecular physiology of the L . asellus eye PRCs may thus be of great interest to sensory biologists .", "Xenopsins are seemingly able to enter cilia .", "This type of opsin is employed in the purely ciliary PRCs of the larval eyes of the brachiopod Terebratalia transversa ( Passamaneck et al . , 2011 ) .", "We find it expressed in eye PRCs in L . asellus , whose microvilli employ r-opsin ( Vöcking et al . , 2015 ) but which also have cilia .", "The expression of Arf4 , rab8 , FIP3 and RPGR provides the possibility that these proteins are involved in xenopsin trafficking , as they are in c-opsin ciliary trafficking in vertebrate rods and cones ( Liu et al . , 1999; Wang and Deretic , 2014 , 2015; Wang et al . , 2012 ) .", "As the C-terminal VxPx motif necessary for the Arf4-mediated transport of vertebrate c-opsins ( Wang and Deretic , 2014 ) is missing in xenopsins , Arf4-mediated transport of xenopsin would require other protein interactions .", "Interestingly , the same holds true for ciliary transport of cnidops and of c-opsins from organisms other than vertebrates and tunicates , as the VxPx motif is only conserved in the latter opsin groups and not in cephalochordate , echinoderm or protostome c-opsins or cnidops , some of which have been shown to be expressed in ciliary PRCs ( Arendt et al . , 2004; Bielecki et al . , 2014; Kozmik et al . , 2008; Vopalensky et al . , 2012 ) .", "It may be that Arf4 binding to c-opsins other than those of vertebrates and tunicates , xenopsins and cnidops relies on other protein interactions as in the case of vertebrate c-opsins .", "As an alternative , Arf4-complex-mediated ciliary opsin trafficking has to be regarded as a chordate invention .", "Our extensive data mining did not change the picture that arthropods and certain annelids are the only organisms in which insights can be obtained on the physiological role of the protostome counterparts of vertebrate visual pigments .", "C-opsins have been found in no other protostomes .", "As outlined above , xenopsin-employing PRCs are promising study subjects for many other questions in sensory biology .", "Furthermore , we propose that data on the employment of xenopsins have significant impact on the understanding of the evolution of animal eyes and of photoreceptor cell types in general .", "As discussed below , we regard the presence of xenopsins in ancestral eyes as a likely scenario .", "This implies that the occurrence of xenopsins in the eyes of protostomes is not due to the flexible recruitment of opsins in existing eyes during evolution or to frequent invasion of new kinds of photoreceptor cells into eyes , but rather to inherited employment in an ancestral eye photoreceptor cell type .", "The wide distribution of r-opsin+ cells in the eyes of protostomes and deuterostomes , alongside the reported similarities in molecular physiology and the process of cell specification , have been interpreted by many studies as a sign of common evolutionary origin and the presence of r-opsin+ rhabdomeric PRCs in the eye of the last common ancestor of bilaterians ( Arendt , 2008; Arendt et al . , 2002a; Koyanagi et al . , 2005; Lamb , 2013; del Pilar Gomez et al . , 2009 ) .", "Data on c-opsins , however , were interpreted in different ways .", "The pivotal role that c-opsins play in the rods and cones of vertebrate eyes is obvious and findings from the cephalochordate frontal eye suggest that c-opsin+ ciliary PRCs were the predominant eye sensory cells already in the last common ancestor of chordates ( Vopalensky et al . , 2012 ) .", "In protostomes , c-opsins were initially described only in the brain ( Arendt et al . , 2004; Beckmann et al . , 2015; Velarde et al . , 2005 ) , which corresponds to their broad occurrence in the brain of basally branching deuterostome c-opsins such as encephalopsins ( Blackshaw and Snyder , 1999; Nissilä et al . , 2012 ) .", "Thus , it has been suggested that c-opsin+ PRCs did originally not form part of eyes , but were rather localized deeply in the brain and not associated with screening pigment ( Arendt , 2008; Arendt et al . , 2004; Lamb , 2013 ) .", "They secondarily invaded the eyes of chordates , but stayed within the brain in protostomes .", "The presence of ciliary PRCs revealed by structural studies of the eyes of some protostome groups ( Purschke et al . , 2006; Salvini-Plawen , 2008; Woollacott and Eakin , 1973; Woollacott and Zimmer , 1972 ) would contradict this scenario if those cells showedmolecular characteristics similar to those of c-opsin+ brain PRCs .", "Accordingly , the discovery of a c-opsin that is expressed in ciliary eye PRCs of a brachiopod put forward alternative explanations favoring a higher lability of PRC function and multiple independent recruitments of r-opsin+ and c-opsin+ PRCs for directional vision in animal eye evolution ( Passamaneck et al . , 2011 ) .", "These data conflicts are resolved by the fact that all c-opsins that were previously reported from the visual cells of protostome eyes turned out to be xenopsins instead of c-opsins , according to Ramirez et al . ( 2016 ) and our study .", "The only exception relies on a PCR-based report of a true c-opsin in an onychophoran eye ( Eriksson et al . , 2013 ) , but this is contradicted by a cellular expression analysis by Beckmann et al . ( 2015 ) showing that c-opsin is absent from the eye retina layer and only present deeply within the brain and the optical ganglia .", "By contrast , all existing data on the expression of xenopsins point towards their presence in eyes .", "The opsin reported from brachiopod larval eyes ( Passamaneck et al . , 2011 ) is a xenopsin .", "We detected xenopsin in eye PRCs of a mollusk and in a few extraocular PRCs , which are regarded as derivatives of cerebral eyes ( Vöcking et al . , 2015 ) .", "RNA-seq data from cephalopods likewise suggest the presence of xenopsins in eyes ( Yoshida et al . , 2015 ) .", "One option to explain the appearance of xenopsin in protostome eyes is to assume repeated recruitment of xenopsins into this context .", "This implies the co-option of xenopsin by ancestral microvillar r-opsin+ eye PRCs in eyes like those of L . asellus .", "In purely ciliary eyes such as those in brachiopods , either the formerly r-opsin+ cells completely changed identity or the original PRCs were replaced by new ciliary xenopsin+ PRCs , or the whole eyes arose completely de novo while the ancestral microvillar eyes were reduced .", "Abrupt changes and new inventions would be important and frequent events in eye evolution .", "Alternatively , assuming the presence of both r-opsin and xenopsin in ancestral eyes provides probably a simpler explanation ( Figure 5 ) .", "Notably , this is not in conflict with the absence of xenopsins in the eyes of well-studied protostomes such as arthropods , or certain annelids , as secondary loss of xenopsin in the respective lineages is clearly evident from sequence resources and opsin phylogeny .", "R-opsin and xenopsin may initially have been employed in different cells , but a very attractive hypothesis is that cellular co-expression of r-opsin and xenopsin , like that which we observed in the eyes of L . asellus , is not an exceptional case but is inherited from ancestral eye PRCs that employed both opsins and exhibited microvilli and cilia ( Figure 5 ) .", "The cerebral eye PRCs of mixed microvillar/ciliary organization described in some organisms ( Blumer , 1996; Hughes , 1970; Zhukov et al . , 2006 ) may have barely maintained the ancestral organization , whereas microvillar eye PRCs may have emerged by reducing the cilia in taxa in which xenopsin was secondarily lost and the only visual pigment that survived is that associated with microvilli .", "Vice versa , purely ciliary eye PRCs , as have been described in several protostomes ( Blumer , 1994 , 1999; Passamaneck et al . , 2011; Woollacott and Eakin , 1973; Zimmer and Woollacott , 1993 ) , may have formed coincidently with downregulation or complete loss of r-opsins .", "The ancestral r-opsin+ and xenopsin+ PRC type may have arisen within the protostome branch leading to lophotrochozoan organisms ( variant A in Figure 5 ) , but even a much earlier origin is conceivable ( variant B in Figure 5 ) as the absence of xenopsins in deuterostome eyes can easily be explained by the obvious secondary loss of this visual pigment in these animals .", "The scenario outlined above links back to older debates about PRC evolution and the ancestral function and significance of vestigial cilia found in many invertebrate microvillar eye PRCs .", "The vestigial ciliary structures described vary in the length and in the structural organization of the microtubular axonem and the ciliary rootlet , and often only basal bodies are found ( Eakin and Westfall , 1964; Eakin , 1972; Turbeville , 1991; Verger-Bocquet , 1992 ) .", "These structures were often interpreted as remnants of the motile cilia of the epidermal cells from which the PRCs initially originated ( Arendt et al . , 2009; Eakin 1979 , 1982; Mayer , 2006 ) .", "Derivation from sensory cilia was only assumed in the context of a hypothesis proposing that animal eye PRCs originated multiple times within the different animal groups from inconspicuous cells enrolled in a diffuse dermal light sense ( von Salvini-Plawen and Mayr , 1977; Salvini-Plawen , 2008 ) .", "This thesis was contradicted by the upcoming molecular physiological and developmental data favoring conserved evolutionary lineages of r-opsin+microvillar and c-opsin+ ciliary PRC types ( Arendt , 2008; Arendt et al . , 2004; Fernald , 2006; Lamb , 2013; Shubin et al . , 2009 ) .", "However , the observed cellular co-expression of r-opsin and xenopsin in cells bearing microvilli and cilia now suggests that vestigial cilia in microvillar PRCs may indeed also go back to cilia with sensory functions .", "It is worth noting that the often inconspicuous primary cilia known from most kinds of vertebrate cells are considered to be sensory organelles that are important for proper cell function , intercellular signalling and development ( Malicki and Johnson , 2017; Singla , 2006; Walz , 2017 ) .", "Last but not least , the distinction between motile and sensory cilia is no longer regarded as clear cut .", "More and more motile cilia that also display sensory capabilities are being reported ( Kleene and Van Houten , 2014; Shah et al . , 2009; Warner et al . , 2014 ) and the distribution across eukaryotes of molecular components relevant for locomotion and sensation suggests that cilia were enrolled in both functions upon their emergence ( Bloodgood , 2010; Johnson and Leroux , 2010; Mitchell , 2007 , 2017; Quarmby and Leroux , 2010 ) .", "The same is conceivable for cilia in ancient photoreceptor cells .", "As current data only show use of xenopsin , but not of c-opsin , in the ciliary structures of protostome cerebral eyes , the described scenario is in line with the hypothesis that the cerebral eyes of animals employ only few different PRC types .", "This scenario does not require the assumption of multiple recruitments of sensory cilia and/or visual pigments or PRCs by cerebral eyes or of multiple origins of those entities .", "Instead , the PRCs employed in prostostome cerebral eyes are rendered as representatives of a highly plastic cell type .", "The ratios of microvilli/cilia and of r-opsin/xenopsin , and in consequence the downstream targets and the physiological properties of the PRCs , may have changed repeatedly within evolutionary lineages unless either the r-opsin or the xenopsin genes were finally lost .", "Subsequent studies on xenopsin+ PRCs may thus strongly impact the understanding of the degree to which conserved cell types and the properties of sensory cells can change over time and of how these changes occur .", "In conclusion , opsin phylogenetic analyses combined with gene-structure analysis strongly confirm the existence of an old clade of xenopsins , which is evolutionarily distinct from c-opsins .", "The physiological role of this opsin type is not yet understood and requires further studies , especially in the interesting case of co-expression with r-opsin .", "The taxonomic distribution and the available epression data suggest that xenopsins may play a pivotal role in tracing PRC and eye evolution in bilaterian animals .", "The dichotomous classification of eye PRCs into ciliary c-opsin+ and rhabdomeric r-opsin+ cell types may no longer be appropriate , and the documented presence of xenopsin in eye PRCs may be due to common ancestry .", "The described evolutionary scenario provides simple explanations for the origin of eye PRCs exhibiting both microvilli and cilia and for how purely ciliary PRCs may have emerged in protostome eyes , and it rises intriguing questions of how much conserved cell types can change over time ." ], [ "Adults of L . asellus were collected close to the Norwegian coastline in Bergen during the period from September to December 2015 and kept in the dark at 8°C .", "They were cultured in groups of female and male animals and fertilized egg balls were collected each morning .", "Larvae were kept on a light/dark cycle of 12h/12 hr at 18°C .", "The assembly of Illumina ( San Diego , California , RRID:SCR_010233 ) HISeq RNA-seq data of 2–11d old larval material described by Vöcking et al . ( 2015 ) was used to identify transcripts of interest via bidirectional blast .", "Whole transcripts and fragments were then amplified by PCR using gene-specific primers and cDNA prepared with Super Script II ( Thermo Fisher Scientific , Waltham , Massachusetts ) RRID:SCR_008452 ) , subsequently ligated into pgemT-easy vector ( Promega , Madison , Wisconsin , RRID:SCR_006724 ) and cloned into Top10 chemically competent E . coli ( Thermo Fisher Scientific ) .", "Sanger sequencing was used to verify the cloned sequences before DIG- and FITC-labeled sense and antisense probes were generated with T7- and SP6-RNA Polymerases ( Roche , Basel , Switzerland , RRID:SCR_001326 ) or with the Megascript Kit ( Thermo Fisher Scientific ) .", "Reciprocal blast yielded unambiguous results for gene orthology assignment of Las-FoxJ1 , Las-MyosinVIIa , Las-rab8a and Las-RPGR .", "In all other cases , public databases ( GenBank ( RRID:SCR_002760 ) , JGI ( RRID:SCR_002383 ) , Uniprot ( RRID:SCR_002380 ) ) and the Leptochiton transcriptome were screened for homologs by text search , blast ( RRID:SCR_004870 ) and HMMER ( RRID:SCR_005305 ) with respective query sequences or domain profiles for subsequent gene tree generation based on maximum likelihood and Bayesian inference .", "Sequence alignments with MAFFT ( RRID:SCR_011811 ) 7 . 221 ( EINSI option ) were manually curated .", "Best fitting substitution models were selected with ProtTest ( RRID:SCR_014628 ) 3 . 4 according to Akaike information , Bayesian information and Decision theory criteria or with the RAXML ( RRID:SCR_006086 ) based ProteinModelSelection script by Alexandros Stamatakis .", "Maximum likelihood analyses were run with RAXML 8 . 2 . 8 on the Cipres Science Gateway ( RRID:SCR_008439 , Miller et al . , 2010 ) with gamma modeling of rate heterogeneity and rapid bootstrapping with automatic stop by majority rule criterion autoMRE .", "Bayesian analyses were run with Phylobayes ( RRID:SCR_006402 ) 4 . 1 or Phylobayes-MPI 1 . 6 using the same substitution models chosen for ML analyses and gamma distribution for rate heterogeneity .", "Generally , four chains were run and tested for convergence with bcomp command discarding the 20% first cycles as burn-in and used for generating majority-rule consensus trees .", "As orthology assignment of the new L . asellus opsin sequence obtained from the RNA-seq data is critical for this study , the general routines for phylogenetic inference described above were further adapted to exclude possible artifacts discussed in recent opsin analyses .", "To evaluate the relationship to c-opsins , ctenopsins , xenopsins , cnidops and anthozoa II opsins sensu Hering and Mayer ( 2014 ) , a broad sampling of these opsin types across metazoans was included .", "For this purpose , publicly available sequence resources ( GenBank , Uniprot , Genoscope ( RRID:SCR_002172 ) , JGI and Macrostomum lignano genome initiative ( http://www . macgenome . org ) ) were screened for homologs by text search , as well as by blast and HMMER searches based on a set of ten query sequences sampled from the above-mentioned opsin groups and the new opsin sequence from L . asellus .", "We kept only those sequences that exhibited the PFAM 7tm_1 domain , contained the residue Lys296 critical for chromophore binding in opsins , and yielded representatives from the above-mentioned opsin groups amongst the best hits after reciprocal blast against GenBank .", "In order to reduce the computation time for downstream analyses , sequences yielding c-opsins as best reciprocal blast hits were down-sampled in taxa such as vertebrates and arthropods , where high numbers of sequences were obtained .", "As the number of sequences retrieved was low for several prostostome taxa , such as annelids , nemerteans , platyhelminths and ecdysozoans others than arthropods , as well as for acoelomorphs , which form a potential sister group to all other bilaterians ( Cannon et al . , 2016 ) , we performed a second round of sampling for these taxa following different strategies .", "Three complete opsin sequences were obtained from the Hypsibius dujardini NCBI TSA archive , matching the published incomplete sequences for c-opsin1 , c-opsin2 , and c-opsin3 , which lacked position 296 and thus were initially removed from the dataset .", "Illumina RNA-seq raw data were downloaded with fastq-dump from the NCBI SRA ( RRID:SCR_004891 ) read archive for the nemerteans Baseodiscus unicolor , Cerebratulus sp .", ", Lineus longissimus , Nipponemertes sp .", ", Paranemertes peregrina , Malacobdella grossa , Cephalothrix hongkongiensis , Hubrechtella ijimai , Tubulanus polymorphus , the flatworms Stenostomum sthenum , Kronborgia amphipodicola , Geocentrophora applanata , Prorhynchus sp .", ", Echinoplana celerrima , Prostheceraeus vittatus , Prosthiostomum siphunculus , Stylochus ellipticus , Itaspiella helgolandica , Microdalylellia schmidti , the acoelomorphs Ascoparia sp .", ", Diopisthoporus longitubus , Eumecyonostomum macrobursalium , Isodiametra pulchra , Meara stichopi , Nemertoderma westbladi , Sterreria sp .", "and the xenoturbellid Xenoturbella bocki .", "From these data and from our own RNA-seq data from several developmental stages from the annelid Owenia fusiformis , transcriptome assemblies were generated using Trimmomatic ( RRID:SCR_011848 ) 0 . 36 for adapter and quality trimming , Rcorrector ( Song and Florea , 2015 ) for read error correction and trinity-2 . 2 . 1 for read assembly .", "Blast databases were generated with the NCBI blast+-suite and screened with tblastn ( RRID:SCR_011822 ) for opsins with a query set of 38 opsin sequences representing all of the major opsin subgroups reported in Hering and Mayer ( 2014 ) and Ramirez et al . ( 2016 ) .", "Only sequences that exhibited the PFAM 7tm_1 domain , contained Lys296 and yielded c-opsins , ctenopsins , cnidops , anthozoa II opsins sensu Hering and Mayer ( 2014 ) and xenopsins amongst best hits were retained .", "The SMEDG Unigene dataset available at the Schmidtea mediterranea Genome Database ( RRID:SCR_007934 ) was screened with the same search strategy .", "Other opsin types , such as r-opsins , Go-opsins , RGR/peropsins , and the recently described chaopsins and bathyopsins were sampled to a lesser degree to reduce computation time of downstream analyses .", "As the choice of outgroup has been shown to be critical for opsin tree inference ( Feuda et al . , 2012; Plachetzki et al . , 2007 ) , we used a broad sampling of opsin sequences as queries to search for closely related GPCRs .", "This led to an outgroup consisting of melatonin , octopamine , dopamine and adrenergic receptors , and placopsins similar to those in the analysis of Hering and Mayer ( 2014 ) and more diverse than those in the analyses of Feuda et al . ( 2012 , 2014 ) and Ramirez et al . ( 2016 ) .", "Sequences were first aligned with MAFFT 7 with option E-INS-i .", "Then , ambiguously aligned N- and C-terminal regions were trimmed , sequences shorter than 160 amino acids removed and the remaining sequences again aligned with MAFFT 7 with option E-INS-I .", "The output was manually edited to remove gappy regions and ambiguously aligned positions , yielding an alignment of 282 positions and 258 sequences .", "Feuda et al . ( 2012 , 2014 ) and Hering and Mayer ( 2014 ) showed that data-set-specific substitution matrices can fit opsin alignments better than precomputed models implemented in tree inference software .", "Thus , we used RAxML to generate such a dataset-specific substitution matrix ( DS-GTR ) by parameter optimization of the general time reversible ( GTR ) model to the trimmed alignment and a precomputed parsimony tree .", "In ProtTest 3 . 4 , the implemented model FLU was exchanged against our DS-GTR , revealing DS-GTR+Γ as best fitting model according to Akaike information , Bayesian information and Decision theory criteria ( Figure 6—figure supplement 1 ) .", "For maximum likelihood analyses , RAxML 8 . 2 was run on the CIPRES Science Gateway ( Miller et al . , 2010 ) .", "A first run was conducted with DS-GTR+Γ and 500 rapid bootstraps , yielding low support for basal branching patterns .", "Hence , a leaf-stability analysis on the bootstrap tree set was conducted with phyutility 2 . 2 and a few sequences with low leaf stability were excluded from the final data-set used for all following analysis .", "A new trimmed alignment was generated from the final data-set as described above and a new RAxML analysis with DS-GTR+Γand 1000 rapid bootstraps was conducted .", "For Bayesian analysis , first a 10-fold cross-validation was run with phylobayes 4 . 1 to choose the most appropriate combination of GTR , LG and DS_GTR with non-parametric ( CAT ) or parametric ( Γ ) modeling of among-site variation or empirical mixture modeling ( C20 , C30 , C40 , C50 , C60 , WLSR5 ) .", "As a result , DS-GTR CAT and GTR CAT were found to be the best non-parametric variants , performing better than the best parametric variant DS-GTR Γ , and all three of these were better than any empirical mixture modeling ( Figure 6 ) .", "For each of the models DS-GTR CAT , GTR CAT and DS-GTR Γ , three chains with 30 , 000 cycles were run , and for each , chain convergence and mixing behavior was evaluated with the tools tracecomp from phylobayes and Tracer from Beast ( RRID:SCR_010228 ) and a burn-in of 6 , 000 cycles .", "Both non-parameteric variants yielded low estimated sample sizes for several model components suggesting poor mixing behavior , which is a known phenomenon of non-parametric models applied to small sequence alignments .", "Thus , only the chains with DS-GTR Γ , which all yielded high estimated sample sizes , were continued for another 30 , 000 cycles .", "Two chains , which exhibited best phylogenetic convergence assessed with bpcomp ( 12 , 000 cycles burn-in , mean deviation 0 . 108 ) , were used to compute the final consensus tree .", "For two opsins ( Platynereis dumerilii ciliary opsin ( AAV63834 . 1 ) and Leptochiton asellus xenopsin ) the whole genes were cloned from genomic DNA for subsequent analysis of exon-intron boundaries .", "Genomic DNA was extracted with the Nucleospin Tissue Kit ( Machery-Nagel , Düren , Germany ) and tested for fragment lengths larger than 20 kb .", "As a starting point , gene-specific primers were designed on the basis of the transcript sequences .", "For genome walking , four libraries were prepared with the Universal Genome Walker Kit ( Takara Bio , Kusatsu , Japan ) by enzymatic digestion and used for sequence elongation starting from exonic fragments .", "In parallel , long amplicons bridging smaller introns were also directly amplified from genomic DNA using Lataq ( Takara Bio ) , iProof ( Biorad , Hercules , California ) and HotStarTaq Plus ( Qiagen , Hilden , Germany , RRID:SCR_008539 ) polymerases .", "Obtained amplicons of up to 8 kb were cloned using a pGem-T easy Vector ( Promega ) TOPO XL PCR cloning kit ( Thermo Fisher Scientific ) , TopTen chemically competent cells ( Thermo Fisher Scientific ) and Sanger sequenced .", "Obtained sequences were used to design further primers for ongoing sequence elongation .", "Read assembly was performed with CLC Main Workstation ( RRID:SCR_000354 ) 7 . 1 .", "WebScipio ( Hatje et al . , 2011 ) was used to determine the gene structure including the respective intron phases of all those opsin sequences included in our phylogenetic analysis for which genomic data were available ( 1 ) at the WebScipio portal , ( 2 ) from other public databases ( Kumamushi genome project in case of Ramazzotius varieornatus , or", "3 ) as we cloned the whole genes from genomic DNA ( Platynereis dumerilii c-opsin and Leptochiton asellus opsin ) .", "The obtained gene structures were than mapped onto the un-curated sequence alignment using Genepainter ( Hammesfahr et al . , 2013 ) in order to identify homologous intron positions and to analyze opsin gene structure conservation .", "To evaluate the stability of the observed patterns against alignment artifacts , gene structures were also mapped onto additional alignments computed with MAFFT ( option G-INS-I ) , ProbCons ( RRID:SCR_011813 ) , Muscle and GLProbs ( RRID:SCR_002739 ) .", "Experiments were performed as described previously ( Vöcking et al . , 2015 ) .", "In brief , animals were fixed in 4% PFA in phosphate buffer and with Tween20 ( PTW; pH 7 . 4 ) and subsequently washed and stored in methanol .", "After rehydration in PTW , samples were briefly digested with Proteinase K , washed and prehybridized in hybridization buffer with 5% Dextran .", "Samples were hybridized with RNA probes for 72 hr and stained with a combination of FastBlue ( Sigma-Aldrich , St . Louis , Missouri , RRID:SCR_008988 ) and Fast Red ( Roche ) .", "The significance of expression signals was evaluated by using sense probes as control experiments .", "All in-situ hybridization experiments were performed on at least 30 specimens per gene for each sense and anti-sense probe and the experiments were repeated at least twice .", "Light microscopic images were taken using Eclipse E800 ( Nikon , Tokyo , Japan ) and AZ100M ( Nikon ) microscopes and adjusted with Adobe ( Mountain View , California ) Photoshop ( RRID:SCR_014199 ) CS5 .", "Confocal images were taken with a SP5 confocal microscope ( Leica , Wetzlar , Germany , RRID:SCR_008960 ) and the image stacks processed with ImageJ ( RRID:SCR_003070 ) and Adobe Photoshop CS5 .", "Experiments were performed as previously described ( Vöcking et al . , 2015 ) .", "Briefly , animals were fixed in 2–5% glutardialdehyde in sodium cacodylate buffer , subsequently postfixed in 1% osmium tetroxide , en-bloc stained with reduced osmium , dehydrated in a graded ethanol series and embedded in Epon/Araldite .", "Serial sections of 70 nm were cut with an ultra 35° diamond knife ( Diatome , Biel , Switzerland ) on an UC7 ultramicrotome ( Leica , Wetzlar , Germany ) and collected on Beryllium-Copper slot grids ( Synaptek , Reston , Virginia ) coated with Pioloform ( Ted Pella , Redding , California ) and counterstained with 2% uranyl acetate and lead citrate .", "Complete series were imaged with STEM-in-SEM as described by Kuwajima et al . ( 2013 ) at a resolution of 4 nm/ pixel in a Supra 55VP ( Zeiss , Oberkochen , Germany ) equipped with Atlas ( Zeiss ) for automated large field of view imaging .", "Acquired images were processed with Photoshop CS5 ( Adobe ) , first registered rigidly followed by affine and elastic alignment ( Saalfeld et al . , 2012 ) with TrakEM2 ( RRID:SCR_008954 , Cardona et al . , 2012 ) implemented in Fiji ( RRID:SCR_002285 ) ." ] ]
[ "Ciliary and rhabdomeric opsins are employed by different kinds of photoreceptor cells , such as ciliary vertebrate rods and cones or protostome microvillar eye photoreceptors , that have specialized structures and molecular physiologies .", "We report unprecedented cellular co-expression of rhabdomeric opsin and a visual pigment of the recently described xenopsins in larval eyes of a mollusk .", "The photoreceptors bear both microvilli and cilia and express proteins that are orthologous to transporters in microvillar and ciliary opsin trafficking .", "Highly conserved but distinct gene structures suggest that xenopsins and ciliary opsins are of independent origin , irrespective of their mutually exclusive distribution in animals .", "Furthermore , we propose that frequent opsin gene loss had a large influence on the evolution , organization and function of brain and eye photoreceptor cells in bilaterian animals .", "The presence of xenopsin in eyes of even different design might be due to a common origin and initial employment of this protein in a highly plastic photoreceptor cell type of mixed microvillar/ciliary organization ." ]
[ "Animal eyes have photoreceptor cells that contain light-sensitive molecules called opsins .", "Although all animal photoreceptor cells of this kind share a common origin , the cells found in different organisms can differ considerably .", "The photoreceptor cells in flies , squids and other invertebrates store a type of opsin called r-opsin in thin projections on the surface known as microvilli .", "On the other hand , the visual photoreceptor cells in human and other vertebrate eyes transport another type of opsin ( known as c-opsin ) into more prominent extensions called cilia .", "It has been suggested that the fly and vertebrate photoreceptor cells represent clearly distinct evolutionary lineages of cells , which diverged early in animal evolution .", "However , several organisms that are more closely related to flies than to vertebrates have eye photoreceptor cells with cilia .", "Do all eye photoreceptors with cilia have a common origin in evolution or did they emerge independently in vertebrates and certain invertebrates ?", "The photoreceptor cells of a marine mollusc called Leptochiton asellus , are unusual because they bear both microvilli and cilia , suggesting they have intermediate characteristics between the two well-known types of photoreceptor cells .", "Previous studies have shown that these photoreceptor cells use r-opsin , but Vöcking et al . have now detected the presence of an additional opsin in the cells .", "This opsin is a member of the recently discovered xenopsin family of molecules .", "Further analyses support the findings of previous studies that suggested this type of opsin emerged early on in animal evolution , independently from c-opsin .", "Other invertebrates that have cilia on their eye photoreceptors also use xenopsin and not c-opsin .", "The findings of Vöcking et al . suggest that , in addition to c-opsin and r-opsin , xenopsin has also driven the evolution of photoreceptor cells in animals .", "Eye photoreceptor cells in invertebrates with cilia probably share a common origin with the microvilli photoreceptor cells that is distinct from that of vertebrate visual cells .", "The observation that two very different types of opsin can be produced within a single cell suggests that the molecular processes that respond to light in photoreceptor cells may be much more complex than previously anticipated .", "Further work on these processes may help us to understand how animal eyes work and how they are affected by disease ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "neuroscience" ]
Neuropilin-2/Semaphorin-3F-mediated repulsion promotes inner hair cell innervation by spiral ganglion neurons
elife-07830-v2
[ [ "The perception of sound in mammals depends on appropriate connectivity between spiral ganglion neurons ( SGNs ) and hair cells ( HCs ) in the cochlea .", "SGNs are bipolar afferents linking the peripheral and central auditory systems by projecting peripheral axons that form ribbon-type synapses with cochlear HCs , and central axons that synapse with neurons in the ipsilateral cochlear nucleus ( Nayagam et al . , 2011 ) .", "It is known that SGN loss is a key aspect of congenital or noise-induced hearing loss , but only a small handful of mechanisms controlling the formation of this part of the auditory system have been identified ( Fritzsch et al . , 2004; Appler and Goodrich , 2011; Green et al . , 2012; Coate and Kelley , 2013 ) .", "During mouse development , otic neuroblasts delaminate from the otocyst around embryonic day 8 . 5 and then differentiate into either vestibular or auditory ( spiral ganglion [SG] ) neurons ( Appler and Goodrich , 2011; Coate and Kelley , 2013 ) .", "Subsequently , the SGN somata extend in a spiral that mirrors the adjacent developing cochlear duct ( ∼E11 . 5-P0 ) while projecting peripheral processes ( sometimes termed ‘peripheral axons’ or ‘dendrites’ ) into the sensory epithelium and central axons into the brainstem .", "The dependence of neurotrophins for the survival and viability of SGNs during these stages has been documented extensively ( Fritzsch et al . , 2004; Green et al . , 2012 ) , but relatively little is known about the mechanisms controlling SGN diversification and wiring with specific cochlear HC regions during development .", "Although SGNs show little morphological variability , they are clearly diverse in several ways .", "First , SGNs are divided into two broad categories: type I and type II .", "Type I SGNs represent 90–95% of the total SGN population , form monosynaptic contacts with individual inner HCs ( IHCs ) , and facilitate the majority of all hearing function .", "Type II SGNs comprise the remaining 5–10% of the SGN population , and , in contrast with Type I SGNs , bypass the IHCs to form en passant synapses with up to 10 outer HCs ( OHCs ) per individual SGN fiber ( Figure 1A ) .", "In addition , type II SGNs are not myelinated by the neural crest derived-Schwann cells that myelinate type I SGNs ( Carney and Silver , 1983; Breuskin et al . , 2010 ) .", "Although their function is not well understood , recent landmark studies have suggested that type II SGNs may facilitate responses to very loud or painful sounds ( Weisz et al . , 2009 , 2012 , 2014 ) .", "SGNs also show differences in protein expression and firing rates that correlate with their location along the tonotopic axis ( Flores-Otero et al . , 2007 ) .", "Finally , type I fibers can be sub-divided into two groups based on spontaneous firing rates and synaptic location at the base of individual IHCs ( Liberman , 1982 ) . 10 . 7554/eLife . 07830 . 003Figure 1 . Development of SGN innervation patterns .", "( A ) Illustration of the innervation pattern of inner hair cells ( IHCs ) and outer hair cells ( OHCs ) by type I and II spiral ganglion neurons ( SGNs ) .", "( B ) Illustrations showing the morphology of the cochlear duct and spiral ganglion ( SG ) for the indicated cross-sections .", "Letters on each illustration correspond to the position of the section in the adjacent panels .", "ce , cochlear epithelium .", "( C–H )", "Cross-sections of the cochlea and associated SG at the indicated time points and positions .", "SGN processes are labeled with Tuj1 ( green ) , hair cells ( HCs ) with anti-Atoh1 ( red ) , and actin with phalloidin ( blue ) .", "SGN processes do not enter the epithelium until HCs being to differentiate ( E14 . 5 base ) but then rapidly extend towards the developing IHCs ( C–E ) .", "As OHCs begin to develop at E15 . 5 , some processes extend past the IHCs to form contacts with OHCs ( F–H ) .", "( I ) Confocal image of flat-mounted cochleae from a Neurog1CreERT2; R26R-tdTomato mouse at P2 .", "HCs are labeled with anti-myosin VI ( blue ) and all SGNs are labeled with Tuj1 ( green ) .", "Asterisks mark the SGN somata , which extend SGN fibers that form into radial bundles ( RB ) prior to forming synapses with HCs .", "( J ) The same preparation as in panel I , but illustrating expression of tdTomato ( anti-dsRed ) to visualize sparsely labeled individual fibers .", "( K ) High-magnification view from panel J illustrating type I SGNs innervating IHCs and type II SGNs passing IHCs to innervate OHCs .", "Scale bar in K: 20 μm , C–H; 45 μm , I and J; 15 μm , K . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 003 How the innervation patterns for type I and II SGNs are established is a fundamental question in auditory neuroscience that has yet to be completely answered .", "One of the most basic issues has been the question of whether individual SGN fibers are specified as either type I or type II prior to the arrival of their peripheral processes in the cochlear duct or if phenotype is established based on interactions within the target environment .", "Echteler used horse radish peroxidase staining to provide evidence that , in the postnatal gerbil cochlea , immature SGNs show unbiased innervation to both HC regions and are subsequently retained in either the IHC or OHC region likely through a process of selective pruning and apoptosis ( Echteler , 1992; Echteler et al . , 2005 ) .", "Similarly , rhodamine-dextran dye-labeling of type I SGNs in mouse suggested that HC innervation was non-specific until about postnatal day 3 when refinement events seemed to commence ( Huang et al . , 2007 ) .", "However , genetic-labeling experiments in which sparse numbers of SGNs expressed alkaline phosphatase under the control of Cre recombinase ( driven by Neurogenin-1 promoter elements; Neurog1CreERT2 ) suggested that ‘mature’ SGN innervation patterns are present by birth ( Koundakjian et al . , 2007 ) .", "These results provide conflicting conclusions regarding the timing and determination of type I and type II SGNs .", "We sought to gain clarity on this issue by combining the same Neurog1CreERT2 allele with a tdTomato reporter in order to characterize the timeline of IHC and OHC innervation by the SGNs .", "In addition , we designed experiments to investigate potential molecular mechanisms controlling the differential innervation of IHCs and OHCs and discovered a prominent chemorepulsive role for Neuropilin-2 and its ligand , Semaphorin-3F ." ], [ "Because existing data were somewhat unclear regarding the developmental timing of type I and type II SGN projection patterns , SGN processes and nascent HCs were visualized in cochlear cross sections from different developmental time points ( Driver et al . , 2013 ) ( Figure 1B–H ) .", "At embryonic day 13 . 5 ( E13 . 5 ) , prior to detectable expression of Atoh1 , SGN processes are present outside of the duct , but do not yet project into the cochlear epithelium ( Figure 1C ) .", "By one day later ( E14 . 5 ) , processes have entered the epithelium at the more mature base of the cochlea while remaining outside in less mature , more mid-modiolar regions ( Figure 1D , E ) .", "Neurite entry correlates with the onset of Atoh1 expression in developing HCs ( Figure 1E; arrow ) .", "At E15 . 5 , the ratio of SGN processes projecting towards the emerging OHC region appears to be closer to 50:50 than to the mature ratio of 5:95 ( Figure 1F , G; see arrowheads ) .", "However , at E16 . 5 , a noticeably higher concentration of processes appears around the IHC region ( Figure 1H; see arrowheads ) .", "These data suggest that the SGNs may assume a type I- or type II-like morphology as early as E16 . 5 , which is both consistent with the time period of hair and supporting cell maturation and earlier than previously suggested .", "Consequently , we focused on these earlier time points in subsequent investigations into the cellular and molecular nature of IHC and OHC innervation .", "Approximately , 10–15 individual SGN peripheral axons project to each IHC , making analysis of changes in individual fibers difficult ( Figure 1I ) .", "To circumvent this problem , mice carrying an inducible Neurog1CreERT2 BAC transgene ( Koundakjian et al . , 2007 ) were crossed with an R26RtdTomato reporter line ( referred to as NCreRTom ) .", "By limiting recombination to a small number of SGNs , individual fibers with type I- and type II-like morphologies can be clearly visualized ( Figure 1J , K ) .", "The intensity of the tdTomato fluorophore also permitted the resolution of small exploratory protrusions on individual SGN processes ( Figure 1K ) .", "To better understand the temporal and spatial dynamics of SGN process development , we visualized sparsely labeled individual SGNs ( see ‘Materials and methods’ for details ) in whole-mount preparations of cochleae from NCreRTom mice at different developmental time points ( Figure 2 ) .", "The mice also carried the Atoh1nGFP transgene ( Lumpkin et al . , 2003 ) , which allows for easy identification of IHCs and OHCs .", "At the mid-point along the cochlea at E15 . 5 , approximately 60% of individual SGN fibers terminated in the IHC region while the remaining 40% extended into the OHC region ( Figure 2A , B , E ) .", "However , in a more basal region of the same cochlea , the number of fibers terminating in the IHC and OHC regions was roughly equivalent ( Figure 2E ) .", "These results were comparable to the distribution of total fibers that was observed in cross-sections ( Figure 1F , G ) .", "In contrast , at E17 . 5 or 18 . 5 , between 70 to 80% of all labeled processes were apposed to IHCs ( Figure 2C–E ) , suggesting that a progressive transition to the mature innervation pattern begins shortly after E15 . 5 .", "Consistent with this trend , at P0 and through P3 , approximately 90–95% of all labeled SGNs fibers terminated in the IHC region with the remaining 5–10% extending into the OHC region ( Figure 2E; see also Figure 1J , K ) .", "Previously , studies using the postnatal gerbil showed that programmed cell death may contribute to the final distribution of type I and II SGNs ( Echteler et al . , 2005 ) .", "To determine if apoptosis could play a role in SGN innervation patterns during embryonic mouse development , we examined cleaved caspase-3 ( CC3 ) immunostaining at E16 . 5 and E17 . 5 ( Figure 2—figure supplement 1; see ‘Materials and methods’ ) .", "In the basal half of the cochlea at these stages , where most of our neuronal-tracing experiments were performed , we detected between 4 and 19 CC3-positive SGNs per cochlea .", "At the apex , between 18 and 50 CC3-positive SGNs were observed per cochlea .", "Given that the number of SGNs is estimated to be greater than twenty thousand at this stage ( Richter et al . , 2011 ) , a role for apopotic cell death in SGN innervation at this stage seems unlikely . 10 . 7554/eLife . 07830 . 004Figure 2 . Sparse SGN labeling reveals retraction of processes from the OHC region during embryonic development .", "( A–D )", "Fixed whole-mount cochleae from Neurog1CreERT2;R26RtdTomato; Atoh1nGFP mice at E15 . 5 ( A , B ) and E18 . 5 ( C , D ) .", "Panels A and C show sparse-labeling of SGN fibers ( tdTomato in magenta ) , while panels B and D show the same regions but include HCs in green .", "Note that at E15 . 5 , the number of projections terminating in the IHC ( arrowheads ) and OHC ( arrows ) regions appears roughly equivalent .", "In contrast , at E18 . 5 , the majority of SGNs terminate in the IHC region .", "( E ) Comparison of the percentage of labeled SGNs that terminate in the IHC ( black bars ) or OHC region ( gray bars ) at different developmental time points ( minimum of 6 cochleae per time point ) .", "Error bars are standard error of the mean ( sem ) .", "( F ) Sequential images at the indicated times from a single time-lapse experiment showing sparsely labeled SGNs with HCs ( left ) or alone ( right ) in an E16 . 5 cochlear explant .", "Yellow arrowheads indicate SGN fiber terminals in the OHC region .", "White arrowheads point to SGNs that are clustered around IHCs that had originally been in the OHC region .", "( G ) Data from four imaging experiments illustrate a decrease in the number of fibers positioned in the OHC region over the course of experimental time .", "Since there was variability in the number of labeled SGNs between experiments , the data for each experiment were normalized to the number of labeled fibers present in the OHC region at time 0 .", "( H ) Illustration of the time-line of cochlear innervation .", "Fibers initially arrive at the IHC region around E14 . 5 .", "At E15 . 5 , when both IHCs and OHCs are present , SGN fiber distribution is roughly equal between the IHC and OHC regions .", "In contrast , by E16 . 5 , many SGN fibers have withdrawn from the OHC region .", "At P0 , the vast majority of SGNs terminate at the IHC region .", "These events occur before the final stages of synapse formation and subsequent fiber pruning .", "Scale bar in D: 15 μm , A–D; 20 μm , F . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 00410 . 7554/eLife . 07830 . 005Figure 2—figure supplement 1 . Programmed cell death ( apoptosis ) is limited in SGNs at E16 . 5 and E17 . 5 . ( A–D ) Confocal images from whole-mount preparations marked with anti-cleaved caspase-3 ( CC3 ) antibodies , Tuj1 antibodies ( to show SGN somata ) , and anti-Sox2 antibodies ( to show the position of the cochlear sensory epithelium ) .", "The arrowheads in these panels point to SGNs that are CC3 positive .", "Scale bar in D , 40 μm .", "Histogram showing average numbers of CC3-positive SGN in the base and apex of cochleae at each stage .", "n = 7 cochleae per group . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 005 These data are consistent with a model in which a percentage of SGN fibers initially extend into the OHC region before retracting to the IHC region starting at approximately E16 . 5 .", "However , an alternate , or possibly , additional hypothesis would be that the population of SGNs observed at E15 . 5 is largely stable and that the change in the ratio of axon distribution is a result of the arrival of new SGN peripheral fibers in the IHC region .", "These new fibers would effectively decrease the percentage of fibers projecting to the OHC region .", "To determine whether existing OHC fibers retract during development , we performed time-lapse imaging using cochleae from NCreRTom;Atoh1nGFP mice for up to 20 hr starting at ages E15 . 5 or E16 . 5 ( see ‘Materials and methods’ ) .", "Analysis of time-lapse videos indicated numerous instances of SGN processes that extended into the OHC region and then retracted back into the IHC region or that were present in the OHC region at the beginning of the experiment and then retracted ( Figure 2F; Video 1 ) .", "We quantified this effect for axons in four independent time-lapse experiments and found that 10–60% of the axons that started in the OHC region had retracted by the end of the acquisition period ( Figure 2G ) .", "Because of the thickness of the tissue , we were not able to resolve the final location of each process that retracted from the OHC region; however , many of them appeared to aggregate around the IHCs ( white arrowheads; Figure 2F ) .", "In addition , although we cannot rule out that it occurs at all , we never identified new SGNs approaching the IHCs in these experiments .", "Overall , these data indicate that many SGN peripheral axons initially extend into the OHC region but subsequently retract to form connections with IHCs .", "Moreover , these results suggest that most SGNs have determined their phenotype ( type I vs type II ) by the late embryonic period or just shortly after birth ( Figure 2H ) . 10 . 7554/eLife . 07830 . 006Video 1 . A cochlea from a mouse carrying the Neurog1CreERT2; Rosa-tdTomato; Atoh1-nGFP alleles that was imaged by spinning disk confocal microscopy for approximately 20 hr . The orange dots mark SGN processes that can be seen projecting into the OHC region and then falling back to the IHC region . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 006 The results described above suggest that development of SGN innervation patterns may be influenced by environmental cues within the developing organ of Corti .", "The Neuropilins are a well-known class of axon-guidance receptors that are activated by secreted Semaphorins ( Sema3s ) and play diverse roles in innervation of both the central and peripheral nervous systems ( Kolodkin and Tessier-Lavigne , 2011 ) .", "Moreover , our previous unpublished studies using RT-PCR demonstrated that Neuropilin-2 ( Nrp2 ) is expressed in whole cochleae at E16 . 5 .", "Therefore , to determine the temporal and spatial distribution of Nrp2 , we localized Nrp2 protein ( Giger et al . , 2000 ) on cross-sections of developing cochleae and counterstained with Tuj1 to mark SGN somata and their processes ( Figure 3A–H ) .", "For the cross-sections shown here , identical fixation , immunostaining , and imaging parameters were used throughout .", "At E14 . 5 , Nrp2 was detectable in the SGNs , but not in surrounding mesenchyme or epithelial tissues ( Figure 3A , B ) .", "At E16 . 5 , when SGNs develop region-specific innervation patterns , Nrp2 is clearly detectable in the somata and peripheral axons of the SGNs ( Figure 3C , D ) .", "The Nrp2 antibody labeled all of the SGN cell bodies , which suggests Nrp2 is expressed by all SGNs .", "Consistent with this conclusion , Nrp2 was visible along all putative type I and II SGN endings in cross-sections at E18 . 5 ( Figure 3E , F ) and in whole-mount preparations at P0 ( Figure 3G , H ) .", "Interestingly , while cross-sections from P0 and P5 cochleae indicated persistent expression of Nrp2 protein in SGN somata , the overall intensity of staining was substantially reduced by comparison with the earlier time points ( not shown ) . 10 . 7554/eLife . 07830 . 007Figure 3 . Nrp2 is expressed in SGNs and is required for normal HC innervation .", "( A–H )", "Cross-sections of the cochlear duct and associated SGNs from the indicated ages .", "Upper panels show both SGNs ( Tuj1 in magenta ) and anti-Nrp2 ( green ) .", "Lower panels show anti-Nrp2 alone .", "( A , B )", "At E14 . 5 , Nrp2 is present in SGN somata and in peripheral axons ( arrow ) projecting into the cochlear epithelium ( ce ) .", "( C , D )", "At E16 . 5 , Nrp2 is detectable in virtually all SGN somata ( arrowheads ) .", "( E , F )", "At E18 . 5 , Nrp2 is detectable in putative type I and type II processes .", "( G , H )", "Whole-mount preparation at P0 indicates expression of Nrp2 in both type I and type II SGNs .", "( I–N )", "Comparison of SGN process distribution at E15 . 5 in WT and Nrp2−/− cochleae .", "Anti-Cdkn1b ( magenta ) labels the developing cochlear prosensory cells prior to HC formation ( I ) while Tuj1 ( green ) marks the SGNs ( J ) .", "The dotted lines in I delineate the IHC and OHC regions .", "( K ) Three-dimensional Y-Z view of the processes in J . Note that roughly equal number of processes terminate in both the IHC and OHC regions .", "In contrast , in a cochlea from an Nrp2−/− mutant , more processes appear to extend into the OHC region ( arrowheads in M , N ) .", "( O ) Quantification of cell bodies indicates no difference in number of SGNs between WT and Nrp2−/− .", "( P–T )", "E17 . 5 whole-mount cochlea stained with markers for all neurons ( Tuj1; blue ) , SGN afferents ( anti-Syt-1; red ) , and olivocochlear efferents ( anti-GAP-43; green ) .", "The high-magnification view in T is from the same preparation shown in P–S .", "( U ) Quantification of number of processes crossing into the OHC region in the base of the cochlea at E16 . 5 .", "Both Nrp2−/− and Nrp2+/− mice show significantly higher numbers of SGN processes in the OHC region as compared to WT .", "***p ≤ 0 . 001 .", "n = 9 , WT; 6 , Nrp2−/−; 4 , Nrp2+/− .", "Error bars , sem .", "Scale bar in H: 35 μm , A–H; 20 μm , F , J , L , M; 10 μm , K , N; 55 μm , P–S; 20 μm , F . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 00710 . 7554/eLife . 07830 . 008Figure 3—figure supplement 1 . Semaphorin3F-Fc recognizes binding partners in vivo . Representative images from wild-type P0 cochleae stained with Fc ( A–H ) or Sema3F-Fc ( I–P ) fusion proteins .", "Samples were counterstained with DAPI ( blue ) to reveal overall morphology and anti-Neurofilament 200 antibodies ( red ) to reveal SGN positions .", "( A–D )", "A low-magnification view shows very little binding by control Fc .", "( E , F )", "However , binding by Sema3F-Fc in a similar preparation is clearly evident in the SGNs ( arrows ) .", "( I–L )", "A high-magnification view from the boxed region in D . Fc control does not show binding to type I or II SGNs .", "( M–P )", "A high-magnification view from the boxed region in H . Sema3F-Fc binds to type I ( arrow ) and type II ( arrowheads ) SGNs .", "Scale bar in P: approximately 450 μm for A–H and 20 μm for I–P . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 008 To demonstrate that the Nrp2 immunoreactivity in both SGN subtypes represents functional receptor distribution , we treated cochlear cross-sections with an Fc-tagged version of Semaphorin-3F ( a known ligand for Nrp2; Sahay et al . , 2003 ) at P0 , a stage when type I and II SGNs are morphologically distinguishable ( as shown in Figure 2 ) .", "The tissue sections were then immunolabeled with anti-Fc antibodies and imaged by confocal microscopy .", "Figure 3—figure supplement 1 shows example preparations counterstained with 4′ , 6-diamidino-2-phenylindole ( DAPI ) and anti-neurofilament antibodies .", "In contrast with control Fc , which showed little to no binding ( Figure 3—figure supplement 1A–D ) , Sema3F-Fc showed substantial binding to SGNs along the basal-to-apical axis of the cochlea ( Figure 3—figure supplement 1E–H; see arrows ) .", "Similar to the anti-Nrp2 antibody staining in Figure 3E–H , Sema3F-Fc was detectable on neurofilament-positive structures that are consistent with the position of peripheral axons of both type I and II SGNs ( Figure 3—figure supplement 1I–P ) .", "These data suggest that functional forms of Nrp2 are present on both type I and II SGNs .", "Given that Nrp2 is important in axon guidance and synaptogenesis in multiple systems ( Giger et al . , 1998; Chen et al . , 2000; Jongbloets and Pasterkamp , 2014 ) and that Nrp2 is expressed by the SGNs , we hypothesized that Nrp2 was necessary for normal cochlear innervation .", "To test this , we analyzed SGN process extensions in cochleae from Nrp2−/− mutant mice ( Giger et al . , 2000 ) .", "As expected , in wild type ( WT ) cochleae at E15 . 5 , SGN processes are nearly evenly distributed between the IHC and OHC regions of the organ of Corti ( Figure 3I–K ) .", "In contrast , in Nrp2−/− cochleae at E15 . 5 , there were large bundles of SGN processes that extended into the OHC region , with far fewer processes terminating near the IHC region ( Figure 3L–N ) .", "These changes were not a result of a decrease in the total number of SGNs , as quantifying SGN somata density indicated no differences between WT and Nrp2−/− cochleae ( Figure 3O ) .", "In order to quantify changes in SGN patterning in Nrp2−/− mutants , it was necessary to discriminate SGNs from associated olivocochlear efferent fibers .", "Since both are labeled by pan-neuronal markers such as neurofilament or beta-III-tubulin ( Tuj1 ) , we screened over a dozen antibodies against known neuronal factors and found that synaptotagmin-1 ( Syt-1 ) antibodies label the SGN afferent axons but do not mark olivocochlear efferents ( Figure 3P–T ) .", "Triple labeling with Tuj1 ( all neurons , blue ) , Syt-1 ( SGN afferent axons , red ) , and GAP-43 ( olivocochlear efferents , green; Simmons et al . , 1996 ) in an E17 . 5 cochlea demonstrates the specificity of the Syt-1 antibody for afferent SGN axons .", "Next , the number of SGN fibers that cross into the OHC region in WT , Nrp2+/− , and Nrp2−/− cochleae was quantified by labeling with Syt-1 antibodies at E16 . 5 .", "Results indicate significant increases in the number of crossing fibers in both Nrp2+/− and Nrp2−/− cochleae ( Figure 3U ) .", "These data are consistent with a model in which Nrp2 plays a role in limiting the number of SGN axons that cross into the OHC region .", "A somewhat unexpected result of the analysis of Syt1+ SGNs was the observation of similar patterning defects in cochleae from Nrp2+/− and Nrp2−/− animals .", "However , phenotypic defects in midline commissural axon path finding have been reported in Nrp2+/− mice , suggesting that heterozygosity for Nrp2 can lead to a significant decrease in Nrp2 protein ( Chen et al . , 2000 ) .", "To determine if this is the case in SGNs , we examined protein levels by Western blot and found a significant decrease in Nrp in Nrp2+/− cochleae ( Figure 4A ) .", "It was surprising that the Nrp2+/− heterozygous cochleae consistently had so little Nrp2 protein , but previous reports have shown positive feedback/autocrine loops associated with Nrp2 expression ( Tyler Hillman et al . , 2011; Goel et al . , 2013 ) .", "Based on these results and the fact that Nrp2−/− mice demonstrate limited survival past birth , we used the Nrp2+/− mice to examine changes in SGN innervation at postnatal ages .", "Moreover , in order to unambiguously visualize potential changes in axonal morphology or path finding , the Nrp2 mutant strain was bred onto the NCre;RTom line .", "This allowed visualization of a limited number of SGN axons in animals with significantly decreased Nrp2 ( Figure 4B–D ) .", "As expected , an increased percentage of SGN fibers extended into the OHC region in Nrp2+/− cochleae ( Figure 4C , D; see arrowheads ) .", "To quantify these changes , we determined percentages of labeled fibers with type I- and type II-like morphologies in cochleae from WT and Nrp2+/− mice .", "In previous studies , anti-peripherin immunolabeling has been used to label type II SGNs ( Huang et al . , 2007; Defourny et al . , 2013 ) , but we and others have found significant peripherin expression in type I SGNs through postnatal day 6 .", "Therefore , type I ( terminating at IHCs ) - and type II-like ( extending into the OHC region and turning towards the base ) fibers were classified based on morphology ( Figure 4C , D ) .", "Results indicated nearly a 40% increase in type II-like SGN fibers ( among the labeled population ) in Nrp2+/− cochleae at P1 ( Figure 4E ) , confirming the phenotype observed in embryonic cochleae from Nrp2 mutants . 10 . 7554/eLife . 07830 . 009Figure 4 . Nrp2 regulates SGN axonal complexity .", "( A ) Western blot demonstrating significantly reduced Nrp2 protein in cochleae from Nrp2+/− mice as compared to WT .", "**p ≤ 0 . 01; n = 9 WT; 7 Nrp2+/− .", "( B ) Low-magnification view of a Neurog1CreERT2; R26R-tdTomato cochlea with boxed regions showing the locations where confocal z-stacks were acquired .", "In all of the micrographs in this figure , white represents anti-tdTomato and blue represents anti-myosin VI .", "( C , D )", "Compared to WT , Nrp2+/− cochleae with sparse SGN labeling show higher numbers of fibers in the OHC region ( yellow arrowheads ) .", "( E ) Quantification of the percentage of sparsely labeled type II-like fibers ( defined as SGNs that had crossed into the OHC region and turned toward the base ) in each genotype .", "***p ≤ 0 . 001; n = 9 WT; 6 Nrp2+/−; ns , not significant .", "( F , G ) 3D reconstructions of type I SGNs ( relative positions are indicated in F ) .", "In Nrp2+/− cochleae , there were many type I SGNs with increased branching ( yellow arrow in G ) , suggesting absence of an inhibitory cue .", "( H ) Quantification of number of filopodia per individual type I SGN in WT and Nrp2+/− cochleae indicates significantly higher numbers of small branches in Nrp2+/− heterozygotes .", "**p ≤ 0 . 01; n = 4 WT; 6 Nrp2+/− .", "( I ) The number of medially and laterally positioned type I SGNs does not change in Nrp2+/− cochleae .", "n = 4 cochleae each genotype .", "( J ) Model illustrating phenotypic changes in cochlear innervation as a result of a decrease or absence of Nrp2 .", "Error bars , sem .", "Scale bar in B: 150 μm , B; 20 μm , C , D , F , G . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 009 Activation of Neuropilins has been shown to alter growth cone motility ( Chen et al . , 1997 ) , an effect that could account for the SGN phenotype .", "Therefore , we wanted to examine whether decreased levels of Nrp2 led to increases in either the number or length of small exploratory processes at the ends of SGN peripheral axons .", "To accomplish this , terminals of individual SGN peripheral axons were examined in cochleae from WT and Nrp2+/− mutants ( with sparse SGN labeling ) at P1 .", "The ends of the type II SGNs showed tremendous morphological variability at these stages , but we were not able to detect any substantial differences between Nrp2+/− and WT .", "However , we were able to systematically measure and count the number of small branches at the ends of type I SGNs ( Figure 4F , G ) .", "WT and Nrp2+/− SGNs did not show differences in filopodial length ( not shown ) , but Nrp2+/− SGNs neurites did show a significant increase in the number of filopodial branches ( Figure 4F–H ) .", "Finally , we examined the synaptic locations of type I SGNs in WT and Nrp2+/− cochleae , as previous work has suggested that low-spontaneous rate fibers synapse on the medial side of IHCs , while high-spontaneous rate fibers synapse on the lateral sides ( Liberman , 1982 ) .", "We tracked over 1500 SGNs between Nrp2+/− and WT cochleae and found no differences in the percentages of medially or laterally positioned SGNs ( Figure 4I ) .", "These data support the hypothesis that Nrp2 mediates an inhibitory signal within SGN fibers that decreases terminal complexity and prevents fibers from extending processes into the OHC region .", "However , Nrp2 does not appear to play a role in the patterning of fibers within the IHC region .", "SGNs and HCs connect via specialized ribbon synapses ( Sobkowicz et al . , 1982 ) .", "Given that decreased Nrp2 results in an increase in the number of SGN processes in the OHC region , we speculated that this should result in an increase in the number of synaptic ribbon bodies in OHCs .", "To investigate this , we labeled and counted Ribeye-positive puncta at the base of IHCs and OHCs in WT and Nrp2+/− cochleae at P8 and P21 ( Figure 5A–D ) .", "Surprisingly , synapse numbers for both IHCs and OHCs were unchanged between WT and Nrp2+/− mutants ( Figure 5E ) .", "For these studies , we were also able to examine both cochleae from one surviving Nrp2−/− mouse at P21 .", "However , no difference in number of ribbon synapses ( as compared to controls ) was observed in these cochleae either .", "Consistent with this result , assessment of hearing sensitivity by auditory brainstem response indicated no significant differences between WT and Nrp2+/− mice ( not shown ) .", "To further examine innervation in Nrp2+/− adult cochleae , we compared numbers of type II SGNs between Nrp2+/− mutants and WT using sparse SGN labeling .", "Although there was a small increase in type II projections in Nrp2+/− cochleae as compared to controls , the difference was not significant .", "Overall , these results suggest that innervation is normal in adult Nrp2+/− cochleae .", "The basis for the change in innervation patterns between embryonic/early postnatal and adult Nrp2+/− animals is unclear .", "However , after birth , the cochlea goes through a significant phase of synaptic pruning in conjunction with innervation by olivocochlear efferents ( Huang et al . , 2012 ) .", "Therefore , it seems likely that this pruning mechanism is independent of Nrp2 and can therefore act to correct any increases in SGN fibers in the OHC region . 10 . 7554/eLife . 07830 . 010Figure 5 . The number of HC ribbon synapses is unchanged in cochleae with reduced Nrp2 . ( A–D ) Visualization of ribbon synapses ( anti-Ribeye in green; white arrows ) on IHCs and OHCs at P21 .", "Nuclei are labeled with DAPI ( blue ) .", "Overall no differences in ribbon synapse numbers were observed between genotypes .", "( E ) Quantification of ribbon bodies per IHCs and OHCs in P8 and P21 cochleae .", "All differences were not significant ( ns ) .", "n = 6 cochleae per genotype per age .", "( F ) Percentage of labeled type II-like fibers ( defined as SGNs that had crossed into the OHC region and turned toward the base ) in each genotype at P21 .", "n = 6 WT; 6 het .", "Although the data trended toward more fibers in the Nrp2+/− cochleae , the difference compared to the control group was not significant ( ns ) .", "Error bars , sem .", "Scale bar , 8 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 010 Nrp2 is thought to be activated by several of the secreted Semaphorins , including Sema3B , Sema3C , Sema3D , and Sema3G ( Sharma et al . , 2012 ) .", "To determine which of these ligands could be activating Nrp2 on SGNs , we localized Sema3 transcripts by in situ hybridization .", "Sema3b ( not shown ) and Sema3c showed a nearly identical expression pattern; mRNA was detectable in the SG , in the greater epithelial ridge ( GER; non-sensory cells ) of the cochlear epithelium , and very faintly in the IHC and OHCs ( Figure 6A–C ) .", "Sema3d mRNA was not detectable in the SG but was detectable in an unusual striping pattern in the GER ( Figure 6D–F; see the arrow in Figure 6E ) .", "In situ hybridization for Sema3g was not performed , but Richardson et al . did not detect Sema3g expression in the E14 . 5 cochlea ( Richardson et al . , 2014 ) .", "In contrast with these results , Sema3f mRNA is expressed in a pattern highly suggestive of a role in activating Nrp2 ( Figure 6G–N ) .", "At E14 . 5 ( not shown ) and E16 . 5 , Sema3f transcripts were detectable in the apical , middle , and basal turns of the cochlea in a pattern that is distinct from the other Sema3s we examined ( Figure 6H ) .", "Expression of Sema3f is present in two regions of the duct: an extreme medial region near the junction with Reissner's membrane and in a lateral domain that correlates with the developing sensory epithelium .", "At higher magnification , Sema3f appears to be expressed in a domain of cells that includes OHCs , pillar cells ( PCs ) , Dieters' cells , and Henson's cells ( Figure 6I ) .", "Moreover , the domain has a sharp medial boundary that appears to correlate with the junction between the IHC and OHC regions .", "To better resolve the spatial distribution of Sema3f mRNA , we post-stained samples with antibodies against myosin VI and Neurofilament light chain ( Figure 6J–L ) .", "The merged image demonstrates that Sema3f expression clearly overlaps with the OHC region and is completely absent from the IHC region .", "In addition , the sharp medial boundary appears to precisely correlate with the limiting extension of developing SGN processes .", "At postnatal day 0 ( P0 ) , Sema3f expression in the cochlea is diminished and the distribution pattern has changed .", "At the apex , Sema3f is detectable in the PCs and faintly present in nearby supporting cells , while at the cochlear base , Sema3f expression is limited to just the outer PCs .", "At P5 , Sema3f was not detectable in the cochlear epithelium ( not shown ) .", "These data suggest that Sema3F protein is distributed in a spatial and temporal pattern consistent with a role in activating Nrp2 on SGNs . 10 . 7554/eLife . 07830 . 011Figure 6 . The expression pattern of Sema3f suggests a role in Nrp2 signaling during cochlear innervation . In situ hybridizations for Sema3c , Sema3d , and Sema3f .", "( A–J )", "Low-magnification sense controls ( A , D , G ) demonstrate specificity for each probe .", "Low ( B , E , H ) and high-magnification views ( C , F , J ) from the boxed regions in B , E , and H for antisense probes indicate unique patterns of expression .", "( B , C )", "Sema3c transcripts are present in the SG , greater epithelial ridge ( GER ) , and faintly in the IHCs and OHCs .", "( E , F )", "Sema3d transcripts are weakly present in the GER and in a striped pattern that includes non-sensory cells within the medial portion of the cochlear epithelium .", "( H , I )", "Sema3f transcripts are present in the lateral portion of the cochlear sensory domain ( bracketed region of I ) , which includes OHCs , outer pillar cells ( PCs ) , Dieters' cells , and Henson's cells .", "( J–L )", "To better resolve the expression pattern for Sema3f , the preparation in panel I was counterstained with anti-neurofilament light chain and anti-myosin VI .", "Results indicate a sharp medial boundary of Sema3f expression that correlates with the lateral edge of type I SGN innervation .", "( M , N )", "Sema3f expression in the apex and base of the cochlea at P0 .", "IHC , inner HC; PC , pillar cell .", "Sema3f levels are diminished at this stage .", "By P5 , Sema3f transcripts are not detectable ( not shown ) .", "Scale bar in N: 350 μm , A , B , D , E , G , H; 20 μm , C , F , I , M , N; 15 μm , J–L . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 011 To test the hypothesis that Sema3F activates Nrp2 in type I SGNs , which would lead to decreased complexity and inhibition of extension , we established cochlear explant cultures from E15 . 5 or E16 . 5 mice and maintained them in media containing control Fc or Sema3F-Fc fusion proteins for 24 hr .", "At the completion of each experiment , all SGN processes were labeled using Tuj1 .", "To observe the fine details of the SGN endings , it was necessary to prepare 3D reconstructions from high-resolution Z-stacks ( Figure 7A–D ) .", "In controls , type I SGN processes located near the IHCs elaborated complex branching patterns with multiple filopodia per process ( Figure 7A , C ) .", "In contrast , SGN processes from cochleae treated with Sema3F-Fc showed mostly blunt endings with very little branching ( Figure 7B , D ) .", "We quantified this effect by counting the total number of SGN branch tips in the IHC region ( Figure 7E ) .", "For experiments initiated at either E15 . 5 or E16 . 5 , the addition of Sema3F-Fc led to nearly a 50% decrease in the number of SGN branch tips . 10 . 7554/eLife . 07830 . 012Figure 7 . Exogenous Sema3f inhibits SGN outgrowth in vitro .", "( A–D )", "Three-dimensional renderings of confocal z-stacks from cultured cochleae treated with either control IgG-Fc or Sema3F-Fc at 20 nM .", "The cultures were stained with anti-myosin VI to show the position of the HCs ( magenta ) and Tuj1 to mark SGNs ( green ) .", "By comparison with an Fc-treated control ( A , C ) in which the SGNs show extensive branching , Sema3F-Fc-treatment significantly inhibited branching ( B , D ) .", "The white dots in C and D mark individual branch points .", "( E ) Quantification of SGN branch numbers normalized to the length of the cochlea within the photographed region .", "For this normalization , we measured the length of the cochlea along the IHC border .", "Addition of Sema3F-Fc led to significantly fewer SGN branches in explants initiated at E15 . 5 ( black bars ) or E16 . 5 ( gray bars ) .", "**p ≤ 0 . 01; *p ≤ 0 . 05 .", "n = 6 cochleae per treatment group .", "( F–K )", "To determine whether Sema3F-Fc reduced the number of SGNs in the cochlear epithelium or reduced the number of secondary branches per SGN , the Sema3F-Fc experiments were repeated using cochleae from Neurog1CreERT2; R26R-tdTomato mice .", "( F , G )", "Low-magnification micrographs from cochleae stained with anti-myosin VI ( HCs , blue ) and tdTomato to mark individual SGNs ( white ) .", "( H ) There were no differences in the percentages of labeled SGNs contacting the cochlear sensory epithelium in either group .", "( I , J )", "High-magnification images of representative individually labeled SGN terminals .", "Yellow dots mark small secondary branches extending from each SGN peripheral process .", "Note the reduced number of secondary branches in Sema3F-Fc-treated explants .", "( K ) Quantification of the number of secondary branches per SGN ending showing the inhibitory effect of Sema3F-Fc .", "***p ≤ 0 . 001 .", "n = 6 cochleae per treatment group .", "Error bars , sem .", "Scale bar in J: 15 μm , A–D; 30 μm , F and G; 7 μm , I and J . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 012 Although there was an obvious effect on the morphology of SGN processes following treatment with Sema3F-Fc , the labeling of all SGNs by Tuj1 prevented a determination of whether this change resulted from a full retraction of some SGNs from the cochlear epithelium or a reduction in the number of branches per individual SGN .", "Therefore , the Sema3F-Fc experiments were repeated using cochleae from NCre;RTom mice ( Figure 7F–K ) .", "While exposure to Sema3F-Fc did not decrease the number of processes that reached the IHC region ( Figure 7F–G ) , there was an obvious decrease in the number of branched protrusions per process ( Figure 7G , I ) .", "To quantify this change , the number of branches per process was determined for greater than 50 SGNs from each treatment group .", "Results indicate a significant reduction in the number of branches in the presence of Sema3F-Fc ( Figure 7K ) .", "These data are consistent with the hypothesis that a localized source of Sema3F in the OHC region acts to activate Nrp2 in SGN growth cones leading to decreased complexity and extension .", "To confirm that Sema3F normally acts to prevent SGN fibers from extending into the OHC domain , the innervation phenotype was examined in Sema3f−/− mutant mice ( Walz et al . , 2007 ) .", "To visualize individual fibers , the sparse-labeling alleles were bred onto the Sema3f mutant line ( Figure 8A , B ) .", "As predicted , Sema3f−/− cochleae showed an increased percentage of SGN processes in the OHC region .", "Interestingly , when we quantified the increase in crossing processes in Sema3f−/− cochleae ( Figure 8C ) , we found that it was comparable to that observed in Nrp2+/− heterozygotes ( Figure 4 ) .", "Compared to littermate controls , we did not observe any changes in either HC or SGN density in Sema3f−/− cochleae ( Figure 8D–G ) , suggesting the extraneous SGN processes in the OHC region are not a secondary effect of other cochlear changes .", "To determine whether the increase in OHC innervation in Sema3f−/− cochleae led to increased ribbon synapse formation , we used the same approach as for Nrp2-deficient cochleae ( Figure 5 ) .", "Results indicated no differences in the density of ribbon synapses between control and Sema3f−/− .", "As was suggested for the Nrp2 animals , these results suggest that synaptic pruning events that occur following innervation probably lead to a correction in the number of SGN fibers present in the OHC region .", "These data support a model in which Sema3F acts as a ligand for Nrp2 to control SGN extension into the OHC region during development . 10 . 7554/eLife . 07830 . 013Figure 8 . Deletion of Sema3f leads to increased SGNs in the OHC region .", "( A , B )", "Representative images from Neurog1CreERT2;R26RtdTom; WT or Sema3f−/− cochleae at P0 stained with anti-myosin VI ( HCs , blue ) and tdTomato to mark individual SGNs ( white ) .", "Increased numbers of crossing fibers ( yellow arrowheads ) projecting into the OHC region are present in the absence of Sema3f .", "IHCs , inner HCs; OHCs , outer HCs .", "( C ) Absence of Sema3f leads to a significant increase in the percentage of labeled type II-like fibers ( defined as SGNs that had crossed into the OHC region and turned toward the base ) .", "Note that the change in percentage in the absence of Sema3f is comparable to the change observed in Nrp2+/− heterozygotes ( Figure 4E ) .", "**p ≤ 0 . 01; *p ≤ 0 . 05 .", "n = 6 WT; 6 Sema3f−/−; error bars , sem .", "( D ) Whole-mount images of HCs in WT and Sema3f−/− cochleae after myosin-VI immunostaining .", "( E ) Quantification of number of IHCs and OHCs indicates no difference between WT and Sema3f−/− .", "n = 7 each genotype .", "( F ) Whole-mount images of SGNs in WT and Sema3f−/− cochleae after Tuj1 immunostaining .", "The white arrows point to individually identifiable SGN somata .", "( G ) Quantification indicates no difference in SGN density between WT and Sema3f−/− .", "n = 5 each genotype ( H–K ) Visualization of ribbon synapses ( anti-Ribeye in green; white arrows ) on IHCs and OHCs at P8 .", "Nuclei are labeled with DAPI ( blue ) .", "Overall , no differences in ribbon synapse numbers were observed between genotypes .", "( L ) Quantification of ribbon bodies per IHCs and OHCs in P8 .", "All differences were not significant ( ns ) .", "Sample sizes: n = 6 for the control group , which is a combination of heterozygous and WT littermates; 4 Sema3f−/− .", "The scale bar in B = 15 μm for A and B; 30 μm , D; 40 μm , F and 12 μm , H–K . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 013 It was shown previously that intracellular-signaling events that occur as a result of Sema3f/Nrp2 interactions depend entirely on the presence of Plexin-A3 ( Yaron et al . , 2005 ) .", "Therefore , we examined Plexin-A3 expression in the SGNs during developmental periods corresponding with IHC and OHC innervation ( Figure 9—figure supplement 1 , Figure 9 ) .", "In situ hybridization data from the Allen Brain Atlas Website clearly show Plxna3 mRNA distribution in the cochleovestibular ganglion at E11 . 5 and in the SGNs at E13 . 5 and E15 . 5 ( http://developingmouse . brain-map . org/gene/show/18610 ) ( Figure 9—figure supplement 1 ) .", "To verify these results , we generated Plxna3 sense and antisense mRNA probes and performed in situ hybridization at E16 . 5 .", "Consistent with the Allen Brain Atlas , Plxna3 antisense probes indicated broad expression in SGNs in both the apical and basal regions of the cochlea ( Figure 9A–C ) .", "Using differential interference contrast to closely examine the SGNs ( Figure 9D–F ) , we did not detect any obvious differences in Plxna3 mRNA within the population , suggesting uniform expression of Plxna3 .", "In contrast , examination of Plexin-A3 protein distribution in whole-mount preparations at E16 . 5 indicated a striking difference in Plexin-A3 levels in SGN peripheral processes .", "At low magnification , Plexin-A3 protein is visible broadly in the SGN processes in a pattern similar to that of Tuj1 ( Figure 9G–I ) .", "However , at high-magnification , Plexin-A3 expression in fibers extending beyond the IHC region is markedly reduced by comparison with substantially higher levels in fibers that terminate near IHCs ( Figure 9G–L ) .", "Since SGNs are actively probing the IHC and OHC regions at this stage ( Figures 1 , 2 ) , individual fibers cannot unambiguously be designated as ‘type I’ or ‘type II’ .", "But , Plexin-A3 expression on fibers located in the OHC region is detectable only on sparse numbers of SGNs as small puncta ( arrowheads ) .", "These data suggest that Plexin-A3 may act as an Nrp2 co-receptor in SGNs , and that differences in Plexin-A3 protein levels could modulate Sema3F-mediated repulsion . 10 . 7554/eLife . 07830 . 014Figure 9 . Differential expression of Plexin-A3 in SGNs and a proposed model .", "( A–F )", "In situ hybridization for Plxna3 at E16 . 5 .", "( A ) A Plxna3 sense control probe does not show any reactivity .", "( B , C )", "Plxna3 transcripts are present in the SG , and possibly faintly in cells throughout the cochlear epithelium ( ce ) .", "( D–F )", "Differential interference contrast ( DIC ) images for A–C were used to identify SGNs by morphology .", "The white dotted lines encircle the SGNs in each panel .", "( G–I )", "Plexin-A3 antibody staining in an E16 . 5 whole-mount cochlea .", "( G–I )", "Expression of Plexin-A3 ( green ) in SGNs appears to overlap with a neuronal marker ( Tuj1 in red ) except in the HC region ( MyoVI staining in blue ) .", "( J–L )", "High-magnification views from the boxed regions in G , H , and I . Plexin-A3 is strongly expressed in SGN peripheral processes located adjacent to the IHC region ( arrows ) but appears markedly reduced in fibers that cross into the OHC region .", "Limited Plexin-A3 is detectable on sparse numbers of SGN processes in the OHC region ( arrowhead ) .", "Scale bar in L: 100 μm , A–I , 25 μm , J–L .", "( M ) Proposed model: Sema3F ( blue shading and blue rectangles ) is expressed by cells in the OHC region of the cochlea with a sharp boundary along the PCs .", "Binding of Sema3F to Nrp2 ( green SGNs and green rectangle ) induces retraction events that inhibit the growth of the type I SGN peripheral processes into the OHC domain .", "PlexinA3 co-receptors , which likely serve a role in this process , are shown in red .", "‘ ? ’ illustrates an outstanding question of how type II SGNs , which express Nrp2 and are confronted by Sema3F , are able to extend into the OHC domain . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 01410 . 7554/eLife . 07830 . 015Figure 9—figure supplement 1 . Plxna3 expression in the inner ear at E11 . 5-E15 . 5 . In situ hybridization using Plxna3 antisense probe in C57BL/6J mice from the Allen Brian Atlas ( http://developingmouse . brain-map . org/gene/show/18610 ) shows that Plxna3 is broadly expressed in the cochleovestibular ganglion neurons ( cvg ) at E11 . 5 and in the SGNs ( sg ) at E13 . 5 and E15 . 5 Specific images can found at: http://developingmouse . brain-map . org/experiment/show/100047035 for E11 . 5 , http://developingmouse . brain-map . org/experiment/show/100049528 for E13 . 5 , and http://developingmouse . brain-map . org/experiment/show/100049508 for E15 . 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 07830 . 015" ], [ "The SGN comprises multiple neuronal phenotypes arranged into precise patterns of innervation .", "Despite initial descriptions dating back more than 100 years , our understanding of the development and organization of this structure remains limited .", "A particularly intriguing issue has been the question of whether type I and type II SGNs are determined as a result of the expression of distinct transcriptional networks , as a result of interactions between peripheral axons and target HCs , or a combination of both .", "Classic studies of developing SGNs by Echteler and others indicated the presence of individual fibers in contact with both IHCs and OHCs , supporting the possibility of target-mediated determination of SGN cell types ( Echteler , 1992 ) .", "However , more recent work using genetic labeling provides strong evidence for the presence of committed type I and type II SGN fibers in the mouse inner ear even at embryonic ages ( Koundakjian et al . , 2007 ) .", "Moreover , detailed morphological and cellular analyses by N Druckenbroad and L Goodrich ( personal communication ) revealed that both type I and II SGNs show stereotyped path-finding behaviors , supporting the idea that each SGN subtype may be predetermined to an extent .", "The results presented here ( and by Druckenbroad and Goodrich ) show that cochlear innervation normally involves the transient extension of SGN fibers into the OHC region between E15 . 5 and E16 . 5 .", "After this brief period and continuing through P0 , the majority of SGN processes converge on the IHC region .", "This occurs through both the arrival of new fibers and the retraction of existing processes from the OHC region .", "Labeling of future type I fibers with transient OHC extensions could appear as individual neurons with indeterminate phenotypes .", "Moreover , considering that the cells within the cochlear epithelium are in the process of both differentiation and active extension between E15 . 5 and E16 . 5 ( Yamamoto et al . , 2009; Wu and Kelley , 2012; Coate and Kelley , 2013 ) , it is not surprising that SGN processes might also be highly dynamic during this time period .", "Nrp2-mediated repulsion in response to Sema3F was identified many years ago and has been documented in multiple events ranging from axon guidance to synaptic pruning ( Adams et al . , 1997; Chen et al . , 1997; Giger et al . , 2000; Demyanenko et al . , 2014 ) .", "Therefore , there is strong precedent for Sema3F-Nrp2 interactions serving a repulsive role in SGN-HC connectivity .", "The observation of active extension and retraction of SGN processes into and out of the OHC region is consistent with the presence of inhibitory signals acting on those processes .", "In these studies , we demonstrate that repulsive signaling mediated by Sema3F in the OHC region and Nrp2 in SGNs controls , at least in part , the number of SGN fibers that terminate on OHCs .", "Our findings support a model in which the embryonic OHC region maintains a compartment of Sema3F protein expression , which activates Nrp2 receptors expressed on SGNs leading to retraction of processes ( Figure 8D ) .", "Since this inhibitory signal is restricted to the OHC domain , this interaction would enforce contacts between SGN fibers and IHCs .", "Type I SGNs were still observed even in the complete absence of either Nrp2 or Sema3f .", "There are several possible explanations for this phenotype .", "First , it is reasonable to assume that compensatory guidance mechanisms are in place to ensure that most type I SGNs terminate on IHCs .", "For example , there are a variety of cell adhesion molecules ( CAMs ) expressed in the cochlea such as the immunoglobulin superfamily of CAMs ( like NCAM ) , cadherins ( E-cad and N-Cad ) , and integrins ( Kelley , 2003 ) , all of which may help attract type I SGNs to the IHC region .", "In addition , it was recently shown that ephrin-A5/Epha4 signaling may also act to repel type I SGNs from OHCs ( Defourny et al . , 2013 ) .", "These studies , however , were limited to early postnatal and adult cochlear samples , thus , the extent to which these factors are involved in targeting events during embryonic development remains uncertain .", "Finally , it is also possible that the developing SGN population is heterogeneous with some neurons demonstrating more dynamic exploratory behavior than others .", "Therefore , some fibers may be less inclined to extend into the OHC region even in the absence of inhibitory signals .", "This hypothesis might be consistent with the idea that some SGNs are initially committed to a type I fate while others rely on target-based interactions to determine their phenotype .", "As discussed , despite the presence of additional type II-like processes in Nrp2+/− cochleae at birth , analysis of adult animals indicated a normal number of type II SGNs and hearing sensitivity .", "These results suggest that the additional type II-like fibers observed at birth are most likely eliminated over time and the factors that mediate this recovery are unknown .", "Given that ‘mature’ type I/type II SGN patterns are established by P0 ( Figure 2 ) and that HC ribbon synapses are visible by P0 ( Huang et al . , 2012 ) , it could be that Eph/ephrin signaling might be involved in synaptic pruning-based refinements postnatally .", "In addition , programmed cell death may also contribute to the final innervation pattern , as apoptosis in SGNs has been reported in the postnatal gerbil ( Echteler et al . , 2005 ) .", "However , we did not detect any appreciable SGN fragmentation or apoptosis in our experiments here ( Figure 2—figure supplement 1 ) or in previous studies ( Coate et al . , 2012 ) .", "Therefore , while we cannot rule it out entirely , we would not predict that apoptosis plays a significant role in shaping cochlear innervation during development .", "Finally , this study also raises several intriguing questions to be addressed in the future .", "First , since Sema3F acts as a repulsive signal for SGN processes , how are type II SGNs , which appear to express Nrp2 ( Figure 2 ) , able to project into the OHC region ( see ‘ ? ’ in Figure 9 ) ?", "We did not detect differences in Nrp2 levels between putative type I and type II SGNs , which could have accounted possibly for differences in responsiveness to Sema3F .", "We are currently in favor of the model that type I and type II SGNs differentially express or target Plexin-A co-receptors , as the absence of these obligatory co-receptors could effectively inactive the Sema3F-Nrp2 inhibitory interaction .", "Plexin-A1 and Plexin-A3 proteins were localized to the SGNs previously ( Murakami et al . , 2001; Katayama et al . , 2013 ) .", "Here , we found Plxna3 mRNA broadly expressed by the SGNs and Plexin-A3 protein enriched in putative type I SGNs ( Figure 9 ) .", "While these data suggest that where type I or II SGNs ultimately terminate may depend on Plexin-A3 protein levels , a more careful analysis of the Plexin-A3 distribution and function is needed .", "A second question relates to the remarkably different patterns of expression exhibited by the different Sema3s ( Figure 6 ) .", "What factor ( s ) control these discrete Sema3 domains ?", "GATA transcription factors have been shown previously to regulate Sema expression and are known players in inner ear morphogenesis and wiring ( Lepore et al . , 2006; Kodo et al . , 2009; Appler and Lu , 2013 ) , so perhaps one role of GATAs in the inner ear is to regulate Sema3 levels .", "Other clues related to this matter may come from previous findings where either the loss of Prox1 or Fgfr3 signaling led to increases in OHC innervation ( Puligilla et al . , 2007; Fritzsch et al . , 2010 ) .", "It is possible , therefore , that Prox1 controls the expression of factors such as Sema3f or that the local distribution of the different Sema3s is controlled by morphogens such as Fgfs .", "Overall , it will be important in future studies to investigate upstream activators of Sema3 expression and mechanisms that ultimately distinguish the morphological differentiation of type I and type II SGNs ." ], [ "All animals used in this study were maintained in accordance with the NIH Animal Care and Use Committee .", "For expression studies and in vitro culture experiments , timed-pregnant CD1 mice ( Charles River Laboratories , Frederick , MD ) were used .", "The Neurog1CreERT2 line used to characterize SGN phenotypes has been described previously ( Koundakjian et al . , 2007 ) .", "These mice were crossed with a Rosa26tdTomato reporter line ( Jackson Laboratories , Bar Harbor , ME; stock# 007914 ) and with an Atoh1nGFP line ( Lumpkin et al . , 2003 ) to generate triple transgenics referred to as ‘NCre;RTom; Atoh1nGFP’ .", "Because a small amount of spontaneous Cre activity within the Neurog1CreERT2 line resulted in recombination and expression of tdTomato in a limited number of SGNs , no tamoxifen was administered to the dams .", "No apparent regional or cell type bias in the pattern of spontaneous recombination was observed along the tonotopic ( basal-to-apical ) axis or between type I and type II SGNs .", "The Nrp2 and Sema3f mutant lines have been described previously ( Giger et al . , 2000; Walz et al . , 2007 ) .", "For immunohistochemical studies of whole mounts , cochleae were removed from the skull and fixed in 4% paraformaldehyle ( PFA; 30 min to 1 hr depending on age ) at RT followed by extensive rinsing in cold 1× phosphate-buffered saline ( PBS ) .", "For tissue sectioning , following fixation , inner ears were exposed to increasing concentrations of sucrose ( up to 30% ) and then embedded and frozen in optimal cutting temperature ( OCT ) medium ( Sakura Finetek , Torrance , CA ) .", "In most cases , OCT blocks were sectioned at 12 μm .", "In situ hybridizations were performed on frozen sections that had been prepared similarly , but with overnight fixation prior to embedding .", "The antibodies and concentrations used in this study were as follows: mouse-anti-Tuj1 ( Covance , Chantilly , CA ) ; 1:500 , rabbit-anti-dsRed ( Clontech , Mountain View , CA ) ; 1:500 , chicken-anti-Atoh1 ( Driver et al . , 2013 ) ; 1:10 , 000 , chicken-anti-NFS ( Aves Labs , Tigard , OR ) ; 1:1 , 000 , rabbit-anti-myosin VI ( Proteus Biosciences , Ramona , CA ) ; 1:1 , 000 , goat-anti-Sox2 ( Santa Cruz Biotechnology , Dallas , TX ) rabbit-anti-Nrp2 for sections ( Giger et al . , 1998 ) ; 1:500 , goat-anti-Nrp2 for whole-mount staining; 1:500 ( R&D Systems , Minneapolis MN ) , mouse-anti-human Fc; 1:100 ( Jackson Immunoresearch , West Grove , PA ) , chicken-anti-synaptotagmin 1 ( Aves Labs ) ; 1:1 , 000 , mouse-anti-GAP43 ( Chemicon , Billerica , MA ) ; 1:1 , 000 , chicken-anti-GFP ( Aves Labs ) ; 1:1 , 000 , mouse-anti-CtBp2 ( BD Biosciences , San Jose , CA ) ; 1:200 , rabbit-anti-cleaved caspase 3; 1:500 , and rabbit-anti-CDKN1B ( formerly p27Kip1; Novus Biologicals , Littleton , CO ) .", "A goat polyclonal antibody against myosin VI was generated ( Pacific Immunology , Ramona , CA ) using an immunizing fusion protein provided by Proteus Biosciences ( Hasson and Mooseker , 1994 ) ; 1:1000 .", "To detect Plexin-A3 , we used a goat-anti-Plexin-A3 antibody from R&D Systems ( catalog# AF4075 ) .", "This antibody was raised against the extracellular domain of mouse and rat Plexin-A3 ( Leu34-Asp150 ) and , according to the manufacturer , shows less than 5% cross-reactivity with other Plexins .", "This antibody was validated by R&D Systems using ELISA , Western blot , and immunofluorescence .", "The Plexin-A3 protein staining we report here ( in SGNs ) is consistent with the Plxna3 mRNA distribution also reported here and noted previously ( Murakami et al . , 2001 ) .", "Actin was detected using phalloidin conjugates ( Life Technologies , Carlsbad , CA ) at 1:100 .", "After fixation , whole-cochleae or tissue sections were permeabilized with PBS supplemented with 0 . 5% Triton-X100 ( Sigma , St . Louis , MO ) for 20 min at RT .", "Tissue samples were then exposed to either 10% normal goat or horse serum ( Vector Labs , Burlingame , CA ) for 1 hr .", "When anti-mouse secondary antibodies were used , anti-mouse FAB fragments ( Jackson Immunoresearch ) were added to the blocking solution at 1:200 .", "When anti-chicken secondary antibodies were used , 10% Blokhen Reagent ( Aves Labs ) was added .", "Following blocking , samples were incubated in primary antibody ( diluted in PBS +0 . 5% Triton ) overnight at 4°C .", "After extensive rinsing the following day , 488- , 555- , or 633-Alexa Fluor secondary antibodies ( Life Technologies ) were used for detection of primary antibody binding .", "Images were acquired using a Zeiss LSM-510 or -710 laser scanning confocal microscope and then further processed using ImageJ and/or Adobe Photoshop software .", "For the experiments looking at Nrp2 expression over developmental time by tissue sectioning in Figure 3 , identical processing , immunostaining and image acquisition procedures and settings were used .", "In situ hybridization was performed as described ( Driver et al . , 2008 ) , but with antisense probes generated from mouse , Sema3f ( NM_011349 ) , Sema3b ( BC150758 ) , Sema3c ( BC066852 . 1 ) , Sema3d ( BC138131 . 1 ) , and Plxna3 ( NM_008883 ) templates .", "For every experiment , sense probes were generated and used as negative controls .", "The images representing Plxna3 from the Allen Brain Atlas database can be found here: http://developingmouse . brain-map . org/gene/show/18610 .", "To localize endogenous binding partners for Sema3F , we used Sema3F-Fc-conjugated protein as previously described ( Sahay et al . , 2003 ) , but with minor modifications .", "Briefly , whole-inner ears were fixed briefly in ice-cold 4% paraformaldehyde to help maintain structural stability and processed for cryosectioning .", "Without permeabilizing , 12-μm sections were rinsed in 1× PBS then incubated with 20 nM human Fc ( Jackson Immunoresearch ) or Sema3F-Fc ( R&D Systems ) overnight at 4°C .", "After extensive rinsing with PBS , the tissue samples were treated with 4% PFA for 15 min , rinsed extensively in 1× PBS , then processed for anti-Fc and anti-NF200 immunostaining .", "P0 cochleae were isolated and digested in radio immunoprecipitation assay ( RIPA ) buffer ( Sigma ) supplemented with protease inhibitors ( Roche , Basel , Switzerland ) .", "Lysates were run on a 4–12% acrylamide gel and then transferred to nitrocellulose membranes .", "Membranes were then blocked with 5% milk block ( Blotto; Santa Cruz Biotechnology , Dallas , TX ) in tris-buffered saline with tween ( TBST ) and then probed with either rabbit-anti-Nrp2 ( 1:2000; R&D Systems; Demyanenko et al . , 2011 ) , or chicken-ant-Tuj1 antibodies ( 1:5000 , Aves ) overnight .", "The following day , following extensive rinsing , the membranes were probed with peroxidase-conjugated secondary antibodies ( 1:5000; Jackson Immunoresearch ) for 45 min , treated with Clarity chemiluminescence substrates ( Bio-Rad , Hercules , CA ) , and imaged using an Imagequant LAS 4000 ( GE Life Sciences , Pittsburgh , PA ) .", "NCre;RTom; Atoh1nGFP-positive embryos or postnatal pups were identified using a dissection microscope with fluorescence illumination .", "Heads were place in chilled 1× Hank's buffered saline solution ( HBSS ) , inner ears were dissected , and the cochlear capsule , stria vascularis , and Reissner's membrane were carefully removed .", "Each semi-intact cochlea was then incubated on a polycarbonate membrane filter ( Sterlitech , Kent , WA ) for 2 hr at 37°C to gently flatten the tissue prior to imaging .", "After this 2-hr period , each cochlea was transferred to a Mattek dish ( Mattek Corporation , Ashland , MA ) and flattened next to the cover glass with a platinum harp strung with nylon filament .", "The imaging medium included Leibovitz medium ( Life Technologies ) , 10% fetal bovine serum , 0 . 2% N2 , 0 . 001% Ciprofloxacin , and 0 . 1 mM Trolox ( as an antioxidant ) .", "Using an Axiovision ( Zeiss , Oberkochen , Germany ) spinning disk confocal microscope , the SGNs and HCs were imaged at a capture speed of 250–400 ms at 10-to 20-min intervals .", "Time-lapse data were subsequently processed using Volocity software ( Perkin Elmer , Waltham , MA ) .", "For experiments using Fc fusion proteins , E15 . 5 or E16 . 5 cochleae from NCre;RTom embryos were prepared as described above , but maintained on polycarbonate filters ( Sterlitech ) for 24 hr .", "In these experiments , the culture medium contained Dulbecco's Modified Eagle Medium ( DMEM ) , 10% fetal bovine serum , 0 . 2% N2 , and 0 . 001% ciprofloxacin .", "Purified human IgG-Fc ( Jackson Immunoresearch ) or Sema3F-Fc ( R&D Systems ) was added directly to the medium to a final concentration of 20 nM .", "Following the culture period , the cochleae were fixed for 15 min in chilled 4% PFA , then rinsed extensively before immunostaining and confocal imaging .", "To quantify the developmental time series for type I and type II projection patterns in Figure 2 , confocal Z-stacks were acquired from the base of each cochlea at positions corresponding to 10–25% of the cochlear length .", "An SGN was considered ‘in the OHC region’ if the most prominent part of the axon ( barrel ) clearly crossed the plane of the medial edge of the first OHC .", "Conversely , an SGN was considered ‘in the IHC region’ if it stopped at the IHC region .", "To quantify the number of Syt+ axons that had crossed into the OHC region in Figure 3 , confocal Z-stacks were acquired at positions corresponding to 50% of the cochlear length .", "The number of Syt+ fibers that crossed into the OHC region were counted and normalized to the length of the region; the length of the region was determined by drawing a line along the medial side of the IHCs .", "For quantifications using the sparse SGN-labeling model ( Figures 4 , 8 ) , a series of high-resolution confocal Z-stacks were acquired along the length of each specimen using an automated-scanning stage .", "The files were subsequently imported to Volocity for quantification of IHC vs OHC position and branching morphometrics .", "To quantify ribbon synapses , cochleae were stained with anti-CtBP2 antibodies and photographed as described previously ( Yu et al . , 2013 ) .", "To quantify E15 . 5 SGN density in Figure 3 , Tuj1-stained whole-mount cochleae were imaged by confocal z-stack and the spherical nuclei within each stack were counted manually .", "For the SGN counts in Figure 8 , similar methods were used , but we were only able to reliably quantify the first few cell layers because of limited tissue penetration by the mouse-anti-Tuj1 antibody at this older stage ( P0 ) .", "For consistency across the P0 samples , we limited our counts to only 10 μm of z depth .", "To quantify HC density in Figure 8 , myosin VI-positive HCs at the base of P0 cochleae were imaged by confocal microscopy .", "For each sample , we acquired 5 confocal z-stacks at 63× in non-overlapping regions along the first 1 mm of cochlear length ( starting from the base ) .", "HCs were counted manually .", "To quantify apoptosis in E16 . 5 and E17 . 5 SGNs , we stained cochleae in whole mount with anti-cleaved caspase-3 ( CC3 ) and Tuj1 antibodies .", "Cells were counted as SGNs undergoing apoptosis if they were ( 1 ) CC3 positive , ( 2 ) within the Tuj1-positive SGN cell body region , and ( 3 ) bipolar cells ( 7–8 μm wide ) with peripheral and central projections .", "We could not rely on Tuj1 co-staining because the Tuj1 antibody did not show identical levels of tissue penetration compared to the CC3 antibodies .", "Other cells ( glial cells , blood vessel cells , mesenchyme cells ) or fragmented debris were not counted .", "Low-magnification views were used to measure cochlear length in order to determine the basal and apical halves .", "Two-tailed t-tests were used throughout .", "Sample numbers are indicated in the figure legends ." ] ]
[ "Auditory function is dependent on the formation of specific innervation patterns between mechanosensory hair cells ( HCs ) and afferent spiral ganglion neurons ( SGNs ) .", "In particular , type I SGNs must precisely connect with inner HCs ( IHCs ) while avoiding connections with nearby outer HCs ( OHCs ) .", "The factors that mediate these patterning events are largely unknown .", "Using sparse-labeling and time-lapse imaging , we visualized for the first time the behaviors of developing SGNs including active retraction of processes from OHCs , suggesting that some type I SGNs contact OHCs before forming synapses with IHCs .", "In addition , we demonstrate that expression of Semaphorin-3F in the OHC region inhibits type I SGN process extension by activating Neuropilin-2 receptors expressed on SGNs .", "These results suggest a model in which cochlear innervation patterns by type I SGNs are determined , at least in part , through a Semaphorin-3F-mediated inhibitory signal that impedes processes from extending beyond the IHC region ." ]
[ "The process of hearing begins when sound waves enter the outer ear , causing the eardrum to vibrate .", "The three small bones of the middle ear pass these vibrations on to the cochlea , a fluid-filled structure shaped like a spiral .", "Tiny hair cells inside the cochlea move in response to the vibrations and convert them into electrical signals , which are transmitted by cells called spiral ganglion neurons ( SGNs ) to the brain .", "Hair cells can be divided into ‘inner’ and ‘outer’ hair cells .", "Inner hair cells transmit most of the information about a sound to the brain , via connections with type I SGNs .", "Outer hair cells are thought to amplify sound and connect to type II SGNs .", "How the type I and II SGNs connect to the correct type of hair cell as the ear develops is not well understood , despite these connections being essential for hearing .", "Coate et al . have now used time-lapse imaging and fixed specimens to follow individually labeled SGNs as they establish these connections within the cochlea of a mouse embryo .", "Although the type I SGNs ultimately formed connections with inner hair cells , many of them made contact with outer hair cells first .", "These contacts were short-lived thanks to a protein found near the outer hair cells , named Semaphorin-3F .", "This protein repels the type I SGNs by activating a receptor on their surface called Neuropilin-2 , and so directs the type I SGNs towards the inner hair cells .", "One of the mysteries that remains to be solved is how type II SGNs are ‘permitted’ to extend into the outer hair cell region , even though they are also confronted by Semaphorin-3F .", "In addition , it will also be important to determine how SGNs adapt to cues from different Semaphorins from different parts of the cochlea as they navigate into different hair cell regions ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "short report", "biochemistry and chemical biology" ]
A reverse transcriptase ribozyme
elife-31153-v2
[ [ "It is widely thought that RNA-based life preceded DNA- and protein-based life during the early history of life on Earth ( Gilbert , 1986; Joyce , 2002 ) .", "Perhaps the strongest evidence in support of this hypothesis is the ribosome , present in all extant life , which is an RNA enzyme that catalyzes the RNA-instructed synthesis of polypeptides ( Nissen et al . , 2000 ) .", "This presumed remnant of the ‘RNA world’ need not have persisted into modern biology , but would have been necessary for the invention of the translation machinery .", "The other key transitional molecule between RNA- and DNA/protein-based life is reverse transcriptase , which catalyzes the RNA-dependent polymerization of DNA and is responsible for maintaining genetic information in the more stable form of DNA .", "The first reverse transcriptase may have been either an RNA or protein enzyme .", "The former seems plausible because such an enzyme could have derived from an RNA-dependent RNA polymerase , which would have been an essential component of RNA-based life .", "It has been argued that the translation machinery requires more heritable information than can be maintained by RNA genomes ( Maynard Smith and Szathmáry , 1995 ) , thus placing the invention of DNA before the invention of proteins .", "Conversely , it has been argued that the biochemical reduction of ribonucleotides to deoxynucleotides is beyond the catalytic abilities of RNA ( Freeland , 1999 ) , placing proteins before DNA , although a photoreductive route to the deoxynucleotides also has been proposed ( Ritson and Sutherland , 2014 ) .", "The present study demonstrates that an RNA enzyme with highly evolved RNA-dependent RNA polymerase activity also can function as a reverse transcriptase , thus providing a bridge between the ancestral and contemporary genetic material without the need for proteins .", "There are several examples of RNA enzymes with RNA-dependent RNA polymerase activity , all of which were obtained by in vitro evolution ( Ekland and Bartel , 1996; Johnston et al . , 2001; McGinness and Joyce , 2002; Sczepanski and Joyce , 2014 ) .", "The most sophisticated of these is the class I polymerase , which derives from an RNA ligase ( Bartel and Szostak , 1993 ) and catalyzes the polymerization of nucleoside 5´-triphosphates ( NTPs ) .", "Over the past two decades , the activity of this enzyme has been greatly improved ( Zaher and Unrau , 2007; Wochner et al . , 2011 ) , most recently acquiring the ability to synthesize a variety of functional RNAs and to catalyze the exponential amplification of short RNAs ( Horning and Joyce , 2016 ) .", "The chemistry of DNA polymerization is more challenging than that of RNA polymerization because the lack of a 2´-hydroxyl in DNA reduces the nucleophilicity of the adjacent 3´-hydroxyl .", "Based on the relative pKa values of the 3´-hydroxyl in either RNA or DNA , this difference is ~100 fold ( Åström et al . , 2004 ) , whereas non-enzymatic addition of activated monomers to either an RNA or DNA primer indicates a difference of ~10 fold ( Wu and Orgel , 1992 ) .", "For all but the most recently evolved form of the RNA polymerase , a single deoxynucleoside 5´-triphosphate ( dNTP ) can be added to a template-bound RNA primer , but subsequent dNTP addition to the 3´-terminal deoxynucleotide does not occur ( Attwater et al . , 2013 ) .", "The most recent form of the polymerase has ~100 fold faster catalytic rate and much greater sequence generality compared to its predecessor ( Horning and Joyce , 2016 ) .", "As reported here , this enzyme is able to compensate for the lower chemical reactivity of a 3´-terminal deoxynucleotide and add multiple successive dNTPs in an RNA-templated manner .", "A second important difference between RNA and DNA is the strong tendency of the former to adopt a C3´-endo sugar pucker , whereas the latter favors a C2´-endo pucker .", "However , when part of a primer bound to an RNA template , both ribo- and deoxyribonucleotides tend to adopt a C3´-endo conformation , so this issue is less likely to be an obstacle in transitioning from an RNA polymerase to a DNA polymerase .", "A third difference between RNA and DNA is the presence of uracil in the former versus thymine in the latter , which provides a means to distinguish thymine from deoxyuridine that results from spontaneous deamination of deoxycytidine .", "Most RNA polymerases , including the class I polymerase ( Attwater et al . , 2013 ) , are indifferent to the presence of a C5-methyl substitution on uracil , so this too is not likely to be an obstacle to the development of a reverse transcriptase .", "Once even a modest level of RNA-dependent DNA polymerase activity arises , it is expected that evolutionary optimization of that activity could occur ." ], [ "Through many successive generations of in vitro evolution , the class I polymerase ribozyme has been progressively refined so that it can add many successive NTPs , operate with a fast catalytic rate , and accept a broad range of template sequences .", "Among the key innovations were: ( 1 ) installation and evolutionary optimization of an accessory domain to increase catalytic efficiency ( Johnston et al . , 2001; Zaher and Unrau , 2007 ) ; ( 2 ) addition of a Watson-Crick pairing domain between the 5´ end of the enzyme and 5´ end of the template to enhance binding of the template-primer complex ( Wochner et al . , 2011 ) ; and ( 3 ) discovery of a constellation of mutations to improve reaction rate and sequence generality ( Horning and Joyce , 2016 ) .", "This most recent form of the enzyme , the ‘24–3 polymerase’ , has an initial rate of NTP addition of >2 min–1 and can copy most template sequences .", "The 24–3 polymerase was tested for its ability to catalyze the RNA-templated addition of dNTPs to the 3´ end of an RNA or DNA primer ( Figure 1A ) .", "The enzyme was found to be capable of multiple successive dNTP additions , which is not the case for its evolutionary predecessors ( Attwater et al . , 2013 ) .", "Employing a 15mer primer that binds to a complementary RNA template , the primer can be extended to generate full-length products , together with a ladder of partial extension products ( Figure 1B ) .", "For short C-rich templates , such as 3´-GCCCCCAC-5´ ( template", "1 ) or 3´-GCCCCCACGCCCCCUC-3´ ( template 2 ) , a substantial fraction of the products are full-length , whereas for templates that are less C-rich and/or contain regions of stable secondary structure ( templates 3 and 4 ) , there is little or no full-length product .", "Long and unstructured C-rich templates , such as 3´-GCCCCCACGCCCCCUCGCCCCCACGCCCCCUC-3´ ( template 5 ) , can give rise to full-length products , in this case requiring the addition of two or more residues of each of the four dNTPs .", "For all templates tested , the reaction proceeds similarly using either an all-RNA primer or an RNA primer that has a single 3´-terminal deoxynucleotides ( Figure 1—figure supplement 1 ) .", "When an all-DNA primer is used , the reaction proceeds similarly for the more favorable templates , but is less efficient for the more challenging templates .", "This behavior presumably reflects the greater difficulty of DNA versus RNA hybridization when primer binding must compete with secondary structure in the primer-binding region of the template .", "The 24–3 enzyme has negligible activity in the DNA-templated polymerization of either dNTPs or NTPs ( Figure 1—figure supplement 2 ) .", "This is true even when an all-RNA primer is used , which enables addition of a single nucleotide , but almost no subsequent nucleotide addition .", "Two approaches were taken to confirm the identity of the reverse transcription products obtained using template 1 .", "First , the presumed full-length materials , initiated by either an all-RNA or an all-DNA primer , were purified by denaturing polyacrylamide gel electrophoresis and subjected to partial digestion with DNase I . This enzyme degrades 3´ , 5´-phosphodiester linkages in DNA but not RNA .", "For the RNA-primed products , the extended portion was degraded by DNase and the primer portion remained intact , whereas for the DNA-primed products the entire molecule was degraded ( Figure 2A ) .", "Authentic standards were treated in a side-by-side manner and gave rise to the same pattern of degradation products .", "The second confirmatory approach involved analysis of the gel-purified , full-length materials by liquid chromatography/mass spectrometry .", "The RNA or DNA primer contained a 5´-fluorescein label to permit visualization in the gel and the reaction involved addition of deoxynucleotides residues having the sequence 5´-CGGGGGTG-3´ .", "For the RNA-primed reaction the calculated mass was 8252 . 4 and the observed mass was 8252 . 4 ( Figure 2B ) ; for the DNA-primed reaction the calculated mass was 8068 . 5 and the observed mass was 8068 . 1 ( Figure 2C ) .", "High-resolution ion trap tandem MS was used to confirm the sequence of the 10mer reverse transcript obtained using template 4 .", "This partial-length product contains all four deoxynucleotides and has the sequence 5´-GCGAGGAGTG-3´ .", "For the RNA-primed reaction , the calculated mass was 8874 . 432 and the observed mass was 8874 . 441 .", "From the parent ion , 3´-terminal fragments were generated that contained 2–9 deoxynucleotides and had observed masses matching the calculated masses for these materials ( Figure 2—figure supplement 1 ) .", "To further investigate the fidelity of reverse transcription , the reaction was carried out either in the presence of all four dNTPs or in a mixture lacking dGTP , dATP , TTP , or dCTP .", "Templates 3 and 4 were tested , both of which contain all four nucleotides and direct the synthesis of partial-length products containing up to 6 or 12 deoxynucleotides , respectively .", "For both templates , the size distribution of the products was the same when employing either all four dNTPs or a mixture that lacked dATP ( Figure 3 ) .", "For reactions without dATP , however , the gel mobility was altered at the site of dATP incorporation , consistent with the misincorporation of dGTP as a G•U wobble pair .", "The 24–3 polymerase is known to tolerate G•U wobble pairing during RNA-templated RNA polymerization ( Horning and Joyce , 2016 ) , so it is not surprising that this is also the case during reverse transcription .", "In contrast , omission of dGTP , TTP , or dCTP resulted in termination of DNA polymerization at the site of the missing dNTP .", "Time-course experiments were carried out to determine the rate of reverse transcription , extending an RNA primer with a single 3´-terminal deoxynucleotides , in comparison to the rate of RNA-dependent RNA polymerization , extending an all-RNA primer .", "These experiments employed 100 nM primer , 125 nM template 1 , 125 nM enzyme , 2 mM each of the four dNTPs or NTPs , and 200 mM MgCl2 , and were carried out at pH 8 . 3°C and 20°C .", "The reaction has a rapid initial burst phase , followed by a second slower phase that continues until >90% of the primer molecules are extended ( Figure 4 ) .", "For DNA polymerization , the rate of the initial burst phase is 1 . 1 min–1 , proceeding to an extent of ~35% , followed by a second phase with a rate of 0 . 029 min–1 .", "For RNA polymerization , the rate of the initial burst is >2 . 0 min–1 , proceeding to an extent of ~20% , followed by a second phase with a rate of 0 . 073 min–1 .", "The reason for biphasic kinetics is unclear .", "The fast phase presumably reflects the fraction of enzyme-template-primer complexes present at the start of the reaction , whereas the slower second phase may reflect the formation of additional reactive complexes .", "Reverse transcription is accelerated at pH 9 . 0 compared to pH 8 . 3 ( Figure 1B ) .", "For reactions initiated by an RNA primer , the higher pH results in some degradation of the primer portion of the extended products .", "The RNA enzyme has a high requirement for Mg2+ , typically 200 mM , which also promotes RNA degradation .", "However , once sequence information has been copied from RNA to DNA , the DNA product can be maintained under high-pH , high-Mg2+ conditions ." ], [ "The RNA-templated synthesis of RNA , as catalyzed by a ribozyme , has been known for 20 years ( Ekland and Bartel , 1996 ) .", "Carrying that activity over to the RNA-templated synthesis of DNA has always seemed plausible ( Joyce , 2002 ) , but required a ribozyme with sufficient polymerization activity to compensate for the inherently lower chemical reactivity of deoxyribose compared to ribose 3´-hydroxyl .", "A similar historical pathway can be imagined for the transition from RNA to DNA genomes during the early history of life on Earth .", "The stability of DNA compared to RNA greatly exceeds the difference in the chemical reactivity of their respective 3´-hydroxyl groups .", "DNA is more prone to depurination compared to RNA , but this weakness is far outweighed by the greater backbone stability of DNA .", "The main chemical vulnerability of DNA is its propensity to undergo spontaneous deamination of cytosine to uracil .", "This shortcoming was presumably addressed by a later evolutionary adaption involving 5-methylation of uracil ( thymine ) and excision-repair of unmethylated uracil residues that derive from cytosine .", "The reverse transcriptase ribozyme has a reasonable catalytic rate , but is significantly limited with regard to the template sequences it can accept .", "It struggles to add multiple A or T residues and is hindered by secondary structure within the RNA template .", "The ribozyme also misincorporates dGTP as a G•U wobble pair when deprived of dATP , although this behavior does not allow the polymerase to traverse U positions on difficult templates ( Figure 1B ) .", "Similar limitations existed for earlier versions of the RNA polymerase ribozyme , which were overcome by many rounds of in vitro evolution .", "It is likely that reverse transcriptase activity also could be improved through evolution .", "A highly optimized RNA-dependent DNA polymerase might be expected to have diminished RNA-dependent RNA polymerase activity , unless both functions were explicitly maintained through selection .", "It also might be possible to use the 24–3 enzyme as a starting point to evolve a DNA-dependent polymerase that synthesizes either RNA or DNA , enabling either forward transcription or DNA replication , respectively .", "The historical emergence of these activities would have marked the end of the RNA world era .", "The starting level of reverse transcriptase activity in the RNA world may have been modest , allowing the copying of only short segments of RNA , as was the case in the present study .", "For this trait to be retained it would need to confer a selective advantage , and for it to be optimized there would need to be further advantage resulting from enhancement of this activity .", "A potential advantage of generating even short segments of DNA might be to protect the termini or other critical regions of RNA , ultimately extending to protection of the entire genome .", "At the outset , it is likely that RNA served as the primer for reverse transcription , perhaps through self-priming by a 3´-terminal hairpin , although priming from an internal 2´-hydroxyl or suitable chemical modification also would have been possible .", "The hybridization of a separate primer would need to compete with the secondary structure encompassing the 3´-terminus of the RNA template , which could be avoided by self-priming .", "All discussion pertaining to the transition from RNA to DNA genomes is speculative , although arguably this event is one of the most significant in the history of life .", "Without the transition to a more stable genetic material , the length of heritable genomes and therefore the complexity of life would have been severely limited .", "The modest chemical difference between ribose and deoxyribose has a profound effect on both the chemical reactivity of mononucleotides and the backbone stability of polynucleotides .", "Genomes having the information content of modern cellular organisms likely would not have been possible without the invention of reverse transcriptase ." ], [ "All oligonucleotides used in this study are listed in supplementary file 1 .", "Synthetic oligonucleotides were either purchased from Integrated DNA Technologies ( Coralville , IA ) or prepared by solid-phase synthesis using an Expedite 8909 DNA/RNA synthesizer , with reagents and phosphoramidites purchased from Glen Research ( Sterling , VA ) .", "RNA templates were prepared by in vitro transcription from synthetic DNA templates .", "Polymerase ribozymes were prepared by in vitro transcription of double-stranded DNA templates generated by PCR from corresponding plasmid DNA .", "All RNA templates and ribozymes were purified by denaturing polyacrylamide gel electrophoresis ( PAGE ) and ethanol precipitation prior to use .", "NTPs were purchased from Sigma-Aldrich ( St . Louis , MO ) and dNTPs were from Denville Scientific ( Holliston , MA ) .", "TURBO DNase I , Superscript II reverse transcriptase , and streptavidin C1 Dynabeads were from ThermoFisher ( Grand Island , NY ) .", "RNA templates were transcribed from 0 . 5 μM single-stranded DNA that had been annealed with 0 . 5 µM of a synthetic oligodeoxynucleotide encoding the second strand of the T7 RNA polymerase promoter .", "Transcription was carried out in a mixture containing 15 U/μL T7 RNA polymerase , 0 . 002 U/μL inorganic pyrophosphatase , 5 mM each NTP , 25 mM MgCl2 , 2 mM spermidine , 10 mM DTT , and 40 mM Tris ( pH 8 . 0 ) , which was incubated at 37°C for 2 hr .", "The DNA then was digested by adding 0 . 1 U/μL TURBO DNase I and continuing incubation for 1 hr .", "Ribozymes were transcribed from fully double-stranded DNA templates ( 20 µg/mL ) that were obtained by PCR amplification of plasmid DNA encoding the 24–3 ribozyme ( courtesy of David Horning ) .", "RNA-templated polymerization of either RNA and DNA was performed using 100 nM ribozyme , 125 nM template , and 125 nM primer .", "The primer , which consisted of RNA , DNA , or RNA with a single 3´-terminal deoxynucleotide , contained both a fluorescein label and biotin moiety at its 5´ end .", "The ribozyme , template , and primer first were heated at 80°C for 2 min , then cooled to 17°C over 5 min and added to the reaction mixture , which also contained 2 mM each NTP or dNTP , 200 mM MgCl2 , 0 . 05% TWEEN20 , and 50 mM Tris ( pH 8 . 3 or 9 . 0 ) .", "Polymerization was carried out at 20°C and quenched by adding 250 mM EDTA .", "The biotinylated primers and extended products were captured on streptavidin C1 Dynabeads , washed twice with alkali ( 25 mM NaOH , 1 mM EDTA , and 0 . 05% TWEEN20 ) and once with TE-urea ( 1 mM EDTA , 0 . 05% TWEEN20 , 10 mM Tris ( pH 8 . 0 ) , and 8 M urea ) , then eluted with 98% formamide and 10 mM EDTA ( pH 8 . 0 ) at 95°C for 15 min .", "The reaction products were analyzed by denaturing PAGE .", "Defined-length extension products for analysis by either DNase digestion or LC/MS were prepared using 1 µM ribozyme , 1 µM template , and 0 . 8 μM RNA or DNA primer .", "The reaction was carried out as described above at pH 8 . 3 for 21 hr .", "Presumed full-length materials were purified by electrophoresis in a denaturing 15% polyacrylamide gel , excised from the gel , eluted with 200 mM NaCl , 1 mM EDTA , and 10 mM Tris ( pH 7 . 5 ) , and ethanol precipitated .", "The purified extension products were subjected to partial DNase digestion in a mixture containing 1 μM oligonucleotide , 0 . 1 U/μL TURBO DNase I , 10 mM MgCl2 , 0 . 5 mM CaCl2 , and 20 mM Tris ( pH 7 . 5 ) , which was incubated at 37°C for 30 min , then quenched with 20 mM EDTA , followed by heat inactivation of the enzyme at 75°C for 10 min .", "The resulting products were analyzed by electrophoresis in a denaturing 15% polyacrylamide gel .", "Liquid chromatography/mass spectrometry analysis was performed by Novatia LLC ( Newtown , PA ) using 50 pmol of purified extension products .", "Standard analyses were performed by electrospray ionization LC/MS on the Oligo HTCS platform , which achieves mass accuracy of 0 . 01–0 . 02% .", "Oligonucleotide sequence confirmation was performed by high-resolution ion trap tandem MS on an LTQ-Orbitrap ion mass spectrometer , which achieves mass resolution of 0 . 003% ( FWHM ) .", "The parent ion was used to generate a fragment spectrum resulting from cleavage at phosphodiester linkages within the DNA portion of the molecule .", "ReSpect deconvolution software ( Positive Probability Ltd . ) was used to deisotope the MS/MS spectrum and to obtain a simplified fragment spectrum with exact masses ." ] ]
[ "A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as a reverse transcriptase , an activity that has never been demonstrated before for RNA .", "This activity is thought to have been crucial for the transition from RNA to DNA genomes during the early history of life on Earth , when it similarly could have arisen as a secondary function of an RNA-dependent RNA polymerase .", "The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate products containing up to 32 deoxynucleotides .", "It is likely that this activity could be improved through evolution , ultimately enabling the synthesis of complete DNA genomes .", "DNA is much more stable compared to RNA and thus provides a larger and more secure repository for genetic information ." ]
[ "All known living things share the same genetic machinery , traditionally called the central dogma .", "According to this dogma , genes in DNA produce messages made from a similar molecule called RNA .", "These RNA messengers provide the instructions to make proteins , which then form structures and act as molecular machines inside cells .", "This process is found in all modern living things , but early life must have been much simpler .", "Many biologists believe that the earliest life only used RNA , which can both store information like DNA and perform tasks like a protein .", "Life evolved from this so-called ‘RNA world’ because DNA provides a more reliable long-term store of information , whilst proteins are more versatile and able to perform more tasks .", "This key step in evolution allowed life to move beyond basic chemistry and develop the size , complexity and diversity we see today .", "Yet , how this transition happened is not well understood .", "In particular , many believe an RNA molecule must have evolved the ability to make DNA from an RNA template , allowing early life to build the first genetic material made from DNA .", "This molecule would be referred to as a reverse transcriptase ribozyme .", "Modern living things do not contain such a molecule .", "Yet based on their previous work using RNA molecules to make copies of other RNAs , Samanta and Joyce attempted to develop an artificial reverse transcriptase ribozyme .", "The goal was to show that these ribozymes can be made and could theoretically have evolved naturally .", "The molecule Samanta and Joyce created was able to reliably produce short sections of DNA , with rare errors .", "This ribozyme is slower and makes more mistakes than molecular systems in modern biology , but it proves that reverse transcriptase ribozymes are possible .", "Using a process called test-tube evolution , which uses the same concepts as natural evolution to improve the qualities of biological molecules , Samanta and Joyce now plan to improve their ribozyme .", "The aim is to confirm that a reverse transcriptase ribozyme could have been a transformative early step in evolution of life on Earth that led to the first DNA genomes .", "This will be a critical addition to scientists’ understanding of how life became more complex and how the first cells formed ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "cancer biology" ]
Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2
elife-37689-v2
[ [ "A major function of apoptosis is to inhibit tumor initiation and progression while its inhibition can result in cancer cell survival and resistance to chemotherapy ( Kirkin et al . , 2004 ) .", "Bcl-2 family proteins regulate intrinsic apoptosis and mitochondrial integrity through direct physical interactions between the anti-apoptotic proteins ( Bcl-2 , Bcl-XL and Mcl-1 ) , the pro-apoptotic proteins ( Bax , Bak ) and the BH3-proteins ( Bad , Bid and Bim ) ( Liu et al . , 2013; Shamas-Din et al . , 2013; Youle and Strasser , 2008 ) .", "In cancer cells , pro-apoptotic BH3 proteins and activated Bax and Bak are sequestered by anti-apoptotic proteins , thereby the cell is protected at the same time as it is primed for death .", "Such cells can be efficiently killed by BH3-mimetic drugs that release bound pro-apoptotic proteins , as shown by the clinical success of the Bcl-2 specific BH3-mimetics venetoclax ( ABT-199 ) and navitoclax ( ABT-263 ) .", "It is commonly accepted that BH3 proteins bind to anti-apoptotic proteins exclusively through a conserved BH3 region ( Chen et al . , 2005 ) .", "ABT-263 is an orally available inhibitor with nanomolar affinity for the BH3-binding sites in Bcl-XL and Bcl-2 that is currently undergoing clinical trials in humans .", "In vitro ABT-263 displaces BH3 peptides derived from different BH3-proteins including Bim ( Tse et al . , 2008 ) from Bcl-2 and Bcl-XL .", "However a discrepancy between this data and the activities of the drugs in live cells was observed independently in three studies .", "1 ) Increased expression of Bcl-XL in human non-Hodgkin lymphomas led to resistance to ABT-737 ( an analogue of ABT-263 ) despite enhanced Bim expression ( Mérino et al . , 2012 ) .", "2 ) Fluorescent Lifetime Imaging Microscopy ( FLIM ) – Förster Resonance Energy Transfer ( FRET ) demonstrated that ABT-263 displaced full-length Bad and tBid ( activated Bid protein ) but not the major isoforms of Bim ( BimEL , BimL and BimS ) from binding to Bcl-XL and Bcl-2 ( Aranovich et al . , 2012; Liu et al . , 2012 ) .", "3 ) ABT-263 resistant binding of Bim to Bcl-XL was observed using bioluminescence resonance energy transfer ( Pécot et al . , 2016 ) .", "However , the molecular mechanism of resistance is not well established .", "Among the Bcl-2 family , Bim is the only BH3-protein that binds all anti-apoptotic proteins with high affinities and that can directly activate Bax and Bak to initiate apoptosis ( Chen et al . , 2005; Letai et al . , 2002 ) .", "Therefore the surprising lack of activity for BH3 mimetics on displacement of Bim could significantly reduce their efficacy in some cancer cells ( Pécot et al . , 2016 ) .", "Understanding this mechanism of resistance may also suggest ways to release specific sequestered BH3-proteins from Bcl-XL and Bcl-2 to kill cancer cells more selectively than broadly inhibiting anti-apoptotic proteins ( Goldsmith et al . , 2006; Ni Chonghaile and Letai , 2008 ) .", "Here we identify the specific residues in Bim responsible for binding to Bcl-XL and Bcl-2 and that confer resistance of the complexes to BH3-mimetic drugs .", "We discovered an unanticipated interaction between the C-terminal sequence ( CTS ) of Bim and both Bcl-XL and Bcl-2 .", "Our data further demonstrate dual yet independent roles for the Bim CTS in binding to Bcl-XL and to membranes .", "Together the two anti-apoptotic protein binding sequences in Bim confer resistance of complexes to the dual Bcl-2 , Bcl-XL inhibitor ABT-263 and to the new generation Bcl-XL inhibitors in clinical development .", "Because the CTS of Bim also functions to activate the pro-apoptotic proteins Bax and Bak , ( Chi et al . , 2019 ) our results suggest that BH3-mimetics may have unpredictable results in cells depending on the Bcl-2 family proteins expressed and that targeting both binding sites on Bcl-XL and/or Bcl-2 may be required to kill some cancer cells that have bypassed apoptosis by inhibiting Bim ." ], [ "New instrumentation for automated time correlated single photon counting ( TCSPC ) from two fluorescence proteins simultaneously enabled automation of FLIM-FRET measurements to evaluate the effects of drugs on the binding of Bcl-2 family proteins fused to mCerulean3 ( mCer3 ) and Venus fluorescence proteins .", "The nomenclature used here indicates proteins fused with a fluorescence protein by a superscripted c for mCer3 and a superscripted v for Venus .", "The superscripted letters precede the protein names for amino-terminal , and follow them for carboxyl-terminal fusions .", "VBH3-proteins were expressed by transient transfection of MCF-7 cells stably over-expressing CBcl-XL or CBcl-2 ( Figure 1 ) .", "Transient expression of VBH3-proteins ensured that there was a large enough range of expression levels within the population of cells to collect the data needed to generate binding curves ( Aranovich et al . , 2012; Kale et al . , 2012; Liu et al . , 2012 ) .", "The expressed fluorescent fusion proteins retained their expected anti- or pro-apoptotic properties ( see below ) .", "To generate quantitative binding curves , TCSPC data were collected for CBcl-XL ( FRET donor ) and VBH3-proteins ( FRET acceptor ) , within regions of interest ( ROIs ) automatically identified in cells based on mCer3 signal intensity ( Figure 1A ) .", "For each ROI the TCSPC data were used to determine the average fluorescence lifetime of the mCer3 donor and the fluorescence intensities of both the donor ( mCer3 ) and the acceptor ( Venus ) .", "Data from more than 8 , 500 ROIs were binned according to the ratio of Venus to mCer3 intensities and together with the corresponding lifetime data used to generate binding curves ( Figure 1B ) .", "BH3-proteins engage the hydrophobic pocket of anti-apoptotic proteins through four conserved hydrophobic residues ( h1-h4 ) within the BH3 region ( Chen et al . , 2005 ) .", "ABT-263 prevents BH3-protein binding by competing for the binding of residues h2 and h4 to Bcl-XL ( Figure 1C ) ( Tse et al . , 2008 ) .", "To quantify the importance of these residues in live cells , we expressed wild-type and mutant VBH3-proteins with alanine mutations in the h2 and h4 positions ( BH3-2A ) and measured binding to CBcl-XL .", "As expected , BH3-2A mutations in VBad and VtBid reduced binding to CBcl-XL dramatically ( Figure 1D–E , compare black and red ) .", "However the BH3-2A mutant of VBimEL bound to CBcl-XL similarly to wild-type VBimEL ( Figure 1F , compare black and red ) , suggesting interactions with other residues contribute to binding Bim to Bcl-XL .", "Consistent with this result ABT-263 displaced both VBad and VtBid , but not VBimEL from CBcl-XL ( Figure 1D–F , compare green and cyan ) .", "To quantify the impact of ABT-263 on VBH3-proteins binding to CBcl-XL and CBcl-2 , and maximize the dynamic range of the assay we estimated the FLIM-FRET efficiency from the fitted binding curves at intensity ratios of Venus to mCer3 of 0 . 5 and 0 . 25 , respectively .", "The FRET signal from the non-binding mutant VBad4E ( in which h1 , h2 , h3 and h4 in the BH3 region were mutated to glutamic acid ) served as a negative control for FRET due to random collisions rather than binding , and was subtracted as background ( Figure 2A ) .", "The percentage of the FRET signal remaining in the presence of ABT-263 is defined here as Resistance to displacement by ABT-263 ( RABT-263 , Figure 2A ) .", "The effects of mutations on the binding of BH3-proteins can be calculated similarly and for the BH3-2A mutation is reported as RBH3-2A .", "RABT-263 and RBH3-2A values for all of the mutants analyzed are provided in Table 1 .", "Plotting RABT-263 for different interactions indicated that >80% of CBcl-XL:VBimEL and CBcl-2:VBimEL complexes were resistant to the addition of ABT-263 ( Figure 2B ) .", "In contrast , CBcl-XL:VBad and CBcl-XL:VtBid complexes were less than 50% resistant to the drug ( Figure 2B-left , black ) while the corresponding Bcl-2 complexes were less than 40% resistant ( Figure 2B-left , blue , Figure 2—figure supplement 1 ) .", "Calculated similarly , RBH3-2A values reflect the minimal effect of BH3-2A mutations on complexes of CBcl-XL and CBcl-2 with VBimEL ( RBH3-2A ~ 80% ) compared to VBad ( RBH3-2A ~ 2% ) and VtBid ( RBH3-2A ~30% ) complexes ( Figure 1D–F , Figure 2B , and Figure 2—figure supplement 1 ) .", "To test the generality of these results in a different cell line that is resistant to the induction of cell death by BH3 proteins , complex formation was also assessed in Baby Mouse Kidney ( BMK ) cells in which the genes for expression of Bax and Bak were knocked out ( DKO ) .", "These cells do not undergo apoptosis in response to expression of BH3-proteins and therefore retain normal cellular morphology .", "As expected , the RABT-263 and RBH3-2A values obtained in BMK-DKO cells for CBcl-XL and VBH3-protein complexes were similar to those observed for MCF-7 cells ( CBcl-XL ( Black ) in Figure 2B compared with Figure 2C , Figure 1D–F and Figure 2—figure supplement 2 ) .", "Similar to the results in MCF-7 cells , the resulting RABT-263 and RBH3-2A values measured for CBcl-XL:VBad and CBcl-XL:VtBid complexes ranged from 40% to 70% lower than those of CBcl-XL:VBimEL complexes confirming that the unusual stability of CBcl-XL:VBim complexes is neither cell line-specific nor a result of molecular crowding that may occur when cells undergo apoptosis ( Figure 2c ) .", "As an additional control to address this latter point directly lifetime measurements were compared for manually selected images of fully adherent MCF-7 cells expressing CBcl-XL and VBim-Bad that retained relatively normal morphology ( alive cells ) from the bottom of the imaging well .", "Images of cells with a more condensed morphology ( dying cells ) were subsequently recorded from a higher imaging plane .", "The distributions of lifetimes measured for the both sets of cells were very similar confirming that the changes in dying cells do not affect the lifetime measurements ( Figure 2—figure supplement 3 ) .", "Compared to ABT-263 ( IC50 >10 μM ) , the Bcl-XL specific inhibitors A-1155463 and A-1331852 more potently disrupted CBcl-XL:VBad complexes with R values of ~45% and 20% respectively , corresponding to an IC50 <1 . 25 μM for these compounds ( Figure 2D and Figure 2—figure supplement 4 ) .", "Nevertheless , their impact on CBcl-XL:VBimEL complexes was limited , confirming the high stability of CBcl-XL:VBimEL complexes .", "Results for displacement of BH3 proteins from Bcl-2 using the specific inhibitor venetoclax were uninterpretable due to interference with the FLIM measurements that resulted from the broad spectrum fluorescence of the drug .", "As a dual inhibitor , ABT-263 enables comparison of the effects of mutations for both CBcl-XL:VBimEL and CBcl-2:VBimEL complexes and was therefore used for subsequent experiments .", "To confirm that the stability of the complexes measured in live cells was due to direct interactions , FRET was measured for donor-fluorophore-labeled recombinant BimL or tBid with acceptor-fluorophore-labeled Bcl-XL in a cell free liposome based system .", "In this assay BimL was used instead of BimEL due to the difficulty in purifying BimEL and the BH3-proteins were added at amounts predetermined to result in complete binding of BimL or tBid to Bcl-XL .", "ABT-263 was then titrated into the samples to compete with the BH3-proteins .", "As expected , a concentration of ABT-263 ~10 fold higher than that of Bcl-XL displaced tBid from Bcl-XL ( IC50 ~400 nM ) yet had little effect on the interaction between Bcl-XL and BimL ( stable at >3 μM ) ( Figure 2E ) .", "Thus similar to what was seen in cells by FLIM FRET , direct binding of Bim to Bcl-XL is more resistant to ABT-263 than is binding of tBid to Bcl-XL .", "To test the functional consequences of this we measured protein binding and liposome permeabilization at the same time .", "Under conditions that support measurement of both , the IC50 for ABT-263 for FRET between cBid and Bim with Bcl-XL was 212 ± 43 and>700 nM , respectively while for liposome permeabilization the corresponding EC50’s were 65 ± 9 nM and 150 ± 16 nM , n = 3 independent duplicates , ±standard error .", "Thus , the change in binding affinity results in a corresponding change in membrane permeabilization .", "The functional properties of the fusion proteins used in FLIM-FRET experiments were examined using multiple assays in MCF-7 cells .", "Transient transfection by the requisite plasmids demonstrated that expression of VBim and VtBid killed MCF-7 cells as assessed morphologically for both cell and nuclear condensation and mitochondrial transmembrane potential ( Figure 3A ) .", "In contrast , expression of VBad had little effect on cell death in this cell line suggesting that inhibition of Bcl-2 and Bcl-XL is not sufficient to induce apoptosis in MCF-7 cells .", "To examine the effect of expression of exogenous CBcl-XL ( confirmed by immunoblotting in Figure 3B ) apoptosis was triggered with TNFα and cycloheximide ( CHX ) in the same cell clones used for FLIM-FRET above .", "Untreated and cells treated with only CHX were used as negative controls .", "As expected , untransfected MCF-7 cells were efficiently killed while cells expressing either CBcl-XL or CBcl-2 were highly resistant to this well-established method for the induction of apoptosis ( Figure 3C ) .", "To determine the importance of other anti-apoptotic proteins expressed endogenously in these cells , they were treated with specific small molecule inhibitors .", "Consistent with the results demonstrating resistance to Bad , MCF-7 cells were largely resistant to inhibition of Bcl-2 and/or Bcl-XL ( Figure 3D ) .", "Neither selective nor dual inhibition of Bcl-2 and Bcl-XL induced substantial cell death in MCF-7 cell lines ( all below 20% Dead ) demonstrating these cells are not highly dependent on expression of either of these anti-apoptotic proteins .", "Nevertheless , expression of CBcl-2 or CBcl-XL reduced cell death further ( Figure 3D ) .", "In contrast , MCF-7 cells were sensitive to inhibition of MCL-1 ( Figure 3E ) .", "Moreover , overexpression of CBcl-XL or CBcl-2 protected MCF-7 cells from the MCL-1 inhibitor S-63845 , likely by inhibiting pro-apoptotic proteins displaced by the BH3-mimetic from MCL-1 ( Figure 3E ) .", "Taken together the data demonstrate that MCF-7 cells are primarily dependent on expression of MCL-1 for survival and that the exogenously expressed fluorescence protein fusions function as predicted in this cell line .", "It is commonly accepted that the only stable binding interaction between Bim and anti-apoptotic proteins is via the BH3-region of Bim ( Bim BH3 ) binding in the hydrophobic groove formed by the BH3 1–2 regions of the anti-apoptotic proteins ( Liu et al . , 2010; Sattler et al . , 1997 ) .", "However , resistance to ABT-263 suggests that in the context of the whole protein either the Bim BH3 region binds differently than BH3-peptides to Bcl-XL or a region not included in previous in vitro experiments , such as the N-terminal and C-terminal sequences shared by all three isoforms , contains sequences conferring ABT-263 resistant binding .", "To test these sequences , mutants harboring deletions in VBimEL and mutants in which the BH3-regions were exchanged between BimEL and Bad ( Figure 4A ) were assayed by FLIM-FRET for resistance to ABT-263 and to the BH3-2A mutation ( Figure 4B–C and Figure 4—figure supplement 1 ) .", "The trends observed for binding of the mutants to CBcl-XL and CBcl-2 were remarkably similar .", "Truncation of the N-terminus of BimEL ( VBimEL-dN ) had little impact on Bim binding ( Table 1 ) , but replacing the BH3 region in Bad with that in BimEL ( VBad-BimEL ) increased RABT-263 compared with VBad .", "Consistent with this result , binding of the reciprocal mutant ( VBimEL-Bad ) to either CBcl-XL or CBcl-2 was more sensitive to the drug and the BH3-2A mutation ( RABT-263 ~60–65% , Figure 4B RBH3-2A ~30% Figure 4C , black and blue ) respectively , compared to VBimEL ( both R values ~ 80% , Table 1 ) .", "As expected , deletion of the entire BH3 region ( BimEL-d20 , Figure 4A ) reduced the interactions with Bcl-XL and Bcl-2 to a level similar to random collisions in the membrane ( Rd20 ~25% , Figure 4—figure supplement 1F and L ) .", "Unexpectedly , deletion of the C-terminal sequence ( CTS ) resulted in significant decreases in RABT-263 and RBH3-2A ( Figure 4B–C and Table 1 ) .", "Combining deletion of the Bim CTS with replacement of the Bim BH3 region by that of Bad ( VBimEL-Bad-dCTS ) , reduced the values of RABT-263 and RBH3-2A to that of VBad ( Figure 4B–C and Table 1 ) .", "Thus , both BH3 and CTS sequences contribute to Bim binding anti-apoptotic proteins .", "To examine the functional importance of the Bim CTS further , the pro-apoptotic activities of selected Bim mutants were examined in MCF-7 cells with and without exogenously-expressed Bcl-XL or Bcl-2 .", "Expression of CBcl-XL or CBcl-2 efficiently protected cells from lower levels of expression of VBimEL and VBimEL-dCTS ( <1000 units of Venus intensity ) however , in neither case was the protection complete at higher levels of expression ( Figure 5A ) .", "Unexpectedly , in MCF-7 cells there was no significant difference in induction of cell death by removing the CTS from BimEL ( Figure 5A ) .", "There are two factors that are likely contributing to this result .", "First .", "Bim not only inhibits anti-apoptotic proteins it also activates the executioner proteins Bax and Bak , potentially confounding analysis .", "As this mechanism may be relevant in multiple cell lines it is examined in more detail elsewhere ( Chi et al . , 2019 ) .", "Second , MCF-7 cells depend on MCL-1 for survival ( Figure 3E ) .", "To avoid the contribution to cell death by inhibition of MCL-1 we made use of the Bim mutant with the Bad BH3 region ( VBimEL-Bad ) as it is not expected to inhibit Mcl-1 .", "Consistent with expectation , VBimEL-Bad was less effective than VBimEL in killing MCF-7 cells ( Figure 5B ) .", "Deletion of the CTS from VBimEL-Bad resulted in a further loss of pro-apoptotic activity suggesting that in VBimEL the effect of the CTS was masked by VBimEL binding to and inhibiting MCL-1 ( Figures 5A and 3b ) .", "Consistent with the results for VBimEL , inhibition of VBimEL-Bad by Bcl-2 and Bcl-XL was limited .", "While this result is also consistent with a role for MCL-1 in binding pro-apoptotic proteins displaced from Bcl-2 and Bcl-XL by VBimEL-Bad there are other possible explanations that will be the subject of future investigations .", "To examine potential molecular mechanisms underlying the effects of deletion of the CTS from Bim in a more controlled system we examined BimL and BimL-dCTS binding to Bcl-XL and displacement of BH3 proteins from Bcl-XL using purified proteins and the FRET based assays similar to those described above ( Figure 2E ) .", "When assayed directly , in incubations containing mitochondria , deletion of the CTS from BimL reduced the apparent affinity for binding to Bcl-XL ( Figure 5C ) .", "As predicted from the reduced affinity of this interaction , BimL-dCTS was also less effective at displacing tBid and activated Bax from Bcl-XL ( Figure 5D and E , respectively .", "Control FRET measurements indicated that at the concentrations used binding of cBid by Bcl-XL was saturated and that all of the proteins other than BimL-dCTS were bound to liposome membranes .", "Moreover , the interactions take place on the membrane surface as both tBid and activated Bax very efficiently bind to liposomes ( Kale et al . , 2014 ) .", "There are five hydrophobic residues/groups named h0-h4 on the hydrophobic side of the BH3 α-helix in pro-apoptotic BH3-proteins hypothesized to mediate BH3-protein binding to anti-apoptotic proteins ( Figure 6A ) .", "To quantify the binding due to each of these residues without potential interference from the additional binding site within the Bim CTS , mutations were introduced into a chimeric fusion protein composed of the Bad sequence with the Bim BH3 region ( VBad-Bim in Figure 4A ) .", "The introduced mutations substitute residues in the Bim BH3 sequence at positions h0 , h1 , h1 +2 ( an R between h1 and h2 ) , and h3 with the corresponding residues in Bad ( Figure 6A ) .", "When measured by FLIM-FRET , the relative effects of most of the individual mutations on the RABT-263 and RBH3-2A values for VBad-Bim binding to CBcl-XL ( black ) and CBcl-2 ( blue ) reduced binding by 10–20% ( Figure 6B , Figure 6—figure supplement 1 and Table 1 ) .", "Among these the h0 and h1 residues contributed to Bim binding to Bcl-2 and Bcl-XL the most .", "Substitution of the h0 sequence with WAA reduced RABT-263 for VBad-Bim to CBcl-XL from 98% ( 95% Confidence Interval ( CI ) 6% ) to 79% ( CI 6% ) .", "Similarly , substitution of I146 with Y decreased RABT-263 for VBad-Bim to 83% ( CI 5% ) .", "The reductions in RABT-263 for VBad-Bim to Bcl-2 were similar ( Table 1 ) .", "However , the RABT-263 for VBad-Bim binding to both Bcl-XL and Bcl-2 was unexpectedly high .", "As Bad also has a CTS sequence that binds to membranes and may have other as yet uncharacterized functions we examined the effect of the I146Y mutation in BimEL-dCTS .", "The RABT-263 for Bcl-XL for this mutant ( BimEL-I146Y-dCTS ) was 38% ( CI 8% ) compared to 69% ( CI 5% ) for VBimEL-dCTS binding to CBcl-XL suggesting a role for I146 in BimEL binding to Bcl-XL ( Figure 6C ) .", "Although a reduction was also seen for CBcl-2 the confidence intervals overlapped suggesting there are subtle differences in Bim binding to Bcl-XL and Bcl-2 .", "The small differences observed for substitutions at other positions within Bim-BH3 suggest either we have reached the limit of sensitivity for the FLIM-FRET assay or they may augment but individually are not critical for ABT-263 resistant binding of Bim to Bcl-XL and Bcl-2 .", "Deletion of the Bim CTS has been shown to abrogate binding to membranes ( O'Connor et al . , 1998 ) .", "We therefore hypothesized that the membrane binding function of this region might affect the resistance of Bcl-XL/Bim and Bcl-2/Bim complexes to either ABT-263 treatment or BH3-2A mutations by increasing the local concentration of the proteins on mitochondria .", "However , instead of an uninterrupted hydrophobic sequence of more than 15 residues typical of tail-anchor proteins , the Bim CTS consists of two short hydrophobic sequences and includes multiple positively charged Arg residues , two of which are near the center of the sequence ( Figure 7A , green ) .", "To examine the importance of sequences within the Bim CTS for ABT-263 resistant binding of VBimEL to CBcl-XL and CBcl-2 , mutants were generated in which hydrophobic ( I181 , L185 , I188 and V192 ) and hydrophilic ( R186 and R190 ) residues of Bim were substituted with the negatively charged amino acid Glutimate ( E ) or the hydrophobic residue Alanine ( CTS-2A ) , respectively ( Figure 7A ) .", "The impact of these mutations on both the subcellular localization of VBimEL at mitochondria ( measured by the Pearson’s r with MitoTracker ) and RABT-263 values were quantified for binding to CBcl-XL and CBcl-2 .", "Compared to VBimEL ( Pearson’s r ~ 0 . 4 ) mitochondrial localization of all of the mutants was similarly poor ( Pearson’s r 0 . 1–0 . 3 ) ( Figure 7B–C ) , and only slightly greater than the Pearson’s r measured for the cytoplasmic Venus control .", "However , the mutants displayed different binding properties to the anti-apoptotic proteins ( Figure 7C and Figure 7—figure supplement 1 ) .", "For example , even though localization at mitochondria for the VBim-CTS2A mutant was impaired , it bound to both anti-apoptotic proteins in an ABT-263 resistant fashion ( RABT-263 ~89% , CI 6% and 81% , CI 11% , for CBcl-XL and CBcl-2 , respectively ) similar to VBimEL ( 85% , CI 5% and 83% , CI 9%; Table 1 and Figure 7C ) .", "In contrast , RABT-263 for VBimEL mutants L185E ( 42% , CI 10% ) , I188E ( 51% , CI 8% ) and V192E ( 70% , CI 8% ) exhibited gradually increasing binding to CBcl-XL ( Table 1 ) without a corresponding increase in binding to mitochondria ( Figure 7C ) .", "When all of the data were analyzed in aggregate using Spearman’s rank-order correlation to include relationships that might not be linear the result was no correlation between localization for Bcl-XL and a weak correlation for Bcl-2 .", "We suspect the latter may be due to the fact that Bcl-2 is constitutively membrane bound while Bcl-XL is located in both the cytoplasm and on the membrane .", "Thus in cells , localization to mitochondria and binding to anti-apoptotic proteins are independent functions of the Bim CTS .", "The effect of the Bim CTS on binding to membranes and Bcl-XL was also assessed directly using purified proteins and liposome membranes .", "As above , binding of BimL and BimL-mutants to Bcl-XL was measured by FRET for incubations with and without liposomes .", "Displacement from Bcl-XL of the BimL mutants I181E , L185E , I188E and dCTS ( numbered according to the BimEL sequence to facilitate comparison ) were measured for different concentrations of ABT-263 ( Figure 7D ) .", "Consistent with our observations in live cells , the binding of BimL-dCTS to Bcl-XL was much more sensitive to ABT-263 ( IC50 ≈ 200 nM ) compared to BimL ( IC50 >>4 µM ) .", "These IC50 values are difficult to compare directly with RABT-263 values because the protein concentrations are different .", "Nevertheless , in this cell free assay using purified BimL and Bcl-XL , mutation of I181 , L185 and L188 in the CTS region all resulted in increased sensitivity to displacement by ABT-263 ( Figure 7D ) .", "As expected ( Pécot et al . , 2016 ) , BimL binding to Bcl-XL in the absence of membranes was more easily displaced by ABT-263 , but still required drug concentrations greater than 1 μM ( compare Figure 7D with E ) .", "In solution , the mutations further decreased resistance of Bcl-XL/BimL complexes to ABT-263 ( Figure 7E ) confirming they augment binding of BimL to Bcl-XL independently of binding to membranes .", "The simplest explanation for ABT-263 resistant binding is a direct interaction between the Bim CTS and Bcl-XL in the context of the full length Bim protein .", "Mutation of the BH3 residue I146 to Y slightly exacerbated the effect of deletion of the entire CTS from Bcl-XL on RABT-263 ( Figure 6C ) .", "However , when the I146Y mutation was combined with mutation of L185E we were unable to record a decrease in RABT-263 ( Figure 7F ) suggesting that the effects of the individual point mutants are not additive .", "Alternatively the effect size may be too small to measure by FLIM-FRET , a result consistent with the L185E mutation having less effect on RABT-263 than deletion of the CTS ( Table 1 ) .", "A direct interaction between the Bim CTS and Bcl-XL is incompatible with current assumptions that the Bim CTS inserts into lipid bilayers as a transmembrane helix similar to other tail-anchor proteins ( Petros et al . , 2004 ) .", "Therefore , we expressed a version of BimEL with Venus fused to the C-terminus ( BimELV ) to prevent the Bim CTS from adopting a transmembrane topology .", "Distance constraints mean that this protein can only undergo FRET with Bcl-XL if the Venus protein is located on the cytoplasmic side of the membrane .", "Fusion of Venus to the C-terminus of BimEL did not abolish localization of BimELV at mitochondria ( Figure 8A and C , red bars ) and did not impair ABT-263 resistant binding to CBcl-XL ( Figure 8B–C ) .", "Moreover , the FLIM-FRET efficiency observed was higher for BimELV than for VBimEL binding to CBcl-XL ( Figure 8B ) a result inconsistent with a transmembrane topology for the Bim CTS , as it suggests that Venus on the C-terminus of Bim is closer than Venus on the N-terminus of Bim to the mCer3 on the N-terminus of Bcl-XL .", "These results not only confirm the two independent functions of the Bim CTS , but also suggest that when bound to membranes the residues within the Bim CTS that bind CBcl-XL , for example L185 , are on the cytoplasmic side of the membrane .", "To examine the topography and Bcl-XL binding of the Bim CTS in solution and membrane bound states directly , we used a well-established procedure in which an environment sensitive fluorescent dye N , N0-dimethyl-N- ( Iodoacetyl ) -N0- ( 7-nitrobenz-2-oxa-1 , 3-diazol-4-yl ) ethylenediamine ( NBD ) was attached to recombinant BimL at specific sites and the environment of the dye was measured by fluorescence spectroscopy ( Kale et al . , 2014 ) .", "A series of mutants were generated in which individual residues across the Bim CTS were replaced with cysteine to enable NBD-labeling .", "As a solvent exposed control , a Bim mutant was prepared with a cysteine located at position 41 .", "The fluorescence intensities of the dye labeled mutants were then recorded in the absence or presence of liposomes , Bcl-XL , and/or the aqueous quencher iodide .", "The fluorescence of NBD increases when the dye inserts into a lipid bilayer or becomes deeply buried in the interior of a protein ( Johnson , 2005 ) .", "When Bim bound to Bcl-XL in solution there were no significant changes in hydrophobicity at any of the positions of the probe suggesting that during complex formation these residues do not become sufficiently buried to be protected from water ( Figure 9A , diagrammed in 9C ) .", "In contrast , when BimL bound to liposomes the NBD fluorescence increased at positions 179–182 and 191–195 suggesting that these two regions anchor the CTS to the lipid bilayer ( Figure 9D , diagrammed in 9F ) .", "Complex formation with Bcl-XL on the membrane did not markedly change the pattern of which amino acids increased in hydrophobicity , suggesting that complex formation does not change the way the Bim CTS interacts with membranes substantially ( Figure 9G diagrammed in 9I ) .", "Iodide quenching was used to identify residues protected by protein-protein interactions in addition to those protected by binding to membranes .", "Due to the relatively large size of iodide , residues involved in protein-protein interactions are often protected from quenching by iodide but unlike residues interacting with lipids the NBD fluorescence does not increase substantially because there is no protection from water ( Johnson , 2005 ) .", "When Bim was incubated with Bcl-XL in solution , comparison with the control position 41 revealed that the NBD probes in the Bim CTS were not protected from iodide ( Figure 9B–C , Figure 9—figure supplement 1 ) , suggesting a relatively low affinity interaction between the Bim CTS and Bcl-XL in solution .", "As expected , when bound to membranes the residues at both ends of the Bim CTS that interact with the membrane were protected from iodide ( Figure 9E compare residues 179–182 and 191–195 ) .", "However , residues in the middle of the sequence surrounding residue 185 exhibited variable levels of protection from iodide with the NBD located at position 185 as accessible to iodide as the soluble control residue 41 ( Figure 9E , Figure 9—figure supplement 1 ) .", "Thus , in membrane bound Bim , the region between the two membrane binding sites in the Bim CTS remains on the surface of the membrane where it is accessible to iodide ( diagrammed in Figure 9F ) .", "In contrast , when both Bcl-XL and liposomes were added the entire Bim CTS was protected from iodide ( Figure 9H , Figure 9—figure supplement 1 ) strongly suggesting that on membranes Bcl-XL binds to or at least covers-up the central region of the Bim CTS ( diagrammed in Figure 9I ) .", "As position L185 was shown to have the largest effect on binding of BimEL to both CBcl-XL and CBcl-2 ( largest decrease in RABT-263 , Figure 7C ) , and is central to the region protected from iodide when BimL bound to Bcl-XL ( compare Figure 9E and H ) we made a series of mutations at this location for VBimEL-BH3-2A .", "In general , exchanging L185 with other amino acids with hydrophobic or polar non-charged side-chains ( G , F , M , and S ) did not affect the response of CBcl-XL:VBimEL-mutant complexes to BH3-2A mutations significantly , except for the L185A mutant that displayed increased binding affinity ( increased RBH3-2A ) ( Figure 9J and Figure 9—figure supplement 2 ) .", "For Bim-L185P , RBH3-2A decreased by about 20% , possibly due to a change in the structure or rigidity of the Bim CTS .", "However , replacing L185 with hydrophilic residues ( H , R , D or E ) decreased RBH3-2A , by ~30–60% , comparable to truncation of the CTS at position 185 ( L185stop ) or deletion of the entire CTS region ( VBimEL-dCTS , Figure 4C ) .", "These results strongly suggest that L185 interacts with Bcl-XL through hydrophobic interactions .", "Overall our data suggest a revised view of the Bcl-XL:Bim interaction ( diagrammed in Figure 9K ) .", "Because Bim is bound to Bcl-XL by two different regions , the Bim BH3 and Bim CTS , the proteins are effectively double-bolt locked together ." ], [ "Using FLIM-FRET and full-length proteins in live cells and in biochemical assays with mitochondria and liposomes , we discovered that the binding interactions between Bcl-XL and Bim are more extensive and result in a much higher affinity interaction than measured previously using peptides or protein fragments ( Chen et al . , 2005 ) .", "Although previous results ascribed an increased apparent affinity to binding to membranes ( Pécot et al . , 2016 ) our results demonstrate that the increased binding affinity of Bim compared to Bad for Bcl-XL is primarily due to the combined effect of interactions with both the Bim BH3 region ( residues 140–160 ) and the central hydrophobic region of the Bim CTS ( residues 184–190 ) .", "While our data also confirm that the interaction occurs optimally at the membrane , the increase in affinity due to binding to membranes is relatively small ( Figure 7D–E ) .", "Together the two Bcl-XL binding sites in Bim confer resistance to the inhibitors ABT-263 , A-1155463 and A-1331852 ( Figure 2D ) and to mutation of Bim BH3 residues at h2 and h4 that we confirmed as the major anti-apoptotic protein binding residues for both tBid and Bad in live cells ( Figure 1D–E , and Figure 2B–C ) .", "The importance of residues within the BH3-region of Bim other than those at the h2 and h4 positions in binding to Bcl-XL and Bcl-2 ( Figure 6 ) was not detected in previous studies using BH3 peptides .", "Moreover , it was not possible to detect the Bcl-XL binding function of the Bim CTS using pull-down strategies , most likely because the required detergents disrupt hydrophobic interactions .", "Therefore , to determine the effect of mutations and drugs on interactions we used an automated system to collect the data required to generate large numbers of FLIM-FRET binding curves each requiring minimally hundreds , but typically made up of thousands of measurements from at least three independent experiments .", "Substitution of candidate residues within the h0-h4 region of the Bim BH3 sequence with the ones in the Bad BH3 revealed that in live cells the h0 group WAA and h1 residue I146 contribute to the binding of Bim to both Bcl-XL and Bcl-2 ( Table 1 ) .", "Because the residues at h2 and h4 are the same in Bad and Bim , the residues at h0 and h1 account for part of the increased affinity of binding of anti-apoptotic proteins by Bim compared to Bad .", "In sum the additional interactions of the Bim BH3 region and the BIM CTS contributed roughly equally to the affinity of the interaction between Bim and Bcl-XL ( Figure 4B , compare VBimEL-Bad and VBimEL-dCTS ) .", "Together deletion of the Bim CTS and mutation of h2 and h4 in the BH3 region of Bim almost eliminated binding to both Bcl-XL and Bcl-2 ( Figure 4C ) .", "Moreover , the Bim CTS directly contributes to interactions with Bcl-XL ( Figure 5 and Figure 7D–E ) and much of this interaction is due to residue L185 ( Figure 9 ) .", "Consistent with this interpretation weak crosslinking was observed between this region of Bim and Bcl-XL ( Chi et al . , 2019 ) .", "Thus unlike conventional lock and key models , the interaction of Bim with Bcl-XL and Bcl-2 is more reminiscent of a double-bolt lock with binding mediated by two distant interacting surfaces ( separated by ~40 residues ) working together .", "The affinity of the CTS region alone for Bcl-XL is low as shown by replacing the BH3 region in Bim with a mutant BH3 region from Bad that does not bind Bcl-XL , ( Figure 4C , BimEL-Bad , RBH3-2A ~26% ) .", "However , even in this construct it appears that the CTS contributes to binding as removing the CTS ( BimEL-Bad-dCTS ) further reduced binding ( RBH3-2A ~10% ) .", "Moreover , double-bolt lock binding even with a reduced affinity BH3 sequence results in a very stable interaction ( BimEL RBH3-2A ~80% ) .", "This mechanism also explains how Bim differs from Bad and tBid , which can be displaced from Bcl-XL and Bcl-2 by ABT-263 .", "Unlike what was concluded from cell free assays using partial proteins , a double-bolt lock model predicts that in cells displacement of Bim from anti-apoptotic proteins will require novel strategies targeting both interacting sites .", "Our re-evaluation of the Bim CTS not only challenges the conventional belief that the sequence only functions to bring and keep Bim at the outer mitochondrial membrane ( O'Connor et al . , 1998; Terrones et al . , 2008 ) but suggests that the Bim CTS does not adopt the previously proposed transmembrane topology ( Petros et al . , 2004 ) .", "Instead , the CTS is pinned to the membrane by hydrophobic sequences at either end while the middle is exposed to the cytoplasmic environment ( Figure 9D–F ) .", "We speculate that this topology allows the central region of the Bim CTS to bind to the membrane surface electrostatically via two Arg residues ( Figure 7B–C and Figure 7—figure supplement 1 ) .", "This hypothesis is consistent with our demonstration that substitution of these Arg residues with Ala decreased localization of the protein to membranes ( Figure 7B–C ) and may explain why there is no correlation between Bim mitochondrial localization and the translocase complex on the outer membrane despite colocalization ( Frank et al . , 2015 ) .", "Binding of this region via hydrophobic residues at each end and electrostatically via arginine side chains is an arrangement that may facilitate Bim CTS binding to Bcl-XL through hydrophobic interactions including residue L185 ( Figure 9J ) .", "This insight suggests the Bim CTS functions as an entirely new type of membrane binding domain .", "Moreover , the nature of the interactions with Bcl-XL and the observation that in healthy cells Bim is not bound to anti-apoptotic proteins suggest it may be possible to generate a pair of small molecules that selectively kill cancer cells by efficiently releasing Bim from or preventing Bim binding to Bcl-2 and Bcl-XL .", "However , given that Bim also activates both Bax and Bak and functional assays in cells demonstrating the importance of MCL-1 in MCF-7 cells ( Figure 3E ) , our data strongly suggest that the effects of BH3 mimetics may be difficult to predict for different cell and tissue types ." ], [ "Plasmids encoding the mCerulean3 ( mCer3 ) , or Venus fluorescence proteins in place of the EGFP coding region in pEGFP-C1 ( Clontech ) were kind gifts from Mark A . Rizzo ( University of Maryland ) and Ray Truant ( McMaster University ) respectively .", "To generate plasmids encoding the fusion proteins the required coding regions were amplified by PCR and inserted into the plasmids encoding the appropriate fluorescence protein .", "All of the plasmids included coding sequences for a GGS linker sequence between the coding regions except for VBad ( linker sequence , SGLRSRGG ) and BimELV ( linker sequence , SRGGGPVAT ) .", "All the swapping and site-directed mutants were obtained through PCR-based mutagenesis using oligonucleotides from Integrated DNA Technologies and Phusion DNA polymerase from New England Biolabs .", "ABT-263 was purchased from Selleckchem , A-1155463 , A-1331852 and ABT-199 were from Chemietek .", "The MCF-7 human breast cancer cell line was cultured in Dulbecco's Modified Eagle Medium ( DMEM , ThermoFisher ) supplemented with 10% of fetal bovine serum ( HyClone ) and non-essential amino acids ( NEAA , ThermoFisher ) .", "Transfections were performed using Fugene HD reagent according to the manufacturer’s standard protocol ( Promega ) .", "MCF-7 cell clones stably transfected with vectors encoding mCer3 , CBcl-XL or CBcl-2 were selected in DMEM supplemented with 10% fetal bovine serum , non-essential amino acids ( NEAA , ThermoFisher ) and 500 μg/ml neomycin .", "After 3 weeks colonies were isolated and cultured as above .", "The baby mouse kidney ( BMK ) cell line in which the genes for Bax and Bak have been deleted ( BMK-DKO ) was a kind gift from the originator Eileen White and was cultured as above .", "The two originating cell lines used in the studies reported here ( MCF-7 and Baby Mouse Kidney cells with both Bax and Bak knocked out ) and all stably transfected clones were shown to be free of mycoplasma using a PCR based test .", "The MCF-7 cells were authenticated by the hospital for Sick Children facility in Toronto .", "Clones that stably express mCer3 or CBcl-XL were selected and cultured with 5 μg/mL Blasticidin S . For microscopy , cells were seeded in 384-well imaging plates ( PerkinElmer ) and cultured as above for 24 hr prior to transient transfection .", "Cells treated with ABT-263 , A-1155463 or A-1331852 were incubated at 37°C for 18–24 hr in a fresh media containing 20 μM drug before imaging unless indicated otherwise .", "MCF-7 cells grown in a 384 well plate ( Cell Carrier Ultra ) were transfected , using the TransIT-X2 ( Mirus ) reagent and manufacturer’s protocol , with plasmids encoding Venus protein alone , or Venus protein fused to BimEL ( VBimEL ) , tBid ( VtBid ) or Bad ( VBad ) .", "Media was changed 3 hr later , and cells were incubated 24 hr .", "Prior to imaging , cells were stained with 10 nM tetramethylrhodamine ( TMRE , to stain mitochondria transmembrane potential , Life Technologies ) and 5 µM DRAQ 5 ( to stain nucleic acids , Biostatus , UK ) and incubated for at least 30 mins at 37°C , 5% CO2 .", "Micrographs of the cells were acquired on an OPERA Phenix ( PerkinElmer ) , using the 20x water immersion lens ( NA 1 . 0 ) .", "Data were acquired within 2 hr after staining and during imaging the cells were maintained at 37°C , 5% CO2 .", "Four channels were collected simultaneously: mCer3 ( Ex 425 nm , Em 435–480 nm ) , Venus ( Ex 488 nm , Em 500–550 nm ) , TMRE ( Ex 561 , Em 570–630 ) , DRAQ 5 ( Ex 640 nm , Em 650–760 nm ) .", "Acquisition was automated and acquisition settings were identical between wells and for each independent replicate ( n = 3 ) .", "Acquisition settings were different for Figures 3 and 5 therefore , the X-axis is not comparable .", "For the data in Figure 5 the cloning vector , pSPUTK ( Stratagene Santa Clara CA ) was used to dilute the plasmid encoding the VBH3 proteins , to reduce overexpression of Venus-tagged protein while ensuring consistent transfection efficiencies .", "Cell images recorded on the OPERA Phenix were imported into Harmony high-content analysis software ( PerkinElmer ) .", "The seven step pipeline built to identify live and dead cells was as follows:", "1 ) Nuclear and total cell areas for each cell were identified using DRAQ five intensity data and a Harmony segmentation algorithm .", "2 ) The cytoplasmic area was obtained by subtracting the nuclear area from the total cell area .", "3 ) Cell images that extended to or beyond the any edge of the micrograph were removed from the analysis as the total area is uncertain .", "4 ) The total intensity of TMRE within each cell image was calculated .", "5 ) Nuclear area and morphology features ( e . g . roundness ) were calculated .", "6 ) Cell area and morphology features were calculated .", "As positive and negative controls , >100 cells treated with 500 ng/ml TNFα and cycloheximide ( apoptotic cells ) and >100 untreated cells , respectively were analyzed similarly for each experiment .", "7 ) The intensity of Venus and mCerulean3 was measured for all selected cells .", "Data from the control cells were used to train a linear classifier in the Harmony software .", "Each cell death curve represents measurements for at least 1000 cells for each of three replicates .", "To confirm that the linear classifier reported data similar to manual analysis a subset of the data was reanalyzed with manual thresholds to score cells as , ‘TMRE negative’ ( TMRE intensity at least two standard deviations lower than the average in an untreated well ) or ‘small nuclear area’ ( Nuclear area at least two standard deviations lower than the average in an untreated well ) .", "Cell death was also measured as a function of the amount of the transiently expressed protein , such as VBimEL , in cells .", "For this analysis individual cells were classified as alive or dead and then results were binned by Venus intensity .", "For these experiments the Venus intensity serves as an estimate of the amount of the BH3 protein produced in the cell .", "The range of intensities within each bin was kept consistent for each transfectant , for all three independent replicates .", "Mean % Dead and standard error was plotted for each intensity bin .", "Steady-state fluorescence images and fluorescence lifetime images were both acquired using a custom-built Alba confocal microscope from ISS ( Champagne , Illinois ) , which measures fluorescence lifetime by time correlated single photon counting ( TCSPC ) .", "Channels of mCer3 and Venus were acquired simultaneously using 445 nm and 514 nm pulse interleaved excitation ( PIE ) through a 442/512/561 multiband filter and emission split by a 520nm-longpass filter was collected through 459–499 nm and 528–555 nm bandpass filters , respectively .", "All images were acquired as 256 pixels x 256 pixels at 25°C using a 60 × 1 . 3 NA PlanApo water immersion objective and laser powers set to minimize photobleaching and photon pileup during acquisition with 0 . 1 ms per pixel dwell time and 5-frame repeat .", "TCSPC-data were processed using VistaVision software ( ISS ) and the average lifetime for each pixel was obtained by fitting FLIM pixels binned to ensure a total photon count >1000 for each set of binned pixels analyzed .", "The lifetime and photon count for each pixel in both channels were exported for additional analysis using ImageJ .", "For Bcl-2 family proteins the problem of measuring protein:protein interactions in live cells ( Herce et al . , 2013 ) is particularly acute as the proteins often function differently at membranes therefore , assays using cell extracts do not capture either the correct or the complete set of activities ( Lovell et al . , 2008 ) .", "Moreover , solubilizing membranes requires detergents artificially increase and decrease Bcl-2 family protein:protein interactions ( Lovell et al . , 2008 ) .", "The use of purified proteins and cell free assays has provided much useful information , however , due to the difficulty of purifying full-length Bcl-2 family proteins , many assays in vitro are based on truncated proteins or peptides and the environment does not well represent that of an intact cell .", "We used FLIM-FRET in live cells using fluorescent protein tagged full-length proteins to quantitatively study protein:protein interactions for Bcl-2 family proteins and to assess the impact of small-molecule inhibitors on these interactions ( Aranovich et al . , 2012; Kale et al . , 2012; Liu et al . , 2012 ) .", "This technique removes the disadvantages of biochemical methods , but has its own limitations .", "For example the over-expression of a fusion protein with a fluorescent protein may result in protein concentrations within cells that are above the Kds for the interactions , affecting the relative Kds measured .", "For these reasons it is difficult to compare interactions between different pairs of proteins , instead the method is best suited to comparing the effects of small molecules and mutations on interactions .", "In our application of FLIM-FRET the ratio of Venus to mCer3 intensities was used as an estimate of the relative amounts of the proteins to plot binding curves .", "Stable expression of mCer3 and using measurements from cells with little variation in amount of mCer3 allows ratio intensities to serve as a reasonable approximation for generating binding curves , but distorts the meaning of the Hill coefficients obtained .", "Moreover , in some cells high expression of Venus reduces the expression of mCer3 and for the some of the proteins analyzed this effect can dominate at high ratios of Venus to mCer3 .", "As a result , in some experiments the data at ratios of Venus to mCer3 greater than 0 . 5 are not well described by a standard binding curve ( e . g . Figure 2—figure supplement 1 , panel C , black ) .", "For this reason quantitative comparisons were restricted to those regions of the curves that are fit using a standard binding curve with a Hill slope .", "For VtBid and VBad it was not possible to measure binding to CBcl-XL at ratios of Venus to mCer3 intensities greater than ~0 . 7 in the presence of ABT-263 because the released VBH3-proteins killed the cells .", "Another complication with using any kind of FRET in live cells is the impact of collisions that can generate false positive signals .", "Fortunately , the latter can be distinguished by measuring binding curves using FLIM-FRET .", "Collisions vary directly with protein concentration and therefore generate straight lines rather than curves that saturate ( e . g . Figure 4—figure supplement 1E , red ) .", "To limit the effect of compounds or mutagenesis potentially changing both the binding affinity and the maximum FRET efficiency to different extents and still generate a single descriptor that can be used to describe the interactions , we introduced the concept of Resistance ( R ) of a complex to a specific compound ( RABT-263 ) or mutation ( RBH3-2A ) ( Figure 2A ) .", "Quantitative analysis based on values of R allows comparison between multiple samples each that results in a unique binding curve and that was generated from measurements of thousands of different intracellular ROIs that would be hard to compare otherwise .", "R values cannot replace the original binding curves for detailed determination of how the interaction is disturbed .", "Binding curves for CBcl-XL and VBad-Bim-I146Y with and without the BH3-2A mutation allowed quantification of the decrease in binding affinity due to the I146Y mutation ( Figure 6 ) .", "To generate binding curves , ROIs containing mitochondria were selected , and the corresponding intensities in both mCer3 and Venus channels and the average lifetime for mCer3 were extracted from the original TCSPC data using a customized ImageJ script .", "ROIs were automatically discarded from the analysis if the CBclXL signal was too low and therefore noisy , or close to saturated , or the Venus signal was close to saturated .", "The ratio of Venus to mCer3 was calculated using the intensities of each channel .", "FLIM-FRET efficiency ( E% ) was calculated for each ROI as: E%= ( 1-τi/τ0 ) ×100% , where τi is the mean lifetime for that ROI and τ0 is the average lifetime for mCer3 for all ROIs not expressing detectable Venus .", "FLIM-FRET efficiency was then distributed into bins of the same size according to Venus:mCer3 intensity ratio ( bin size 0 . 1 for ratios lower than 0 . 5 , 0 . 25 for ratios between 0 . 5 and 1 . 25 , 0 . 5 for ratios higher than 1 . 25 ) and plotted ( ±se ) against Venus:mCer3 ratio .", "The binding curves were fitted using GraphPad Prism version 5 . 0d for Macintosh ( GraphPad Software , San Diego California USA , www . graphpad . com ) with the function: E% = Emax × ( IVenus÷ImCerulean3 ) h/[Kdh+ ( IVenus÷ImCerulean3 ) h] .", "Emax is the maximum FLIM-FRET efficiency corresponding to saturation of donor binding sites by an acceptor; ImCerulean3 and IVenus are intensities of mCer3 and Venus , respectively; Kd is the relative equilibrium dissociation constant; h is the Hill slope .", "BMK-DKO cells transiently expressing Venus-tagged constructs of interest were stained with 5 µM DRAQ 5 ( BioStatus ) to stain nucleic acids , and 500 nM MitoTracker-Red to stain mitochondria , ( Life Technologies ) in DMEM at 37°C and 5% CO2 , 30 min .", "One positive control well was additionally stained with 500 nM MitoTracker-Green ( to stain mitochondria , Life Technologies ) .", "Cells were incubated at 37°C , 5% CO2 during sample imaging .", "For each well , 60 images at 256 × 256 pixels resolution were recorded using a 40x water immersion ( NA 1 . 1 ) lens on an Opera Phenix confocal microscope .", "DRAQ 5 ( Ex 640 nm , Em 650–760 nm ) , Venus ( Ex 488 nm , Em 500–550 nm ) and MitoTracker-Red ( Ex 561 nm Em 570–630 nm ) channels were acquired sequentially , automatically switching between channels at each field of view .", "An analysis pipeline was created in Cell Profiler 2 . 2 . 0 .", "In this pipeline , the DRAQ five channel was used to identify nuclei , and total cell area with smoothing to ensure the entire cell is selected as one region .", "Cells that extend to the border of the image were rejected from , the analysis .", "The cytoplasm for each cell was identified as an object ( Cytoplasm = ‘Total cell area’ – ‘nuclear area’ ) .", "Objects suitable for analysis were identified by mean Venus intensity to identify cells expressing appropriate levels of the Venus-tagged proteins of interest .", "A median Venus intensity ( low threshold ) was used to discard any cells that passed the mean Venus filter due to improper segmentation ( ie . overlap between an untransfected cell and a bright transfected cell ) .", "Objects were further selected by mean MitoTracker-Red intensity ( high and low threshold ) , to ensure appropriate staining .", "A size filter was used to remove small dead cells or debris and any abnormally large ( possibly improperly segmented ) cells .", "Background was subtracted for each image and a Pearson’s correlation coefficient ( Pearson’s", "r ) between Venus and MitoTracker-Red signals was calculated for all remaining objects .", "In each replicate , a minimum of 20 cells were identified per protein of interest and mean Pearson’s r was calculated .", "In sum , at least 150 cells were analyzed per sample .", "The average and standard error of the mean Pearson’s r values determined for the three replicates was plotted using GraphPad Prism .", "Full length and single cysteine mutants of Bcl-XL and tBid were purified as described previously ( Kale et al . , 2014 ) .", "For Bim , the cDNA encoding full-length wild-type murine BimL was introduced into pBluescript II KS ( + ) vector ( Stratagene , Santa Clara CA ) .", "Sequences encoding a polyhistidine tag followed by a TEV protease recognition site ( MHHHHHHGGSGGTGGSENLYFQGT ) were added to create an in-frame fusion to the N-terminus of BimL .", "BimL-dCTS was constructed by introducing a stop codon ( TAA ) after position P121 to delete the entire CTS .", "Single cysteine mutants were generated by PCR-based mutagenesis using oligonucleotides from Integrated DNA Technologies and Phusion DNA polymerase from New England Biolabs .", "The recombinant proteins were expressed in Arabinose Induced ( AI ) Escherichia coli strain ( Life Tech , Carlsbad , CA ) .", "E . coli cells were lysed by mechanical disruption with a French press .", "The cell lysate was separated on a Nickel-NTA column ( Qiagen , Valencia CA ) to bind the recombinant His-tag fused proteins and after washing a buffer containing 300 mM imidazole was applied to elute the proteins .", "This elution was then adjusted to 150 mM NaCl and applied to a High Performance Phenyl Sepharose ( HPPS ) column .", "BimL was eluted with a no salt buffer and dialyzed against a buffer containing 10 mM HEPES pH7 . 0 , 20% Glycerol , and then flash-frozen and stored at −80°C .", "Single cysteine mutants of Bcl-XL and tBid were labeled with the indicated maleimide-linked fluorescent dyes as described previously ( Kale et al . , 2014; Lovell et al . , 2008 ) .", "Single cysteine mutants of BimL were labeled with the same protocol as tBid with the exception that the labeling buffer also contained 4M urea .", "Single cysteine mutants of BimL ( 41C ) and tBid ( 126C ) were purified and labeled with Alexa 568-maleimide .", "A single cysteine mutant of Bcl-XL ( 152C ) was purified and labeled with Alexa 647-maleimide .", "Alexa568 labeled BimL or tBid was incubated with either Alexa647-labeled or as a control unlabeled Bcl-XL along with the indicated concentrations of ABT-263 .", "The intensity of Alexa568 fluorescence with unlabeled or Alexa647-labeled Bcl-XL was measured as Funlabeled or Flabeled respectively .", "FRET , indicating protein-protein interaction , was quantified using the decrease of Alexa568 fluorescence when BimL or tBid bound to Alexa647-labeled Bcl-XL compared to unlabeled Bcl-XL .", "FRET efficiency was calculated as: FRET efficiency ( % ) = ( 1-Flabeled/Funlabeled ) *100% as described previously for cBid-Bcl-XL .", "Binding was measured after incubation at 37°C for 1 hr .", "To compare the inhibitory effect of ABT-263 on different BimL mutants or tBid binding to Bcl-XL , ABT-263 was titrated into a solution containing 10 nM BH3 protein ( BimL or tBid ) and 40 nM Bcl-XL .", "At these concentrations BH3 protein binding to Bcl-XL had saturated ( BH3 protein bound = 100% ) and represents the maximum FRET efficiency ( Fmax ) .", "Inhibition of BH3 protein binding to Bcl-XL by ABT-263 was measured based on the decrease of FRET ( BH3 protein bound = F/Fmax*100% ) .", "Data were fitted to a standard inhibitor dose response equation with a Hill slope using GraphPad Prism ( V6 . 02 ) .", "For BH3 proteins in which binding was poorly inhibited ( BimL ( Figure 2E and Figure 7D–E ) , BH3 protein bound was assumed to be 0% at an infinite concentration of ABT-263 .", "Loss of protein on the cuvettes and other surfaces at low concentrations required use of protein at concentrations higher than the Kds of binding .", "For this reason the dissociation constant is not independent of protein concentration and the data in Figure 2E , Figure 7D–E report relative displacements comparable between figures rather than absolute affinity .", "The single cysteine mutants of Bim were purified and labeled with NBD ( N , N0-dimethyl-N- ( Iodoacetyl ) -N0- ( 7-nitrobenz-2-oxa-1 , 3-diazol-4-yl ) ethylenediamine; Molecular Probes , Cat .", "#: D-2004 ) .", "The NBD fluorescence assay and iodide quenching of NBD-labeled Bim mutants was performed as described previously for Bax ( Kale et al . , 2014 ) , with the exception that the assays were performed in 100 ul volume in low protein binding 96-well plates and fluorescence was measured using a plate reader ( Tecan M1000 ) .", "In the experiments reported here , 20 nM NBD-labeled Bim alone or 20 nM Bim plus 50 nM of Bcl-XL were incubated with 0 . 2 mg/mL ( final lipid concentration ) of liposomes .", "For the ‘In solution’ control , the protein", "( s ) were incubated with an equal volume of assay buffer instead of liposomes ." ] ]
[ "Tumor initiation , progression and resistance to chemotherapy rely on cancer cells bypassing programmed cell death by apoptosis .", "We report that unlike other pro-apoptotic proteins , Bim contains two distinct binding sites for the anti-apoptotic proteins Bcl-XL and Bcl-2 .", "These include the BH3 sequence shared with other pro-apoptotic proteins and an unexpected sequence located near the Bim carboxyl-terminus ( residues 181–192 ) .", "Using automated Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer ( FLIM-FRET ) we show that the two binding interfaces enable Bim to double-bolt lock Bcl-XL and Bcl-2 in complexes resistant to displacement by BH3-mimetic drugs currently in use or being evaluated for cancer therapy .", "Quantifying in live cells the contributions of individual amino acids revealed that residue L185 previously thought involved in binding Bim to membranes , instead contributes to binding to anti-apoptotic proteins .", "This double-bolt lock mechanism has profound implications for the utility of BH3-mimetics as drugs .", "​" ]
[ "The body can get rid of cells that are unnecessary , infected or damaged by instructing them to go through a process known as apoptosis , which results in the cell killing itself .", "These orders are given in the form of pro-death molecules , such as the BH3 proteins , which kick start apoptosis .", "In order to survive and multiply , cancer cells can increase the levels of anti-death proteins which bind and ‘trap’ BH3 proteins , preventing them from triggering apoptosis .", "At the molecular level , the process involves the anti-death proteins recognizing and attaching to a specific ‘BH3 sequence’ in the pro-death signals .", "A new type of anti-cancer treatment works by tricking the anti-death proteins into binding a drug rather than BH3 proteins , which are then free to induce apoptosis .", "These decoy drugs mimic the BH3 sequences that the anti-death proteins recognize and attach to .", "However , studies have shown that the BH3-protein Bim stays bound to the body’s anti-death proteins rather than being displaced by the drugs .", "In patients , this means that the cancer cells may resist and survive the treatment .", "Here , Liu et al . try to understand why Bim keeps on attaching to anti-death proteins by developing an advanced microscopy technique to dissect the interactions between the two types of molecules .", "This revealed that Bim has a second sequence for binding to anti-death proteins , which is located far away from the ‘normal’ BH3 sequence .", "Together , the two sequences allow Bim to double-bolt lock to anti-death proteins , which explains why it cannot be displaced by drugs that mimic only the BH3 sequence .", "In the future , the new binding sequence may serve as a target for anti-cancer drugs .", "The microscopy technique developed by Liu et al . can also be used to study other pairs of interacting proteins ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "tools and resources", "immunology and inflammation" ]
T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse
elife-32417-v2
[ [ "Calcium ( Ca2+ ) is an essential second messenger responsible for a wide variety of cellular functions ( Berridge et al . , 2000; Clapham , 2007; Berridge , 2012 ) .", "Through the use of synthetic small molecule Ca2+ indicators such as fura-2 and fluo-4 , imaging studies have greatly expanded our understanding of Ca2+ signaling dynamics ( Tsien et al . , 1982; Grynkiewicz et al . , 1985 ) .", "However , such indicators cannot be targeted to specific subcellular compartments or cell populations , and are unsuitable for long-term studies due to leakage out of cells .", "Moreover , they often do not faithfully report pure cytosolic Ca2+ signals , because of diffusion into cellular compartments such as the nucleus .", "One alternative to overcoming these limitations is with genetically encoded Ca2+ indicators ( GECIs ) , first developed two decades ago as FRET-based fluorescence probes ( Miyawaki et al . , 1997; Romoser et al . , 1997; Pérez Koldenkova and Nagai , 2013 ) .", "Key advantages to GECIs include the capability for genetic targeting to specific cell types or subcellular organelles , measuring local Ca2+ levels by direct fusion to a protein of interest , modulation of expression levels by inclusion of an inducible promoter , and long-term studies due to continuous expression of the genetic indicator ( Miyawaki et al . , 1997; Pérez Koldenkova and Nagai , 2013 ) .", "Despite these inherent advantages , the initial FRET-based GECI probes were not widely used as their performance fell far behind small molecule Ca2+ indicators , particularly in Ca2+ sensitivity , brightness , and dynamic range .", "Since then , successive rounds of design and contributions from multiple research groups have resulted in numerous variants of GECIs with high dynamic range and dramatically improved performance ( Baird et al . , 1999; Nakai et al . , 2001; Tian et al . , 2009; Zhao et al . , 2011; Akerboom et al . , 2012; Akerboom et al . , 2013; Chen et al . , 2013 ) .", "Single fluorescent protein-based GECIs containing a circularly permutated green fluorescent protein ( GFP ) exhibit high brightness , fast response kinetics , and offer multiple color variants , including the GECO and the GCaMP series ( Tian et al . , 2009; Zhao et al . , 2011; Akerboom et al . , 2012; Chen et al . , 2013 ) .", "FRET-based GECIs have continued to evolve as well , with sequential improvements including incorporation of circularly permuted yellow fluorescent proteins ( cpYFPs ) to improve dynamic range in the yellow cameleon ( YC ) family ( Nagai et al . , 2004 ) , use of troponin C as the Ca2+ sensing element in the TN indicator family ( Heim and Griesbeck , 2004 ) , computational redesign of the calmodulin-M13 interface to increase the range of Ca2+ sensitivity and reduce perturbation by native calmodulin in the DcpV family ( Palmer et al . , 2006 ) , and complete redesign of the troponin C domain to increase response kinetics and reduce buffering of cytosolic Ca2+ in the TN-XXL family ( Mank et al . , 2006; Mank et al . , 2008 ) .", "The latest generation of GECIs have crossed key performance thresholds previously set by small-molecule indicators , enabling GECIs to be widely applied in diverse Ca2+ imaging studies without sacrificing performance .", "Members of the GCaMP6 family are capable of tracking cytosolic Ca2+ changes from single neuronal action potentials , with higher sensitivity than small-molecule indicators such as OGB-1 ( Chen et al . , 2013 ) .", "The availability of multicolored variants in the GECO family and the RCaMP series allowed for simultaneous measurement of Ca2+ dynamics in different cell populations in the same preparation , or in different subcellular compartments within the same cell ( Zhao et al . , 2011; Akerboom et al . , 2013 ) .", "These variants can be integrated with optogenetics to simultaneously evoke channel rhodopsin activity while monitoring localized Ca2+ responses in independent spectral channels ( Akerboom et al . , 2013 ) .", "Moreover , individual GECIs can be tagged onto membrane Ca2+ channels to directly measure Ca2+ influx through the target channel of interest , enabling optical recording of single channel activity without the need for technique-intensive patch clamping ( Dynes et al . , 2016 ) .", "Another advantage of GECIs is their capability to be incorporated into transgenic organisms .", "Although several GECI-expressing transgenic mouse lines have already been reported , many of these studies used older variants of GECIs that are expressed only in selected tissues ( Hasan et al . , 2004; Ji et al . , 2004; Tallini et al . , 2006; Heim et al . , 2007 ) .", "The Ai38 mouse line overcomes these issues by combining GCaMP3 with a robust and flexible Cre/lox system for selective expression in specific cell populations ( Zariwala et al . , 2012 ) .", "Based on a series of Cre-responder lines designed for characterization of the whole mouse brain ( Madisen et al . , 2010 ) , the Ai38 mouse line contains GCaMP3 targeted to the Rosa26 locus but requires Cre recombinase for expression .", "By crossing Ai38 with various Cre mouse lines , GCaMP3 can be selectively expressed in specific cell populations .", "Thus , target cells may be endogenously labeled without invasive procedures , avoiding potential off-target side effects reported in GECI transgenic lines with global expression ( Direnberger et al . , 2012 ) .", "The newly released PC::G5-tdT mouse line provides improved functionality by targeting a Cre-dependent GCaMP5G-IRES-tdTomato transgenic cassette to the Polr2a locus ( Gee et al . , 2014 ) .", "However , in the PC::G5-tdT mouse line , GCaMP5G and tdTomato are expressed individually , and localize to different cell compartments .", "Moreover , because expression of tdTomato is driven by an internal ribosomal entry site , the expression level is highly variable and weaker than GCaMP5G , limiting identification of positive cells and preventing accurate ratiometric measurements .", "Although single fluorescent protein-based indicators have high brightness and fast response kinetics , as non-ratiometric probes they are problematic for Ca2+ imaging in motile cells where fluorescence changes resulting from movement may be indistinguishable from actual changes in Ca2+ levels .", "Here , we introduce a novel genetically encoded Ca2+ indicator - that we christen ‘Salsa6f’ - by fusing green GCaMP6f to the Ca2+-insensitive red fluorescent protein tdTomato .", "This probe enables true ratiometric imaging , in conjunction with the high dynamic range of GCaMP6 .", "We further describe the generation of a transgenic mouse enabling Salsa6f expression in a tissue-specific manner , and demonstrate its utility for imaging T lymphocytes in vitro and in vivo ." ], [ "In order to develop a better tool to monitor Ca2+ signaling in T cells both in vivo and in vitro , we first evaluated the latest generation of genetically encoded Ca2+ indicators ( GECIs ) ( Zhao et al . , 2011; Chen et al . , 2013 ) .", "We transiently expressed and screened a variety of single fluorescent protein-based GECIs in HEK 293A cells ( Figure 1A ) , and selected GCaMP6f based on fluorescence intensity , dynamic range , and Ca2+ affinity suitable for detecting a spectrum of cytosolic Ca2+ signals ( Kd = 375 nM ) .", "To enable cell tracking even when basal Ca2+ levels evoke little GCaMP6f fluorescence , we fused GCaMP6f to the Ca2+-insensitive red fluorescent protein tdTomato , chosen for its photostability and efficient two-photon excitation ( Drobizhev et al . , 2011 ) .", "A V5 epitope tag ( Lobbestael E et al . , 2010 ) served to link tdTomato to GCaMP6f ( Figure 1B ) .", "The resultant ratiometric fusion indicator , coined ‘Salsa6f’ for the combination of red tdTomato with the green GCaMP6f , was readily expressed by transfection into HEK 293A cells and human T cells .", "Salsa6f exhibited a ten-fold dynamic range , with a brightness comparable to GCaMP6f alone ( Figure 1A , C ) .", "For two-photon microscopy , both components of Salsa6f can be visualized by femtosecond excitation at 900 nm ( Figure 1D ) .", "GCaMP6f produces increased green fluorescence during elevations in cytosolic Ca2+ , while tdTomato provides a stable red fluorescence that facilitates cell tracking and allows for ratiometric Ca2+ imaging ( Figure 1D; Video 1 ) .", "Salsa6f is excluded from the nucleus , ensuring accurate measurement of cytosolic Ca2+ fluctuations ( Figure 1D , E ) .", "When expressed by transfection in human T cells , Salsa6f reported Ca2+ oscillations induced by immobilized αCD3/28 antibodies with a high signal to noise ratio and time resolution ( Figure 1E , F ) .", "Guided by the transgenic targeting strategy for the Ai38 mouse line ( Zariwala et al . , 2012 ) , we inserted Salsa6f into a Gt ( ROSA ) 26Sor5’-pCAG-FRT-LSL-Salsa6f-WPRE-bGHpA-AttB-FRT-NeoR-AttP-Gt ( ROSA ) 26Sor3’ cassette , then targeted it to the Rosa26 locus in JM8 . N4 mouse embryonic stem ( ES ) cells ( Figure 2A ) .", "Cells positive for the allele Gt ( ROSA ) 26SorpCAG-FRT-LSL-Salsa6f-WPRE-bGHpA-AttB-FRT-NeoR-AttP were selected by neomycin resistance , and correctly targeted clones were screened by Southern blot ( Figure 2B ) , then injected into C57BL/6J blastocysts for implantation .", "Chimeric pups carrying the Salsa6f transgene were identified by PCR screening for the Nnt gene , as the initial JM8 . N4 ES cells were Nnt+/+ while the C57BL/6J blastocysts were Nnt-/- ( Figure 2C ) .", "Positive chimeras were bred to R26ΦC31o mice to remove the neomycin resistance gene and to produce LSL-Salsa6f F1 founders that are heterozygotic for the Gt ( ROSA ) 26SorpCAG-FRT-LSL-Salsa6f-WPRE-bGHpA-AttB/P allele , then further bred to generate homozygotic mice which we term LSL-Salsa6f ( Hom ) .", "LSL-Salsa6f ( Hom ) mice were bred to Cd4Cre+/+ mice to obtain reporter mice heterozygous for Salsa6f , designated as Cd4-Salsa6f ( Het ) mice from here on , that selectively express Salsa6f in T cells ( Figure 3A ) .", "Mice homozygous for Salsa6f are designated as Cd4-Salsa6f ( Hom ) .", "Salsa6f was detected by tdTomato fluorescence on flow cytometry .", "88% of these Salsa6f+ cells in thymus were double positive for Cd4 and Cd8 ( Figure 3B ) .", "This is due to the double-positive stage during development , in which developing thymocytes will express both Cd4 and Cd8 before undergoing positive and negative selection to become either mature Cd4+ or Cd8+ T cells .", "Salsa6f was readily detected by the red tdTomato signal in cells from spleen ( 40% ) , lymph node ( 57% ) , and thymus ( 93% ) ( Figure 3C ) .", "As expected , double positive cells were not detected in the spleen ( Figure 3D ) .", "More than 98% of Cd4+ and Cd8+ T cells from these reporter mice were positive for Salsa6f .", "Salsa6f was also detected in 5% of Cd19+ cells and 3% of Cd11b+ cells ( Figure 3E ) .", "A small fraction of B cells express Cd4 mRNA , which may explain the presence of Salsa6f in Cd19+ cells ( Zhang and Henderson , 1994 ) .", "Cd11b+ cells positive for Salsa6f may be splenic resident dendritic cells that also express Cd4 ( Vremec et al . , 2000; Turley et al . , 2010 ) .", "The total number and relative frequencies of Cd4+ , Cd8+ , Cd19+ , and Cd11b+ cells were similar to the Cd4Cre controls ( Figure 3F , G ) .", "To determine whether expression of Salsa6f might affect functional responses downstream of Ca2+ signaling in T cells , we first purified Cd4+ T cells and monitored cell proliferation in vitro during TCR engagement of αCd3 and co-stimulating αCd28 antibodies attached to activating beads .", "Both hetero and homozygotic Salsa6f-expressing Cd4+ T cells proliferated similar to the Cd4Cre controls ( Figure 4A , B ) .", "To further probe functional responses , we differentiated naive Cd4+ T cells using polarizing cytokine stimuli to generate Th1 , Th17 and induced regulatory T cells ( iTregs ) .", "Salsa6f-positive naive Cd4+ T cells from both Cd4-Salsa6f ( Het ) and Cd4-Salsa6f ( Hom ) mice readily differentiated into various helper T cell subtypes similar to the Cd4Cre controls ( Figure 4C–E and Figure 4—figure supplement 1 ) .", "In addition , as described in the companion paper , adoptively transferred Salsa6f-positive cells readily homed to lymph nodes and exhibited normal motility characteristics ( Dong et al . , 2017 ) .", "In summary , our results demonstrate normal T-cell functions of Salsa6f-expressing T lymphocytes with respect to development , cellular phenotype , cell proliferation , differentiation , homing , and motility .", "Cd4+ T cells were purified from Cd4-Salsa6f ( Het ) reporter mice , stimulated with plate-bound αCd3/28 antibodies for 2 days , and imaged by confocal microscopy while still in contact with immobilized antibodies ( Figure 5A , Video 2 ) .", "Activated Cd4+ T cells expressing Salsa6f exhibited stable red fluorescence and wide fluctuations in green fluorescence due to Ca2+ oscillations resulting from T-cell receptor engagement ( Figure 5B ) .", "Despite variability in total fluorescence between cells due to individual differences in cell size , the basal and peak green/red Salsa6f ratios ( referred from now on as G/R ratio for GCaMP6f/tdTomato intensity ) were comparable between cells and showed up to six-fold increases during peaks in Ca2+ fluctuations ( Figure 5C ) .", "Flow cytometric analysis of Salsa6f mouse T cells revealed a 13-fold increase in G/R ratio , by pretreatment with ionomycin in Ca2+-free medium to deplete cytosolic Ca2+ followed by addback of extracellular Ca2+ , further emphasizing the high dynamic range of Salsa6f ( Figure 5D ) .", "Finally , to test if increasing the genetic dosage can improve the brightness of Salsa6f , we compared Cd4+ T cells from Cd4-Salsa6f ( Het ) and Cd4-Salsa6f ( Hom ) mice .", "T cells from homozygous mice with two allelic copies of the Salsa6f reporter cassette exhibited almost a two-fold increase in tdTomato fluorescence compared to heterozygous mice ( Figure 5E ) , allowing for genetic control of Salsa6f expression level when brightness is an issue .", "We first examined the localization of Salsa6f in naïve Cd4+ T cells isolated from Cd4- Salsa6f ( Het ) mice and in Cd4+ T cells activated for 2 days on plate-bound αCd3/28 .", "Line scans of the confocal images of cells plated on poly-L-lysine-coated coverslips showed that Salsa6f is primarily localized to the cytoplasm and is excluded from the nucleus ( Figure 6A , B ) .", "Increasing the cytosolic Ca2+ levels using thapsigargin ( TG ) in 2 mM Ca2+ Ringer’s solution caused a selective increase in the GCaMP6f signal , without altering the localization of Salsa6f probe .", "In contrast , the small-molecule dye Ca2+ indicators fluo-4 or fura-2 loaded into Cd4+ T cells from Cd4Cre mice are distributed throughout the cell , including the nucleus ( Figure 6C and data not shown ) .", "A different transgenic mouse , PC::G5-tdT utilizes an internal ribosomal entry site to express both tdTomato and GCaMP5G as separate proteins that localize differently ( Gee et al . , 2014 ) , with tdTomato distributed throughout the cell including the nucleus and GCaMP5G predominantly in the cytosol ( Figure 6—figure supplement 1 ) .", "In contrast , our tandem probe , Salsa6f , results in both red and green fluorescent proteins co-localized in the cytosol , allowing true ratiometric Ca2+ imaging and facilitating tracking of cells .", "Overlap of GCaMP6f emission and tdTomato excitation spectra raises the possibility of FRET ( Forster resonance energy transfer ) which would decrease G/R ratio and reduce dynamic range .", "If there were significant FRET , we would expect acceptor ( tdTomato ) signal to increase with increase in donor ( GCaMP6f ) intensity , as shown for GECI probes utilizing GCaMP and mCherry dependent on the rigidity of the linker used ( Cho et al . , 2017 ) .", "We treated Salsa6f-positive T cells with Ringer’s buffer containing Ionomycin and different concentrations of Ca2+ and measured the steady-state GCaMP6f and tdTomato fluorescence signals .", "The GCaMP6f signal increased as expected with increasing Ca2+ concentrations , but the tdTomato signal remained unchanged ( Figure 6D ) .", "This suggests that the V5 epitope tag is effective in reducing FRET between GCaMP6f and tdTomato which otherwise may compromise probe performance .", "We next characterized the in situ Ca2+ affinity of Salsa6f in Cd4+ T cells isolated from Cd4-Salsa6f ( Het ) mice , using fura-2 as a calibration standard .", "Fura-2 has an in situ Kd ~225 nM at 25° C ( Lewis and Cahalan , 1989 ) , which is somewhat lower than in vitro Kd values reported for GCaMP6f ( Chen et al . , 2013; Badura et al . , 2014 ) .", "We used time-lapse imaging to record G/R ratio in response to ionomycin applied with stepwise increases in the external Ca2+ concentration and , using identical protocols , compared it to fura-2 Ca2+ signals in control Cd4+ T cells from Cd4Cre mice ( Figure 6E , F ) .", "To facilitate comparison , fura-2 and Salsa6f ratios were normalized with Rmin = 0 and Rmax = 1 .", "Salsa6f responded with faster rise and decay kinetics than fura-2 to progressive increases in cytosolic Ca2+ levels , especially at lower external Ca2+ concentrations .", "Salsa6f responses also saturated at lower cytosolic Ca2+ levels than fura-2 responses .", "This is not altogether surprising given that genetically encoded Ca2+ indicators have been reported to have a steeper Hill coefficient than chemical indicators ( Badura et al . , 2014 ) .", "Assuming that Cd4-Salsa6f T cells reach similar Ca2+ levels as control Cd4Cre cells , we plotted peak and the steady state G/R ratios against the cytosolic Ca2+ concentrations obtained from the fura-2 experiment which yielded an estimated in situ Kd for Salsa6f in the range of 160–300 nM ( Figure 6G ) .", "Based on these results , we conclude that Salsa6f is sensitive in detecting cytosolic Ca2+ from 100 nM - 2 μM , which is in the range of physiological cytosolic Ca2+ signals .", "TCR engagement activates a canonical Ca2+ signaling pathway in T cells , characterized by IP3-induced Ca2+ release from the endoplasmic reticulum , leading to store-operated Ca2+ entry ( SOCE ) through Orai1 channels ( Cahalan and Chandy , 2009; Prakriya and Lewis , 2015 ) .", "Past studies on T cell Ca2+ signaling have largely relied on indicators like fura-2 and fluo-4 which have the potential drawback of being distributed into the nucleus , thus confounding the measurement of pure cytoplasmic Ca2+ signals , a problem particularly notable in T cells with their large nuclear to cytoplasmic volume ratio .", "Salsa6f , with its high dynamic range , ratiometric readout and targeted localization in the cytosol , is thus well suited to record physiological cytosolic Ca2+ signals .", "To this end , we recorded Ca2+ signals from 2-day-activated Cd4+ T cells from Cd4-Salsa6f ( Het ) mice in response to a variety of stimuli .", "To study SOCE more directly , we depleted ER Ca2+ stores in activated and in naïve T cells by applying TG in Ca2+-free solution .", "We observed a small but sharp initial peak indicating ER store release followed by a sustained Ca2+ signal upon restoring Ca2+ to the external bath , indicative of SOCE ( Figure 7A , Video 3 and Figure 7—figure supplement 1A ) .", "Almost all cells responded to this supra-physiological stimulus ( Figure 7A , right panel ) .", "In contrast , T cells plated on αCd3/28 to activate TCR-induced signaling showed heterogeneous responses , with some exhibiting asynchronous Ca2+ oscillations of varying frequencies and durations whereas others failed to respond ( Figure 7B , Video 4 ) .", "Past studies have attributed these Ca2+ oscillations to SOCE from repetitive opening and closing of Orai1 channels allowing Ca2+ to enter T cells in a periodic and asynchronous manner ( Lewis and Cahalan , 1989; Dolmetsch and Lewis , 1994 ) , unlike the sustained activation with TG treatment .", "Cells plated on αCd3 alone also showed rhythmic oscillatory Ca2+ signals; however , the percentage of responding cells was significantly lower than with αCd3/28 , resulting in a lower average signal compared with αCd3/28 stimulation ( Figure 7C , Video 5 ) These results suggest that co-stimulatory signaling through Cd28 enhances the response to TCR activated Ca2+ signals , in alignment with previous observations ( Chen and Flies , 2013 ) .", "Finally , we focused on a novel Ca2+ signaling pathway involving mechanosensitive Ca2+-permeable channels .", "We examined activation of mechanosensitive channels in Salsa6f+ T cells that were plated on αCd3/28-coated glass coverslips , by flowing external solution rapidly past the cells .", "Perfusion produced a transient rise in the cytosolic Ca2+ signal , likely a result of flow shear stress ( Figure 7D and Figure 7—figure supplement 1B ) .", "The Piezo family of mechanosensitive channels plays a vital role in cell motility and development ( Nourse and Pathak , 2017 ) , but it is not known whether these channels are expressed and play a role in immune cell function .", "Perfusion of medium including Yoda1 , a selective small molecule activator of Piezo1 ( Syeda et al . , 2015 ) , resulted in robust Ca2+ signals in activated and naive T cells that were larger and more sustained than those activated by perfusion of solvent or media alone ( Figure 7D and Figure 7—figure supplement 1C ) .", "In summary , we show Ca2+ signals in T cells in response to an activator of Piezo1 channels .", "These results illustrate the utility of Salsa6f for screening agents that modulate Ca2+ signaling in T cells and open the possibility for further exploration of the functional role of Piezo1 channels in T cell function .", "We also monitored Ca2+ signaling in response to TCR activation by αCd3/28 in various subsets of T cells from Cd4-Salsa6f ( Het ) mice , including naive T cells , Th17 cells and iTregs ( Figure 8A–C ) .", "All subtypes of T cells responded to plate-bound stimulation of αCd3/28 with oscillatory changes in their cytosolic Ca2+ levels , very similar to the Ca2+ responses in 2-day-activated T cells illustrated in Figure 7B .", "The responses were heterogeneous , with some cells showing multiple peaks of varying durations and amplitudes , occasional sustained signals and other cells failing to respond .", "Whereas the overall average responses were not very different between the three subtypes examined , some individual Th17 cells and iTregs showed higher amplitude signals than any naive T cells , but with a greater percentage of non-responding cells .", "The Cd4-Salsa6f mouse thus opens up new avenues to study the fundamental nature of Ca2+ signals in T cell subsets generated in response to variety of stimuli , and to explore the relationship between patterns of Ca2+ signals and specific downstream functions .", "Using two-photon microscopy to image lymph nodes from Cd4-Salsa6f ( Hom ) mice under steady-state conditions , we observed sporadic localized elevations of green fluorescence indicative of intracellular Ca2+ signaling ( Figure 9A , B ) .", "Imaging conditions were optimized for single-wavelength excitation of both TdTomato and GCaMP6f components of Salsa6f ( Figure 9—figure supplement 1 ) .", "Some of the green Salsa6f signals were T cell-sized , and the pattern of red Salsa6f fluorescence observed is consistent with the exclusion of the Salsa6f indicator from the nucleus ( Figure 9C ) .", "In addition , we observed numerous bright , transient green fluorescent signals which were much smaller in area ( about 2 µm2 in area ) ( Figure 9B , D; Video 6 ) .", "We term these fluorescent transients ‘sparkles’ , because during rapid playback of time-lapse image streams cells appear to sparkle .", "Because T cells move rapidly and are not uniformly distributed in lymph nodes , we developed an image processing approach in order to minimize fluctuations in background fluorescence and in order to sensitively identify cell-wide Ca2+ signals and sparkles ( Figure 9—figure supplement 2 ) .", "Based on the one-to-one correspondence of tdTomato and GCaMP6f , we estimated and subtracted out fluctuations in green fluorescence due to cell movement and distribution .", "After processing , sparkles were found to occur widely across the imaging field ( Figure 9E–G ) , and many reached a characteristic maximum intensity ( compare Figure 9F and G ) .", "The brightness of sparkles and the uniformity of background fluorescence allowed us to use a stringent threshold of signals exceeding 5 . 4 SD of the background noise level to systematically identify bright sparkles .", "Hundreds of events with a mean amplitude of 6 . 5 SD above background were observed in each 25 min imaging session ( one image stack every five seconds ) , whereas fewer than one event exceeding our threshold was expected to occur from random noise fluctuations .", "Sparkles were more frequent than cell-wide transients ( Figure 9H ) , extended over a median area of 1 . 9 µm2 ( n = 441 sparkles from three lymph nodes; Figure 9I ) , and usually lasted for only one or two consecutive frames ( Figure 9J ) .", "Sparkle trace shape differs from that expected for autofluorescent cell processes drifting into the imaging field ( Figure 9K ) .", "Taken together , these observations suggest that sparkles correspond to local Ca2+ signals restricted to small subcellular domains of T cells migrating through the lymph node .", "The high density of expressing fluorescent T cells in the lymph nodes of Cd4-Salsa6f ( Hom ) mice makes it difficult to visualize and study subcellular Ca2+ signals within individual cells .", "We thus adoptively transferred Cd4-Salsa6f T cells into wild-type recipients so these cells could be viewed in isolation at low density .", "Small green fluorescence transients seen in lymph nodes after adoptive transfer were similar in intensity to transients seen in Cd4-Salsa6f ( Hom ) lymph nodes ( Figure 10A ) .", "When small , bright , and brief green fluorescence signals were traced back , they were found to originate in red fluorescent Cd4-Salsa6f ( Hom ) T cells ( Figure 10B–M ) .", "Cell movement was used to define the front and back of labeled T cells for mapping the subcellular location of Ca2+ signals .", "Some sparkles , identified in processed green channel images , were found to selectively localize to the front or back of motile T cells in which both the cell front and back were clearly seen ( Figure 10D , E , J , K ) .", "Green-red channel ratiometric images , enabled by Salsa6f labeling , confirmed differences in Ca2+ concentration between front and back , although regions of elevated Ca2+ were also observed flanking the nucleus ( Figure 10F , L ) .", "Differences in green-red channel ratiometric pixel intensities between front and back were highly significant ( Figure 10G , M; p<0 . 0001 , Mann–Whitney test ) .", "Thus , motile T cells exhibit Ca2+ signals that are largely restricted to subregions of the cytoplasm , and these Ca2+ signals – sparkles – were identified with high signal-to-noise ratio by Salsa6f expression .", "In a companion paper ( Dong et al . , 2017 ) , we use Salsa6f transgenic mice to consider the relationship between Ca2+ signals , both cell-wide and local , and T-cell motility in the lymph node ." ], [ "We introduce Salsa6f , a novel , ratiometric genetically-encoded Ca2+ probe .", "Salsa6f is a fusion of the high-performing green fluorescent GECI GCaMP6f and the bright red fluorescent tdTomato .", "This simple modification imparts powerful capabilities , which facilitate tracking cells in the absence of Ca2+ signaling , enable ratiometric imaging to eliminate motility artifacts , and permit convenient single-wavelength femtosecond excitation for two-photon microscopy .", "Salsa6f addresses a key weakness of single fluorescent protein-based GECIs by enabling tracking of motile cells and identification of cell morphology , even at basal Ca2+ levels when GCaMP6f fluorescence is very weak .", "We generated a transgenic reporter mouse with Cre-dependent expression of Salsa6f , enabling Ca2+ signals to be imaged in specific , genetically defined cell types .", "Transgenic expression of Salsa6f brings the power of ratiometric chemical Ca2+ indicators to imaging cellular Ca2+ signals amid the complex tissue environments found in vivo .", "Salsa6f preserves the exceptional performance of GCaMP6f , which in the presence of high levels of Ca2+ is as bright as the standard high-performing green fluorescent protein , EGFP ( Chen et al . , 2013 ) .", "We find that Salsa6f possesses a dynamic range similar to GCaMP6f , and both are superior to FRET-based GECIs ( Heim et al . , 2007; Thestrup et al . , 2014 ) .", "The Ca2+ affinity of Salsa6f , 160–300 nM , is well suited to detecting a variety of cellular Ca2+ signals .", "Inclusion of tdTomato in Salsa6f enables ratiometric imaging , calibration , and measurement of Ca2+ concentrations within cells .", "Moreover , Salsa6f is distributed uniformly throughout the cytosol; its exclusion from the nucleus provides reliable and selective reporting of cytosolic Ca2+ signaling .", "This is in contrast to the recently developed PC::G5-tdT mouse strain in which the tdTomato is found throughout the cell but the separately expressed GCaMP5G is excluded from the nucleus ( Gee et al . , 2014 ) .", "We created a transgenic mouse strain in which Salsa6f is expressed under genetic control using the Rosa26Cre recombinase system , and we used this system to label immune cells that express Cd4 .", "Salsa6f labeling enables readout of cytosolic Ca2+ dynamics in T cells in vitro with high dynamic range , without the handling and potential toxicity associated with loading of chemical Ca2+ indicators .", "Indeed , a concern with any Ca2+ indicator is whether it may perturb cell function by buffering free Ca2+ ions or other means .", "In Cd4+ immune cells , we found no effects of Salsa6f expression , even in Cd4-Salsa6f ( Hom ) mice , with respect to cellular phenotype , cell proliferation , differentiation , and , in our companion paper ( Dong et al . , 2017 ) , homing and T-cell motility .", "Salsa6f was used to detect Ca2+ influx due to direct activation of SOCE , TCR stimulation , and an activator of Piezo1 channels – the latter observed in T cells for the first time to our knowledge .", "We also detected differences in patterns of Ca2+ signaling between naive T cells , Th17 cells , and iTregs .", "These experiments demonstrate the sensitivity , brightness , uniformity of labeling , and ease of detecting dynamic Ca2+ signals using Salsa6f .", "A primary advance of this work is to take the in vitro capabilities of an excellent Ca2+ indicator and bring these into the realm of in vivo imaging .", "Within tissues , nearby cells exhibit differences , ranging from subtle to dramatic , in morphology , connectivity , and molecular profile .", "The red fluorescence of Salsa6f , combined with genetic Salsa6f labeling , associates these characteristics with readout of cellular Ca2+ signaling .", "Both the tdTomato and GCaMP6f in Salsa6f are excited well by a 920 nm femtosecond pulsed wavelength for two-photon imaging , enabling visualization hundreds of micrometers deep into lymph nodes .", "The immune system poses additional challenges for imaging because the constituent cells are highly motile .", "Indeed , direct cell-to-cell interactions of motile immune cells form the basis of immune surveillance ( Mrass et al . , 2010; Krummel et al . , 2016 ) .", "We were able to readily identify red fluorescent Salsa6f T cells easily in intact lymph nodes following adoptive transfer .", "Our images reveal uniform red fluorescence labeling by Salsa6f with clear subcellular morphology in imaging sessions encompassing hundreds of time lapse images .", "Moreover , Salsa6f offers the opportunity not only to record fluctuations in relative Ca2+ levels over time , but also to read out absolute Ca2+ concentrations within cells .", "We have measured the affinity of Salsa6f in intact cells; in principle , use of this approach will allow other microscope systems to be calibrated for measuring absolute Ca2+ concentrations with Salsa6f .", "Knowledge of absolute Ca2+ concentrations is necessary to develop quantitative models of Ca2+ signaling and cell behavior .", "Indeed , we demonstrate that clear Salsa6f ratio images can be generated from motile T cells in intact lymph nodes .", "Our Salsa6f transgenic mouse line enables more sophisticated experimental approaches .", "One is the ability to detect rare Ca2+ signaling events .", "The high brightness and dynamic range of modern GECIs like Salsa6f contribute to detection of rare Ca2+ signaling events inside intact tissues or even whole transgenic animals ( Kubo et al . , 2014; Portugues et al . , 2014 ) .", "Ca2+ signals have been previously recorded within lymph nodes in adoptively transferred T cells transgenically expressing doxycycline-inducible mCameleon ( Le Borgne et al . , 2016 ) , GCaMP3 and dsRed as separate proteins ( Shulman et al . , 2014 ) , and virally transduced YC-nano-50CD ( Liu et al . , 2015 ) .", "Potential disadvantages of these probes include low dynamic range , nuclear expression of mCameleon and dsRed , the high Ca2+ affinity of YC-nano-50CD ( 50 nM ) , and lack of ratiometric measurement .", "Imaging T cells transgenically expressing Salsa6f helps to overcome many of these limitations .", "Detecting rare events is made harder by inhomogeneities in cell populations of the lymph node as well as the movement of immune cells therein .", "Because of the one-to-one correspondence of tdTomato and GCaMP6f in Salsa6f , we were able to estimate and subtract resting GCaMP6f fluorescence even in motile cells .", "This approach substantially improves the uniformity of the fluorescence background upon which rare Ca2+ signaling events can be detected .", "Reliable and uniform cytosolic labeling contributes as well .", "Combined , these factors enabled us to detect not only sporadic cell-wide Ca2+ elevations , but also sparkles , much smaller sporadic local Ca2+ signals .", "The sensitivity and resolution of these images are sufficient to map local signals from intact lymph nodes to sub-regions of T cells .", "Moreover , while we focused upon the brightest local Ca2+ signals to demonstrate their existence , we expect that Salsa6f will enable even lower intensity Ca2+ signals to be linked to subcellular mechanisms and , ultimately , resulting cell behaviors .", "In a companion paper ( Dong et al . , 2017 ) , we capitalize on the unique properties of Salsa6F to relate Ca2+ signals , both global and local , to T-cell motility in the intact lymph node ." ], [ "Plasmids encoding GECIs ( GECO and GCaMP6 ) were obtained from Addgene for screening in live cells .", "HEK 293A cells ( Invitrogen- Life Technologies # R705-07 ) were screened for viruses and mycoplasma , split and frozen into working stocks , cultured using aseptic techniques , and used to evaluate candidate genetically encoded Ca2+ indicators .", "Each probe was cotransfected with Orai1 and STIM1 into HEK 293A cells using Lipofectamine 2000 ( Invitrogen , Carlsbad , CA ) for 48 hr before screening on an epifluorescence microscope .", "For construction of Salsa6f , a plasmid for tdTomato ( Addgene , Cambridge , MA ) and the pEGP-N1 vector ( Clontech , Mountain View , CA ) was used as a backbone .", "GCaMP6f was amplified via PCR with N- and C-terminal primers ( 5’ CACAACCGGTCGCCACCATGGTCGACTCATCACGTC 3’ and 5’ AGTCGCGGCCGCTTTAAAGCTTCGCTGTCATCATTTGTAC 3’ ) and ligated into pEGFP-N1 at the AgeI/NotI sites to replace the eGFP gene , while tdTomato was amplified via PCR with N- and C-terminal primers ( 5’ ATCCGCTAGCGCTACCGGTCGCC 3’ and 5’ TAACGAGATCTGCTTGTACAGCTCGTCCATGCC 3’ ) and ligated into the backbone at the NheI/BglII sites .", "An oligo containing the V5 epitope tag was synthesized with sense and antisense strands ( 5’ GATCTCGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACG 3’ and 5’ GATCCGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCCGA 3’ ) and ligated into the backbone at the BglII/BamHI sites , linking tdTomato to GCaMP6f and creating Salsa6f .", "The amplified regions of the construct were verified by sequencing ( Eton Bioscience Inc . , San Diego , CA ) .", "This plasmid , driven by the CMV promoter , was used for transient transfections in HEK 293A cells with Lipofectamine 2000 and in primary human T cells with Amaxa Nucleofection .", "The transgenic cassette in Figure 2B was generated by inserting Salsa6f , from the plasmid described above , into the Ai38 vector ( Addgene Plasmid #34883 ) and replacing GCaMP3 .", "The final targeting vector included the CAG ( cytomegalovirus early enhancer/chicken β-actin ) promoter , an LSL sequence with LoxP-STOP-LoxP , the Salsa6f probe ( tdTomato-V5-GCaMP6f ) , the woodchuck hepatitis virus posttranscriptional regulatory element ( WPRE ) , and a neomycin resistance gene ( NeoR ) , all flanked by 5’ and 3’ Rosa26 homology arms of 1 . 1 and 4 . 3 kb .", "The targeting vector was linearized with PvuI and electroporated into JM8 . N4 mouse embryonic stem ( ES ) cells of C57BL/6N background .", "Following selection with G418 , clones carrying the Gt ( ROSA ) 26SorpCAG-FRT-LSL-Salsa6f-WPRE-bGHpA-AttB-FRT-NeoR-AttP allele were screened by Southern blotting after digestion with HindIII for the 5’ end or BglI for the 3’ end .", "Four correctly targeted clones were expanded and checked by chromosome counting , then two clones with >90% euploidy were further expanded and injected into C57BL/6J blastocysts for implantation into pseudopregnant foster mothers .", "Presence of the Salsa6f transgenic cassette was detected in the resulting chimeric pups by PCR screening for the Nnt gene , as the initial JM8 . N4 ES cells are Nnt+/+ while the C57BL/6J blastocysts are Nnt-/- .", "Finally , positive chimeras were bred to R26ΦC31o mice ( JAX #007743 ) to remove the neomycin resistance gene flanked by AttB and AttP sites in the original transgenic cassette , and to produce F1 founders carrying the allele Gt ( ROSA ) 26SorpCAG-FRT-LSL-Salsa6f-WPRE-bGHpA-AttB/P at the Rosa26 locus .", "These F1 founders were then bred to homozygosity to generate LSL-Salsa6f ( Hom ) mice , and subsequently crossed to homozygotic Cd4Cre mice ( JAX #017336 ) to generate Cd4-Salsa6f ( Het ) mice expressing Salsa6f only in T cells .", "Cd4-Salsa6f mice were further bred to generate homozygotic Cd4-Salsa6f ( Hom ) mice for increased Salsa6f expression and fluorescence .", "For T-cell proliferation: Cd4 T cells were isolated from spleen and lymph nodes of 6–10 week old mice using negative selection ( StemCell Technologies , Cambridge , MA ) .", "CellTrace Violet ( CTV ) -labeled T cells were co-cultured with αCd3/Cd28 coated dynabeads ( Life Technologies Corp . , Grand Island , NY ) at 1:1 ratio according to the manufacturer’s protocol in a U bottom 96 well plate .", "For T cell differentiation: Naive Cd4 T cells were differentiated on activating polystyrene surface ( Corning Inc . , Corning , NY ) with plate-bound αCd3 ( 2 . 5 µg/ml ) and αCd28 ( 2 . 5 µg/ml ) in the presence of cytokines for 6 days ( Yosef et al . , 2013 ) .", "For Th1 differentiation: 25 ng/mL rmIL-12 ( BioLegend , San Diego , CA ) , 10 µg/mL αmouse IL4 ( Biolegend ) .", "For Th17 differentiation: 2 . 5 ng/mL rhTGF-β1 ( Tonbo Biosciences , San Diego , CA ) , 50 ng/mL rmIL-6 ( Tonbo Biosciences ) , 25 ng/ml rmIL-23 ( BioLegend ) , and 25 ng/ml rmIL-β1 ( BioLegend ) .", "For iTreg differentiation: 10 ng/mL rhTGF-β1 , 100 units/mL of rmIL-2 ( BioLegend ) , 5 µM Retinoic Acid ( Sigma , St . Louis , MO ) .", "CTV dilution assay was performed in live cells ( Fixable Viability Dye eFluor 780 negative gating; Thermofisher Scientific Inc . , Grand Island , NY ) .", "To detect intracellular cytokines , 6 day differentiated cells were stimulated in with 25 ng of phorbol 12-myristate 13-acetate ( PMA ) , 1 µg ionomycin ( Sigma ) , and monensin ( Golgistop BD biosciences ) for 4 hr at 37°C .", "Dead cells were labeled with Ghost dye 780 ( BioLegend ) , then washed , fixed , permeabilized using FoxP3 staining buffer set ( Thermofisher Inc ) .", "The following antibodies were used to detect intracellular cytokines: IL-17A-APC ( clone TC11-18H10 . 1 , BioLegend ) ; IFNγ-Pacific Blue ( clone XMG1 . 2 , BioLegend ) ; Foxp3-PE ( clone FJK16s , Thermofisher Scientific Inc . ) ; in permeabilization buffer ( eBioscience ) .", "Data were acquired using NovoCyte flow cytometer ( ACEA Biosciences ) and analyzed using FlowJo .", "Cd4 T cells were activated by plating on six-well plates coated overnight with 2 . 5 μg/mL αCd3/αCd28 ( Invivogen , San Diego , CA ) at 4°C .", "Cells were cultured in RPMI medium ( Lonza ) containing 10% FCS , L-glutamine , Non-essential amino acids , Sodium pyruvate , β-mercaptoethanol and 50 U/mL of IL-2 at 37°C in 5% CO2 incubator .", "Following 2 days of culture , cells were plated on either poly-L-lysine or 1 μg/mL α-Cd3/α28 coated 35-mm glass chambers ( Lab-Tek , Thermofisher Inc . ) for imaging .", "RPMI medium with 2% FCS and L-glutamine containing 2 mM Ca2+ was used for imaging experiments .", "For experiments involving calibration and characterization of the Salsa6f probe in Cd4-Salsa6f cells , Ringer solution containing various concentrations of Ca2+ was used .", "For Ca2+ imaging of different T-cell subsets , Th17 cells and iTregs were differentiated as described above .", "For Ca2+ imaging of Cd4+ T cells from Cd4-Salsa6f mice , we used an Olympus Fluoview FV3000RS confocal laser scanning microscope , equipped with high-speed resonance scanner and the IX3-ZDC2 Z-drift compensator ( Olympus Corp . , Waltham , MA ) .", "Diode lasers ( 488 and 561 nm ) were used for excitation , and two high-sensitivity cooled GaAsP PMTs were used for detection .", "Cells were imaged using the Olympus 40x silicone oil objective ( NA 1 . 25 ) , by taking five slice z-stacks at 2 µm/step , at 5 s intervals , for up to 20 min .", "Temperature , humidity , and CO2 were maintained using a Tokai-Hit WSKM-F1 stagetop incubator .", "Data were processed and analyzed using Imaris and ImageJ software .", "Calcium imaging experiments were done at 37°C on 2-day-activated Cd4+ T cells from Cd4-Salsa6f ( Het ) mice , unless otherwise indicated .", "Salsa6f calibration experiments were done at room temperature .", "Lymph nodes images were acquired using a custom-built two photon microscope based on Olympus BX51 upright frame , Motorized ZDeck stage ( Prior , Rockland , MA ) , with excitation generated by a tunable Chameleon femtosecond laser ( Coherent , Santa Clara , CA ) ( Miller et al . , 2002 ) .", "The following wavelengths were used to excite single or combination of fluorophores: 920 nm to excite tdTomato and GCaMP6f; 1040 nm to excite tdTomato alone .", "495 nm and 538 nm dichroic filters were arranged in series to separate blue , green and red signals .", "Two-photon excitation maxima of tdTomato and GCaMP6f are 1040 and 920 nm , respectively ( Drobizhev et al . , 2011; Chen et al . , 2013 ) .", "Using 1040 nm excitation , tdTomato signals were readily detected up to 300 µm depth; however , 1040 is not ideal to image Salsa6f because:", "1 ) Collagen fibers generate second harmonic at 520 nm when excited with 1040 nm , which interferes with simultaneous detection of GCaMP6f ( emission maxima , 509 nm ) and", "2 ) 1040 nm does not excite GCaMP6f ( Figure 9—figure supplement 1A , top row ) .", "Alternatively , 920 nm optimally excites GCaMP6f , and excites tdTomato sufficiently , and Salsa6f signals were detected up to 300 µm depth , while second harmonic collagen signals ( 460 nm ) can be easily separated into blue channel ( Figure 9—figure supplement 1A , bottom row ) .", "Additionally , autofluorescent structures ( LN resident DCs and fibroblastic reticular cells ) show up as yellow bodies when excited with 920 nm , which serve as a guide to locate the T cell zone ( Figure 9—figure supplement 1B ) .", "Therefore , 920 nm is the ideal two-photon excitation wavelength for simultaneous imaging of tdTomato and GCaMP6f as component parts of Salsa6f .", "Lymph nodes were oriented with the hilum away from the water dipping microscope objective ( Nikon 25x , NA 1 . 05 ) .", "The node was maintained at 36–37°C by perfusion with medium ( RPMI ) bubbled with medical grade carbogen ( 95% O2 and 5% CO2 ) using a peristaltic heated perfusion system ( Warner Instruments ) , with thermocouple-based temperature sensors placed next to the tissue in a custom built chamber .", "3D image stacks of x = 250 μm , y = 250 μm , and z = 20 or 52 μm ( 4 μm step size ) were sequentially acquired at 5 or 11 s intervals , respectively , using image acquisition software Slidebook ( Intelligent Imaging Innovations ) as described previously ( Matheu et al . , 2015 ) .", "This volume collection was repeated for up to 40 min to create a 4D data set .", "Graphpad Prism was used for statistical analysis and generating figures .", "p values are indicated in figures: ns p>0 . 05 , *p<0 . 05; **p<0 . 01; ***p<0 . 001; and ****p<0 . 0001 .", "Stacks of six optical sections 4 µm apart from the T-zone of Cd4-Salsa6f ( Hom ) lymph nodes were acquired once every 5 s at a resolution of 0 . 488 or 0 . 684 µm per pixel .", "Maximum intensity projections of 1 pixel radius median-filtered images were used for subsequent processing and analysis .", "Autofluorescent cells were identified by averaging the red or green time lapse image stacks and automated local thresholding ( Bernsen five pixel radius ) using the public domain image processing program ImageJ .", "Autofluorescent cell masks were dilated by four pixels , regions exhibiting less contrast and detail due to light scattering manually masked to produce the final time lapse image mask .", "Red ( tdTomato ) channel fluorescence from Salsa6f corresponding to green ( GCaMP6f ) channel resting state fluorescence was determined to be fivefold higher using our standard two-photon microscope acquisition settings .", "Final green images were produced by subtracting a 0 . 2x scaled red channel image , and subsequently subtracting out the average of all green channel time lapse images .", "The standard deviation ( SD ) of each masked green channel time lapse image stack was used to determine thresholds for local ( sparkle ) and cell-wide Ca2+ events .", "Thresholds for detection of local and cell-wide Ca2+ events were 5 . 4 and 2 . 1 SD and 1 . 4 µm2 and 25 µm2 , respectively .", "Local Ca2+ events were excluded from the set of sparkles if they coincided with a cell-wide event .", "Frequency of background events was calculated using a standard normal distribution with a Z-score corresponding to the average intensity of local events ( 6 . 5 SD ) , which was 1 in 2 × 1010 pixels ( WolframAlpha ) .", "The front and back of motile T cells were traced manually from red channel TdTomato images .", "Individual pixel intensities from the front and back regions of interest were plotted and compared using the non-parametric Mann–Whitney test in Graphpad Prism ." ] ]
[ "Calcium is an essential cellular messenger that regulates numerous functions in living organisms .", "Here , we describe development and characterization of ‘Salsa6f’ , a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+ , and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types .", "The development and function of T cells was unaffected in Cd4-Salsa6f mice .", "We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naive T cells , helper Th17 T cells and regulatory T cells , and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels .", "Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo .", "Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients ( ‘sparkles’ ) as cells migrate ." ]
[ "To help protect the body from disease , small immune cells called T lymphocytes move rapidly , searching for signs of infection .", "These signs are antigens – processed pieces of proteins from invading bacteria and viruses – which are displayed on the surface of so-called antigen-presenting cells .", "To visit as many different antigen-presenting cells as possible , T cells move quickly from one to the next in an apparently random manner .", "How T cells are programmed to move in this way is largely unknown .", "The entry of calcium ions into cells , through channel proteins , triggers characteristic actions in many cells throughout the body .", "As such it is possible that the T cells’ movements are related to calcium signals too .", "However , it was technically challenging to directly measure the amount of calcium in moving cells within the body .", "To overcome this issue , Dong , Othy et al . genetically engineered mice to produce a new calcium-sensitive reporter protein in their T cells .", "The reporter , which was named Salsa6f , consisted of a red fluorescent protein fused to another protein that glows green when it binds to calcium ions .", "Measuring the ratio of red and green fluorescence gives a measure of the concentration of calcium ions inside the cell .", "In the absence of calcium signaling , the cells can still be tracked via the red fluorescence of Salsa6f .", "Importantly , the reporter did not affect the development or activity of the T cells in the mice .", "In a related study , Dong , Othy et al . then used their transgenic mice to ask whether calcium signals guide moving T cells as they search for antigens .", "Future studies could use these transgenic mice to track the calcium ion concentration in numerous cell types .", "This would enable new approaches to relate the inner workings of cells to their behaviors in many different organ systems throughout the body ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "ecology", "plant biology" ]
Natural genetic variation in Arabidopsis thaliana defense metabolism genes modulates field fitness
elife-05604-v1
[ [ "High levels of standing variation have often been observed among many natural plant and animal populations .", "This is particularly true for the model species Arabidopsis thaliana , which exhibits variation both within and among natural populations and/or accessions ( Pigliucci and Marlow , 2001; Atwell et al . , 2010; Bomblies et al . , 2010; Chan et al . , 2010; Platt et al . , 2010; Cao et al . , 2011; Debieu et al . , 2013; Joseph et al . , 2013; Long et al . , 2013; Anwer et al . , 2014; Li et al . , 2014 ) .", "Models based on mutation-selection balance theory predict that this observed variation will be due to rare alleles at many loci introduced through random mutations that evolution acts on to eliminate through persistent purifying natural selection ( Kimura , 1968; Turelli , 1984 ) .", "In agreement , studies of nucleotide variation in Arabidopsis have found an excess of low frequency polymorphisms relative to expectation ( Purugganan & Suddith , 1998 , 1999 ) .", "However , other studies cloning causal genetic variants from natural Arabidopsis accessions have found several intriguing examples of intermediate frequency alleles maintained at polymorphic loci ( Johanson et al . , 2000; Long et al . , 2000; Li et al . , 2014 ) .", "This variation among loci has led to a long-standing interest in elucidating to what extent this genetic variation is neutral in origin or , alternatively , maintained through selective forces ( Levene , 1953; Hedrick et al . , 1976; Bull , 1987; Stahl et al . , 1999; Prasad et al . , 2012 ) .", "The neutral theory posits that the majority of genetic polymorphisms have no effect on fitness and that stochastic evolutionary processes , such as genetic drift and migration , are sufficient to explain the genetic and phenotypic variation observed within and among populations ( Darwin , 1859; Kimura , 1968; Duret , 2008 ) .", "This hypothesis has generated numerous modeling studies demonstrating that the standing level of genetic variation in traits can be explained by the demographic history of a species not linked to fitness of an individual ( Wolf et al . , 2000; Barton and Turelli , 2004; Hufford et al . , 2012; Pyhajarvi et al . , 2013 ) .", "However , for many ecologically important traits , phenotypic variation has been shown to empirically impact fitness in natural populations , suggesting that natural selection also plays an important role in the evolution of such traits ( Mothershead and Marquis , 2000; Adler et al . , 2001; Tian et al . , 2003; Korves et al . , 2007; Milla et al . , 2009 ) .", "A key step necessary to begin to resolve these discrepancies between theory and empirical observations requires the validation of fitness consequences of variation at specific loci or pathways in the field ( Turelli and Barton , 2004; Fournier-Level et al . , 2011; Hancock et al . , 2011 ) .", "Determining the impact of polygenic variation upon fitness in the field informs our understanding of the potential selective and non-selective evolutionary processes that protect or maintain phenotypic variation within a species , such as genetic drift and balancing selection ( Kimura , 1968; Hedrick et al . , 1976; Mitchell-Olds et al . , 2007; Mojica et al . , 2012 ) .", "However , most population level studies of evolution and selection in the field have focused on polygenic populations and have been unable to validate the link between variation at specific underlying genes and the resulting fitness consequences of this variation ( Lande and Arnold , 1983; Mitchell-Olds and Rutledge , 1986; Gillespie and Turelli , 1989; Orr , 1998 ) .", "Studies using structured mapping populations , such as Arabidopsis RILs , can only associate large genomic regions , rather than individual genes , with quantitative variation in fitness ( Weinig et al . , 2003; Stinchcombe et al . , 2004; Juenger et al . , 2005; Malmberg et al . , 2005 ) .", "More recently , genome wide studies using A . thaliana accessions have been able to associate SNPs to fitness in the field and even predict relative fitness of accessions grown in a common garden ( Fournier-Level et al . , 2011; Hancock et al . , 2011 ) .", "However , these associations between loci and fitness need more refining to validate the effect of individual genes .", "Testing if individual genes impact fitness in the field first requires identifying and cloning the causal genes underlying the phenotypic variation of interest ( Mitchell-Olds , 1995; Tian et al . , 2003; Mitchell-Olds et al . , 2007 ) .", "Then , these natural alleles need to be recreated as single gene lines , which can require approaches such as chemical mutation ( e . g . , EMS ) , generation of transgenic individuals via Agrobacterium-mediated transformation , and/or generation of isogenic lines through successive rounds of backcrossing .", "Therefore , empirical field testing of individual causative polymorphic genes has only been done rarely , and we do not yet have a good understanding of the extent to which individual genes impact fitness in the field ( Tian et al . , 2003; Schuman et al . , 2012 ) .", "A . thaliana has become a key model system and is extremely suitable for characterizing , cloning and validating genes influencing the fitness consequences of underlying natural variation .", "This is due , in part , to the ease of transformation as well as the abundance of genomic resources available for this organism , including an extensive library of T-DNA insertion lines and natural accessions ( Alonso et al . , 2003 ) .", "Arabidopsis persists in many different environments and experiences selection from both abiotic pressures , such as temperature and precipitation , and biotic pressures , such as insect and pathogen populations that vary temporally and spatially ( Meyerowitz , 1987; Richards et al . , 2009 ) .", "Potentially to maximize fitness across a broad range of biotia , Arabidopsis has evolved high levels of natural variation among accessions for many important phenotypic traits , including the defense compounds , glucosinolates ( GSLs ) ( Stahl et al . , 1999; Atwell et al . , 2010; Chan et al . , 2010 ) .", "GSLs constitute a diverse set of plant-made defensive metabolites restricted primarily to the Brassicales that are partitioned into three classes , indolic , aliphatic and aromatic , depending on their amino acid precursor .", "These N and S containing compounds are stored in the vacuoles of plant cells until they are activated through tissue damage , which can occur through insect feeding and pathogen attack .", "Natural genetic polymorphisms found among a suite of aliphatic GSL genes in Arabidopsis are responsible for the majority of GSL diversity observed in the leaf tissue ( Figure 1 ) .", "These aliphatic GSL genes encode enzymes , transcription factors and activation co-factors that have been identified , cloned and validated in a laboratory setting ( Table", "1 ) ( Haughn et al . , 1991; Li and Quiros , 2003; Hansen et al . , 2007 , 2008; Hirai et al . , 2007; Li et al . , 2008; Neal et al . , 2010 ) .", "Previous studies have uncovered links between GSL variation and ecologically important traits in Arabidopsis , such as resistance to insect/pathogen damage , flowering time , and growth , suggesting that GSLs play an important role in determining plant fitness ( Mauricio , 1998; Kliebenstein et al . , 2002; Bidart-Bouzat and Kliebenstein , 2008; Hansen et al . , 2008; Burow et al . , 2010; Kerwin et al . , 2011; Züst et al . , 2011 ) .", "Since the genes responsible for the majority of natural polymorphism in aliphatic GSL have been well characterized in a laboratory setting , the GSL pathway in Arabidopsis provides a good system for understanding the impact that individual genes might have on fitness in the field ( Kliebenstein et al . , 2001b; Halkier and Gershenzon , 2006; Hansen et al . , 2008 ) .", "In this study , we tested the fitness consequences of aliphatic GSL variation in the field by utilizing a collection of lines that vary at specific GSL genes in Arabidopsis ( Col-0 ) , which recreated observed natural variation in the aliphatic GSL pathway found among accessions ( Table", "2 ) ( Mauricio , 1998; Kliebenstein et al . , 2002; Bidart-Bouzat and Kliebenstein , 2008; Hansen et al . , 2008; Burow et al . , 2010; Kerwin et al . , 2011; Züst et al . , 2011 ) . 10 . 7554/eLife . 05604 . 003Figure 1 . Overview of aliphatic GSL biosynthesis and activation in Arabidopsis thaliana . Arrows represent the steps involved in aliphatic glucosinolate ( GSL ) biosynthesis that have been validated through laboratory experiments and are naturally variable within A . thaliana .", "Gene names are listed next to or above the arrows .", "( A ) Regulation of aliphatic GSL biosynthesis .", "The transcription factors MYB28 and MYB29 control accumulation of aliphatic GLS .", "A double knockout in these genes results in no aliphatic GLS accumulation , while a single knockout in these genes leads to a 50% reduction in aliphatic GSL ( myb28 ) or a 25% reduction in aliphatic GSL ( myb29 ) , compared to WT Col-0 .", "The biosynthetic enzymes MAM1 and AOP2 also influence aliphatic GLS accumulation and a non-functional allele at either locus leads to decreased GSL accumulation .", "( B ) Amino acid chain elongation .", "During chain elongation , carbons are added to a methionine precursor through a series of reactions producing an elongated amino acid .", "Variation at the Elong locus controls the number of carbons added to the amino acid precursor and therefore , the length of the GSL side chain , R . A functional allele at this locus , MAM1 , leads primarily to accumulation of GSL with four carbon ( 4C ) length side chains , whereas a non-functional allele , gsm1 leads to accumulation of GSL with three carbon ( 3C ) length side chains .", "The elongated amino acid is subsequently converted into a GLS via the core pathway ( not shown ) .", "( C ) Side chain modification .", "The GSL compounds produced can then undergo a series of side chain modifications that lead to the suite of diverse GSL compounds found in Arabidopsis .", "Side chain modification is controlled by variation of GSOX1 , GSOX3 , AOP2 , AOP3 and GSOH .", "GSOX1 & GSOX3 oxygenate a methylthio ( MT ) to methylsulfinyl ( MSO ) GSL .", "AOP2 converts MSO to alkenyl , such as allyl and but-3-enyl .", "AOP3 , on the other hand converts only 3C length MSO to OH-propyl GSL and cannot act on the 4MSO GSL .", "GSOH oxygenates the 4C but-3-enyl to the OH-alkenyl GSL , OH-but-3-enyl .", "Since GSOH acts on but-3-enyl GSL , which is a product of AOP2 , a functional AOP2 is necessary for GSOH to function and AOP2 is said to be epistatic to GSOH .", "Col-0 is functional for MAM1 and the GSOX's , null for both AOP2 and AOP3 , and functional for GSOH , resulting in accumulation of primarily 4MSO GLS .", "See Figure 1—figure supplements 1–17 for images of GSL traces for each GSL genotypes in our mutant laboratory population .", "( D ) GSL Activation .", "Once produced , GLS are stored in the vacuole in their stable , unreactive form until activation occurs .", "Upon cellular disruption , such as occurs during pathogen attack , insect herbivory or even wind damage , GLS come into contact with their own plant-made activating enzyme , myrosinase .", "After production , myrosinase is stored in vacuoles of idioblastic cells called myrosin bodies .", "Myrosinase activates the GSL compound by cleaving the glucose moiety , yielding an unstable aglycone structure that non-enzymatically rearranges to either nitriles or isothiocyanates , depending on the presence of the co-activators ESM1 and ESP . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00310 . 7554/eLife . 05604 . 004Figure 1—figure supplement 1 . HPLC trace of Arabidopsis thaliana accession Columbia-0 wild-type genotype . Shown is an HPLC trace from a representative individual of field-grown Col-0 that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , 6MSO = 6-Methylsulfinylhexyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00410 . 7554/eLife . 05604 . 005Figure 1—figure supplement 2 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb28 genotype . Shown is an HPLC trace from a representative individual of field-grown myb28 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00510 . 7554/eLife . 05604 . 006Figure 1—figure supplement 3 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb29 genotype . Shown is an HPLC trace from a representative individual of field-grown myb29 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , 6MSO = 6-Methylsulfinylhexyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00610 . 7554/eLife . 05604 . 007Figure 1—figure supplement 4 . HPLC trace of Arabidopsis thaliana accession Columbia-0 gsm1 genotype . Shown is an HPLC trace from a representative individual of field-grown gsm1 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00710 . 7554/eLife . 05604 . 008Figure 1—figure supplement 5 . HPLC trace of Arabidopsis thaliana accession Columbia-0 gsox1 genotype . Shown is an HPLC trace from a representative individual of field-grown gsox1 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , 6MSO = 6-Methylsulfinylhexyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00810 . 7554/eLife . 05604 . 009Figure 1—figure supplement 6 . HPLC trace of Arabidopsis thaliana accession Columbia-0 gsox3 genotype . Shown is an HPLC trace from a representative individual of field-grown gsox3 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , 6MSO = 6-Methylsulfinylhexyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 00910 . 7554/eLife . 05604 . 010Figure 1—figure supplement 7 . HPLC trace of Arabidopsis thaliana accession Columbia-0 AOP2 genotype . Shown is an HPLC trace from a representative individual of field-grown AOP2 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , ( R ) OH-But-3-enyl = ( R ) OH-But-3-enyl GSL , ( S ) OH-But-3-enyl = ( S ) OH-But-3-enyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , But-3-enyl = But-3-enyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01010 . 7554/eLife . 05604 . 011Figure 1—figure supplement 8 . HPLC trace of Arabidopsis thaliana accession Columbia-0 ESP genotype . Shown is an HPLC trace from a representative individual of field-grown ESP genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , 6MSO = 6-Methylsulfinylhexyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01110 . 7554/eLife . 05604 . 012Figure 1—figure supplement 9 . HPLC trace of Arabidopsis thaliana accession Columbia-0 gsoh genotype . Shown is an HPLC trace from a representative individual of field-grown gsoh genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 5MSO = 5-Methylsulfinylpentyl GSL , But-3-enyl = But-3-enyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01210 . 7554/eLife . 05604 . 013Figure 1—figure supplement 10 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb28/myb29 genotype . Shown is an HPLC trace from a representative individual of field-grown myb28/myb29 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01310 . 7554/eLife . 05604 . 014Figure 1—figure supplement 11 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb28/gsm1 genotype . Shown is an HPLC trace from a representative individual of field-grown myb28/gsm1 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01410 . 7554/eLife . 05604 . 015Figure 1—figure supplement 12 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb29/gsm1 genotype . Shown is an HPLC trace from a representative individual of field-grown myb29/gsm1 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01510 . 7554/eLife . 05604 . 016Figure 1—figure supplement 13 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb28/AOP2 genotype . Shown is an HPLC trace from a representative individual of field-grown myb28/AOP2 genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , ( R ) OH-But-3-enyl = ( R ) OH-But-3-enyl GSL , ( S ) OH-But-3-enyl = ( S ) OH-But-3-enyl GSL , But-3-enyl = But-3-enyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01610 . 7554/eLife . 05604 . 017Figure 1—figure supplement 14 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb28/gsoh genotype . Shown is an HPLC trace from a representative individual of field-grown myb28/gsoh genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01710 . 7554/eLife . 05604 . 018Figure 1—figure supplement 15 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb29/AOP2/gsoh genotype . Shown is an HPLC trace from a representative individual of field-grown myb29/AOP2/gsoh genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , Allyl = Allyl ( Propenyl ) GSL , 5MSO = 5-Methylsulfinylpentyl GSL , But-3-enyl = But-3-enyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01810 . 7554/eLife . 05604 . 019Figure 1—figure supplement 16 . HPLC trace of Arabidopsis thaliana accession Columbia-0 AOP2/gsoh genotype . Shown is an HPLC trace from a representative individual of field-grown AOP2/gsoh genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "3MSO = 3-Methylsulfinylpropyl glucosinolate ( GSL ) , 4MSO = 4-Methylsulfinylbutyl GSL , Allyl = Allyl ( Propenyl ) GSL , 5MSO = 5-Methylsulfinylpentyl GSL , But-3-enyl = But-3-enyl GSL , 7MSO = 7-Methylsulfinylheptyl GSL , 8MSO = 8-Methylsulfinyloctyl GSL , I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 01910 . 7554/eLife . 05604 . 020Figure 1—figure supplement 17 . HPLC trace of Arabidopsis thaliana accession Columbia-0 myb28/myb29/gsoh genotype . Shown is an HPLC trace from a representative individual of field-grown myb28/myb29/gsoh genotype that depicts the GSL profile found within the leaves of this genotype .", "The mixture of various GSL structures present within the leaf and the relative amounts of each constitute the GSL profile in an individual .", "I3M = Indol-3-ylmethyl GSL , 4MI3M = 4-Methoxyindol-3-ylmethyl GSL , NMI3M = 1-Methoxyindol-3-ylmethyl GSL . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 02010 . 7554/eLife . 05604 . 021Table 1 . Polymorphic genes involved in aliphatic GSL synthesis and activationDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 021Gene nameLocusATG #Gene typeGene functionMutation type in Col-0MYB28MYB28At5g61420TFPositive regulator of aliphatic GSL ( Sønderby et al . , 2007 , 2010 ) T-DNAMYB29MYB29At5g07690TFPositive regulator of aliphatic GSL ( Sønderby et al . , 2007 , 2010 ) T-DNAMAM1MAM1At5g23010EnzymeControls 3C–4C chain elongation ( Haughn et al . , 1991; de Quiros et al . , 2000; Kroymann et al . , 2003 ) EMSGSOX1GSOXAt1g65860EnzymeConverts MT to MSO GSL ( Hansen et al . , 2007; Li et al . , 2008 ) T-DNAGSOX3GSOXAt1g62560EnzymeConverts MT to MSO GSL ( Hansen et al . , 2007; Li et al . , 2008 ) T-DNAAOP2AOPAt4g03060EnzymeConverts MSO to alkenyl GSL ( Kliebenstein et al . , 2001c ) 35S OXAOP3AOPAt4g03050EnzymeConverts 3MSO to hydroxy-propyl GSL ( Kliebenstein et al . , 2001c ) n/aGSOHGSOHAt2g25450EnzymeConverts butenyl to OHB ( Hansen et al . , 2008 ) T-DNAESPESPAt1g54040Co-factorGuides formation of activated GSL to nitriles ( Lambrix et al . , 2001 ) 35S OXShown are the identities , functions and mutation types of nine genes representing seven loci important for aliphatic GSL synthesis and activation .", "These genes were chosen for mutant laboratory population of Arabidopsis thaliana accession Col-0 due to the fact that they represent the majority of aliphatic GSL variation observed in Arabidopsis .", "Each of these genes is naturally polymorphic among Arabidopsis accessions . 10 . 7554/eLife . 05604 . 022Table 2 . Allelic variation of polymorphic aliphatic GSL loci in structured populationDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 022GenotypeMYB28MYB29MAM1GSOX1GSOX3AOP2GSOHESPCol-0+++++−+−myb28−++++−+−myb29+−+++−+−gsm1++−++−+−gsox1+++−+−+−gsox3++++−−+−AOP2++++++−−AOP2/gsoh++++++−−Gsoh+++++−−−Myb28/gsoh−++++−+−Myb28/gsm1−+−++−+−Myb28/AOP2−++++++−Myb29/gsm1+−−++−+−Myb29/AOP2/gsoh++++++−−Myb28/myb29−−+++−+−Myb28/myb29/gsoh−−+++−−−ESP+++++−++Shown are the genotypes in the mutant laboratory GSL population used in this study and the allelic state of each gene within each of them .", "Each gene in our population is naturally polymorphic among Arabidopsis accessions .", "See Table 1 for gene functions .", "For each gene listed , a ‘+’ indicates a functional allele and a ‘−’ indicates a non-functional allele .", "The loss-of-function and gain-of-function mutant lines shown in Table 1 were manually crossed to generate this population of genotypes , each of which vary from Col-0 at only these eight genes , including single , double , and triple mutants ." ], [ "The GSL profile of a plant is characterized by the presence and relative abundance of the various GSL structures it produces .", "Among Arabidopsis accessions , GSL profiles show extensive phenotypic variation across the species geographic distribution ( Figure 2 ) ( Chan et al . , 2010 ) .", "While previous studies have linked GSL profile variation to insect resistance , as well as correlated the geographic distribution of insect populations with GSL profile-type across Europe , it is still not known to what extent , if at all , individual GSL genes affect fitness in the field ( Mauricio , 1998; Bidart-Bouzat and Kliebenstein , 2008; Züst et al . , 2012 ) .", "To test if standing genetic variation within the aliphatic GSL defensive pathway of A . thaliana impacts fitness in the field , we utilized an existing set of genotypes that recreate natural variation at eight specific GSL loci , with the reference accession , Col-0 , as the genetic background .", "These transgenic lines consisted of loss-of-function T-DNA insertion lines , an EMS mutant and gain-of-function overexpression lines that were originally created to validate individual genes as causal for GSL natural variation ( Table 1 ) .", "For example , the AOP2 gene was found to encode an enzyme that converts methylsulfinyl ( MSO ) GSL into alkenyl GSL ( Figure 1 and Figure 1—figure supplement 7 ) ( Kliebenstein et al . , 2001c ) .", "Importantly , the AOP2 gene is polymorphic among Arabidopsis accessions , with Col-0 accession containing a natural knockout that abolishes its function .", "Therefore , introducing the functional allele back into Col-0 created a single gene mimic of the natural variation found in Arabidopsis ( Figure 1 and Table 1 ) ( Kliebenstein et al . , 2001c ) .", "The natural variation at the other causal genes has been similarly mimicked as described in the listed citations ( Table 1 ) .", "This was facilitated by the fact that all of these genes contain natural presence/absence polymorphisms ( citations in Table 1 ) . 10 . 7554/eLife . 05604 . 023Figure 2 . Globally distributed collection of Arabidopsis thaliana accessions that vary with respect to GSL haplotype . Shown are the geographic origins of 144 Arabidopsis accessions across ( A ) Europe and Northern Africa , ( B ) North America and ( C ) Japan , as well as their corresponding GSL haplotypes and chemotypes .", "GSL haplotype names correspond to allelic identity at six polymorphic loci involved in aliphatic GSL production , based on GSL profile data collected from each accession .", "Haplotype names use Col-0 as a reference , which is functional at four or the six loci .", "Symbol shape , color and size indicate GSL chemotype ( i . e . , phenotype based on GSL profile ) .", "Red = 3C ( non-functional MAM1 ) , green = 4C GSL ( functional MAM1 ) , triangle = MSO ( non-functional AOP ) , square = alkenyl ( functional AOP2 ) , circle = OH-alkenyl ( functional GSOH ) , star = OH-Propyl ( functional AOP3 ) , point size 1 = 100% accumulation of aliphatic GSL ( compared to Col-0 ) , point size 0 . 5 = 50% accumulation of aliphatic GSL ( non-functional MYB28 ) and point size 1 . 5 = 75% accumulation of aliphatic GSL ( non-functional MYB29 ) .", "See Figure 2—source data 1 for table of accession geographic information , Figure 1 for schematic of biosynthetic pathway and Figure 9 for more details on the allelic state at each locus for all 18 GSL haplotypes .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 02310 . 7554/eLife . 05604 . 024Figure 2—source data 1 . Geographic origin and GSL haplotype information for a collection of 144 Arabidopsis thaliana accessions . Shown are the province/city and country of origin and corresponding latitude and longitude coordinates for 144 Arabidopsis accessions along with their GSL haplotypes .", "See Figure 2 legend for full details . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 024 Each of these transgenic lines had been backcrossed to Col-0 several times to remove unlinked polymorphisms in the original studies ( Table 1 ) .", "For this study , the transgenic lines were manually crossed to each other to represent the phenotypic variation in GSL profiles found among Arabidopsis accessions ( Table 2 , Figures 1 , 2 ) .", "This synthetic laboratory population varies at specific genes controlling aliphatic GSL variation within a single common genetic background .", "Utilizing this synthetic laboratory population , we can explicitly measure the impact of variation in a suite of aliphatic GSL genes on fitness components in the field without confounding variation in other regions of the genome .", "We tested our population in multiple environments , which allowed us to separate the effects of genotype from environment , to determine if traits measured in the field are environmentally controlled .", "This could be particularly important if selection pressures fluctuate across environments .", "We transplanted 2 week old , greenhouse-germinated replicates of the synthetic laboratory population into the field at the University of California , Davis in Davis , CA in Spring 2012 and the University of Wyoming in Laramie , WY in Summer 2011 and Summer 2012 .", "In each of our three field trials , which represent three environments , genotypes were replicated in 40 randomized blocks in the field , for a total of 120 blocks/replicates .", "To distinguish the effects of GSL variation alone from the interaction of GSL variation with field herbivory as well as assess the effects of leaf damage in the field , half of the blocks in each field trial were treated with pesticides and the other half were not ( Figure 3 ) ( Mauricio , 1998 ) . 10 . 7554/eLife . 05604 . 025Figure 3 . Split-plot field trial setup . Shown is the field trial setup used in all three environments .", "In each environment , 40 blocks were arranged into rows of 10 blocks and each row was called a plot .", "Within each block , the complete set of 17 genotypes was randomly organized , for a total of 40 genotype replicates per environment .", "Each plot ( four per environment ) was placed into one of two treatment groups .", "The ‘− Herbivory’ treatment group received pesticide application to prevent leaf damage ( shown in blue ) .", "The ‘+ Herbivory’ or control treatment group did not receive pesticide application ( shown in red ) .", "This setup was repeated in each of the three environments , for a total of 120 blocks/genotype replicates and 12 plots , split between the two treatment groups .", "Environment and treatment were nested within plot , making this field trial setup a split-plot design .", "Seedlings were transplanted from the greenhouse into the field at 2 weeks of age where they were allowed to flower and then subsequently harvested for further analysis in the laboratory . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 025 Since the genes underlying variation in the aliphatic GSL pathway investigated in this study have been previously validated using lab techniques , we have a solid working knowledge of the resulting laboratory GSL profiles ( Beekwilder et al . , 2008; Hansen et al . , 2008 ) ( Figure 1—figure supplements 1–17 ) .", "However , these GSL genotypes have not previously been tested in the field to determine if they produce the same GSL profiles as when grown in the laboratory .", "We particularly wanted to assess if variation at individual aliphatic GSL genes has the same impact on GSL profile in the field as predicted from published lab experiments when the plants are grown in different complex environments , and therefore measured GSL on all the plants grown in each of our three field trials .", "A mixed model analysis of field GSL revealed that the majority of variation in GSL profiles in the field was controlled by the GSL genotypes that we generated ( Table 3 ) .", "Importantly , the majority of the GSL genotypes produced the expected GSL profiles in the field , consistent with the lab studies ( Figure 4 and Figure 1—figure supplements 1–17 ) .", "To quantify the similarity in profiles between field and lab grown samples , we conducted a PCA analysis using the GSL profiles of these genotypes grown in a growth chamber .", "The first four vectors from our PCA were able to explain >99% of the variation in GSL profile .", "We utilized the loadings from the chamber PCA to estimate PCA scores of the first four vectors using the chamber GSL and field GSL .", "The scores for the field grown genotypes were highly correlated with the lab grown genotypes , showing that the GSL genetic variation leads to highly similar field and lab profiles ( Table 4 ) . 10 . 7554/eLife . 05604 . 026Table 3 . Mixed model table of leaf damage , flowering time and GSL in the fieldDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 02610 . 7554/eLife . 05604 . 027Table 3—source data 1 . Mixed model table of phenotypes measured in the fieldDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 02710 . 7554/eLife . 05604 . 028Table 3—source data 2 . LSMeans of phenotypes measured in the fieldDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 028Fixed effectsLeaf damageFlowering timeSource of variationdfSSMSFp valuedfSSMSFp valueGenotype16280183 . 77 . 6E-071674714679 . 45 . 8E-23Environment22071047 . 50 . 0223660318302464 . 68 . 6E-08Treatment117170 . 3–11071077 . 30 . 04Geno:Env32617194 . 07 . 2E-13322678843 . 05 . 3E-08Geno:Trt167551 . 0–16655411 . 3–Env:Trt232161 . 9–25262636 . 50 . 03Geno:Env:Trt3220161 . 3–321115351 . 3–Random effectsLeaf damageFlowering timeSource of variationdfSSMSχ2p valuedfSSMSχ2p valuePlot ( Trt:Env ) 1008 . 70 . 0031000 . 0–Residual190450NA–1750260NA–Fixed effectsTotal aliphatic GSLTotal indole GSLSource of variationdfSSMSFp valuedfSSMSFp valueGenotype16250927315683034 . 63 . 0E-95163343220907 . 31 . 1E-16Environment21458872941 . 6–215207600 . 6–Treatment1143014300 . 3–1220822084 . 3–Geno:Env3230599395622 . 13 . 3E-0432141394421 . 50 . 03Geno:Trt1610013962591 . 4–1674884681 . 6–Env:Trt2393819690 . 4–26103051 . 0–Geno:Env:Trt3211626936330 . 8–3295312981 . 0–Random effectsTotal aliphatic GSLTotal indole GSLSource of variationdfSSMSχ2p valuedfSSMSχ2p valuePlot ( Trt:Env ) 119319372 . 65 . 6E-161151567 . 52 . 2E-16Residual149043422NA–14912690NA–A linear mixed model was fitted to phenotypes measured on plants grown in the field .", "Variation was partitioned among the fixed effects , Genotype , Environment , and Treatment as well as a random factor , Plot , inside which Treatment and Environment were nested .", "Phenotypic data used in the model was collected on 17 genotypes from three environments and two treatment groups .", "df = degrees of freedom , SS = Type II Sums of Squares variation , MS = Mean Squared variation , F = F statistic ( for fixed factors ) , χ2 = chi squared statistic ( for random factors ) .", "p value = probability value from either an F test or a chi squared test , depeding on the source of variation .", "Non-significant p values ( >0 . 05 ) are represented by a dash . 10 . 7554/eLife . 05604 . 029Figure 4 . Average GSL profiles from select laboratory population genotypes grown in the field . Shown are the genotype averages of various aliphatic GSL chemical structures from GSL genotypes grown in all three environments in the field .", "The GSL structures present and the corresponding abundances makeup the GSL profile of an individual .", "Results are based on single leaf analysis of 4 week old plants ( see Table 2 for full list of GSL genotypes used in this study ) .", "Each color represents a different aliphatic GSL genotype .", "Error bars represent standard error of the mean .", "Letters represent significantly different genotypes based on Tukey's HSD .", "See Figure 4—source data 1 for full list of GSL genotypes used in this study and the corresponding LSMeans and SE of GSL structures produced by all GSL genotypes used in study averaged across field trials .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 02910 . 7554/eLife . 05604 . 030Figure 4—source data 1 . LSMeans and SE of GSL structures produced by all GSL genotypes used in study averaged across field trials . Shown are the LSMeans and SE for GSL structures produced by GSL genotypes used in this study , averaged across the three field trials .", "Letters correspond to significant differences based on Tukeys's HSD .", "See Fgiure 4 legend for full details . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 03010 . 7554/eLife . 05604 . 031Table 4 . PCA comparison of GSL profiles produced by GSL genotypes from synthetic laboratory population grown in the chamber and all three environmentsDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 031EnvironmentPCA1 = 48 . 5%PCA2 = 29%PCA3 = 16%PCA4 = 6%Rp valueRp valueRp valueRp valueChamber1 . 001 . 001 . 001 . 00UCD20120 . 97<0 . 0010 . 97<0 . 0010 . 82<0 . 0010 . 96<0 . 001UWY20110 . 91<0 . 0010 . 95<0 . 0010 . 74<0 . 0010 . 97<0 . 001UWY20120 . 90<0 . 0010 . 91<0 . 0010 . 85<0 . 0010 . 86<0 . 001Glucosinolate analysis was conducted on the 17 genotypes within a Long-day growth chamber ( 16 hr light ) set to match the median light regime for the three environmments .", "Principal component analysis was conducted on the mean glucosinolate accumulation for the aliphatic glucosinoles within the chamber environment .", "This creates a set of mathematical descriptors of the chemotype variation across the 17 genotypes .", "The first three eigenvectors were used to generate scores from the lsmeans of the glucosinolates across the 17 glucosinolate genotypes independently for the chamber and three different field environments values .", "These scores were then correlated to test if the GSL profiles were similar or not across the environments .", "The R of the correlation to the Chamber scores for the 17 genotypes for each of the three PCA vectors are shown in conjunction with the p value as determined by Pearson correlation .", "To the right of each PCA vector label is shown the fraction of total variance approximated by the given vector .", "In total , the four vectors describe >99% of the GSL profile variance .", "In addition to the quantitative comparison of profiles , we also investigated the specificity of each locus in producing particular GSL structures to ensure that its field behavior mimicked the lab behavior .", "We found that , for the most part , each GSL gene produced the expected GSL phenotype in the field .", "For example , all lines harboring a functional AOP2 gene produce alkenyl GSL ( e . g . , but-3-enyl GSL ) ( Figures 1 , 4 ) .", "Additionally , the functional/non-functional allelic state at the MAM1 locus was always predictive of the chain-length of the GSL in the field as predicted from lab experiments .", "The lines with a functional MAM1 , like Col-0 , produced more 4C GSL than 3C GSL , while genotypes with a non-functional MAM1 always produced more 3C GSL than 4C GSL ( Figure", "4 ) ( Haughn et al . , 1991 ) .", "A functional copy of GSOH , the gene encoding the enzyme to create 2-OH-but-3-enyl , always leads to the production of 2-OH-but-3-enyl GSL from but-3-enyl GSL ( Figures 1 ,", "4 ) ( Hansen et al . , 2008 ) .", "In addition to the biosynthetic genes , the MYB genes , which encode transcription factors that control accumulation of aliphatic GSLs , showed similar field phenotypes as were found in the lab ( Hirai et al . , 2007; Sønderby et al . , 2007 , 2010 ) .", "Specifically , a non-functional MYB28 leads to an almost complete reduction in long chain ( 8C ) GSL and a 60% reduction in short chain ( 3C and 4C ) GSL ( Figure", "4 ) ( Hirai et al . , 2007; Sønderby et al . , 2007 , 2010 ) .", "A non-functional MYB29 leads to a 40% reduction in short chain GSL with no significant reduction in long chain GSL ( Figure", "4 ) ( Hirai et al . , 2007; Sønderby et al . , 2007 , 2010 ) .", "A double mutant in MYB28 and MYB29 lead to an almost complete loss of all aliphatic GSLs , as expected ( Figure", "4 ) ( Hirai et al . , 2007; Sønderby et al . , 2007 , 2010 ) .", "The only genes for which the field and laboratory GSL profile data differ are GSOX1 and GSOX3 , which are two tightly linked genes at the GSOX locus that also contains two additional genes , GSOX2 and GSOX4 .", "In the lab , gsox1 and gsox3 mutants accumulate higher levels of methylthio ( MT ) GSL than does Col-0 , due to reduced expression of a flavin-monooxygenase that converts the MT to MSO GSL ( Figure 1 ) ( Hansen et al . , 2007; Li et al . , 2008 ) .", "In the field there was no measureable accumulation of MT GSL in any line , likely due to the redundant function of the GSOX2 and GSOX4 genes ( Kerwin et al . , 2011 , 2012; Li et al . , 2008 ) .", "Thus , the field results show that the laboratory work on GSL genotypes and their associated GSL profiles are translatable and predictive of the GSL profiles found in naturally fluctuating environments .", "Conducting field trials in multiple environments enabled us to test the effect of different environmental conditions on our field traits .", "The specific composition of GLSs within a genotype largely did not change across the environments ( Table 4 ) .", "In contrast , the total amount of aliphatic GSL content , that is , the sum of all aliphatic GSLs per sample , showed a significant genotype by environment effect , indicating that impact of environment on total aliphatic GSL accumulation varied among the different GSL genotypes in this study ( Table 3 and Figure 5 ) .", "For example , the AOP2 genotype showed a dramatic variation in total aliphatic GSL across the three field trials ( Figure 5 ) .", "In contrast , a number of other genotypes tended to show similar accumulation across the environments .", "For example , genotypes with a myb28/myb29 double knockout accumulated virtually no GSL in all three environments .", "Thus , the GSL genotype is the dominant determinant of GSL profile in the field while total aliphatic GSL accumulation is determined by an interaction of genotype and environment within our laboratory population . 10 . 7554/eLife . 05604 . 032Figure 5 . Total aliphatic GSL accumulation of GSL genotypes from the laboratory population grown in the field . Shown are the genotype averages in all three environments of total aliphatic GSL from individuals grown in the field .", "Results are based on single leaf analysis of 4 week old plants .", "Bar color based on Dunnett's multiple comparison procedure .", "Within each environment , dark grey bars = Col-0 genotype , black bars = genotypes that accumulate significantly more or less total aliphatic GSL than Col-0 ( p value ≤ 0 . 05 ) , light grey bars = genotypes that accumulate suggestively more or less total aliphatic GSL than Col-0 ( p value = 0 . 05–0 . 1 ) and white bars = genotypes that are not significantly different than Col-0 ( p value >0 . 1 ) .", "Error bars represent standard error of the mean . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 032 A critical way in which plant environments fluctuate is with respect to insect populations that vary both temporally and spatially in a manner that could have a profound impact on variation in plant damage ( Mauricio , 1998; Richards et al . , 2009 ) .", "To assess if changes in environment impact herbivory levels , we measured leaf damage on a scale from 0–10 in all three field trials , with and without a pesticide treatment ( Figure 6 ) .", "A mixed model analysis showed that leaf damage significantly varied across the three environments but that the pesticide application did not significantly alter leaf damage in the field ( Table 3 ) .", "The UWY2012 field trial ( mean = 2 . 610 ) had significantly higher levels of leaf damage than both UWY2011 ( mean = 1 . 17 , p value <1e-04 ) and UCD2012 ( mean = 1 . 50 , p value <1e-04 ) , though UCD2012 and UWY2011 environments did not differ significantly for leaf damage ( p value = 0 . 44 ) .", "Field plots were treated with pesticides once every 2 weeks , which did not entirely eliminate leaf damage on the treated individuals .", "A more aggressive pesticide treatment regime would have been necessary to abolish leaf damage in the treated group .", "In addition , the levels of leaf damage measured in our study are low relative to other field studies in Arabidopsis ( Bidart-Bouzat and Kliebenstein , 2008 ) .", "The field site was located adjacent to other experimental field sites and greenhouses that also treated for pests , which may or may not have had an impact on the relative levels and/or pesticide resistance of herbivores in the vicinity .", "This combination of low overall leaf damage levels and the fact that the pesticide treatment did not eliminate leaf damage in the treated group is likely the cause for this lack of a treatment effect .", "However , there is a significant environment effect for leaf damage , indicating that this trait varied across the three field trials .", "In fact , we see no significant correlation of leaf damage across the three environments ( Table 5 ) .", "This suggests the three environments experienced differing herbivory pressures .", "Since we did not measure herbivore levels , we cannot determine whether the differences in leaf damage are the direct result of differences in insect populations .", "It is interesting to note that the UWY field site showed both the highest and lowest leaf damage levels , demonstrating that there can be potentially large temporal fluctuations in herbivory at a single location ( Table 3—source data 2 ) . 10 . 7554/eLife . 05604 . 033Figure 6 . Mean normalized leaf damage of GSL genotypes from the laboratory population grown in the field . Shown are the mean normalized genotype averages in all three environments of leaf damage from GSL genotypes grown in the field .", "Mean normalization was conducted by first dividing the genotype average of each GSL genotype within an environment to the corresponding environment average .", "Then , each normalized value was multiplied by the grand mean across all three environments .", "This was done in order put the leaf damage estimates in each environment on the same order of magnitude to ease visual comparisons of genotypes across environments and to highlight the fact that relative leaf damage of a given GSL genotype varies across environments . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 03310 . 7554/eLife . 05604 . 034Figure 6—source data 1 . Mean normalized values for leaf damage in the field . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 03410 . 7554/eLife . 05604 . 035Table 5 . Environmental correlations for leaf damage in the fieldDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 035UCD2012-UWY2012UWY2012-UWY2011UCD2012-UWY2011Rp valueRp valueRp value−0 . 25–0 . 01–0 . 22–Shown are Pearson's correlations for leaf damage between the different environments .", "R = correlation coefficients; p value = probability statistic .", "Non-significant p values are represented by a dash .", "GSL variation is known to affect leaf damage incurred by insect herbivory within a controlled lab setting and we wanted to test if this could also be observed within a naturally fluctuating field setting ( Lambrix et al . , 2001; Kliebenstein et al . , 2002; Beekwilder et al . , 2008; Hansen et al . , 2008 ) .", "Within a field environment , levels of leaf damage significantly varied across GSL genotypes , in agreement with the role of GSL in deterring herbivory ( Table 3 ) .", "However , the extent of leaf damage incurred upon different GSL genotypes in the field fluctuated among environments , such that no particular GSL genotype showed a consistent maximal or minimal level of leaf damage across the three field trials ( Figure 6 ) .", "For example , the myb28/AOP2 and AOP2 genotypes had similar herbivory in UCD2012 ( mean = 1 . 30 and 1 . 95 , respectively ) and UWY2011 ( mean = 1 . 29 and 0 . 80 , respectively ) but strongly diverged in UWY2012 ( mean = 1 . 45 and 5 . 64 , respectively ) ( Figure 6 and Figure 6—source data 1 ) .", "It has been shown , in a laboratory setting that the extent to which GSL profile provides resistance varies across different herbivore species ( Kroymann et al . , 2003; Pfalz et al . , 2007; Hansen et al . , 2008 ) .", "In addition , GSL have been shown to provide resistance to fungi , bacteria and nematodes , which may have also been present and variable between our environments ( Manici et al . , 1997; Tierens et al . , 2001; Aires et al . , 2009; Witzel et al . , 2013 ) .", "It is likely that the composition of the herbivore communities differed between the two field sites .", "Though we did not conduct a complete survey of the herbivores present at UWY and UCD , we did observe differences in leaf damage patterns between the two locations , suggesting that there would be differences in the composition of herbivores species present .", "Together , these results show that GSL variation controls differential leaf damage in each field trial but the specific directions of effect for individual GSL genotypes is subject to environmental conditions , such as the composition of herbivores , which can vary temporally and spatially .", "Since our laboratory population contains single gene variants , we have the ability to test the fitness consequences of individual genotypes in a field setting , an important step in connecting the GSL variation observed among Arabidopsis accessions with potential selective and non-selective evolutionary processes .", "To test if the GSL genotypes alter plant fitness in the three environments , we measured fecundity of each individual grown in the field .", "Plants were harvested from the field at maturity and the numbers of fruits , flowers and buds per plant were counted in the laboratory to yield total fruit count ( TFC ) .", "TFC has previously been shown to be a good proxy for fecundity in Arabidopsis where total number of seeds per plant is highly correlated with total number of siliques ( i . e . , fruits ) ( Wolf et al . , 2000; Kliebenstein et al . , 2001c ) .", "Among the GSL genotypes we observed variation in silique length .", "Arabidopsis siliques contain two rows of seeds in a linear conformation , so that silique length strongly correlates with seed number at maturity , assuming uniform seed size .", "Therefore variation in silique length or seed size could affect our fecundity estimates .", "Silique length and seed size were measured from digital images of GSL genotypes harvested from the field and seed size showed no significant variation ( data not shown ) .", "However , there was significant variation in silique length across GSL genotypes as well as a significant genotype by environment interaction ( Table 3—source data 1 ) .", "We concluded that the significant differences in silique lengths are likely reflective of fecundity and adjusted our fitness measurements using this information .", "Estimates of absolute fitness were therefore obtained for each individual as TFC multiplied by silique length both including and excluding individuals that did not survive to harvest .", "Survivorship was included in fitness estimates to avoid obtaining artificially inflated fitness estimates from GSL genotypes with higher death rates that would result from removing individuals that do not survive to fruiting and have a fitness of zero .", "In this study , GSL genotype had a significant impact on absolute field fitness ( Table 6 ) .", "There was also a significant interaction effect between GSL genotype and environment for absolute fitness both including and excluding survivorship , suggesting that the impact that GSL genotype has on fitness is conditioned upon the environment ( Table 6 ) .", "Environment did not show a significant main effect on either measure of absolute fitness , suggesting that the population mean for absolute fitness may have been comparable across the environments and instead it is the fitness of GSL genotypes relative to each other within an environment that varies .", "Thus , these GSL genotypes that recreate natural variation within a single common genetic background influence field fitness of A . thaliana in an environmentally dependent manner . 10 . 7554/eLife . 05604 . 036Table 6 . Mixed model of fitness phenotypes in the fieldDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 036Fixed effectsAbsolute fitness ( w/survivorship ) Absolute fitness ( w/out survivorship ) Source of variationdfSSMSFp valuedfSSMSFp valueGenotype16455453284662 . 24 . 9E-0316397186248242 . 32 . 0E-03Environment288326441632 . 2–2127177635884 . 6–Treatment1194819480 . 2–1204220420 . 3–Geno:Env32706781220871 . 70 . 0132508962159051 . 50 . 04Geno:Trt1613779586120 . 6–16169036105651 . 0–Env:Trt2291814590 . 1–2488324420 . 2–Geno:Env:Trt32348291108840 . 8–3223522473510 . 7–Random EffectsAbsolute fitness ( w/survivorship ) Absolute fitness ( w/out survivorship ) Source of variationdfSSMSChi . sqp valuedfSSMSChi . sqp valuePlot ( Trt:Env ) 126402640216 . 50133113311279 . 30Residual1692125817NA–1451100617NA–Fixed effectsRelative fitnessSurvivorshipSource of variationdfSSMSFp valuedfSSMSFp valueGenotype162822 . 73 . 9E-0416504 . 96 . 9E-10Environment2211 . 0–2219 . 30 . 01Treatment1000 . 2–1001 . 2–Geno:Env323911 . 84 . 2E-0332903 . 83 . 8E-12Geno:Trt16810 . 8–16100 . 8–Env:Trt2000 . 1–2000 . 1–Geno:Env:Trt321710 . 8–32301 . 1–Random effectsRelative fitnessSurvivorshipSource of variationdfSSMSChi . sqp valuedfSSMSChi . sqp valuePlot ( Trt:Env ) 10 . 10 . 1184 . 501000–Residual169213 . 8E-04NA–19000 . 13 . 5E-05NA–A linear mixed model was fitted to phenotypes measured on plants grown in the field .", "Variation was partitioned among the fixed effects , Genotype , Environment , and Treatment as well as a random factor , Plot , inside which Treatment and Environment were nested .", "Phenotypic data used in the model was collected on 17 genotypes from three environments and two treatment groups .", "df = degrees of freedom , SS = Type II Sums of Squares variation , MS = Mean Squared variation , F = F statistic .", "Non-significant p values ( >0 . 05 ) are represented by a dash .", "To visualize if the rank in absolute fitness of GSL genotypes fluctuates among the three environments and to compare the patterns of fluctuation of GSL genotypes across environments , we plotted the mean normalized fitness of all GSL genotypes in all environments for both absolute fitness measures , including and excluding survivorship ( Figure 7 and Figure 7—figure supplement 1 ) .", "Absolute fitness varied greatly between the highest and lowest ranked GSL genotypes within each of the environments ( Figure 7 and Figure 7—source data 1 ) .", "In addition , the performance of different GSL genotypes relative to each other varied across environments , so that no GSL genotype outperformed all the others in all three environments .", "For example , myb28/AOP2 shows the greatest fitness in the UCD2012 environment and the lowest fitness in UWY2012 .", "In contrast , myb28/gsoh shows an opposite pattern while other genotypes showing a diversity of other patterns ( Figure 7 ) .", "This fluctuation in rank of GSL genotypes across environments can also be observed if we look at fluctuations of TFC with and without survivorship across the three environments , though the patterns for specific GSL genotypes vary across the different fitness measures ( Figure 7—figure supplement 1 ) .", "Thus , it appears that the significant interaction of GSL genotype by environment controlling fitness is caused by fluctuations in the fitness rank of different genotypes across environments ( Figure 7 and Figure 7—source data 1 ) . 10 . 7554/eLife . 05604 . 037Figure 7 . Mean normalized absolute fitness of GSL genotypes from the laboratory population grown in the field . Shown are the mean normalized genotype averages of absolute fitness from GSL genotypes grown in all three environments calculated either including or excluding survivorship , as indicated .", "Absolute fitness including survivorship was calculated as total fruit count ( TFC ) × silique length × survivorship , whereas absolute fitness excluding survivorship was calculated as TFC × silique length for individuals that survived to harvest .", "Mean normalization was conducted for each phenotype by first dividing the average of each GSL genotype within an environment to the corresponding population mean for each environment .", "Then , each normalized value was multiplied by the grand mean across all three environments .", "This was done in order put the phenotype estimates in each environment on the same order of magnitude to ease visual comparisons .", "Solid lines represent distinct patterns that GSL genotypes display across the environments and are meant as a visual aid . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 03710 . 7554/eLife . 05604 . 038Figure 7—source data 1 . Mean normalized values for phenotypic traits in the field . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 03810 . 7554/eLife . 05604 . 039Figure 7—figure supplement 1 . Mean normalized total fruit count of GSL genotypes from the laboratory population grown in the field . Shown are the mean normalized genotype averages of total fruit count ( TFC ) from GSL genotypes grown in all three environments calculated either including or excluding survivorship , as indicated .", "TFC was measured in the laboratory as the total number of fruits , flowers and buds per individual .", "Mean normalization was conducted for each phenotype by first dividing the average of each GSL genotype within an environment to the corresponding population mean for each environment .", "Then , each normalized value was multiplied by the grand mean across all three environments .", "This was done in order put the phenotype estimates in each environment on the same order of magnitude to ease visual comparisons of genotypes across environments .", "Solid lines represent distinct patterns that GSL genotypes display across the environments and are meant as a visual aid . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 039 Within an environment , individuals compete against their neighbors for resources during their lifetime and natural selection favors those with higher performance relative to others .", "Therefore , in addition to absolute fitness , we also analyzed the effect of the GSL genotype on relative fitness in the field , both with and without survivorship .", "We calculated relative fitness of each GSL genotype within each environment as absolute fitness divided the population mean within that environment .", "Even more strongly than with our absolute fitness measurements , we found that GSL genotype and the interaction between GSL genotype and environment both had a significant impact on relative fitness in the field both including and excluding survivorship ( Table 6 ) .", "For example , myb28 has a higher than average relative fitness in UWY2011 but shows an average and slightly lower than average relative fitness in UWY2012 and UCD2012 , respectively ( Figure 8 ) .", "In other cases , relative fitness of a GSL genotype is similar among the UWY field trials but differs in the UCD field trial .", "Two examples , with opposite patterns are myb28/AOP2 , that has low relative fitness in both UWY field trials but higher relative fitness in UCD and gsm1 , that has high relative fitness in both UWY field trials but lower relative fitness in UCD .", "This indicates that temporal and spatial fluctuations in fitness can both occur and are dependent on genotypic differences . 10 . 7554/eLife . 05604 . 040Figure 8 . Relative and absolute of GSL genotypes from the laboratory population grown in the field . Heatmaps with hierarchical clustering of GSL genotypes representing the model corrected means of ( A ) absolute fitness including survivorship and ( B ) relative fitness of each genotype in each environment .", "Absolute fitness was calculated for each individual as the total fruit count × silique length × survivorship .", "Relative fitness was calculated by normalizing absolute fitness for each genotype against the population mean within an environment . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 040 Interestingly , heatmaps of absolute fitness and relative fitness reveal unexpected hierarchical clustering of the environments between the two traits ( Figure 8 ) .", "In both cases , UCD2012 clusters with UWY2011 and the two UWY field trials do not cluster together , showing that within an environment across years there is the potential for greater variability than across environments .", "In our analysis , we measured flowering time and total indole GSL in the field .", "In a laboratory setting , GSL genes have been observed to pleiotropically alter these traits ( Kerwin et al . , 2011 ) .", "In the field , both of these phenotypes were significantly affected by the GSL genetic variation in our synthetic population , indicating that aliphatic GSL genes can have pleiotropic impacts beyond the aliphatic GSL pathway that can be observed in natural settings ( Table 3 and Table 3—source data 1 ) .", "Therefore , there is the possibility that either of these phenotypes might be driving the observed variation in fitness of GSL genotypes across these environments .", "To test this , we conducted genetic correlations using the genotypic means for absolute fitness , flowering time and total indole GSL within each environment ( Table 7 ) .", "We did not observe a significant correlation between absolute fitness and our pleiotriopic traits , using either parametric or non-parametric approaches , in any of our three environments ( Table 7 ) .", "This indicates that while the GSL genes are causing pleiotropic effects , these pleiotropic effects are probably not driving the observed fitness consequences of the GSL genotypes in our field trials . 10 . 7554/eLife . 05604 . 041Table 7 . Genetic correlations between fitness and Pleitropic traits in the fieldDOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 041Absolute fitnessFlowering timeTotal indole GSLTrait ( UCD2012 ) Absolute fitness–−0 . 40−0 . 21 Flowering time−0 . 22–0 . 06 Total indole GSL−0 . 05−0 . 23–Trait ( UWY2011 ) Absolute fitness–−0 . 24−0 . 27 Flowering time−0 . 19–−0 . 05 Total indole GSL−0 . 430 . 12–Trait ( UWY2012 ) Absolute fitness–0 . 22−0 . 25 Flowering time0 . 29–0 . 59* Total indole GSL−0 . 210 . 38–Shown are genetic correlations between absolute fitness and traits pleiotripically controlled by GSL genes .", "Pearson's correlation coefficients are on the top half of the tables and Spearman rank correlations are on the bottom .", "*p value < 0 . 05 , **p value < 0 . 01 , ***p value < 0 . 001 .", "To test if natural Arabidopsis accessions show a pattern of variation consistent with fluctuating selection , we determined the GSL haplotype for a global collection of accessions using their GSL profile ( Figure 2 ) .", "Using the validated GSL phenotype caused by genetic variation at the eight causal genes for the aliphatic GSL pathway , we assigned a GSL haplotype to each Arabidopsis accession , given its GSL profile ( Table 1 and Figure 1 ) .", "Using the available GSL profile information , the underlying allelic state at each of the eight genes assigned functional or non-functional , based on presence or absence of different GSL structures as well as the relative abundances of different structures , that is , based on the GSL profile of the accession .", "This identified 18 distinct aliphatic GSL haplotypes among the set of 144 natural Arabidopsis accessions , observed at different frequencies ( Figure 9 and Figure 9—source data 1 ) .", "Using the observed single locus allelic frequencies , we calculated the expected GSL haplotype frequencies for each of the 18 multi-locus genotypes ( Figure 9—source data 1 ) .", "These expected frequencies for the GSL genotypes represent theoretical frequencies that would be expected if no selection gradient acted upon GSL variation and no genetic drift , migration or other non-selective effect upon population structure biased the allele distribution .", "Comparing the population of observed vs expected frequencies was highly non-random ( p < 0 . 001 ) ( Figure 9 and Figure 9—source data 1 ) .", "Further , specific multi-locus GSL genotypes occurred significantly more or less often than expected ( Figure 9 and Figure 9—source data 1 ) .", "Thus , the non-random variation of GSL haplotypes within the Arabidopsis accessions supports the observations from the empirical field trials .", "It is similarly possible that this observed non-random variation is caused by non-selective processes such as migration , population structure and/or local bottleneck .", "Significant future efforts will be required to test the extent to which this non-random variation is caused by neutral demographic processes vs potential fluctuating selection . 10 . 7554/eLife . 05604 . 042Figure 9 . GSL haplotype frequencies of Arabidopsis thaliana accessions based on GSL profile data from chamber-grown individuals . Shown are the GSL haplotypes observed among a population of 144 Arabidopsis accessions for which our lab had existing GSL data .", "Seven loci important in the aliphatic GSL pathway were called based on GSL profile data from the lab as ‘+’ = functional , ‘−’ = non-functional or ‘NA’ = unobservable due to epistasis ( see Figure 1 for an explanation of epistasis in the GSL biosynthetic pathway ) .", "Bar length represents the observed GSL genotype frequencies among 144 Arabidopsis accessions .", "Bar color represents the difference , for a given GSL haplotype , between expected and observed genotype frequencies , based on Chi Squared distribution ( significant p values shown ) .", "Blue = GSL genotypes found more frequently than expected ( p value ≤ 0 . 05 ) , red = GSL genotypes found less frequently than expected ( p value ≤ 0 . 05 ) and grey = GSL genotypes found as frequently as expected ( p value >0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 04210 . 7554/eLife . 05604 . 043Figure 9—source data 1 . χ2 analysis comparing observed and expected GSL haplotype frequencies observed among a globally distributed population of 144 Arabidopsis thaliana accessions . DOI: http://dx . doi . org/10 . 7554/eLife . 05604 . 043" ], [ "Based on our measures of fitness in the field , we showed that GSL variation can control fitness within the field .", "These fitness effects were not driven by pleiotropic phenotypes like flowering , but the specific selective pressures driving these fitness differences remain to be identified .", "Identifying these pressures will require vastly larger surveys of natural populations and long-term field trials .", "Using the empirical values for fitness , we could show that the GSL system within these environments fits models where fluctuating selection can maintain standing polygenic variation .", "Further trials are required to test if this is more broadly applicable across a broader range of environments .", "This would require more field trials using our synthetic population to provide the capacity to empirically evaluate models of maintenance of standing variation and its influence on adaptation ( Gillespie and Turelli , 1989; Orr , 1998; Agrawal , 2001 ) .", "It remains to be directly tested if similar evolutionary processes drive evolution of other ecologically important traits that must respond to fluctuating environmental conditions such as pathogen populations and water availability ." ], [ "The following eight loci in the aliphatic GSL pathway were modified in the synthetic laboratory population of A . thaliana genotypes: AOP2 ( At4g03060 ) , ESP ( At1g54040 ) , MYB28 ( At5g61420 ) , MYB29 ( At5g07690 ) , GSOH ( At2g25450 ) , MAM1 ( At5g23010 ) , GSOX1 ( At1g65860 ) , GSOX3 ( At1g62560 ) .", "The following knockout or complementation lines for the following loci in A . thaliana Col-0 were used to generate the lab population: AOP2 = 35S:AOP2 ( Li and Quiros , 2003 ) , ESP = 35S:ESP ( Burow et al . , 2006 ) , MYB28 = SALK_136312 , ( Sønderby et al . , 2007 ) , MYB29 = SM . 34316 ( Hirai et al . , 2007 ) , GS-OH = SALK_09807 ( Hansen et al . , 2008 ) , MAM1 = EMS mutant line gsm1 ( Haughn et al . , 1991 ) , GSOX1 = SALK_079493 ( Li et al . , 2008 ) , GSOX3 = CSHL_GT13906 ( Li et al . , 2008 ) .", "Mutant lines were manually crossed to each other to generate a population of plants containing homozogyous combinations of mutations in the different genes mentioned above , representing a subset of the potential variation in the aliphatic GSL pathway observed among Arabidopsis accessions ( Table 2 ) .", "Individuals were genotyped via PCR using the primers and reaction conditions listed below . PCR primer sets and reaction conditions for genotypingLocusPrimers sequenceGroupMYB29 genemyb29-1 RP 5′-TATGTTTGCATCATCTCGTCTTC-3′1myb29-1 LP 5′-TTGTAGATTGCGATGGGCTA-3′MYB29 T-DNAmyb29-1 RP 5′-TATGTTTGCATCATCTCGTCTTC-3′1myb29-1 LB 5′-ATATTGACCATCATACTCATTGC-3′AOP2 geneAOP2 FOR ODD13 5′-AACAGCGAAACGATCCAGAAGA-3′1AOP2 REV ODD24 5′-GTGCTTCTCGTCCACAA-3′MAM1 genegsm1-2 FOR 5′-TCATCGCTTCTGACATCTTCC-3′1gsm1-2 REV 5′-GTCTTGGCGATGGTCTTAATG-3′GSOX3 genegsox3 RP ( P3P ) 5′-TCGTCCTGACAAGACTGCTG-3′2gsox3 LP ( P3P ) 5′-GAGGGTCCAGTCGAAAAACTC-3′GSOX3 T-DNAgsox3 RP ( P3P ) 5′-TCGTCCTGACAAGACTGCTG-3′2LB2 5′-GCTTCCTATTATATCTTCCCAAATTACCAATACA-3′GSOH geneGSOH RP1 5′-GCTTCGGGATTAGGAGGAAC-3′2GSOH LP 5′-ATGAAGATTGGCGTGAAAGG-3′GSOH T-DNAGSOH RP1 5′-GCTTCGGGATTAGGAGGAAC-3′2LBb1 . 3 5′-ATTTTGCCGATTTCGGAAC-3′GSOX1 genegsox1 RP ( P3P ) 5′-CTAGCGCGGGTAGAAAGACAT-3′3gsox1 LP ( P3P ) 5′-GCATTCCAAAAATACCATAACG-3′GSOX1 T-DNAgsox1 RP ( P3P ) 5′-CTAGCGCGGGTAGAAAGACAT-3′3LB2 5′GCTTCCTATTATATCTTCCCAAATTACCAATACA-3′MYB28 genemyb28-1 RP 5′-TGTATAAACCAGCTTTTTGGGG-3′3myb28-1 LP 5′-TTTTTCATTATGCGTTTGCAG-3′MYB28 T-DNAmyb28-1 RP 5′-TGTATAAACCAGCTTTTTGGGG-3′3LBa1 5′-TGGTTCACGTAGTGGGCCATCG-3′Reaction conditions for group 1Initial melting32 cyclesFinal extension94°C94°C60°C72°C72°C30 s30 s45 s90 s10 minReaction conditions for group 2Initial melting30 cyclesFinal extension94°C94°C61°C72°C72°C30 s30 s45 s90 s10 minReaction conditions for group 3Initial melting30 cyclesFinal extension94°C94°C65°C72°C72°C45 s45 s45 s90 s10 min Field trials were conducted in two locations , the latter over 2 years , giving three separate environments total .", "The first field trial was performed at the University of Wyoming ( UWY ) in Laramie , WY during Summer 2011 , the second at UC Davis in Davis , CA Spring 2012 , and the third at UWY during Summer 2012 .", "Seeds were sown into flats with 2 inch 50-celled inserts using Sunshine #5 ( Sungro , Agawam , MA ) potting soil containing slow release fertilizer and stratified at 4°C for 4 days before being transferred into the greenhouse at either the University of Wyoming in Laramie ( UWY ) or the University of California at Davis ( UCD ) to facilitate germination synchrony .", "In the UWY greenhouse , plants received 15 hr light/9 hr dark natural phototoperiod with temperatures fluctuating diurnally from 10°C to 30°C .", "In the UCD greenhouse , plants received 14 hr light/10 hr dark natural photoperiod with temperatures fluctuating from 15°C to 35°C .", "Further , starting all the plants in the greenhouse minimizes variation in the initial seedling conditions .", "After germination , seedlings were thinned to one per pot and GSL genotypes were randomly organized into 40 blocks per field trial , for a total of 120 blocks total and also 120 GSL genotype replicates total .", "Individuals were transplanted from the greenhouse into the field 2 weeks post germination .", "A single plant of each genotype was present in each block in all three environments and blocks were arranged into four rows of ten blocks each ( Figure 3 ) .", "Each row of 10 blocks is referred to as a plot , so that there were four plots per field trial and 12 plots total .", "Within each plot is nested a treatment by environment combination .", "Every 14 days , two plots ( 20 blocks total ) per environment were treated with pesticides to decrease leaf damage due to herbivory .", "At UWY , plants were sprayed with the insecticide Sevin ( GardenTech , Palatine , IL ) to repel flea beetles .", "At UCD , plants were treated with Marathon 1% granular ( OHP , Mainland , PA ) and Lily Miller Slug , Snail & Insect Killer Bait ( Lily Miller Brands , Walnut Creek , CA ) .", "The plants were allowed to grow in the field for 4 weeks before being harvested .", "At harvest , the aerial portion of each plant was collected from the field , placed into a quart sized freezer bag and transferred into 4°C for temporary storage .", "After the harvest completion , the UCD field plants were immediately placed into −80°C for storage .", "The UWY field plants were shipped to UC Davis overnight on dry ice and then placed in −80°C for storage .", "GSL were measured on all field trial plants to assess field effects of the genotypes on GSL accumulation .", "At approximately 4 weeks of age , a single , fully expanded , green leaf was collected from each plant .", "In order to measure leaf area of each sample , leaves from twelve plants at a time were placed on a white sheet of paper with a grid overlay .", "A ruler was placed on the sheet of paper below the leaves and digitally imaged using a Nikon D3100 ( Tokyo , Japan ) .", "The photographed leaves were then placed directly into 96 deep well plates containing 400 μl 90% methanol and stored in the freezer until extraction .", "For the UWY field trial , the leaves were stored at −20°C for 3–4 weeks and shipped overnight to Davis , CA on dry ice .", "For the Davis field trial , all plates were stored at −20°C until extraction .", "After harvest , desulfoglucosinolates were extracted from all samples using a high-throughput protocol briefly described below ( Kliebenstein et al . , 2001a ) .", "One gram of Sephadex DEAE A-25 ( Sigma–Aldrich , St . Louis , MO ) was added to each well of a 96 well filter plate using a column loader .", "To hydrate the Sephadex , 300 μl of H2O was transferred to each well using a multichannel pipet and allowed to incubate at room temp 1 hr .", "Excess H2O was removed from the Sephadex by placing filter plate on top of a 96 deep well discard plate ( used to catch the flow through ) and centrifuged at 1000 rpm for 2 min .", "To homogenize the samples , 96 deep well plates containing a single A . thaliana leaf , two 2 . 3 mm ball bearings and 400 μl of 90% methanol in each well were shaken in a Harbil 5-Gallon Mixer ( Fluid Management Co . , Wheeling , IL ) for 3–5 min .", "Plates were centrifuged at 4000 rpm for 20 min .", "To bind GSL to Sephadex , 150 μl of supernatant from each well ( containing the extracted organic compounds ) was transferred to the corresponding well of the 96 well filter plate containing hydrated Sephadex and centrifuged at 1200 rpm for 3 min on top of the 96 deep well discard plate .", "To wash away all the non-binding organic compounds from the Sephadex , 150 μl of 90% methanol was added to each well and the plate was centrifuged at 1200 rpm for 3 min .", "To remove excess methanol , two wash steps were conducted by adding 150 μl of H2O to the plate followed by centrifugation at 1200 rpm for 3 min .", "To release the GSL from the Sephadex , 10 μl of Sulfatase ( Sigma–Alrich ) and 100 μl of water were added to each well of the 96 well filter plate then incubated overnight in the dark .", "The desulfoglucosinolates were then eluted into a 96 well round bottom plate via centrifugation at 1200 rpm for 3 min .", "For each GSL sample , 50 μl of the 110 μl of extract was injected on an Agilent 1100 HPLC ( Agilent , Santa Clara , CA ) using a Lichrocart 250–4 RP18e column ( Hewlett–Packard , Palo Alto , CA ) .", "Individual GSL compounds were detected at 229 nm and separated utilizing the following program with an aqueous acetonitrile gradient: a 6-min gradient from 1 . 5% to 5 . 0% ( vol/vol ) acetonitrile , followed by a 2-min gradient from 5% to 7% ( vol/vol ) acetonitrile , a 7-min gradient from 7% to 25% ( vol/vol ) acetonitrile , a 2-min gradient from 25% to 92% ( vol/vol ) acetonitrile , 6 min at 92% ( vol/vol ) acetonitrile , a 1-min gradient from 92% to 1 . 5% ( vol/vol ) acetonitrile , and a final 5 min at 1 . 5% ( vol/vol ) acetonitrile ( Kliebenstein et al . , 2001a ) .", "For each peak , the GSL structure was determined by comparing the retention time and UV absorption spectrum with known standards .", "The integral under each peak was automatically calculated and this value in mili-absorption units was converted to picamoles/mm2 tissue using response factor slopes determined from purified standards and area of leaf tissue used per sample as measured by ImageJ ( Kliebenstein et al . , 2001a; Reichelt et al . , 2002 ) .", "Leaf damage estimates were visually taken in the field at 4 weeks of age , just before tissue collection for GSL extraction .", "A scale from 0–10 was used to determine amount of pest damage incurred on each plant , with 0 representing no damage and 10 representing complete destruction of the plant ( i . e . , the plant completely eaten ) .", "Absolute fitness was calculated as total fruit count ( TFC ) × silique length × survival .", "TFC was measured as the count of fruits ( siliques ) + flowers + buds per individual .", "Silique length was measured in ImageJ from digital images of harvested field plants taken using a Nikon D3100 as follows: each plant was placed flat on a white sheet of paper next to a ruler and pictures were taken using auto focus .", "After setting the scale in ImageJ using the ruler placed in each image , the segmented line tool was used to draw a line from the pedicle to the tip of the silique .", "For each plant , eight siliques were measured at random and these values were averaged to get a value for each plant .", "Survival was scored on a binary ( 0–1 ) scale .", "Plants that germinated , were transplanted into the field and survived to harvest were given a survival score of 1 and plants that germinated and were transplanted but did not survive to harvest were given a score of 0 .", "Individuals that did not germinate or did not survive to transplantation were given an NA .", "Relative fitness was calculated for each GSL genotype within each environment relative to Col-0 .", "To do this , average absolute fitness of a GSL genotype was divided by the average absolute fitness of Col-0 within a environment .", "Col-0 was chosen as the reference genotype given that it is the background genotype .", "All statistical analyses were carried out using the R statistical computing language ( Team , 2014 ) .", "The field trial was conducted in a split plot design with each plot nested within treatment by environment .", "We used a restricted maximum likelihood ( REML ) approach to fit a linear mixed effects model to the field traits and partition the variation of each among the fixed effects , genotype , environment , treatment and the random factor , plot nested within treatment and environment .", "There were 17 genotypes , which refers to the GSL genotype in the synthetic laboratory population .", "There were three environments: Wyoming 2011 , Wyoming 2012 and Davis 2012 .", "The two treatments were control and pesticide treated .", "We had 4 plots per environment ( 2 in each treatment group ) for a total of 12 plots .", "We used the following formula to fit this model using the lme4 package in R ( Baayen et al . , 2008 ) : lmer ( Trait ∼ Genotype*Environment*Treatment + ( 1|Plot ( Treatment:Environment ) ) ) .", "The Anova function from the car package in R was utilized to determine which fixed effects variables significantly altered the mean of each trait ( p value <= 0 . 05 ) ( Fox and Weisberg , 2011 ) .", "We estimated population means ( i . e . , LSMeans ) of each field trait for all genotypes across treatment and environment using the LSMeans function from the doBy package in R ( Højsgaard et al . , 2013 ) .", "Dunnett's multiple comparison testing was performed on the traits to determine which genotypes had significantly different means than Col-0 , our reference genotype using the glht function from the multcomp package in R ( Hothorn et al . , 2014 ) .", "Additionally , Tukey's multiple comparison was performed on the traits to compare all the genotypes to all the other genotypes for significant differences using the same glht function from the multcomp package in R ( Hothorn et al . , 2014 ) .", "PCA was conducted using the princomp function from the base package ( Team , 2014 ) ." ] ]
[ "Natural populations persist in complex environments , where biotic stressors , such as pathogen and insect communities , fluctuate temporally and spatially .", "These shifting biotic pressures generate heterogeneous selective forces that can maintain standing natural variation within a species .", "To directly test if genes containing causal variation for the Arabidopsis thaliana defensive compounds , glucosinolates ( GSL ) control field fitness and are therefore subject to natural selection , we conducted a multi-year field trial using lines that vary in only specific causal genes .", "Interestingly , we found that variation in these naturally polymorphic GSL genes affected fitness in each of our environments but the pattern fluctuated such that highly fit genotypes in one trial displayed lower fitness in another and that no GSL genotype or genotypes consistently out-performed the others .", "This was true both across locations and within the same location across years .", "These results indicate that environmental heterogeneity may contribute to the maintenance of GSL variation observed within Arabidopsis thaliana ." ]
[ "‘Genetic variation’ describes the naturally occurring differences in DNA sequences that are found among individuals of the same species .", "These genetic differences arise from random mutations and may be passed on to their offspring .", "Some of these mutations may improve the ability of an individual to survive and reproduce—known as fitness—and are likely to become more common in the population .", "Other mutations may reduce an individual's fitness and are likely to be lost .", "However , it is believed that most of the mutations will have no effect on the fitness of individuals .", "It is not known why many of these ‘neutral’ genetic differences are maintained in populations .", "Some researchers have proposed that they are kept by chance and that there is no direct advantage to the population of keeping them unless these neutral mutations later become beneficial .", "However , other researchers think that the genetic variation itself may improve the fitness of the population by allowing it to quickly adapt to changes in the environment .", "Arabidopsis thaliana is a small plant that lives in many different environments and has high levels of genetic variation in many of its physical traits .", "One of these traits is the production of molecules called glucosinolates , which help the plants to defend against herbivores and infection by microbes .", "Previous studies have suggested that variation in the genes that make glucosinolates may improve the fitness of A . thaliana populations .", "To test this idea , Kerwin et al . carried out a field trial using A . thaliana plants that were genetically identical except for some of the genes involved in the production of glucosinolates .", "Kerwin et al . grew the plants in several different environments over several years .", "The field trial shows that variation in these genes affected the fitness of the plants in each of the different environments .", "However , the fitness benefit depended on the environment , and no single gene variant provided the best fitness across all environments , or over all the years of the trial .", "Kerwin et al . 's findings suggest that changes in the environment may contribute to the maintenance of genetic variation in the genes that make glucosinolates .", "This raises the questions of how many other genes in plants ( or other species such as humans ) have genetic variation that contributes to fitness across varied environments; and how can this link be tested in natural settings ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Widespread correlation patterns of fMRI signal across visual cortex reflect eccentricity organization
elife-03952-v1
[ [ "Within the visual system , a prominent organizational principle is that of retinotopy: adjacent neurons along the cortical surface typically receive input from adjacent points on the surface of the retina .", "Each retinotopic map can be divided along two orthogonal axes: polar angle ( i . e . , angular distance ) and eccentricity ( i . e . , radial distance ) .", "Early electrophysiological recordings in monkeys and cats identified several representations of the contralateral visual field in and around the calcarine sulcus ( Daniel and Whitteridge , 1961; Zeki , 1969; Allman and Kaas , 1971; Van Essen et al . , 1984 ) .", "Through the use of functional magnetic resonance imaging ( fMRI ) , it has now become evident that the human visual system contains over two-dozen visual maps ( for review of original mapping studies , see Silver and Kastner , 2009; Wandell and Winawer , 2011; Abdollahi et al . , 2014; Wang et al . , 2014 ) .", "The topographic organization of individual areas is thought to provide an infrastructure for the integration of information across areas and along the visual hierarchy ( Kaas , 1997; Wandell et al . , 2007 ) .", "Anatomical studies in primates have demonstrated that neurons with overlapping receptive fields ( RFs ) are interconnected ( Cragg , 1969; Zeki , 1969; Van Essen and Zeki , 1978; Maunsell and Van Essen , 1983 ) .", "Similarly , fMRI connectivity studies in humans have demonstrated topographically-local correlations between regions with overlapping visual field representations ( Heinzle et al . , 2011; Haak et al . , 2012; Butt et al . , 2013; Donner et al . , 2013; Gravel et al . , 2014; Raemaekers et al . , 2014 ) .", "In addition , widespread functional correlation patterns have been observed across visual cortex in macaques ( Leopold et al . , 2003; Vincent et al . , 2007 ) and humans ( Nir et al . , 2006 , 2008; Yeo et al . , 2011; Donner et al . , 2013 ) .", "These patterns contain broad differences between foveal and peripheral cortex ( Raemaekers et al . , 2014 ) , though may also be tied to the fine-scale organization of individual retinotopic maps .", "In this study , we used fMRI to investigate the relationship between the spatial pattern of correlated BOLD signal and the retinotopic organization of eight visual areas ( V1–hV4 , VO1–2 , V3A–B ) .", "Correlation analyses were performed on data collected during task-free conditions ( eyes-closed and fixation ) , and during movie viewing .", "Correlation patterns were consistent across subjects and experiments .", "In addition to finding patterns that support well-established anatomical connectivity between areas with overlapping RFs , our analyses revealed a widespread correlation pattern based on eccentricity representation , in which the BOLD signal was correlated in areas with non-overlapping visual field representations , but with matching eccentricity representations .", "This eccentricity-based correlation pattern was observed between upper and lower visual field representations , within and across visual areas , and between hemispheres .", "Moreover , correlation patterns were similar in the presence and absence of bottom-up visual input .", "Finally , the measured correlation pattern could not be accounted for by overlapping RFs , inter-hemisphere homotopic connections , anatomical distance , eye movements , subject motion , or physiological noise .", "Our results demonstrate that functional coupling between visual areas reflect both local and widespread topographical patterns .", "We propose that this widespread pattern is part of an intrinsic functional architecture of the visual system that could reflect eccentricity-dependent processing ." ], [ "To investigate the topography of functional correlations across visual cortex , Pearson correlation coefficients were computed between the timeseries of all surface data points ( nodes ) within the left and right visual cortices .", "For any individual node , correlation coefficients with all other nodes within visual cortex typically ranged between −0 . 10 and 0 . 75 in individual subjects ( after cerebrospinal fluid and white matter signal regression ) .", "For illustration of the raw functional correlation results , we present fixation resting-state correlation maps for four example seed locations in subject S1 ( Figure 1A ) , eyes shut resting-state correlation maps for four example seed locations in subject S2 ( Figure 1B ) , and group average resting-state maps for four example seed locations ( Figure 2A ) .", "In each of the four seed locations , the BOLD timeseries was sampled from a single node ( black dot ) within right dorsal V2 , and the seed location was gradually shifted from foveal ( <1 . 0° ) to peripheral-most ( ∼11 . 5° ) representations as defined from a separate eccentricity localizer experiment ( rightmost panel ) . 10 . 7554/eLife . 03952 . 003Figure 1 . Seed-based correlations on resting state in individual subjects . Correlation maps in both hemispheres of ( A ) subject S1 for resting fixation and ( B ) subject S2 for resting eyes shut at four seed locations ( <1 . 0° , ∼2 . 5° , ∼5 . 5° , ∼11 . 5°; left to right ) in dorsal V2 of the right hemisphere .", "For each seed , the strongest correlations ( red / yellow ) span several visuotopic areas within an eccentricity range roughly corresponding to that of the seed area ( black dot ) in both the ipsilateral and contralateral hemispheres .", "The correlations have a similar organization to eccentricity maps ( far right ) .", "To facilitate visual comparisons between hemispheres , the left hemisphere images have been horizontally reflected .", "Solid white bars mark borders between visual field maps .", "White dashed bars outline three bands of iso-eccentricity . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 00310 . 7554/eLife . 03952 . 004Figure 1—figure supplement 1 . Seed-based correlations on resting state eyes shut in individual subjects . Correlation maps in both hemispheres of ( A ) subject S3 at four seed locations ( <1 . 0° , ∼2 . 5° , ∼5 . 5° , ∼11 . 5°; left to right ) in ventral V3 of the right hemisphere and ( B ) of subject S4 in ventral V2 of the right hemisphere .", "For each seed , the strongest correlations ( red / yellow ) span several visuotopic areas within an eccentricity range roughly corresponding to that of the seed area ( black dot ) .", "The correlations have a similar organization to eccentricity maps ( far right ) .", "To enable comparisons between hemispheres , the left hemisphere images have been horizontally reflected .", "Conventions the same as Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 00410 . 7554/eLife . 03952 . 005Figure 1—figure supplement 2 . Seed-based correlations on resting state eyes shut in individual subjects . Correlation maps in both hemispheres of ( A ) subject S5 at four seed locations ( <1 . 0° , ∼2 . 5° , ∼5 . 5° , ∼11 . 5°; left to right ) in dorsal V3 of the right hemisphere and ( B ) of subject S6 in ventral V2 of the right hemisphere .", "For each seed , the strongest correlations ( red / yellow ) span several visuotopic areas within an eccentricity range roughly corresponding to that of the seed area ( black dot ) .", "The correlations have a similar organization to eccentricity maps ( far right ) .", "To enable comparisons between hemispheres , the left hemisphere images have been horizontally reflected .", "Conventions the same as Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 00510 . 7554/eLife . 03952 . 006Figure 2 . Group average seed-based correlations on resting state data .", "( A ) Correlation maps in both hemispheres of group average data for resting fixation at four seed locations ( <1 . 0° , ∼2 . 5° , ∼5 . 5° , ∼11 . 5°; left to right ) in dorsal V2 .", "For each seed , the strongest correlations ( red / yellow ) span several visuotopic areas within an eccentricity range roughly corresponding to that of the seed area ( black dot ) in both the ipsilateral and contralateral hemispheres .", "The correlations have a similar organization to eccentricity maps ( far right ) .", "To facilitate visual comparisons between hemispheres , the left hemisphere images have been horizontally reflected .", "Solid white bars mark borders between visual field maps .", "White dashed bars outline three bands of iso-eccentricity .", "( B ) Seed-based correlations plotted as a function of visual field representation for four seed locations .", "Eccentricity values are derived from a log-scaled stimulus ( see ‘Materials and methods’ ) .", "Black , dashed circles denote distance from fixation in visual degrees for each seed location . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 006 In all cases , we observed a combination of topographically local and widespread correlation patterns ( with respect to visual field representations ) .", "Each of the four seed locations was strongly correlated ( red / yellow ) with adjacent cortex .", "In addition , the strongest correlations with each seed extended across visual cortex and spanned several visual areas , from V1 to V3A–B , dorsally , and to VO1–2 ventrally .", "Strong correlations with nodes adjacent to the seed and within ipsilateral dorsal cortex likely reflect well-established connectivity based on overlapping visual field representations ( see Heinzle et al . , 2011; Haak et al . , 2012; Butt et al . , 2013 ) , but may also reflect the intrinsic spatial spread of the BOLD signal ( Engel et al . , 1997; Parkes et al . , 2005 ) .", "Interestingly , correlations were seen within both dorsal and ventral occipital cortex , comprised of lower and upper visual field representations respectively ( Figure 1 ) .", "Despite anatomical distance and representing a different part of visual space , the eccentricity representation ( distance from fovea ) of peak correlations within ventral occipital cortex corresponded to that of the seed location .", "These four seed locations also yielded comparable correlation patterns in the contralateral ( left ) hemisphere , comprised of right visual field representations .", "Local and widespread correlation patterns were observed in most individual subjects and the group average data for dorsal and ventral cortex seeds in V2 and V3 , regardless of seeding near the horizontal or vertical meridians ( see Figure 1—figure supplements 1 , 2 for additional individual subject data ) .", "The BOLD signals in areas with eccentricity preferences similar to the seed were correlated in the presence and absence of visual input , even in cases where the spatial receptive fields ( RFs ) were non-overlapping ( i . e . , across lower and upper or right and left visual field representations ) .", "To summarize the group average V2-seeds correlation results , we projected the correlation maps in Figure 2A into visual field coordinates , and averaged across areas V1 , V2 , V3 , V3A–B , hV4 , and VO1–2 ( Figure 2B ) .", "The correlation patterns highlight both visuotopically local and widespread correlation patterns ( Figure 2B ) .", "Peak correlations ( red ) were evident in parts of the visual field around each seed location with strong correlations ( red / yellow ) also extending across the visual field along an eccentricity ring corresponding to that of the seed location .", "Similar local and widespread eccentricity-based correlation patterns were observed in data from the movie viewing experiment ( Figure 3 ) .", "Individual subject and group average correlation patterns were similar to previously reported group average correlation patterns ( Yeo et al . , 2011 ) .", "Below , we formally tested the relation of eccentricity representations to the spatial pattern of correlated BOLD signal between individual brain areas and across tasks . 10 . 7554/eLife . 03952 . 007Figure 3 . Group average seed-based correlations on movie viewing data .", "( A ) Correlation maps in both hemispheres of group average data for movie viewing at four seed locations ( <1 . 0° , ∼2 . 5° , ∼5 . 5° , ∼11 . 5°; left to right ) in dorsal V2 .", "For each seed , the strongest correlations ( red / yellow ) span several visuotopic areas within an eccentricity range roughly corresponding to that of the seed areas ( black dot ) .", "The correlations have a similar organization to the eccentricity maps ( far right column of Fig . 2 ) .", "Conventions the same as Figure 2 .", "( B ) Seed-based correlations plotted as a function of visual field representation for the four seed locations .", "Black , dashed circles denote the seed area . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 007 To characterize the widespread eccentricity-based correlation pattern that was observed in raw correlation maps , individual subject timeseries data were grouped by visual area and then partitioned into 12 bins between 0 . 50° and 12 . 50° of eccentricity .", "Data binning was used as a form of averaging to increase signal-to-noise within bins , while preventing the spread of signal between bins .", "Within-subject pairwise correlations were calculated between the mean timeseries of all bins for visual areas V1 , V2 , V3 , hV4 , V3A–B ( combined ) , and VO1–2 ( combined ) , each of which has sufficient surface area to allow for fine-scale binning of eccentricity data .", "Areas V1–V3 were separated into quadrants for most analyses .", "No additional extrastriate areas were included in these analyses .", "Correlations between dorsal and ventral bins of V1 , V2 , and V3 were strongest for iso-eccentricity representations .", "These correlations were seen across the vertical and horizontal meridians at both foveal and peripheral-most bins .", "We first illustrate the binning analysis by examining the raw correlations between the ventral and dorsal quadrants in V3 ( Figure 4 ) .", "This pair was chosen for illustration because correlations computed across quadrants are less susceptible to the influence of overlapping receptive fields ( RFs ) or cortical proximity .", "For regions that represent the same visual quadrant ( e . g . , the dorsal portions of visual areas V1 , V2 , and V3 ) , it is difficult to dissociate effects due to shared eccentricity representations from overlapping receptive fields .", "Similarly , for adjacent dorsal regions , the shortest cortical distances are typically at corresponding eccentricity representations , making it difficult to dissociate eccentricity-related correlations from cortical ( and volumetric ) distance-based correlations .", "The dorsal and ventral portions of visual area V3 ( as well as V2 ) , however , only anatomically border each other at the fovea , represent different parts of the visual field ( lower and upper , respectively ) , and thereby minimize both the overlapping receptive field and anatomical adjacency concerns .", "Thus , correlation analyses between these areas allowed us to test for widespread eccentricity-based correlation patterns in cases where effects of cortical distance and overlapping RFs are minimal .", "As seen for subject S4 , the exploratory seed-based analyses showed strong correlations ( red/yellow ) between dorsal and ventral V3 at corresponding eccentricity representations ( Figure 4A ) .", "Binned data showed the same correlation pattern .", "For each of the selected dorsal V3 bins , correlations with ventral V3 bins were strongest at ( and around ) corresponding iso-eccentricities ( Figure 4B ) .", "For example , peripheral-most V3d bins correlated most with peripheral-most V3v bins , and the correlations gradually decreased moving towards the foveal bin ( Figure 4B , right ) .", "The entire correlation matrix for all eccentricity locations between dorsal and ventral V3 ( Figure 4C , center panel ) , revealed a similar pattern , where correlations were strongest among ventral and dorsal bins with iso-eccentricity representations ( i . e . , the diagonal ) , and were weaker for bins with large radial distances ( e . g . , foveal vs peripheral-most ) . 10 . 7554/eLife . 03952 . 008Figure 4 . Illustration of eccentricity binning correlations on resting state .", "( A ) Correlation maps in left hemisphere of subject S4 for 3 seed bin locations ( <0 . 5–0 . 84° , 3 . 71–4 . 63° , 10 . 36–12 . 50°; left to right ) in dorsal V3 and subject S4's eccentricity map ( right ) .", "Grayscale dots mark approximate seed bin locations .", "( B ) Correlations with all ventral V3 bins are plotted for the three dorsal seed locations .", "The strongest correlations between dorsal and ventral V3 were at , and around , iso-eccentricity representations .", "( C ) The entire correlation matrix for all eccentricity locations between dorsal and ventral V3 ( center ) revealed a similar pattern where correlations were strongest at or near iso-eccentricity ( i . e . , the diagonal ) , and weaker for bins with large radial distances ( e . g . , foveal vs peripheral-most ) .", "Radial distance ( left ) was strongly correlated with the measured group connectivity ( r = 0 . 84 ) and was uncorrelated to the cortical distance ( right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 008 The pattern of correlations reflects radial distance , not anatomical distance .", "We computed the ( ranked ) radial distance between eccentricity bins .", "A radial bin distance of zero corresponds to bins with the same ( iso- ) eccentricity representation , while a radial bin distance of 11 corresponds to bins furthest from each other on the eccentricity axis ( i . e . , foveal vs peripheral-most ) .", "Figure 4C provides the spatial pattern of correlations between ventral and dorsal quadrants in area V3 as well as the predicted patterns based on radial distance ( left gray panel ) and based on cortical distance ( right gray panel ) .", "Note that both the radial bin distance and the cortical distance ( as well as an overlapping RF model ) predict strong correlations between foveal bins ( Figure 4C ) .", "However , whereas cortical distance predicts that correlations will be weaker between cortically–distant iso-eccentricity bins ( e . g . , ventral and dorsal V3 peripheral-most ) , the radial bin distance predicts a strong correlation between iso-eccentricities ( Figure 4C ) .", "Indeed , the group average bin data was strongly correlated with the predicted eccentricity pattern ( r = 0 . 84 ) and was not positively correlated with cortical distance ( r = −0 . 04 ) or volume distance ( r = −0 . 19 ) ( Figure 4C ) .", "Individual subject matrices were also correlated with radial distance ( mean r = 0 . 45 ) , and the Fisher-transformed correlations significantly differed from zero across subjects ( one-sample t-test , t ( 13 ) = 6 . 37 , p < 0 . 0001 ) .", "Correlations with radial bin distance were apparent within and across all visual areas tested , including areas with overlapping and non-overlapping visual field representations ( both within and across hemispheres ) .", "We computed intra- and inter-hemisphere correlations as a function of eccentricity bin within and across visual areas V1 , V2 , V3 , hV4 , V3A–B ( combined ) , and VO1–2 ( combined ) in each condition ( resting eyes closed , resting eyes open , and movie fixation; Figure 5 ) .", "In Figure 5 , gray frames denote comparisons between regions with overlapping visual field representations while black frames denote comparisons between regions with minimal or no overlap in visual field representations ( i . e . , across ventral and dorsal visual fields and across hemispheres ) .", "For all comparisons in each condition the strongest correlations ( red/yellow ) were consistently between bins at comparable eccentricity locations ( i . e . , along the diagonal of each sub-matrix in Figure 5 ) .", "This was the case even for correlations between dorsal and ventral visual portions of V1 , V2 , and V3 , and between hemispheres , which represent mostly discrete parts of visual space .", "Across area pairs , the average individual subject correlation coefficient with radial distance ranged between 0 . 17 and 0 . 80 with a median of 0 . 39 and an interquartile range of 0 . 12 .", "Fisher-transformed individual subject correlations significantly differed from zero for all pairs in each experiment ( one-sample t-test , t ( 13 ) > 2 . 31 , ps < 0 . 05 , FDR-corrected ) except for two pairs of inter-hemisphere correlations with hV4 in the movie viewing condition ( ps = 0 . 13 and 0 . 07 ) .", "In all experiments , individual subject correlations with radial distance were generally strongest for early visual areas ( V1 , V2 , and V3 ) .", "This could reflect the relatively smaller , more spatially focal receptive fields in early visual areas , but also the larger surface area ( i . e . , more data points ) . 10 . 7554/eLife . 03952 . 009Figure 5 . Correlation matrices for resting state and movie viewing conditions . Intra- ( bottom-left matrix triangle ) and inter-hemisphere ( top-right matrix triangle ) correlations are shown for all pairwise comparisons between visual areas V1 , V2 , V3 , hV4 , VO1-2 , and V3A-B for resting fixation ( left matrix ) , resting eyes shut ( center matrix ) , and movie viewing ( right matrix ) experiments .", "For each pair of visual areas , the strongest correlations ( red ) are at corresponding eccentricity bins and its neighbors ( diagonal in each sub-matrix ) .", "Grey and black boxes bound area pairs with overlapping and non-overlapping visual field representations , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 009 For each area pair , correlation coefficients were positive at 0–radial distance , and decreased at larger radial distances across the visual field .", "To assess the general effect of radial distance for each area pair , eccentricity bin correlations were grouped as a function of radial bin distance and averaged .", "Averaged correlations were plotted relative to iso-eccentricity ( Figure 6A , left panel ) .", "In each quadrant , the radial mid-arc ( Figure 6A , white outline ) corresponds to the average correlation of the 0-distance bin ( iso-eccentricity ) .", "Radial arcs further away from the mid-arc correspond to larger radial distances .", "For each radial plot , for example , V2–V3 , radial arcs inside of the mid-arc illustrate correlations between relatively more foveal representations of V2 and relatively more peripheral representations of V3 .", "Radial arcs outside of the mid-arc illustrate correlations between relatively more peripheral representations of V2 and relatively more foveal representations of V3 .", "Inner and outer radial points will be identical for within area comparisons ( e . g . , V2–V2 ) , but could differ for between area comparisons ( e . g . , V2–V3 ) .", "As illustrated by resting state correlations within and between areas V2 and V3 ( Figure 6A ) , iso-eccentricity bins were positively correlated , regardless of visual field quadrant .", "This was observed in all experiment conditions and area comparisons , and individual subject Fisher-transformed correlations significantly differed from zero ( ps < 0 . 05 , FDR corrected ) .", "There were clear differences in overall correlation magnitude between quadrants .", "As expected , correlation coefficients decreased at larger radial distances for foveal and peripheral-most bins in each quadrant , regardless of this magnitude difference ( Figure 6A ) . 10 . 7554/eLife . 03952 . 010Figure 6 . Intra-run eccentricity-based connectivity analyses for resting-state and movie viewing conditions .", "( A ) Radial bin distance correlation plots between resting-state data bins for within quadrant/within hemisphere ( upper left ) , within quadrant/between hemisphere ( upper right ) , between quadrant/within hemisphere ( lower left ) , and between quadrant/between hemisphere ( lower right ) comparisons .", "In each quadrant , the mid-radial arc ( white outline ) corresponds to the average correlation at iso-eccentricity with outer and inner arcs corresponding to average correlations at increasingly larger radial distances .", "( B ) Average individual subject correlations are plotted for areas V2 and V3 as a function of radial distance between the upper and lower visual fields ( top ) , right and left visual fields ( middle ) , as well as across both left and right and upper and lower visual fields ( bottom ) for all conditions .", "Correlations are normalized to the average correlations at iso-eccentricity ( 0-radial distance ) .", "All correlations were strongest at the 0-radial distance and steadily decreased at larger distances for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 01010 . 7554/eLife . 03952 . 011Figure 6—figure supplement 1 . Intra-run eccentricity-based connectivity analyses for resting-state and movie viewing conditions without meridian data . Average individual subject correlations are plotted for areas V2 and V3 as a function of radial distance between the upper and lower visual fields ( top ) , right and left visual fields ( middle ) , as well as across both left and right and upper and lower visual fields ( bottom ) for all conditions .", "Meridian data were removed prior to binning .", "Data corresponding to 60° of polar angle centered on the horizontal meridian was removed for comparisons between dorsal and ventral regions .", "Data corresponding to 90° of polar angle centered on each vertical meridian was removed for inter-hemisphere comparisons .", "All correlations were strongest at the 0-radial distance ( iso-eccentricity ) and steadily decreased at larger distances for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 01110 . 7554/eLife . 03952 . 012Figure 6—figure supplement 2 . Intra-run angular-based connectivity analyses for resting-state and movie viewing conditions .", "( A ) Average individual subject correlations are plotted for areas V2 and V3 as a function of angular distance within an area ( top ) , between the upper and lower visual field ( second row ) , right and left visual fields ( third row ) , as well as across both left and right and upper and lower visual fields ( bottom ) for all conditions .", "( B ) Average individual subject correlations are plotted for mirror symmetric connections between upper and lower visual fields ( top ) and between right and left visual fields ( bottom ) .", "See Figure 6 for conventions . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 01210 . 7554/eLife . 03952 . 013Figure 6—figure supplement 3 . Real data vs artificial data . Comparison of average individual subject correlations for real ( blue ) and artificial ( red ) data .", "Data are plotted for areas V1 , V2 , and V3 as a function of radial distance between the upper and lower visual fields ( top ) , right and left visual fields ( middle ) , as well as across both left and right and upper and lower visual fields ( bottom ) for all conditions .", "The pattern of correlations between real and artificial data differed for all comparisons .", "For the real data , correlations decreased as a function of radial distance in every comparison .", "For the artificial data , correlations decreased as a function of radial distance only for within quadrant and ( partially ) for between anatomically adjacent areas .", "For comparisons between dorsal and ventral regions and between hemispheres , artificial data correlations did not vary as a function of radial distance .", "Even for within quadrant , the slope of the correlations across radial distances was markedly different between real and artificial data .", "These plots demonstrate that the effect of radial distance on correlation strength cannot be explained by spatial correlations due to intrinsic properties of BOLD imaging or our preprocessing as these were preserved in the artificial data . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 013 The relative decrease in correlation strength as a function of radial distance was similar across the visual field and across experiments ( Figure 6B ) .", "We plotted the average correlation as a function of radial bin distance ( relative to iso-eccentricity; see ‘Materials and methods’ ) between the upper and lower visual fields , right and left visual fields , as well as across both left and right and upper and lower visual fields separately ( see graphic illustrations in Figure 6B left panels ) .", "Data are presented for comparisons between quadrants within and between areas V2 and V3 .", "As discussed in the eccentricity binning section , these comparisons were chosen for illustration because overlap in visual field representation is minimal .", "Consistent with the radial arc plots ( Figure 6A ) , correlations between dorsal and ventral portions of V2 and V3 were strongest at 0–radial distance , and steadily decreased at larger radial distances for all conditions ( Figure 6; top row ) .", "We observed similar correlation patterns as a function of radial bin distance across hemispheres , both along the horizontal plane ( e . g . , RH V2v and LH V2v; middle row ) and diagonally across the upper and lower visual fields ( e . g . , RH V2v and LH V2d; bottom row ) .", "The patterns were observed even after removal of horizontal and vertical meridian data , further demonstrating that these effects are unlikely to be driven by local , overlapping visual field representations ( Box 1; Figure 6—figure supplement 1 ) .", "The decrease in correlation coefficients at larger radial bin distances yielded negative slopes from linear fits in each subject for all V2 and V3 pairs .", "For any given pair , negative slopes were similar across condition .", "Across V2 and V3 pairs , the average individual subject slope of the linear fit ranged between −0 . 22 and −0 . 49 .", "Further , the average individual subject slope for each area pair was greater than 97 . 5% of a permuted distribution ( mean 97 . 5% across areas , conditions = −0 . 06 ) where the labels of radial distances were scrambled before deriving individual subject radial distance correlations and slopes .", "As seen in the radial plots ( Figure 6B ) , the slopes were similar across regional comparisons , though were slightly shallower for comparisons that spanned both quadrants and hemispheres ( i . e . , diagonal; bottom row ) .", "Finally , we observed these effects during resting fixation and eyes closed conditions , as well as during the processing of the movie , attesting to the robustness of the effect , and excluding many potential confounds ( see ‘Control analyses’ ) .", "Similar results were found in all other comparisons between visual areas .", "The magnitude of correlation coefficients linearly decreased as a function of radial bin distance for all area comparisons ( V1 , V2 , V3 , hV4 , VO1–2 , V3A–B ) , though slopes were generally steeper for comparisons between V1 , V2 , and V3 .", "Across all areas , average individual subject slopes ranged between −0 . 14 and −0 . 80 , and each was greater than 97 . 5% of the permuted distribution ( mean 97 . 5% across areas , conditions = −0 . 09 ) .", "For comparisons between dorsal areas , between ventral areas , and between mirror-symmetric ( across vertical meridian ) areas , correlations were positive at 0–angular distance , and decreased at larger angular distances ( Figure 6—figure supplement 2 ) .", "To assess the general effect of angular distance for each area pair , data were grouped into 12 equally spaced polar angle bins .", "Correlations between bins were computed and grouped as a function of angular bin distance .", "For within-quadrant , within-hemisphere and mirror symmetric ( across vertical meridian ) within-quadrant , between-hemisphere comparisons , correlations steadily decreased at larger angular distances .", "Taken together , these results suggest that angular connectivity reflects overlapping RF between areas and mirror-symmetric connections between hemispheres .", "No effect of angular distance was observed between dorsal and ventral comparisons based on actual angular distance or when reflecting across the horizontal meridian ( i . e . , mirror symmetry ) .", "As with any negative finding , we cannot conclude with certainty that such angular-based connectivity does not exist , though , to our knowledge , no study has shown angular connectivity between regions with non-overlapping RFs .", "For each area pair , the spatial pattern of correlations ( across eccentricity bins ) was similar between experimental conditions .", "Across all areas , the average individual subject correlation of these spatial patterns between experimental conditions was 0 . 86 for intra-hemisphere comparisons and 0 . 75 for inter-hemisphere comparisons .", "The average individual subject correlation between resting-state and movie viewing conditions was 0 . 84 for intra-hemisphere comparisons and 0 . 71 for inter-hemisphere comparisons .", "Overall , these data are consistent with the exploratory seed based analyses , and suggest that the spatial pattern of correlations between visual areas tested were similar in the presence and absence of a strong bottom-up input .", "Given the similarity of correlation matrices across all three conditions , this widespread eccentricity-based correlation pattern appears to reflect an eccentricity bias that is inherent to the organization of the visual system ( i . e . , stable during rest ) and may support processing during active perception of the visual environment ( e . g . , during movie viewing ) .", "We conducted correlations between individual runs to directly test whether the patterns observed during the movie viewing condition reflected processing of the stimulus input .", "Intrinsic neural dynamics during the resting and movie conditions that are not related to the processing of visual stimuli , as well as non-neuronal artifacts ( e . g . , respiratory rate , motion ) , can only influence the pattern of correlations within each run , but should not induce correlations between runs .", "Indeed , inter-run Fisher-transformed correlations on resting state data showed no effect of radial distance ( all ps > 0 . 05 ) , validating the assumption that noise correlations should not be reliable across runs ( Figure 7 , blue and black lines ) .", "In contrast , inter-run correlations during movie watching showed radial distance effects similar to intra-run correlations ( Figure 7 , red lines ) .", "Inter-run correlations were statistically significant for 96% of all comparisons ( ps < 0 . 05 , FDR corrected ) .", "The slopes of coefficients as a function of radial distance for movie data were , generally , weaker for the inter-run relative to intra-run analyses ( compare Figures 6B , 7 ) .", "However , the inter-run analyses inherently had less data and spatial attention was not controlled , making the differences between intra- and inter-run analyses difficult to interpret .", "The inter-run results indicate that the observed widespread eccentricity-based correlations can reflect the processing of the incoming information during viewing of real life stimuli , and further suggest that these correlations patterns are unlikely to be driven by non-neuronal artifacts . 10 . 7554/eLife . 03952 . 015Figure 7 . Inter-run eccentricity-based connectivity analyses for resting-state and movie viewing conditions . For the movie viewing condition , average individual subject inter-run correlations between hemispheres as well as between dorsal and ventral portions of V2 and V3 were strongest at the 0-radial distance ( iso-eccentricity ) and steadily decreased at larger distances .", "For resting state data , correlations did not vary as a function of radial distance .", "See Figure 6 for conventions . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 015 We performed a variety of control analyses to rule out possible confounds .", "The observed widespread eccentricity-based correlation pattern could not be attributed to non-neuronal artifacts ( e . g . , physiological noise , motion , BOLD spatial autocorrelation , eye movements ) .", "Global artifacts did not drive our results as correlation patterns were strengthened by the removal of non-neuronal signals ( i . e . , motion and white matter ) .", "The eccentricity-based correlation pattern was not due to inherent spread of the BOLD signal , anatomical distance , or any biases in our analyses , as artificial data that preserved such biases were not correlated with radial distance ( see Box 2; Figure 6—figure supplement 3 ) .", "Subject motion also could not explain the observed correlation patterns .", "We minimized the overall influence of movement in our data by using highly trained MRI subjects and by removing signal correlated with subject motion via regression .", "Further , the strength of eccentricity correlation did not significantly co-vary with degree of subject movement .", "We performed Spearman rank correlations between each subject's total amount of movement and the slope of his or her radial bin distance coefficients ( i . e . , effect of eccentricity on correlation strength ) .", "There was no significant correlation across subjects between the mean slope ( averaged across all area pairs ) and any of the six motion parameters ( all rs < 0 . 24 , ps > 0 . 05 ) .", "In addition , there were no significant correlations across subjects between the slope and total amount of movement for any individual area pair ( ps > 0 . 05 , FDR-corrected ) .", "These analyses strongly suggest that the observed correlation patterns were not driven by subject motion .", "It is unlikely that eye movements drove the eccentricity-based correlation pattern as similar results were observed at rest during both fixation and eyes closed conditions .", "Finally , significant inter-run eccentricity-based correlation patterns were observed during movie viewing , but not during rest , further ruling out the possible influence of non-neuronal intrinsic artifacts ( e . g . , respiration rate , cardiac rate , motion ) , which should not be correlated across runs .", "Both local and widespread connectivity patterns contributed to the spatial pattern of observed correlations .", "Each visual hemifield map was separated into 36 bins: six divisions of eccentricity ( E1–E6 ) that each contained six divisions of polar angle ( A1–A6 ) such that each bin represented a unique part of visual space ( Figure 8—figure supplement 1 ) .", "To visualize the pattern of correlations across all bins with respect to local ( overlapping RF ) and widespread ( eccentricity and polar angle ) connectivity patterns , data were plotted as a function of radial and angular distance ( Figure 8 ) .", "Correlations were qualitatively strongest at the intersection of iso-polar angle and iso-eccentricity , and were generally elevated along iso-eccentricity representations , irrespective of polar angle distance ( Figure 8 ) .", "To quantify this , we assessed the relationship between the measured correlations across visual areas V1 , V2 , V3 , and hV4 and several possible connectivity patterns using linear , least squares regression ( see ‘Materials and methods’ ) .", "For any pair of visual areas , we modeled correlations between all bin pairs ( 36 × 36 ) as the linear weighted sum of four sources of connectivity .", "Two sources reflect topographically local connectivity between regions that are in close anatomical proximity or contain overlapping receptive fields: ( 1 ) instrumental ‘noise’ connectivity: correlation pattern based on an assessment of the spatial auto-correlation and preprocessing in our data; ( 2 ) overlapping RF connectivity: correlation pattern based on the overlap of estimated population receptive fields ( pRF; Dumoulin and Wandell , 2008; Amano et al . , 2009; Harvey and Dumoulin , 2011 ) .", "The two other sources reflect topographically widespread connectivity that span much of the visual field and are not specifically tied to receptive fields or anatomical proximity: ( 1 ) eccentricity connectivity: correlation pattern based on radial distance with correlations strongest at iso-eccentricity representations; ( 2 ) polar angle connectivity: correlation pattern based on angular distance with correlations strongest at iso-polar angle representations . 10 . 7554/eLife . 03952 . 017Figure 8 . Radial and angular distance plots . Resting state correlation bins plotted as a function of average radial and angular distance ( ΔE and ΔA , respectively ) for areas V1 , V2 , V3 , and hV4 .", "Intra- and inter- hemisphere correlations are plotted in the left and right hemifields , respectively .", "For intra-hemisphere correlations ( left hemifield ) , the mid arc ( oval; see legend ) corresponds to the average correlation at the intersection of iso-eccentricity and iso-polar angle .", "There is no iso-polar angle for inter-hemisphere comparisons ( right hemifield ) , so plotted data begin off the horizontal meridian at an iso-polar distance of one , and correspond to the correlations between bins adjacent to the vertical meridian .", "Outer and inner arcs correspond to larger radial distance correlations ( same conventions as symmetry plots in Figure 6A ) .", "Arcs closer to the vertical meridian correspond to larger angular distances ( see legend ) .", "For each plot , for example , V2–V3 , arcs above the mid arc ( +ΔA ) illustrate correlations between relatively more upper visual field representations of V2 and relatively lower visual field representations of V3 ( reverse for below mid arc , −ΔA ) .", "For comparisons with area hV4 , there was no data for some radial and angular distances ( black arcs ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 01710 . 7554/eLife . 03952 . 018Figure 8—figure supplement 1 . Connectivity patterns for model fitting .", "( A ) For areas V1 , V2 , V3 , and hV4 , data were divided into six bins of eccentricity ( E1–E6 ) , each containing six bins of polar angle ( A1–A6 ) .", "( B ) Illustration of the conversion from visual field representation to matrix representation for all pairwise ( 36 × 36 ) bin correlations .", "( C ) Illustrations of four predicted patterns of intra-hemisphere connectivity between areas V2 and V3 ( from left to right ) : ( 1 ) Noise , ( 2 ) RF overlap , ( 3 ) Eccentricity , and ( 4 ) Polar angle . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 01810 . 7554/eLife . 03952 . 019Figure 8—figure supplement 2 . Homotopic radial and angular distance plots . Correlation bins are plotted as a function of average radial and angular distance for V1 , V2 , V3 , and hV4 .", "Intra- and inter- hemisphere correlations are plotted in the left and right hemifields , respectively .", "For inter-hemisphere comparisons , the visual field position of bins in one hemifield is mirror reflected across the vertical meridian ( homotopic angular distances denoted as A′ and eccentricity distances as E′ , see top-right legend ) .", "In each hemifield , the mid arc ( oval; see legend ) corresponds to the average correlation at the intersection of iso-eccentricity and iso-polar angle .", "All other conventions the same as Figure 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 019 In general , the strongest overlapping RF effects were observed for intra-areal comparisons whereas eccentricity effects were consistently observed across all comparisons and were much larger than overlapping RF and polar angle effects for inter-hemisphere comparisons .", "To quantify the apparent connectivity patterns , we conducted an initial regression analysis using a model that included all four sources of connectivity as predictors .", "For individual subjects , the combination of local and widespread predictors well fit intra-area correlation patterns , and moderately fit inter-area and inter-hemisphere patterns .", "The average individual subject variance explained by the full model ranged between 40% and 68% for intra-areal comparisons and between 11% and 28% for inter-areal comparisons .", "Across area pairs , there were consistent effects of both local and widespread predictors ( Figure 9—figure supplement 1 ) .", "Across subjects , the coefficients for the overlapping RF predictor were significantly positive for all intra-hemisphere , intra-areal comparisons as well as for most other comparisons ( ps < 0 . 05 , FDR-corrected ) .", "Coefficients for the eccentricity predictor were significantly positive for all but one comparison ( V1–hV4 inter-hemisphere ) ( ps < 0 . 05 , FDR corrected ) .", "Coefficients for the polar angle predictor were significantly positive for most intra-hemisphere comparisons , but only for a few inter-hemisphere comparisons ( ps < 0 . 05 , FDR corrected ) .", "Though these results suggest some effect of both local and widespread connectivity , we found that both eccentricity and polar angle predictors were strongly correlated with the overlapping RF predictor for most intra-hemisphere area pairs ( mean r = 0 . 54; STD = 0 . 08 ) .", "Since the local and widespread predictors have a non-trivial degree of collinearity , the coefficient estimates for individual predictors from the full model may not be accurate for intra-hemisphere comparisons , which had the strongest correlations between local and widespread predictors .", "From these results , it is possible that some of the observed eccentricity and polar angle effects are due to shared variance with the local , overlapping RF model .", "However , it is very unlikely that all of the widespread effects can generically be explained by the overlapping receptive field model since significant correlations were observed in the eccentricity bin analyses between iso-eccentricity representations in distinct parts of visual space ( Figure 6; e . g . , between V2d RH and V2v LH ) .", "When removing the shared variance between local and widespread connectivity predictors , significant effects of eccentricity connectivity were still observed .", "To assess the contribution of widespread connectivity on the measured correlation patterns while controlling for the shared variance with the local connectivity model , we first removed the variance explained by local connectivity ( i . e . , overlapping RF and instrumental predictors ) , then calculated correlations between the residuals and the widespread connectivity models in each subject .", "The average correlation coefficient between the residual pattern and the eccentricity predictor was similar across most areas for both within and across hemispheres ( Figure 9 , medium gray bars ) .", "The Fisher-transformed individual subject eccentricity residual correlations were reliably different from zero for 56/60 visual area pairs across rest and movie viewing experiments ( ps < 0 . 05 , FDR corrected ) .", "Consistent with the binning analyses , residual correlations that included hV4 were weaker than residual correlations between areas V1 , V2 , and V3 .", "In contrast , there was no clear , consistent effect of the polar angle predictor across areas ( Figure 9 , lightest gray bars ) .", "The polar angle residual correlations were only significantly positive for 4/60 pairs ( ps < 0 . 05 , FDR corrected ) .", "For all other comparisons , residual polar angle correlations tended to be negative .", "The residual pattern was more correlated with the eccentricity predictor than with the polar angle predictor for 49/60 pairs ( ps < 0 . 05 , FDR corrected ) ( Figure 9 ) . 10 . 7554/eLife . 03952 . 020Figure 9 . Residual correlations with eccentricity and polar angle predictors .", "( A ) Intra- and ( B ) inter- hemisphere average individual subject correlations between the unexplained variance from an overlapping RF model fit and eccentricity ( medium gray bars ) and polar angle ( light gray bars ) predictors are plotted for all pairs of visual areas V1 , V2 , V3 , and hV4 .", "Residual correlations were significantly above 0 for 56/60 eccentricity comparisons ( ps < 0 . 05; FDR corrected; one-sample t-test ) and were significantly greater than polar angle correlations for 49/60 comparisons ( ps < 0 . 05 , FDR corrected; paired t-test ) .", "Correlations between the residuals and the eccentricity predictor were comparable within and across areas , as well as within and across hemispheres , though were generally weaker for comparisons with hV4 .", "Notations above each bar denote significance relative to the null hypothesis ( one-sample t-test ) and brackets denote significant differences between conditions ( two-sample t-test ) .", "* ps < 0 . 05; ∼ps < 0 . 10 ( FDR corrected ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 02010 . 7554/eLife . 03952 . 021Figure 9—figure supplement 1 . Intra- and inter- hemisphere effects of local and widespread connectivity .", "( A ) Intra- and ( B ) inter- hemisphere group mean coefficients plotted from the linear regression with a model that included two predictors of local connectivity ( Noise and overlapping RF ) and two predictors of widespread connectivity ( Eccentricity and polar angle ) .", "Significant effects of overlapping RF ( dark gray ) and polar angle ( white ) were generally observed for intra-hemisphere comparisons .", "Significant effects of eccentricity ( light gray ) were generally observed for both intra- and inter- hemisphere comparisons .", "*ps < 0 . 05 ( FDR corrected ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 02110 . 7554/eLife . 03952 . 022Figure 9—figure supplement 2 . Residual correlations with eccentricity and polar angle predictors after accounting for potential effects of BOLD signal spread .", "( A ) Intra- and ( B ) inter- hemisphere group mean correlations between the unexplained variance after simulating the spread of the BOLD signal across RFs ( via Cholesky analysis ) and eccentricity ( medium gray bars ) and polar angle ( light gray bars ) predictors are plotted for all pairs of visual areas V1 , V2 , V3 , and hV4 .", "The Cholesky analysis accounts for any additional effects of the point spread function on overlapping RF connectivity pattern .", "Residual correlations were significantly above 0 for 58/60 eccentricity comparisons ( ps < 0 . 05; FDR corrected; one-sample t-test ) and were significantly greater than polar angle correlations for 46/60 comparisons ( ps < 0 . 05; FDR corrected; paired t-test ) .", "Overall , the results were little changed from the initial residual correlations .", "Notation conventions the same as Figure 9 .", "*ps < 0 . 05; ∼ps < 0 . 10 ( FDR corrected ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 02210 . 7554/eLife . 03952 . 023Figure 9—figure supplement 3 . Effect of homotopic connectivity .", "( A ) Coefficients are plotted for inter-hemisphere comparisons from a linear regression with a model that included homotopic RF connectivity , eccentricity , and polar angle predictors .", "In contrast to the overlapping RF predictor , significant effects of homotopic RF connectivity were observed for some inter-hemisphere comparisons .", "Conventions the same as Figure 9—figure supplement 1 .", "( B ) Inter- hemisphere group mean correlations between the unexplained variance from a homotopic RF connectivity model fit and eccentricity ( medium gray bars ) and polar angle ( lightest gray bars ) predictors are plotted for all area pairs of visual areas V1 , V2 , V3 , and hV4 .", "Residual correlations were significantly above 0 for 26/30 eccentricity comparisons ( ps < 0 . 05; FDR corrected; one-sample t-test ) and were significantly greater than polar angle correlations for 28/30 comparisons ( ps < 0 . 05; FDR corrected; paired t-test ) .", "Overall , the results were similar to the initial residual correlations .", "Notation conventions the same as Figure 9 .", "*ps < 0 . 05; ∼ps < 0 . 10 ( FDR corrected ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03952 . 023 These residual widespread effects were robust to scaling of pRF sizes in the local connectivity model .", "We used the average pRF values reported across several studies to derive our model of overlapping RF connectivity , however the size of pRFs for some areas varied as much as 2× across these studies .", "To make sure that the variance in pRF estimates across studies didn't significantly affect the results , we re-ran the model analyses using pRF estimates from individual studies to generate the model of overlapping RFs ( see ‘Materials and methods’ ) .", "Using these parameters to derive the overlapping RF model had no appreciable effect on the magnitude or significance of residual correlations .", "Further , the magnitude and significance of the eccentricity-based effects were also largely unchanged when we scaled pRF estimates to account for any additional effects of the point spread function in the BOLD signal not captured in the original pRF estimate ( see ‘Materials and methods’; Figure 9—figure supplement 2 ) .", "Overall , these control analyses provide additional evidence for a widespread eccentricity-based connectivity that cannot be accounted for by local connectivity patterns .", "Widespread eccentricity-based connectivity effects were consistently observed before and after removal of local connectivity variance .", "Even when attributing all shared variance between local and widespread predictors to the local predictor , eccentricity-based effects were still observed .", "In contrast , consistent polar angle effects were generally only observed prior to removal of local connectivity variance .", "Eccentricity effects were generally stronger than polar angle both before and after removal of local connectivity variance , suggesting that this difference is driven by asymmetries in widespread , not local , connectivity patterns .", "In further support of this , the variance explained by the local connectivity model was strongly correlated with both eccentricity ( mean r across areas = 0 . 50; STD = 0 . 08 ) and polar angle ( mean r across areas = 0 . 49; STD = 0 . 07 ) predictors , and there was little difference between them ( mean r difference across areas = 0 . 01; STD = 0 . 06 ) .", "Taken together , these data suggest that connectivity effects based on overlapping RFs are comparable along eccentricity and polar angle axes ( i . e . , isotropic with respect to visual field ) , which is consistent with anatomical studies in macaques that suggest visual field coverage of local , lateral connections is generally isotropic ( Angelucci et al . , 2002 ) .", "Importantly , the pattern of widespread correlated BOLD signal beyond overlapping receptive fields reflects eccentricity organization .", "Our data suggests little effect of polar angle connectivity beyond overlapping RFs .", "As with any negative finding , we can only report that we failed to see an effect of angular connectivity between regions with non-overlapping RFs , but do not conclude with certainty that it does not exist .", "It is important to note that significant polar angle correlations were observed in the full model analysis for several area pairs .", "However , these correlations were almost entirely explained by the overlapping RF connectivity model .", "As discussed above , the variance explained by the local connectivity model was strongly correlated with both the eccentricity and polar angle models , and there was little difference between them .", "Second , the residual widespread effects were not biased by any imbalance in the number of data-points ( nodes ) across data bins .", "We conducted a control analysis where we equalized the number of nodes in each bin of the topographic model analysis .", "We used bins with 20 , 30 , and 50 surface nodes .", "For bins that contained more than the maximum number of nodes , we subsampled the data .", "In some subjects , a few bins contained less than the set number of nodes .", "We conducted the control analyses both including and excluding these bins with few nodes .", "Eccentricity and ( lack of ) polar angle residual correlations were observed in these analyses and were consistent with the original results shown in Figure 9 , and the interpretation of the results did not change .", "Thus , our analyses were sensitive enough to reveal polar angle connectivity effects , and the lack of consistent residual polar angle correlations between regions with non-overlapping RFs cannot be explained by such biases in our data .", "One of the more robust effects consistently observed in resting state correlations is that of homotopic connectivity; the BOLD signals of homologous cortical regions between hemispheres are highly correlated ( Biswal et al . , 1995 ) .", "Recent studies have demonstrated homotopic connectivity in visual cortex with respect to visual field representation ( Heinzle et al . , 2011; Butt et al . , 2013 ) .", "Homotopic connectivity was evident in our data when plotting inter-hemisphere correlations with respect to the radial and angular distance after reflecting the position coordinates of one hemisphere across the vertical meridian onto the other hemifield ( Figure 8—figure supplement 2 ) .", "Indeed , for inter-hemisphere comparisons , the homotopic RF predictor generally captured more variance than the overlapping RF predictor .", "We tested whether homotopic connectivity ( with respect to the vertical meridian ) could account for the inter-hemisphere eccentricity-based connectivity effects .", "We removed all variance explained by the homotopic RF predictor then recomputed the residual correlations .", "The eccentricity effects were still present in these residual correlations ( Figure 9—figure supplement 3 ) , though intra-areal hV4 correlations were notably weaker .", "This was to be expected as the eccentricity and overlapping RF predictors were highly correlated for intra-areal hV4 , and removal of the local connectivity variance also removed a large portion of the variance attributable to the eccentricity predictor .", "Overall , these data show that the eccentricity-based connectivity effects were not driven by homotopic RF connectivity .", "The variance explained by the overlapping RF model ( after accounting for patterns attributable to the ‘noise’ model ) was generally greater than the variance explained by the model of residual widespread connectivity ( after removal of overlapping RF connectivity effects ) for within hemisphere comparisons ( mean ratio across areas , conditions = 1 . 69:1 . 00 ) .", "For between hemisphere comparisons , variance explained by the overlapping RF model ( after accounting for patterns attributable to the ‘noise’ model ) was weaker than the variance explained by the model of residual widespread connectivity without accounting for mirror symmetrical connections ( mean ratio across areas , condition = 1 . 00:5 . 65 ) , but was generally greater when accounting for these connections ( mean ratio across areas , condition = 1 . 27:1 . 00 ) .", "In summary , these data demonstrate that both topographically local and widespread connectivity patterns significantly contributed to the observed correlation patterns across V1 , V2 , V3 and hV4 .", "Critically , the eccentricity correlation patterns explained an additional , significant amount of variance in our data , which was not accounted for by the local connectivity model .", "For most inter-hemispheric comparisons , adding the eccentricity predictor more than doubled the variance explained by the local connectivity model , suggesting that it was the major component of the correlation patterns between hemispheres .", "Notably , after accounting for overlapping pRF connectivity , the strength of eccentricity correlations were similar within and across hemispheres for most comparisons between V1 , V2 , and V3 , suggesting that this widespread connectivity pattern uniformly spans the whole visual field ." ], [ "We investigated the spatial pattern of functional correlations across eight human early visual and extrastriate cortical areas using fMRI during conditions in which there was little or no external input ( resting-state ) and during conditions in which there was a dynamic external input ( movie viewing ) .", "Local and widespread ( spatial ) patterns of correlated BOLD signal were observed in all experiments , both in the presence and absence of visual input .", "In agreement with prior reports , we observed topographically-local correlation patterns based on overlapping representations of visual space .", "In addition , we found strong evidence for topographically-widespread correlation patterns based on eccentricity within and across hemispheres , which spanned the entire visual field .", "The eccentricity-based correlation patterns were strongest between early visual areas ( V1–V3 ) .", "The effects observed in extrastriate areas ( hV4 , V3A–B , and VO1–2 ) were generally weaker than early visual areas , consistent with the coarser topographic organization of visual space and smaller surface area in these areas .", "Our data extend recent findings of topographic connectivity within visual cortex using fMRI .", "At a fine-scale , correlation patterns reflect overlapping RFs within hemispheres and homotopic connections between hemispheres ( Heinzle et al . , 2011; Haak et al . , 2012; Butt et al . , 2013 ) .", "By modeling V1 inter-areal connections based on overlapping RFs , Gravel and colleagues ( 2014 ) generated polar angle and eccentricity maps for V2 and V3 .", "However , Raemaekers and colleagues ( 2014 ) reported that such fine-scaled connectivity was only observable after filtering coarse-scale components .", "At a coarse scale , there has been evidence for a general foveal-peripheral distinction ( Vincent et al . , 2007; Nallasamy and Tsao , 2011; Smith et al . , 2012; Raemaekers et al . , 2014 ) , but these studies did not report a systematic topography of correlation patterns that reflects the underlying eccentricity organization , nor were effects of overlapping RFs and cortical distance controlled .", "Evidence for a more systematic relation to eccentricity was reported between V1 and ventral V3 ( Yeo et al . , 2011 ) , though such data are also consistent with overlapping RF connectivity as well as cortical distance since only ventral V3 was probed .", "Here , we explicitly and quantitatively combined these connectivity phenomena in a fine-grained analysis across a wider range of brain regions , replicating previous findings of overlapping RF and homotopic correlation patterns without filtering coarse spatial components ( Heinzle et al . , 2011; Haak et al . , 2012; Butt et al . , 2013; Gravel et al . , 2014 ) .", "Even after accounting for these patterns , we found a robust widespread correlation pattern that reflects the eccentricity organization across much of visual cortex .", "Such systematic correlation patterns suggest orderly integration processes across the whole visual field at multiple levels of the processing hierarchy , not just a foveal-peripheral dichotomy .", "Eccentricity-based correlation patterns may reflect an intrinsic functional organization of visual cortex .", "Our results during rest demonstrate that regions with iso-eccentricity representations are likely to be co-active , even in the absence of visual input .", "Our results during the movie viewing condition demonstrate that the temporal dynamics yielding these eccentricity-based correlation patterns are also present during strong bottom-up input , and indicate that this organization is relevant for the processing of incoming visual input .", "In agreement with these findings , studies in macaques and cats have shown that the activity of neurons with similar response properties are correlated in the presence and absence of external input , suggesting that spontaneous neuronal activity is tightly linked to intrinsic cortical networks ( Arieli et al . , 1995; Tsodyks et al . , 1999; Goldberg et al . , 2004; Ghuman et al . , 2013 ) .", "Further , both overlapping RF and eccentricity-based connectivity patterns were observed in the presence and absence of external input , suggesting that topographically-local and widespread patterns are both part of this intrinsic functional architecture .", "The observed patterns of functional connectivity may reflect both direct and indirect anatomical connectivity ( Vincent et al . , 2007; Honey et al . , 2009 ) .", "Local connectivity is likely supported by direct anatomical connections between overlapping RFs ( Cragg , 1969; Essen and Zeki , 1978; Maunsell and Van Essen , 1983 ) .", "Such wiring is necessary for the integration of information within focal points of our visual environment .", "Direct intra-areal anatomical connections between dorsal and ventral visual cortex at the horizontal meridians ( Jeffs et al . , 2009 ) and between both hemispheres at the vertical meridians ( Hubel and Wiesel , 1967; Essen and Zeki , 1978; Newsome and Allman , 1980; Cusick et al . , 1984; Kennedy et al . , 1986 ) could support widespread functional connectivity across the visual field , though we are not aware of anatomical studies explicitly reporting eccentricity-based patterns of intra-areal connectivity .", "Further , while labeled cells of lateral connections in macaque striate and extrastriate cortex exhibit some anisotropy with respect to the cortical surface , this is thought to reflect cortical magnification factor , and yield isotropic visual field coverage ( Angelucci et al . , 2002 ) .", "Consistent with the anatomical connectivity , we found that correlation patterns between regions with overlapping RFs were comparable along eccentricity and polar angle dimensions .", "Beyond overlapping RFs , correlation patterns were anisotropic ( with respect to visual field coverage ) and reflected the underlying eccentricity organization .", "Alternatively , the observed eccentricity-based correlation patterns may actually reflect a broader-scale anatomical organization of direct ( and indirect ) connections , facilitated via differences in intra-areal projections between cortical sites representing central and peripheral space ( Colby et al . , 1988; Nakamura et al . , 1993; Gattass et al . , 2005; Ungerleider et al . , 2008 , 2014 ) .", "Such a distinction has been characterized in the patterns of supra-areal anatomical connections between early visual and extrastriate cortex in non-human primates ( Rosa , 2002; Gattass et al . , 2005; Rosa and Tweedale , 2005; Rosa et al . , 2009; Buckner and Yeo , 2014 ) .", "It is not known whether these anatomical connectivity patterns are ‘bi-modal’ , and only distinguish central and peripheral space , or reflect a finer-scale organization where connectivity patterns with intermediate eccentricity representations are distinguishable from central and peripheral connectivity profiles .", "Our results predict that these anatomical connections should reflect a gradient , though this remains to be explored .", "In particular , feedback projections from extrastriate areas with receptive fields covering wide swaths of the visual field to early and intermediate visual areas could facilitate such widespread , eccentricity-dependent correlation patterns .", "It is interesting to note that when comparing the profile of anatomical connectivity between V2/V4 and higher order cortex ( e . g . , Figure 7 , Gattass et al . , 2005 ) with the organization of eccentricity across visual cortex in macaques ( Brewer et al . , 2002; Kolster et al . , 2009; Arcaro et al . , 2011 ) , it is clear that higher order areas connected with peripheral parts of V2 and V4 ( e . g . , PO , PIP , LIP , DP , TF ) have a large representation of the periphery , and higher order areas connected with foveal parts of V2 and V4 ( e . g . , TEO and TE ) have a large representation of the fovea .", "The exact relationship between the observed correlation patterns and anatomical pathways will need to be further investigated .", "Our data link the functional organization of early and higher order visual cortex .", "Previous studies have proposed eccentricity as a large-scale functional organizing principle for higher order visual cortex ( Levy et al . , 2001; Hasson et al . , 2002; Malach et al . , 2002 ) .", "Higher order areas with foveal biases tend to be specialized in face and object recognition , and areas with peripheral biases tend to be involved in scene analysis ( Levy et al . , 2001; Hasson et al . , 2002; Malach et al . , 2002 ) .", "Perceptually , these recognition processes require different visual acuities .", "For example , while fine acuity is needed for the featural discrimination among similar face and object exemplars ( Fiorentini et al . , 1983; Goffaux et al . , 2005; Keil , 2008 ) , a coarser acuity is needed for mapping the surrounding layout necessary for navigation in space ( Oliva and Schyns , 1997; Oliva and Torralba , 2006 ) .", "Further , eye movement patterns during scene perception are related to the types of information within a scene ( Buswell , 1935; Henderson and Hollingworth , 1999 ) .", "People tend to foveate on faces while orienting their peripheral vision at landscape features and room contours ( Yarbus , 1967 ) .", "These eccentricity biases are also reflected in the connectivity patterns between face and place category-selective regions in ventral temporal cortex with extrastriate visual area hV4 ( Baldassano et al . , 2012 ) .", "Our data show that this divergence in the computational processes necessary for foveal and peripheral recognition is evident even in early visual cortex .", "Our results underscore the importance of relating functional connectivity data to known functional ( or anatomical ) organization ( Jbabdi et al . , 2013; Sporns and Honey , 2013; Wang et al . , 2013 ) .", "The detailed retinotopic organization of visual cortex allowed for a unique opportunity to systematically compare patterns of correlated BOLD activity with the known underlying functional organization of the visual system .", "Across subjects and experiments , correlations were stronger between areas at matched eccentricities than within areas at large eccentricity distances , suggesting that functional connectivity analyses on BOLD data are more sensitive at revealing widespread , inter-areal connectivity patterns than the localization of individual retinotopic areas ( see also Yeo et al . , 2011 ) .", "Alternative connectivity approaches not based on similarity may prove useful at revealing area boundaries ( Wig et al . , 2014 ) , though this remains to be tested more thoroughly beyond the V1/V2 border .", "Thus , we propose that relating correlation patterns to known functional and anatomical data will prove important for identifying the functional pathways for the integration of information across individual , functionally specialized areas ." ], [ "14 subjects ( aged 24–34 years , six females ) participated in the study , which was approved by the Institutional Review Board of Princeton University ( Resting State & Retinotopy Experiments: IRB#4616 , Movie Viewing Experiments: IRB#5516 ) .", "All participants were in good health without history of psychiatric or neurological disorders and gave their informed written consent to participate in the study and consent to publish in accordance with ethical standards set out by the Federal Policy for the Protection of Human Subjects ( or ‘Common Rule’ , U . S . Department of Health and Human Services Title 45 DFR 46 ) .", "Subjects had normal or corrected-to-normal visual acuity .", "All participants were experienced MRI subjects that were well trained to maintain central fixation for several minutes at a time while lying still during scans .", "All subjects participated in three scanning sessions , during which resting state scans were collected , high-resolution structural images were acquired for cortical surface reconstructions , and polar angle and eccentricity measurements were obtained to delineate retinotopic areas .", "11 of these subjects viewed movie clips in a single additional scanning session .", "Each subject participated in two versions of resting state: ( 1 ) fixation and ( 2 ) eyes closed .", "During the fixation scans , subjects were instructed to maintain fixation on a centrally presented dot ( 0 . 3° diameter ) overlaid on a mean grey luminance screen background for 10 min .", "During the eyes closed scans , the projector was turned off and subjects were instructed to keep their eyes closed for 10 min .", "Two runs were collected per resting condition .", "11 subjects viewed an audiovisual movie clip from the film Dog Day Afternoon .", "Subjects were instructed to attend to the movie , but maintain fixation on a centrally presented dot ( 0 . 3° diameter ) .", "Movie stimuli subtended 20° horizontally and 16° vertically .", "Two runs were collected per condition with each run lasting 5 min 45 s .", "Polar angle and eccentricity representations were measured using a standard traveling wave paradigm consisting of a colored checkerboard wedge or annulus , respectively ( Swisher et al . , 2007; Arcaro et al . , 2009 , 2011 ) .", "For eccentricity mapping , the annulus increased on a logarithmic scale over time in size and rate of expansion to approximately match the human cortical magnification function in early visual cortex ( Horton and Hoyt , 1991; Swisher et al . , 2007 ) .", "Using a logarithmic scale yields a roughly even distribution of eccentricity phases across the cortical surface for early visual areas V1 and V2 ( Hansen et al . , 2007; Swisher et al . , 2007; Schira et al . , 2009 ) .", "Stimuli mapped the central 15° of the visual field .", "Due to limitations of the scanner bore size and viewing angle , peripheral representations beyond 15° were not mapped nor included in any analyses .", "Each run consisted of eight 40 s cycles .", "For each subject , 4–5 polar angle runs and 2–3 eccentricity runs were collected .", "Early visual and extrastriate areas V1 , V2 , V3 , hV4 , V3A–B , VO1–2 were defined using standard criteria reported previously ( Sereno et al . , 1995; DeYoe et al . , 1996; Engel et al . , 1997; Brewer et al . , 2005; Wandell et al . , 2007; Arcaro et al . , 2009 ) .", "For more details , see Arcaro et al . ( 2009 , 2011 ) .", "Data were acquired with a 3T Skyra magnetic resonance imaging ( MRI ) scanner ( Siemens , Munich , Germany ) using a 16-channel head coil .", "All functional acquisitions used a gradient echo , echo planar sequence with a 64 square matrix ( slice thickness of 4 mm , interleaved acquisition ) leading to an in-plane resolution of 3 × 3 mm2 ( field of view [FOV] , 192 × 192 mm2; GRAPPA iPAT = 2; 32 slices per volume for resting state and 27 for movie stimuli; repetition time [TR] = 1 . 8 s for resting state and 1 . 5 s for movie scans; echo time [TE] = 30 ms; flip angle = 72° ) .", "High-resolution structural scans were acquired in each scan session for registration to surface anatomical images ( MPRAGE sequence; 256 matrix; 240 × 240 mm2 FOV; TR , 1 . 9 s; TE 2 . 1 ms; flip angle 9° , 0 . 9375 × 0 . 9375 × 0 . 9375 mm3 resolution ) .", "Data were analyzed using AFNI ( Cox , 1996 ) ( http://afni . nimh . nih . gov/afni/ ) , SUMA ( http://afni . nimh . nih . gov/afni/suma/ ) , MATLAB ( The MathWorks Inc . , Natick , MA ) , and FreeSurfer ( Dale et al . , 1999; Fischl et al . , 1999a ) ( http://surfer . nmr . mgh . harvard . edu/ ) .", "Functional data were slice-time and motion corrected .", "Motion distance ( estimated by AFNI's 3dvolreg ) did not exceed 1 . 0 mm ( relative to starting head position ) in any of the six motion parameter estimates ( three translation and three rotation ) during any run for any subject .", "In preparation for correlation analyses , several additional steps were performed on the data: ( 1 ) removal of signal deviation >2 . 5 SDs from the mean ( AFNI's 3dDespike ) ; ( 2 ) temporal filtering retaining frequencies in the 0 . 01–0 . 1 Hz band; ( 3 ) linear and quadratic detrending; and ( 4 ) removal by linear regression of several sources of variance:", "( i ) the six motion parameter estimates ( three translation and three rotation ) and their temporal derivatives ,", "( ii ) the signal from a ventricular region , and", "( iii ) the signal from a white matter region .", "Removal of ventricular and white matter signal resulted in a general , broad decrease in the raw correlation values by about 0 . 2 , though subsequent eccentricity-specific effects were slightly increased .", "These are standard preprocessing steps for resting-state correlation analyses ( e . g . , Vincent et al . , 2007; Yeo et al . , 2011 ) , though our results were not dependent on these preprocessing steps , and correlation analyses on the raw data yielded qualitatively and statistically similar results .", "Global mean signal ( GMS ) removal was not included in the analysis reported here given concerns about negative correlations ( Fox et al . , 2009; Murphy et al . , 2009; Saad et al . , 2012 ) , though inclusion of GMS removal yielded statistically similar results .", "To minimize the effect of any evoked response due to the scanner onset , the initial 21 . 6 s and 19 . 5 s were removed from each rest and movie scan , respectively .", "All voxels that fell between the gray and white matter boundaries were mapped to surface model units ( nodes ) .", "Only for figure illustrations from single seed correlation analyses , data were spatially filtered using a Gaussian filter to a maximum smoothness of 4 mm full-width at half-max ( FWHM ) ( by estimating the FWHM before spatial filtering ) , ensuring uniformity across the surface and maintaining spatial specificity while increasing the signal-to-noise ratio ( SNR ) ( Chung et al . , 2005 ) .", "No such spatial filtering was applied on data used for eccentricity bin or topographic model regression analyses .", "The timeseries from all surface nodes spanning early visual and extrastriate areas V1 , V2 , V3 , hV4 , VO1–2 ( combined ) , V3A–B ( combined ) , were extracted into MATLAB for correlation analyses .", "Eccentricity measurements were coarse for the surrounding visual cortex , so no additional extrastriate visual areas ( e . g . , LO1/2 , TO1/2 , PHC1/2 , IPS0-5 ) were included in the analyses .", "Each subject's reconstructed cortical surface was warped to the Buckner40 template in Freesurfer ( Fischl et al . , 1999b ) and then resampled in SUMA using an icosahedral shape to generate a standard mesh with a constant number of co-registered nodes ( Argall et al . , 2006 ) .", "Phase and correlation maps were converted from individual surface space to the standard-mesh surface to generate group average data .", "Co-registered correlation maps were averaged across subjects to derive group average maps for the four seed locations .", "To visualize correlations as a function of visual field representation , individual subject radial and angular position data were converted to a 30 × 30 Cartesian grid space .", "Correlations were grouped as a function of visual field position on the grid ( rounded to the nearest whole number ) .", "Multiple correlation values for the same visual position in individual subjects were averaged , and then correlation maps were averaged across subjects .", "For each subject , nodes were grouped by visual area ( V1 , V2 , V3 , hV4 , V3A , V3B , VO1 , VO2 ) for right and left hemispheres separately .", "Visual areas V1 , V2 , and V3 were separated into dorsal and ventral parts .", "Given the smaller surface area relative to V1 , V2 , and V3 , visual areas V3A and V3B ( as well as VO1 and VO2 ) were grouped together to increase the total number of samples ( nodes ) .", "Due to the cortical magnification factor of early visual cortex , there were a limited number of voxels representing the periphery beyond 12 . 50° .", "Therefore , we restricted our analyses to the central 12 . 50° , and we refer to representations >10° eccentricity as peripheral-most .", "To increase signal-to-noise and control the extent of spatial signal blur , nodes were subdivided into 12 bins spanning 0 . 50°–12 . 50° eccentricity for each visual area .", "Eccentricity values from a log-scaled stimulus were used for the current analyses because the cortical magnification factor in early visual cortex is accounted for , yielding an approximately uniform distribution of nodes ( i . e . , data points ) across eccentricity bins .", "The boundaries between bins corresponded to: 0 . 50° , 0 . 84° , 1 . 24° , 1 . 71° , 2 . 27° , 2 . 93° , 3 . 71° , 4 . 63° , 5 . 73° , 7 . 02° , 8 . 55° , 10 . 36° , and 12 . 50° .", "For each visual area , the timeseries of all nodes within each eccentricity bin were averaged to derive a mean timeseries for each eccentricity bin .", "In each subject , Pearson correlation coefficients were calculated between the mean timeseries of all eccentricity bins within as well as between visual areas , both within and between hemispheres .", "For each pair of visual areas , matrices were created containing all possible correlations between eccentricity bins .", "For each subject , these correlation matrices were created for each run separately and then averaged .", "Group average correlation matrices were also calculated for each area pair in each task ( resting-fixation , resting-eyes shut , and movie-fixation ) .", "The magnitude of coefficients varied considerably between area pairs ( e . g . , correlation coefficients between V1 and V2 were larger than between V1 and hV4 at matched eccentricity bins ) .", "In order to illustrate the consistency in the pattern of eccentricity bin correlations across matrices ( Figures 2C , 3 only ) , coefficients were z-score normalized for each area pair separately .", "This preserved the relative differences in correlations between eccentricity bins within each matrix , but removed large magnitude differences between matrices .", "Non-normalized correlation matrices were used for all subsequent analyses and statistics .", "To ensure that the log-scaled eccentricity stimulus was not confounding the results , analyses were also run using eccentricity values that were converted into visual degrees .", "Comparable eccentricity-based effects were observed in this control analysis , though larger eccentricities had relatively fewer nodes per bin , and correlations with these bins were therefore more variable .", "Next , a ranked radial distance was calculated for each bin pair such that a radial distance of 0 corresponded to bin pairs with the same eccentricity value ( iso-eccentricity ) and 11 corresponded to pairs containing the foveal and peripheral-most bins .", "A radial distance matrix was created containing the differences between all eccentricity bin pairs ( Figure 2C , left ) .", "To assess the relationship between the eccentricity structure and correlation patterns within the visual system , individual subject correlation matrices were correlated with this radial distance matrix .", "Correlation coefficients within each matrix were then grouped as a function of radial distance .", "This yielded several correlation estimates for each radial distance value .", "Grouped correlation coefficients were then averaged to yield a single , mean correlation coefficient for each radial distance from 0–11 .", "Correlations were Fisher z-transformed for statistical tests .", "For each visual area pair , two-tailed t-tests were performed on the Fisher-transformed 0–distance correlations to assess whether the subject population reliably differed from", "0 . False Discovery Rate ( FDR ) corrections were applied for each condition separately ( Benjamini and Hochberg , 1995 ) .", "A linear regression was performed across all distance correlations ( 0–11 ) for each visual area pair in each subject .", "For within area correlations ( e . g . , V1 to V1 ) , 0 distance coefficients were excluded from the regression since the coefficients reflected correlations between identical timeseries ( i . e . , mean coefficients were always a value of 1 ) .", "The slopes were used to evaluate the strength of eccentricity-based correlations between area pairs and across conditions .", "Correlation coefficients and slopes were then averaged across subjects to derive group mean distance correlations and group mean slopes for each visual area pair .", "Statistical significance of the group mean slopes was tested using a non-parametric permutation test in which the radial distance values were shuffled prior to grouping of individual correlations .", "For each iteration , the same label shuffling was applied to all subjects .", "Linear regression analyses were performed on each subject's permuted data and the mean slopes were calculated .", "This permutation was run 10 , 000 times .", "For all area pairs , the mean slopes ( averaged across subjects ) from the non-permuted data were larger than 97 . 5% of slopes from the mean permuted data ( i . e . , significant for a two-tailed test with α = 0 . 05 ) .", "As observed with the binning analysis , the overall magnitude of mean radial distance coefficients broadly varied across visual area pairs ( e . g . , coefficients between V1 and V2 were much larger than between V1 and hV4 at matched eccentricity differences ) and across conditions .", "Such variability in correlation strength was orthogonal to the focus of the current study .", "To minimize this magnitude variability , but preserve the relation of correlations across radial distances for illustration purposes ( Figures 6 , 7 ) , correlation coefficients were normalized to the mean correlation coefficient at 0–distance ( i . e . , iso-eccentricity ) in individual subjects as follows:dX=1− ( r0distance−rXdistance ) , where r0 is the average correlation value at iso-eccentricity and dX is the correlation normalized to the average correlation at iso-eccentricity .", "This yielded a scale where 1 equals the correlation value of 0–distance .", "Values smaller ( or larger ) than 1 indicate that the correlations decrease ( or increase ) with greater radial distance .", "Since this was a simple subtraction , the relative coefficient differences between radial distances were identical to the non-normalized coefficients ( i . e . , preserves the slope ) .", "Importantly , the non-normalized coefficient values at and near the 0–distance were always significantly positive .", "Slight negative coefficients ( between 0 . 00 and −0 . 15 ) were only observed for a few visual area pairs at large radial distance ( i . e . , between foveal and peripheral-most ) .", "The relation of local and widespread connectivity models to the observed correlation patterns was quantified across visual areas V1 , V2 , V3 , and hh using regression .", "V3A–B and VO1–2 were excluded from these analyses due to the lack of published data on their population receptive fields ( pRFs; Dumoulin and Wandell , 2008 ) .", "To compare the widespread eccentricity connectivity with other models of connectivity , each hemifield map was separated into six divisions of eccentricity , each containing six divisions of polar angle , yielding a total of 36 bins for each hemifield representation .", "Two spatial patterns of topographically local connectivity and two patterns of topographically widespread connectivity were generated:A .", "Topographically local connectivity:Instrumental ‘noise’ connectivity ( NSE ) : subject-specific predicted ‘noise’ correlations that are assumed to be non-neuronal in nature , and could result from any biases introduced from data acquisition and analyses , as well as the intrinsic spatial signal spread in the BOLD imaging ( Bandettini , 2009 ) .", "The point spread function at 3T has been estimated to be about 3 . 5 mm ( Engel et al . , 1997 ) .", "To simulate the spatial autocorrelation in BOLD imaging at 3T , the timeseries of each voxel was replaced with a randomly generated , un-correlated timeseries for each subject's data , and a Gaussian filter with a kernel of 3 . 5 mm was applied to the data .", "These artificial data were passed through the same processing steps as the real data to derive estimated ‘noise’ correlations for each subject . Point-to-point RF connectivity ( RF ) : predicted correlations based on overlap of receptive fields in visual space .", "Due to the log-scaling used for eccentricity mapping , phase measurements were converted to visual degrees for this analysis .", "The receptive field of each node was calculated using a two-dimensional circular Gaussian spread ( Dumoulin and Wandell , 2008 ) :g ( x , y ) =Aexp−[ ( x−x0 ) 2+ ( y−y0 ) 2]/2σ2 , where ( x0 , y0 ) is the visual field representation of a given node ( in Cartesian coordinates ) , A is normalization constant to ensure integration unity , and σ is the Gaussian spread inferred from previously published pRF measurements ( Dumoulin and Wandell , 2008; Amano et al . , 2009; Harvey and Dumoulin , 2011; Heinzle et al . , 2011 ) such that the integral of g ( x , y ) is", "1 . Specifically , a linear relationship was estimated between pRFs and eccentricity from the minimum and maximum eccentricities reported for each area .", "For any given area , pRF sizes varied across studies .", "To best approximate the pRF sizes from these prior reports , the average slope and intercept across reports was used , though analyses using individual slopes and intercepts from each of the prior reports yielded qualitatively and statistically similar results .", "On average , the size of the pRFs for 0 . 5° and 12 . 5° eccentricities were calculated as 0 . 4° and 1 . 6° for V1 , 0 . 48° and 2 . 3° for V2 , 1 . 0° and 4 . 15° for V3 .", "For hV4 , the size of the pRFs for 0 . 5° and 12 . 5° eccentricities were calculated solely from Harvey and Dumoulin ( 2011 ) as 1 . 2° and 5 . 8° .", "pRFs were constructed for each node .", "Individual node pRFs were binned and averaged to construct a response field for each bin .", "Signal spread between bins was then calculated as the amount of response field overlap between bins relative to the total response field area of the bin pair . B .", "Topographically widespread connectivity:Eccentricity connectivity ( Ecc ) : predicted correlations based on radial distance .", "Bin pairs were assigned a value between 0 and 1; correlations were then assumed to be linearly proportional to the difference in eccentricity representations with iso-eccentricity representations assigned a value of 1 . Polar angle connectivity ( Pol ) : predicted correlations based on angular distance .", "Bin pairs were assigned a value between 0 and 1; correlations were then assumed to be linearly proportional to the difference in polar angle representations with iso-polar angle representations assigned a value of", "1 . The contribution of these four spatial patterns on the measured correlations was assessed using linear least-squares regression:C ( x , y ) =A+β1∗NSE ( x , y ) +β2∗RF ( x , y ) +β3∗Ecc ( x , y ) +β4∗Pol ( x , y ) +ε ( x , y ) , such that for any two area pairs ( x , y ) , the correlation pattern C ( x , y ) is the linear weighted sum of four modeled sources , NSE ( x , y ) , RF ( x , y ) , Ecc ( x , y ) & Pol ( x , y ) , with separate parameter coefficients ßx , a constant A , and some measured error ε .", "Intra- and inter-hemisphere patterns were assessed separately .", "The results did not statistically differ using iterative reweighted least squares regression ( bi-square ) , and so we only report the ordinary least squares regression results .", "The contribution of widespread predictors ( eccentricity and polar angle ) on the measured correlation patterns was re-assessed after first removing any shared variance with the topographically local connectivity .", "In each subject , topographically local connectivity ( overlapping RF and instrumental ‘noise’ correlation ) was removed from the data ( via linear least-squares regression ) , and then the unexplained variance in the data ( residuals ) was correlated with eccentricity and polar angle predicted patterns , separately .", "Two-tailed t-tests were performed on the Fisher-transformed residual correlations to assess whether the subject population reliably differed from", "0 . Paired t-tests were performed between eccentricity and polar angle residual correlations .", "False Discovery Rate ( FDR ) corrections were applied to each condition separately ( Benjamini and Hochberg , 1995 ) .", "Previously published pRF measurements were used to create our local connectivity model .", "Though these pRF size estimates were likely influenced by the point spread function of BOLD imaging , any unaccounted effect of the point spread function could lead to an underestimation of pRF coverage and thus of overlapping RF connectivity .", "We tested whether the observed connectivity patterns could be explained by such an underestimation of overlapping pRF effects .", "For each area pair , artificial data were generated with the correlation structure of the local connectivity via Cholesky decomposition .", "Artificial data were simulated for each run of resting and movie scans in individual subjects .", "These artificial data were then spatially smoothed with a 3 . 5 mm kernel Gaussian filter to approximate the point-spread function ( Engel et al . , 1997 ) , and correlations were computed between all 36 bins to generate a new local connectivity model .", "Though the instrumental noise predictor ( NSE ) in the local connectivity model accounts for the same point spread function , this connectivity model extends the effects of increased spatial blur for inter-areal , overlapping RF connectivity .", "To create a model of homotopic RF connectivity ( with respect to the vertical meridian ) , the sign of the x coordinate was flipped for the RFs in one hemisphere .", "Overlap was calculated in the same manner as for local overlapping RF connectivity .", "Topographically local connectivity and homotopic effects were removed from the data ( via linear least-squares regression ) .", "The unexplained variance in the data ( residuals ) was then correlated with eccentricity and polar angle predictors , and statistical tests were performed on the Fisher-transformed residual correlation coefficients ." ] ]
[ "The human visual system can be divided into over two-dozen distinct areas , each of which contains a topographic map of the visual field .", "A fundamental question in vision neuroscience is how the visual system integrates information from the environment across different areas .", "Using neuroimaging , we investigated the spatial pattern of correlated BOLD signal across eight visual areas on data collected during rest conditions and during naturalistic movie viewing .", "The correlation pattern between areas reflected the underlying receptive field organization with higher correlations between cortical sites containing overlapping representations of visual space .", "In addition , the correlation pattern reflected the underlying widespread eccentricity organization of visual cortex , in which the highest correlations were observed for cortical sites with iso-eccentricity representations including regions with non-overlapping representations of visual space .", "This eccentricity-based correlation pattern appears to be part of an intrinsic functional architecture that supports the integration of information across functionally specialized visual areas ." ]
[ "Imagine you are looking out over a scenic landscape .", "The image you perceive is actually made up of many different visual components—for example color and movement—that are processed across many different areas in a region of the brain called the visual cortex .", "An important question for neuroscience is how the visual system combines information from so many different areas to create a coherent picture of the world around us .", "Many areas of the visual cortex have their own map of what we see ( the visual field ) .", "These maps allow the brain to maintain its representation of the visual field as the information passes from one processing area to the next .", "Areas that process corresponding parts of the visual field are physically interconnected , and tend to be active at the same time , which suggests that they are working together in some way .", "In addition , areas of the visual cortex that process different sections of the visual field can be activated at the same time , but it is not clear how this works .", "Here , Arcaro et al . used a technique called functional magnetic resonance imaging ( fMRI ) to image the brains of people as they watched movies and while they rested .", "The images showed that seemingly unrelated areas of the visual cortex respond in similar ways if they are processing sections of the visual field that are the same distance from the center of the person's gaze .", "For example , if you look directly at the center of a computer screen parts of the brain that process the top of the screen are active at the same time as parts that process the bottom .", "Arcaro et al . 's findings suggest that the brain uses the distance from the center of our gaze to bring together information from different areas of the visual cortex .", "This offers a new insight into how the brain assembles the many pieces of the visual jigsaw to make a complete picture .", "Future work will investigate how the architecture of the visual cortex is able to support this coupling of different areas , and how it might influence our perception of the visual world ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "biochemistry and chemical biology", "tools and resources" ]
Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin
elife-11621-v1
[ [ "Wnt proteins constitute a large family of secreted glycoproteins that control diverse aspects of embryonic development and adult homeostasis ( Logan and Nusse , 2004 ) .", "As they influence the balance between proliferation and differentiation in many cell types , they are fundamentally implicated in the biological processes with great medical importances , including bone formation ( Regard et al . , 2012 ) , immune regulation ( Yu et al . , 2010 ) , cancer ( Zimmerman et al . , 2012 ) , and stem cell renewal ( ten Berge et al . , 2011 ) .", "At least 19 Wnt proteins are present in mammals , each serving different but potentially overlapping functions ( Holstein , 2012 ) .", "All mammalian Wnts are predicted to be covalently lipidated at a conserved Ser residue ( Takada et al . , 2006; Willert et al . , 2003 ) , a modification essential to their biological activity because of its contribution to the interaction with Frizzled receptors ( Janda et al . , 2012 ) .", "Due to the strong hydrophobic property granted by the lipid moiety , however , purified Wnt proteins tend to lose their biological activity , probably due to the aggregation even in the presence of detergents ( Dhamdhere et al . , 2014 ) .", "The most general source for soluble Wnt ligands used in a paracrine-type experiments ( i . e . , exogenous addition of ligands ) is the culture supernatants of cells producing Wnts .", "In fact , L cells ( mouse fibroblastic cell line ) stably expressing Wnt3a ( Willert et al . , 2003 ) or Wnt5a ( Chen et al . , 2003 ) have been established and are already available from a public cell bank .", "Curiously , it has been known that Wnt secretion from cultured cells require the presence of bovine serum in the media ( Willert , 2008 ) .", "As a result , Wnt-containing culture media would always have to contain ~10% serum , precluding the use of such sample in biological assays that are sensitive to the factors contained in bovine serum ( e . g . , growth factors and proteases ) .", "The serum component ( s ) that support Wnt secretion has not been identified , and it is generally assumed that serum lipids render the hydrophobic Wnt proteins soluble .", "Willert et al had found that the inclusion of a detergent CHAPS at 1% can solubilize Wnt proteins and successfully developed a method to purify Wnt proteins from serum-containing media , which opened a way to apply purified Wnts to various experimental systems ( Willert , 2008 ) .", "By using this technology , several Wnt ligands are now commercially available in a purified format .", "However , the Wnt ligands thus purified contain CHAPS and are incompatible with detergent-sensitive cell-based assays .", "Moreover , CHAPS-solubilized Wnt quickly loses its biological activity upon incubation at 37°C , unless it is reconstituted into lipid vesicle ( Dhamdhere et al . , 2014 ) .", "In the present report , we identified the serum component responsible for the aqueous partitioning of Wnt proteins as a serum glycoprotein afamin rather than lipid molecules .", "Afamin and Wnt apparently makes 1:1 complex in the culture media , which is highly soluble in detergent-free buffers upon isolation but is decomposed upon the addition of 1% CHAPS .", "Afamin-Wnt complex is biologically active , indicating that afamin can deliver Wnt ligands to its receptors on cell surface .", "By using afamin as a carrier , we succeeded in preparing large quantity of biologically active Wnt3a , Wnt5a , and Wnt3 proteins with no other components , which can be stored at 4°C ." ], [ "We first systematically explored the tagging condition for the expression and secretion of mouse Wnt3a in HEK cells , and found that N-terminal addition of the 21-residue 'TARGET ( Tg ) ' tag developed in our lab ( Tabata et al . , 2010 ) ( see schematics in Supplementary file 1 ) was compatible .", "As shown in Figure 1A , conditioned medium ( CM ) from HEK cells stably expressing Tg-Wnt3a contained Wnt3a antigen at a level comparable to that from L-3a cells .", "The Wnt reporter assay revealed that both CM possessed similar activity ( Figure 1B ) , indicating that the N-terminal tagging did not impair the biological activity of Wnt3a that had been successfully secreted into the media .", "The TARGET tag system takes advantage of the specific recognition of concatenated YPGQ sequence by a monoclonal antibody P20 . 1 ( Nogi et al . , 2008 ) , allowing the rapid one-step purification of tagged protein by a buffer containing competing peptide or 40% propylene glycol .", "Affinity purification of Tg-Wnt3a from the CM was conducted on P20 . 1-Sepharose , resulting in the ~37 kDa protein band corresponding to the tagged Wnt3a appearing in the peptide-eluted fractions ( Figure 2 , lanes 7–9 ) .", "Near-complete removal of the bovine serum albumin ( BSA ) , the largest protein constituent present in the CM , was achieved during the purification , although very small amount of BSA is still contaminated in the eluted fractions .", "However , we noticed that protein bands with relative molecular masses of 70 and 25 kDa were always present in the fractions containing Wnt3a ( Figure 2 ) .", "Among them , the 70-kDa protein was reproducibly co-purified with Wnt3a at near stoicheometric fashion , while the amount of 25-kDa protein was generally much lower than Wnt3a and varied among different experiments .", "As these proteins do not appear in the elution fractions from the P20 . 1-Sepharose incubated with the control CM lacking Wnt3a , they are likely to associate and be co-purified with Wnt3a .", "The N-terminal sequence of the 25-kDa band ( GDDPQSSWDRV ) revealed that it is the bovine apolipoprotein A1 ( ApoA1; NP_776667 ) .", "This is consistent with the report by Neumann et al that a part of secreted Wnt3a is released onto ApoA1-containing HDL particles ( Neumann et al . , 2009 ) .", "In contrast , N-terminal Edman sequencing of the 70-kDa band derived a sequence of LPTQPQDVDD , which was in 100% match with the first 10 residues of a relatively unknown protein called afamin ( NP_001179104 ) . 10 . 7554/eLife . 11621 . 003Figure 1 . N-terminally tagged Wnt3a secretion from HEK cells .", "( A ) Indicated amounts of CM from the confluent L cells stably expressing untagged Wnt3a ( L-3a ) or HEK293S GnT1- cells stably expressing TARGET-tagged Wnt3a ( Tg-Wnt3a/HEK ) were subjected to a Western blotting using anti-mouse Wnt3a antibody .", "Note that Tg-Wnt3a migrate slower than the untagged Wnt3a due to the presence of extra 35-residue ( ~4 kDa ) tag sequence .", "( B ) The stable TCF reporter cells were incubated with the indicated concentration of CM for 6 hr .", "Luciferase activities in the cell lysates were determined and expressed as the relative increase from the control value obtained in the mock-treated cells .", "Data are mean ± SD of three independent experiments , in which quadruplicate determinations were made .", "See also Figure 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 00310 . 7554/eLife . 11621 . 004Figure 1—source data 1 . The Excel spreadsheet source file for Figure 1B .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 00410 . 7554/eLife . 11621 . 005Figure 2 . Affinity purification of tagged Wnt3a from the culture supernatants . A small column of Sepharose coupled with anti-TARGET tag antibody P20 . 1 ( ~3 ml ) that had been incubated with ~220 ml of CM from the confluent culture was washed with TBS , followed by elution with TBS containing 0 . 2 mg/ml competing C8 peptide .", "Ten µl sample from each fraction ( fraction size = 3 ml ) was subjected to 5–20% SDS-PAGE under nonreducing condition and stained with Coomassie Blue .", "A portion of the same gel ( fractions 7–11 ) was stained with Oriole fluorescent stain to visualize minor contaminating bands . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 005 Afamin ( AFM ) , also known as α-albumin , is a plasma glycoprotein that belongs to the albumin superfamily proteins ( Lichenstein et al . , 1994 ) .", "Little is known about the physiological function of this protein except for its potential role as a carrier of vitamin E ( Kratzer et al . , 2009 ) , and there have been no reports that suggest functional linkage between AFM and Wnts .", "In order to confirm that AFM and Wnt3a are associated in the CM , we raised a monoclonal antibody against bovine AFM and performed immunoprecipitation from the Wnt3a-containing CM .", "As clearly shown in Figure 3A , both tagged and untagged Wnt3a can be co-immunoprecipitaed with bovine AFM from the CM , indicating that Wnt3a itself but not the tag portion has ability to specifically bind to AFM .", "This result prompted us to investigate whether serum AFM represents the agent responsible for the ability of serum to allow soluble Wnt3a secretion into the medium .", "To this end , we produced and purified recombinant human AFM protein using mammalian expression system ( Figure 3—figure supplement 1 ) and tested its ability to facilitate Wnt3a secretion into the serum-free media by L-3a and stable Tg-Wnt3a-expressing HEK cells .", "As reported previously , Wnt3a was successfully secreted into the media when 5% fetal calf serum ( FCS ) is present , but is nearly absent in the supernatant when the same cells are cultured in a plain DMEM ( Figure 3B ) .", "Remarkably , addition of purified recombinant human AFM into the media restored the Wnt3a secretion in a dose dependent manner , even though the medium contained no other proteins or special additives ( Figure 3B ) .", "Albumin superfamily comprises of four members , including serum albumin , AFM , α-fetoprotein ( AFP ) , and vitamin D binding protein ( VDBP ) , all present in blood at different concentrations ( Lichenstein et al . , 1994 ) .", "We prepared all of them in recombinant form ( Figure 3—figure supplement 1 ) and tested their ability to support Wnt3a secretion .", "As shown in Figure 3C , only AFM was able to enhance Wnt3a secretion , suggesting that AFM is the major serum factor responsible for this activity . 10 . 7554/eLife . 11621 . 006Figure 3 . Serum afamin binds to Wnt3a secreted from cells .", "( A ) Serum-containing CM from the mock , L-3a , or Tg-Wnt3a/HEK cells were incubated with Sepharose beads immobilized with an anti-bovine AFM monoclonal antibody ( clone B91 ) and the immunoprecipitated materials were separated by SDS-PAGE under reducing condition , followed by Oriole Fluorescent protein stain ( left ) and anti-Wnt3a immunoblotting ( right ) .", "Positions for bovine AFM and tagged/untagged Wnt3a are shown in the right .", "Asterisks denote nonspecific serum-derived proteins .", "( B ) L-3a or Tg-Wnt3a/HEK cells were cultured in DMEM containing various concentrations of serum or purified recombinant human AFM for 5 days , and the resultant CMs ( 3 µl ) were subjected to the anti-Wnt3a immunoblotting .", "( C ) Effect of various albumin family proteins to support Wnt3a secretion from L-3a cells is evaluated as in ( B ) .", "HSA , human serum albumin; AFM , human afamin; AFP , human α-fetoprotein; VDBP , mouse vitamin D binding protein . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 00610 . 7554/eLife . 11621 . 007Figure 3—figure supplement 1 . Purified recombinant albumin family proteins . Purities of the recombinant albumin family proteins used in the experiment shown in the Figure 3C were checked by running on 5–20% SDS-PAGE under reducing ( left ) and nonreducing ( right ) conditions , followed by Coomassie Blue staining . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 007 Although many researchers have purified Wnt proteins from serum-containing culture media , no one had reported the presence of serum afamin in their preparations .", "As the critical trick for the successful Wnt purification discovered by Willert et al is the addition of 1% CHAPS at the beginning of the purification steps ( Willert , 2008 ) , we speculated that AFM-Wnt interaction may be susceptible to a detergent treatment .", "In order to test this hypothesis , the partially pure Wnt3a preparation containing AFM was subjected to size exclusion chromatography ( SEC ) on a Superdex 200 column in the presence or absence of 1% CHAPS .", "Under both conditions , the majority of the proteins formed large high-molecular weight ( HMW ) aggregates eluting at a void peak around 8–10 ml ( Figures 4A and B ) .", "In the detergent-free condition ( panels labeled as “'– CHAPS”' in Figure 4 ) , however , there is a second peak eluting at 13 . 6 ml corresponding to the size of ~100 kDa .", "Coomassie staining ( Figure 4C ) and anti-Wnt3a immunoblot ( Figure 4D ) revealed that Wnt3a protein is contained in this 100-kDa peak as well as in the HMW fractions .", "When the same set of samples were probed with anti-AFM antisera , AFM immunoreactivity was solely present in the 100-kDa peak and not in the HMW fractions ( Figure 4E ) .", "As the apparent size of this peak ( 100 kDa ) is close to the sum of the molecular masses of Wnt3a ( 37 kDa ) and AFM ( 70 kDa ) , we conclude that this peak represents the stable and monodispersed 1:1 complex formed between Wnt3a and AFM .", "In contrast to AFM , the 25-kDa ApoA1 antigen was broadly present in the HMW fractions but not in the 13 . 6-ml peak ( Figure 4F ) , suggesting that the Wnt3a trapped in HDL particles accounts for at least part of the HMW peak .", "It also became evident that the HMW fractions are enriched by Wnt3a oligomers that are covalently crosslinked by disulfide bonds , while such oligomers are absent in AFM-containing fractions ( Figure 4D ) .", "In sharp contrast to the results described above , the gel filtration profile obtained in the presence of 1% CHAPS ( panels labeled as +CHAPS ) shows completely different pattern; the Wnt3a do not co-elute with AFM any more but present in a broad range of fractions centered around ~50 kDa , with a concomitant increase in the covalent oligomer formation ( Figures 4B–D ) .", "In addition , elution positions for ApoA1 is shifted to the fractions with much smaller size ( Figure 4F ) , suggesting that the HDL particles can no longer exist under this condition .", "Based on these observations , we conclude that the specific interaction between Wnt3a and AFM is lost in the presence of 1% CHAPS , hence evaded from the discovery of its presence in the previous studies . 10 . 7554/eLife . 11621 . 008Figure 4 . AFM and Wnt3a form stable 1:1 complex in the absence of detergents .", "( A , B )", "SEC profiles of affinity-purified Wnt3a preparation in the absence ( A , –CHAPS ) or presence ( B , +CHAPS ) of 1% CHAPS .", "Graphs are expanded view of the areas indicated by dotted line box in the whole chromatograms ( insets ) .", "Elusion positions for molecular mass standards including thyroglobulin ( 669 kDa ) , ferritin ( 440 kDa ) , aldorase ( 158 kDa ) and ovalbumin ( 44 kDa ) are indicated at the top .", "Sixteen fractions collected from each chromatography are subjected to nonreducing SDS-PAGE on 5–20% gradient gels , followed by Coomassie Blue staining ( C ) or immunoblotting with anti-Wnt3a ( D ) , anti-bovine AFM ( E ) , or anti-bovine ApoA1 ( F ) .", "In ( C ) ~ ( F ) , analysis of the samples from ( A ) and ( B ) are shown in the left ( –CHAPS ) or right ( +CHAPS ) panels , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 008 We next checked if the Wnt3a complexed with AFM retains its biological activity .", "When the purified AFM-Wnt3a complex dissolved in a CHAPS-free buffer was tested in the Wnt/β-catenin-responsive TCF reporter assay using HEK293 cells stably expressing luciferase gene under the control of TCF/LEF , concentration-dependent induction of reporter was observed ( Figure 5A , left ) .", "The positive signal was detected at concentration as low as 0 . 56 nM ( 20 ng/ml ) , and reached the saturation at around 15 nM ( 500 ng/ml ) .", "In contrast , commercially available carrier-free Wnt3a protein ( R&D Systems , purified according to the method by Willert et al . ( Willert , 2008 ) ) exhibited much lower activity in the same assay condition ( Figure 5A , right ) .", "Because of the concentration range employed for this sample ( <45 nM ) to minimize the amount of CHAPS brought into the cell culture condition , it is not clear if the signal reached the saturation .", "Assuming that the maximum reporter induction achievable by two different Wnt3a preparations is identical , the EC50 value for the AFM-Wnt3a complex is estimated to be 5~10-fold lower than that for the purified Wnt3a .", "Furthermore , AFM-Wnt3a complex exhibited similar activity before and after the storage at 4°C for 1 month in PBS ( Figure 5—figure supplement 1 ) , indicating the stable nature of this preparation in the absence of any detergents . 10 . 7554/eLife . 11621 . 009Figure 5 . Wnt3a in complex with AFM is biologically active .", "( A ) Comparison between the purified Wnt3a from a commercial source and the AFM-Wnt3a complex .", "Increasing concentrations of Wnt3a proteins are added to TCF reporter cells and the Wnt signaling activities are evaluated as in Figure 1B .", "The data are mean ± SD of three independent experiments , in which quadruplicate determinations were made .", "See Figure 5—source data", "1 . ( B ) AFM-Wnt3a complex supports self-renewal of human intestinal stem cells .", "The ability of single-cell dissociated human intestinal organoids to expand was evaluated by culturing in the absence ( control ) or presence of various forms of Wnt3a preparations .", "Photographs are taken every 7 days at a low ( x2 , entire view of one 48-well , upper panels ) and the high ( x 10 , lower panels ) magnifications .", "Bar: 200 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 00910 . 7554/eLife . 11621 . 010Figure 5—source data 1 . The Excel spreadsheet source file for Figure 5A .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01010 . 7554/eLife . 11621 . 011Figure 5—source data 2 . The Excel spreadsheet source file for Figure 5—figure supplement", "1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01110 . 7554/eLife . 11621 . 012Figure 5—source data 3 . The Excel spreadsheet source file for Figure 5—figure supplement", "2 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01210 . 7554/eLife . 11621 . 013Figure 5—figure supplement 1 . Stability of the AFM-Wnt3a complex . The AFM-Wnt3a complex preparation was flash-frozen and stored at -80°C ( left panel , white bars ) or stored in a refrigerator at 4°C for 1 month ( right panel , black bars ) after the purification , and subjected to the reporter assay using HEK293T cells transiently transfected with reporter plasmids .", "Briefly , HEK293T cells seeded in 24-well plates were transfected with 50 ng of TOPFlash plasmid ( Upstate ) together with 5 ng of the Renilla luciferase construct , pRL-TK ( Promega ) .", "Eighteen hours after the transfection , culture media were gently replaced with the test reagents described above diluted in a fresh DMEM containing 10% FCS .", "The test reagents were added and incubated with the reporter-transfected cells for an additional 18 hours prior to the harvest and cell lysis .", "Reporter activities were measured using a dual luciferase reporter assay system ( Promega ) .", "The data are mean ± SD ( n = 4 ) .", "See Figure 5—source data 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01310 . 7554/eLife . 11621 . 014Figure 5—figure supplement 2 . Quantitative analysis of the human intestinal organoid growth and expansion . The organoid growth was evaluated by measuring the total areas of viable organoids per well from the photographs taken at each experimental point shown in Figure 5B .", "The data are mean ± S . E . M . from triplicates .", "See Figure 5—source data 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 014 The biological activity of AFM-Wnt3a complex was also evaluated using the human intestinal organoid culture .", "Active Wnt3a signal is required for the stem cells present at the bottom of the intestinal crypt to self-renew ( Sato and Clevers , 2013 ) , and it is established that human colonic organoids require a set of growth factors ( including Wnt3a , EGF , noggin , and R-spondin-1 ) for sustained expansion ( Sato et al . , 2011 ) .", "Incubation of the single-cell dissociated human intestinal organoids with the serum-containing CM of Wnt3a-expressing cells together with other components successfully supported the continued growth and expansion of the organoids ( Figure 5B , 'Wnt3a CM ) , while in the absence of Wnt3a ( Figure 5B , control ) the cells ceased proliferation after the second passage .", "However , purified Wnt3a obtained from a commercial source failed to show this activity when added at 300 ng/ml to the culture ( Figure 5B , Wnt3a ( R&D ) ) .", "This result is consistent with the finding by Dhamdhere et al that CHAPS-purified Wnt3a lose its activity upon prolonged incubation at 37°C ( Dhamdhere et al . , 2014 ) .", "It seems likely that continued presence of active Wnt3a during the long culture ( 7 days per passage ) is required for the expansion of the organoid .", "In contrast , AFM-Wnt3a complex was able to support organoid expansion at the same concentration ( Figure 5B , Wnt3a:AFM , and Figure 5—figure supplement 2 ) .", "When supplemented with the same concentration of AFM-Wnt3a complex in the medium for each passage , organoids were able to expand for more than 18 passages over a period of 4 months ( data not shown ) .", "As this culture media does not include any detergents , high salt , or serum components , it provides ideal experimental condition to evaluate growth , renewal , and differentiation of the intestinal or other type of stem cells for various studies .", "To check if the ability of AFM to solubilize and stabilize Wnt protein in aqueous environment is specific to Wnt3a , we established a stable HEK cell line expressing N-terminally tagged mouse Wnt5a and purified it from the CM .", "As shown in Figure 6A , Tg-Wnt5a co-purified with serum afamin by the anti-tag immunoaffinity chromatography , just like Wnt3a ( compare with Figure 2 ) .", "Furthermore , the SEC profile of the immunopurified Wnt5a preparation was essentially the same as that of Wnt3a , showing the presence of monodispersed AFM-Wnt5a complex peak eluting at around 150 kDa , in addition to the HMW aggregate fractions ( Figure 6B and Figure 6—figure supplement 1 ) .", "Finally , the AFM-Wnt5a complex thus purified was biologically active , because it induced phosphorylation of Dvl2 in NIH3T3 cells at concentrations above 10 ng/ml ( Figure 6C ) .", "Importantly , the activity of AFM-Wnt5a complex was indistinguishable with that of AFM-free Wnt5a protein freshly purified from the CM using the conventional method ( Kurayoshi et al . , 2007 ) .", "As an exogenous addition of AFM in the serum-free culture condition promoted secretion of Wnt3a from cells ( Figure 3B ) , we hypothesized that AFM may be able to enhance the production of Wnts when present during their biosynthesis within cells .", "Therefore , we evaluated the effect of AFM co-expression on the transient expression of Wnt3a in Expi293F cells .", "In order to increase the yield during the immunoaffinity purification , we changed the tag sequence attached to the N-terminus of Wnt3a to a recently developed PA tag ( construct #3 in Supplementary file 1 ) , which has shorter sequence than the TARGET tag and yet recognized by its antibody ( NZ-1 ) with very high affinity ( Fujii et al . , 2014 ) .", "Transient transfection of PA-Wnt3a alone into the suspension culture of Expi293F cells did not result in the secretion of Wnt3a ( Figure 7A . lane 1 ) , which was expected because the culture medium for this cell line did not contain serum .", "In contrast , when the cells were co-transfected with PA-Wnt3a and human AFM , ~37-kDa Wnt3a band became visible in the NZ-1 immunoprecipitates along with the associated AFM ( Figure 7A , lane 2 ) , confirming the ability of AFM to enhance Wnt production/secretion by co-expression .", "Using this co-expression system , we further evaluated the effect of AFM on the expression and secretion of various Wnt family proteins other than mouse Wnt3a and Wnt5a .", "To this end , expression constructs for all 19 members of human Wnt proteins containing N-terminal PA tag were prepared from the Open Source Wnt Project DNA collection ( Najdi et al . , 2012 ) .", "Out of the 19 PA-tagged human Wnts , 12 members ( Wnt1 , Wnt2b , Wnt3 , Wnt3a , Wnt5a , Wnt7a , Wnt7b , Wnt8a , Wnt9a , Wnt9b , Wnt10a , and Wnt10b ) could be secreted at detectable level from HEK293T cells when transfected alone in the presence of serum ( Figure 7—figure supplement 1 ) .", "These 12 Wnts were then subjected to the AFM co-expression experiments .", "As in the case of mouse Wnt3a , 12 human Wnt proteins exhibited no or very little secretion into the serum-free culture media when singly transfected into the Expi293F cells ( Figure 7B , lanes marked with – ) .", "However , their expression was enhanced upon the co-transfection with human AFM ( Figure 7B , lanes marked with + ) .", "Although the extent of the enhancement varied among different Wnts , all Wnts directly associated with AFM as evidenced by the co-immunoprecipitation experiments ( Figure 7C ) , suggesting that AFM can serve as a secretion vehicle for most if not all Wnt proteins to aid their simple purification .", "The culture media containing various Wnts in complex with AFM thus obtained were tested for their ability to activate Wnt/β-catenin signaling in TCF reporter cells .", "As shown in Figure 7D , only Wnt3 and Wnt3a showed significant activity in this assay system .", "This result contradicts with that obtained by Najdi et al who reported that , in addition to Wnt3 and Wnt3a , other Wnts including Wnt1 , Wnt7a , Wnt7b , Wnt8a , Wnt9b , and Wnt10b were capable of activating Wnt/β-catenin signaling in HEK cells to some extent ( Najdi et al . , 2012 ) .", "However , they evaluated the activity of untagged Wnts in an autocrine format by directly transfecting each Wnt into the reporter cells rather than exogenously adding the secreted Wnt proteins .", "We confirmed that some of our PA-tagged Wnts including Wnt1 , Wnt9b , and Wnt10b also showed some activity when tested in the autocrine type of assay ( Figure 7—figure supplement 2 ) .", "It is therefore possible that the discrepancy reflects the requirement of the cell-associated Wnt presentation for some Wnt subtypes to signal , which has been postulated for Wnt1 ( Green et al . , 2013 ) .", "Still , there is a possibility that the signaling property of these Wnts are attenuated by the complex formation with AFM . 10 . 7554/eLife . 11621 . 015Figure 6 . Serum AFM binds and solubilize Wnt5a .", "( A ) Purification of Tg-Wnt5a from the CM of stable HEK cell line was conducted as in the case for Tg-Wn3a , and the resulting column fractions were analyzed by SDS-PAGE and Coomassie Blue staining .", "Positions for the 70-kDa bovine AFM and the 40-kDa mouse Wnt5a are shown at the right .", "( B ) SEC profile of the tag-purified Wnt5a .", "Note that the ~150-kDa peak ( fractions 9–13 ) contains roughly equimolar amounts of AFM ( A ) and Wnt5a ( W ) proteins as judged by the non-reducing SDS-PAGE ( inset ) .", "The 40-kDa Wnt5a was present in both HMW and monodispersed fractions .", "See also Figure 6—figure supplement", "1 . ( C ) Biological activity of AFM-Wnt5a complex .", "NIH3T3 cells were incubated with either AFM-free , untagged Wnt5a ( lanes 1–4 ) or the purified Tg-Wnt5a:AFM complex ( lanes 5–8 ) at increasing concentrations .", "The level of cellular Dvl2 phosphrylation was assessed by the mobility shift of Dvl2 band in the anti-Dvl2 immunoblots using the whole cell lysates . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01510 . 7554/eLife . 11621 . 016Figure 6—figure supplement 1 . Identification of the 40-kDa band as Wnt5a . The SEC fractions 1–14 in Figure 6B were subjected to an immunoblotting with anti-mouse Wnt5a monoclonal antibody .", "The minor band migrating at ~20 kDa likely represents a truncated fragment of Wnt5a . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01610 . 7554/eLife . 11621 . 017Figure 7 . Increased production/secretion of various Wnt proteins by the co-expression with AFM .", "( A ) Expi293F cells were transiently transfected with PA-tagged mouse Wnt3a ( lane 1 ) , PA-tagged mouse Wnt3a + Tg-tagged human AFM ( lane 2 ) , or mock ( lane", "3 ) plasmids under the serum-free condition and the resultant culture supernatants were subjected to the immunoprecipitation with anti-PA tag antibody NZ-1 and analyzed on a nonreducing 5–20% SDS-PAGE gel followed by Coomassie Blue staining .", "A , Tg-hAFM; W , PA-mWnt3a .", "( B–D )", "AFM co-expression facilitates various Wnt secretion .", "N-terminally PA-tagged human Wnts are transiently co-transfected with Tg-tagged human AFM into Expi293F cells , and the culture media are immunoprecipitated with either anti-PA NZ-1 ( B ) or anti-Tg P20 . 1 ( C ) , followed by immunoblotting with biotinylated NZ-1 to visualize PA-tagged Wnts .", "In ( D ) , the same set of culture media are subjected to the TCF reporter assay as in Figure 1B , to see if they can induce Wnt/β-catenin signaling .", "The data are mean ± SE ( n = 4 ) from a representative experiment .", "See Figure 7—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01710 . 7554/eLife . 11621 . 018Figure 7—source data 1 . The Excel spreadsheet source file for Figure 7D .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01810 . 7554/eLife . 11621 . 019Figure 7—source data 2 . The Excel spreadsheet source file for Figure 7—figure supplement", "2 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 01910 . 7554/eLife . 11621 . 020Figure 7—figure supplement 1 . Expression/secretion profile of all human Wnt constructs in the presence of serum . N-terminally PA-tagged human Wnt constructs ( constructs #4 - #22 shown in Supplementary file 1 ) are singly transfected into HEK293T cells in the presence of 10% bovine serum and the resultant culture supernatants were subjected to the immunoprecipitation with NZ-1 followed by immunoblotting with biotinylated NZ-1 as in Figure 7B .", "Asterisks denote nonspecific bands present in all samples including that obtained with mock-transfected culture supernatant . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 02010 . 7554/eLife . 11621 . 021Figure 7—figure supplement 2 . Signaling activity of 19 PA-tagged Wnts in an autocrine type assay . HEK293T cells seeded in 96-well plates were simultaneously transfected with 10 ng of TOPFlash plasmid , 0 . 5 ng of the Renilla luciferase construct , and 100 ng of N-terminally PA-tagged human Wnt constructs ( constructs #4 - #22 shown in Supplementary file 1 ) using Lipofectamine2000 ( ThermoFisher Scientific , Inc . ) .", "Eighteen hours after the transfection , culture media were gently removed and the cells were washed once with PBS , followed by cell lysis and the determination of the luciferase activity using a dual luciferase reporter assay system ( Promega ) .", "The data are mean ± SD ( n = 4 ) .", "See Figure 7—source data 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 021 Taking advantage of the scalable nature of the transient expression using suspension cells , we performed large-scale expression/purification of mouse Wnt3a and human Wnt3 .", "As shown in Figure 8A , both Wnt proteins can be purified from the culture media of transfected Expi293F cells using anti-PA tag immunoaffinity resin , solely in complex with the co-expressed human AFM .", "Typically , we obtained ~170 µg mouse Wnt3a and ~120 µg human Wnt3 from a 300 ml culture media .", "Although it is difficult to tell whether AFM and Wnt can form complex before the secretion ( e . g . , in the secretary compartments ) or the complex formation is exclusively a post-secretion event ( i . e . , associated after both proteins are secreted separately ) , this result indicates that AFM does not need to be added as protein but can be simultaneously produced with Wnts in the same cells to support its secretion .", "As in the case of AFM-Wnt complex formed in the serum-containing culture media ( i . e . , Figures 2 and 6 ) , the AFM-Wnt3a/3 complex produced from cells upon co-expression primarily behaved as a monodispersed molecular species ( Figure 8B , C , fraction II ) .", "Furthermore , both Wnts are not present in the HMW peak ( fraction I ) , suggesting that the absence of serum components in the culture media is advantageous to obtain cleaner Wnt samples devoid of contaminating proteins such as HDL .", "Although the estimated molecular size for these complexes ( ~140 kDa ) is significantly different from that obtained in the previous experiment ( 100 kDa , Figure 4A ) , the recombinant human AFM used in the co-expression experiments carries one more N-glycosylation site and a long ( 36 residues ) N-terminal tag peptide compared to the serum-derived , natural bovine AFM , which may have contributed to the larger Stokes radius of the complex .", "Finally , we confirmed that AFM-Wnt3a as well as AFM-Wnt3 complexes thus produced were active in the Wnt/β-catenin signaling reporter assay , exhibiting similar concentration dependency with that of AFM-Wnt3a complex purified from serum-containing CM ( Figure 8D , E ) .", "Overall , the AFM co-expression strategy combined with the use of transient transfection of suspension cells is an excellent choice of method when the production of biologically active Wnt ligands at large quantity is required . 10 . 7554/eLife . 11621 . 022Figure 8 . Large scale production of Wnt3a and Wnt3 by using AFM co-expression method .", "( A ) A 300 ml culture of Expi293F cells were used to co-express Tg-tagged human AFM and PA-tagged mouse Wnt3a or human Wnt3 .", "The AFM-Wnt complexes were purified from the co-transfected culture media using NZ-1-immunoaffinity resin and checked for its purity on a nonreducing 5–20% SDS-PAGE gel followed by Coomassie Blue staining .", "( B , C )", "SEC profile of the NZ-1-purified AFM-Wnt3a ( B ) or AFM-Wnt3 ( C ) complex .", "The eluted material from the NZ-1 immunoaffinity chromatography ( input , same as the sample shown in ( A ) ) was subjected to the SEC on Superdex 200 , showing two peaks .", "The void peak ( fraction I ) comprised only of HMW aggregates that barely enter the top of separation gel , while the ~140-kDa peak ( fraction II ) contained AFM and Wnt3a/Wnt3 at similar amounts .", "( D , E )", "Signaling activity of AFM-Wnt complex prepared by AFM co-expression method .", "The NZ-1-purified AFM-Wnt3a ( D ) or AFM-Wnt3 ( E ) complex was subjected to the TCF reporter assay as in Figure 5A .", "The data are mean ± SD of three independent experiments , in which quadruplicate determinations were made .", "See Figure 8—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 02210 . 7554/eLife . 11621 . 023Figure 8—source data 1 . The Excel spreadsheet source file for Figure 8D and E . DOI: http://dx . doi . org/10 . 7554/eLife . 11621 . 023" ], [ "Despite their fundamental involvement in general metazoan development as well as various human diseases , Wnt proteins and their mechanism of action had evaded biochemical scrutiny for many years .", "The most enigmatic character of Wnt proteins is their hydrophobic nature due to the covalent lipid modification discovered by Willert et al ( Willert et al . , 2003 ) , which was later identified by Takada and colleagues as an acylation of conserved Ser residue with palmitoleic acid ( Takada et al . , 2006 ) .", "Although the Wnt purification method established by Willert et al set an important milestone in the Wnt research , its application to the structure-function studies had been slow due to both the technical difficulties associated with the method that involves multiple steps of large-scale chromatography , and the unavoidable presence of detergent in the final sample .", "The next breakthrough in the Wnt preparation was brought by Garcia and colleagues , who determined the crystal structure of Xenopus Wnt8 ( Janda et al . , 2012 ) .", "They expressed Wnt8 protein as a stable and tight complex with a fragment of its receptor Frizzled8 ( Fz8 ) , which sequestered the problematic acyl chain and increased the solubility of the complex .", "The structure clarified the essential role of the fatty acid moiety in the receptor recognition , and offered a first molecular view about how Wnt initiate signal outside the cell .", "However , this kind of soluble Wnt proteins cannot be used in the functional assays , because the important active site ( i . e . , the receptor binding site ) is already masked in the complex and unavailable .", "Another way of making Wnt proteins soluble in aqueous solution is to remove the acyl chain that renders the insolubility .", "In fact , Kakugawa et al have recently reported an extracellular deacylase called Notum , which removes the palmitoleic acid attached to the conserved Ser residue in Wnt3a ( Kakugawa et al . , 2015 ) .", "However , the resultant deacylated Wnt , although it may be water-soluble , lacks biological activity and its utility in the structure-function analysis is limited .", "Probably the best known method to present purified active Wnt proteins in the absence of potentially cell toxic compounds is to reconstitute them in a liposome ( Morrell et al . , 2008 ) .", "Although the liposome-packaged Wnt3a have long life at 37°C and may potentially be suitable for the in vivo delivery ( Dhamdhere et al . , 2014 ) , one still needs to produce large quantity of CHAPS-solubilized Wnt proteins , which remains technically demanding .", "We discovered that AFM has unexpected ability to solubilize/partition the lipophilic Wnt proteins in the culture media of Wnt-producing cells .", "AFM can specifically and stoicheometrically interact with several Wnt proteins , allow them to stay dispersed in aqueous solution , and keep them from inactivation/aggregation during the storage .", "As AFM is an endogenous protein abundantly present in mammalian body , complexation with AFM is an ideal format of Wnt preparation suitable for the use in various biochemical , cell biological , or even therapeutic applications .", "AFM is a member of albumin superfamily plasma proteins , which comprises of four proteins existing in blood at widely different concentrations ranging from as low as 50 ng/ml for AFP to extraordinarily high 40 mg/ml for serum albumin ( Lichenstein et al . , 1994 ) .", "AFM is present in normal adult blood at 10–30 µg/ml but its physiological function remains unknown , except for its potential role as a vitamin E transport protein ( Dieplinger and Dieplinger , 2015 ) .", "Interestingly , AFM is able to bind hydrophobic substances like other albumin family proteins , but the binding specificity seems to be unique ( Voegele et al . , 2002 ) .", "The facts that", "1 ) only AFM but not other three albumin family proteins can capture Wnts and", "2 ) the interaction between AFM and Wnt is completely disrupted by 1% CHAPS seem to indicate that AFM uses its unique lipid-binding pocket to bind and sequester the palmitoleic moiety of Wnt proteins .", "If such lipid binding specificity of AFM is responsible for this special ability , all Wnt ligands , or even non-Wnt proteins that are modified by palmitoleic acid may be protected by AFM .", "In fact , we found that 12 different Wnt proteins were able to associate with AFM , leading to the secretion from cells into a serum-free medium upon co-expression with AFM .", "The idea that AFM binds and sequester fatty acid chain of Wnts may seem to contradict with the fact that AFM-Wnt complex is highly active in cell-based assays , because the acyl chain moiety has to be available for the receptor ( i . e . , Fz ) binding to initiate signaling .", "We speculate that the affinity of AFM to the fatty acid is strong enough to make stable complex with lipid-modified Wnt but weaker than that of Fz CRD , enabling it to 'deliver' the ligand to the receptor at the cell surface .", "Although the experimental/technological importance of the activity of AFM as a Wnt carrier protein is clear , its relevance in physiology remains to be explored .", "AFM , as other albumin family proteins , is primarily synthesized in the liver and present not only in blood but also in other adult body fluids including cerebrospinal , ovarian follicular , and seminal fluids ( Dieplinger and Dieplinger , 2015 ) .", "However , nothing is known about its expression pattern or potential function during mammalian development .", "As invertebrates do not have albumin family proteins , AFM does not seem to play essential roles in the evolutionary ancient function of Wnt singaling pathway .", "Nevertheless , the Wnt-binding activity is likely to be conserved among mammalian AFM , since bovine ( Figure 3A ) , human ( Figures 3B and C ) , and mouse ( data not shown ) AFM are all capable of supporting Wnt secretion from cells .", "Brack et al reported that Wnt or Wnt-like molecule present in the serum from aged mice may be responsible for the conversion of muscle stem cells from myogenic to fibrogenic phenotypes ( Brack et al . , 2007 ) .", "It is possible that Wnt ligands present in adult body fluids play unidentified ( patho ) physiological roles under certain condition , which may be regulated by AFM through its carrier function .", "In Drosophila , there is a known Wnt carrier protein called Swim , which is a secreted glycoprotein of ~50 kDa belonging to the lipocalin family transport proteins ( Mulligan et al . , 2012 ) .", "Like AFM , Swim binds Wg ligand in an acyl chain-dependent manner , and maintains the solubility and activity of purified Wg .", "Reduction of Swim leads to a more confined distribution of extracellular Wg around the producing cells , suggesting its role in establishing the proper long-range Wg gradient .", "There are a number of other factors that can mediate transport of Wnt ligands from the secreting cells to the responding cells , including Wnt-containing exosome assembled by the Evi/Wntless-mediated Wnt trafficking ( Gross et al . , 2012 ) , large lipoprotein particles that carry not only Wnt but also other lipid modified morphogen Hedgehog ( Panakova et al . , 2005 ) , and cell surface heparan sulfate proteoglycans that serve as the reservoir for Wnt to shape the extracellular gradient ( Han et al . , 2005 ) .", "AFM may be considered as a new candidate factor involved in the regulation of Wnt distribution and presentation in mammals .", "Genetic manipulation of AFM gene in mice and their phenotypic analyses are eagerly waited ." ], [ "cDNAs coding for mouse Wnt3a and Wnt5a , monoclonal antibodies against mouse Wnt3a and Wnt5a , and L cell stably expressing mouse Wnt3a ( L-3a ) ( Shibamoto et al . , 1998 ) were all kindly provided by Shinji Takada ( Okazaki Integrated Bioscience Center , Japan ) .", "The Open Source Wnt Project plasmids containing ORF clones for human Wnt proteins ( Najdi et al . , 2012 ) were obtained from Addgene .", "A cDNA coding for the human afamin was a generous gift from Luc Bélanger ( Research Center , L’Hotel-Dieu de Québec , Canada ) .", "cDNAs coding for the following proteins were obtained by DNA synthesis based on their reported nucleotide sequences; bovine AFM ( NM_001192175 ) , human serum albumin ( BC034023 ) , human AFP ( BC027881 ) , and mouse VDBP ( AK010965 ) .", "A highly pure , carrier-free recombinant mouse Wnt3a protein was purchased from R&D Systems ( catalogue No . 1324-WNP-010/CF ) .", "HEK293T , HEK293S GnT1 ( kindly provided by G . Korhana ) , and L-3a cells are maintained in basal media containing DMEM ( for HEK lines ) or DMEM/F12_1:1 ( for L-3a ) , supplemented with 10% fetal calf serum ( FCS ) .", "All cell transfection experiments were performed using X-tremeGENE HP ( Roche ) .", "Expression constructs for mouse and human Wnt proteins with various N-terminal tags were designed to include tobacco etch virus ( TEV ) protease cleavage sequence after the tag as shown in Supplementary file 1 , and constructed by extension PCR .", "Expression plasmids for various albumin family proteins with N-terminal tag ( either Tg or PA tag ) were also constructed by the same strategy .", "The coding region for Tg-Wnt3a was subcloned into a mammalian episomal expression vector pEBMulti-Hyg ( Wako Pure Chemical , Japan ) and used to transfect HEK293S GnT1- cells .", "Transfected cells were selected against medium containing 0 . 2 mg/ml hygromycin B , and the resistant polyclonal cell population confirmed to maintain high Wnt3a-producing ability over the repeated passages were used for the protein production .", "HEK293S GnT1- cells stably producing Tg-Wnt5a were established in a similar manner .", "For the co-expression of Wnt and AFM , expression plasmids for Tg-hAFM and N-terminally PA tagged mouse or human Wnts were co-transfected at a DNA ratio of 10:1 using the Expi293 Expression System ( Life Technologies Inc . Tokyo Japan ) , according to the procedures recommended by the manufacturer .", "The culture medium was harvested at 90 hr post-transfection , and immediately used for the Wnt/β-catenin reporter assay or the protein purification .", "Purification of the TARGET-tagged Wnt proteins was conducted according to the general procedure described previously ( Nogi et al . , 2008 ) .", "Briefly , precleared CM was incubated with P20 . 1-Sepharose at 1~2 ml beads/100 ml CM at 4°C for 3 hr , followed by packaging into a small column .", "The resin was washed with 5 column volume of 20 mM Tris , 150 mM NaCl , pH 7 . 5 ( TBS ) and then eluted with 0 . 2 mg/ml C8 peptide ( PRGYPGQV ) in TBS .", "Elution peak fractions were combined and concentrated by a centrifugal ultrafiltration devise ( Spin-X UF , MWCO 10 , 000 , Corning ) .", "For further purification , if necessary , the sample was subjected to size exclution chromatography on Superdex 200 10/300GL column ( GE Healthcare ) equilibrated with TBS .", "When used in cell-based activity assays , purified samples were dialyzed against phosphate-buffered saline ( PBS ) and filter-sterilized before use .", "PA-tagged albumin family proteins were produced using the Expi293 Expression System , and purification of these proteins as well as the Tg-AFM:PA-Wnt complexes from the CM using NZ-1-Sepharose affinity chromatography was performed based on the protocol reported by Fujii et al ( Fujii et al . , 2014 ) .", "The concentration of the Wnt3a or Wnt3 in the purified AFM complex was estimated by a densitometric analysis of protein bands in the SDS-PAGE gel using BSA as standard .", "Untagged Wnt5a was purified from the CM of stable L cell line as described previously ( Kurayoshi et al . , 2007 ) .", "Monoclonal antibodies against bovine AFM were prepared by a conventional hybridoma method using the mouse myeloma cell line P3U1 and Balb/C mice immunized with the tag-cleaved bovine AFM protein .", "Of total of six independent clones , a clone B91 ( IgG1 , κ ) recognized native bovine AFM with high affinity and was compatible with the immunoprecipitation experiments ( see below ) .", "The same bovine AFM protein was used as immunogen to raise antisera in rabbits .", "To raise antisera against bovine ApoA1 , two peptide segments ( residues 1–12 , DDPQSSWDRVKD , and residues 161–174 , QLAPYSDDLRQRLT ) were selected based on their potential antigenicity and used as immunogens after conjugation with KLH .", "Antibodies were purified from culture supernatants ( for monoclonal antibodies ) or rabbit sera ( for polyclonal antibodies ) by using affinity chromatography on Protein A-Sepharose ( GE Healthcare ) .", "Purified anti-TARGET tag P20 . 1 and anti-PA tag NZ-1 antibodies ( Fujii et al . , 2014; Nogi et al . , 2008 ) were obtained from Wako Pure Chemical Co .", "Purified B91 IgG was coupled to CNBr-activated Sepharose beads ( GE Healthcare ) at a density of ~2 mg IgG/ml beads by the protocol provided by the manufacturer .", "For the immunoprecipitation of bovine AFM from the serum-containing media , a 20 µl of B91-Sepharose beads were incubated with 1 ml of various CM at 4°C for 3 h , washed three times with TBS , and boiled after the addition of 30 µl of SDS sample buffer .", "The samples were subjected to SDS-PAGE and visualized by either Oriole fluorescent protein stain ( BIO-RAD ) or anti-Wnt3a immunoblotting .", "For anti-Wnt3a and anti-Wnt5a immunoblottings , the samples were separated by SDS-PAGE , transferred to PVDF membrane , and incubated with 5 µg/ml anti-Wnt antibodies that had been biotinylated by using EZ-Link NHS-LC Biotin ( PIERCE Chemical Co . ) .", "After washing , the membranes were probed with peroxidase-conjugated Streptavidin ( VECTOR Laboratories , SA-5004 ) , followed by visualization with enhanced chemiluminescence reagent .", "All other immunoblotting experiments were conducted in a similar manner , except for the use of unlabeled primary antibodies ( 30 µg/ml for rabbit polyclonal anti-bovine AFM , 1/500 dilution for rabbit antisera against ApoA1 ) and peroxidase-conjugated secondary anti-rabbit IgG ( Sigma A-6154 , 0 . 4 µg/ml ) .", "Pull-down experiments of various Wnt proteins co-expressed with Tg-hAFM in Expi293F cells were conducted using NZ-1- or P20 . 1-Sepharose as described previously ( Fujii et al . , 2014; Nogi et al . , 2008 ) , followed by biotinylated NZ-1 immunoblotting to visualize PA-tagged Wnts .", "For the luciferase reporter assays , HEK293 cells stably integrated with a firefly luciferase gene under the control of TCF/LEF response element ( BPS Bioscience ) were used .", "Cells were suspended at ~8 105 cells/ml and seeded in 96-well plates at 50 µl/well , followed by an overnight culture before the assay .", "For the preparation of CM containing Wnt3a , stable Wnt3a-producing cells were cultured for 3~4 days until they reach 80–90% confluency .", "The harvested CM were diluted with the control CM from the similarly cultured HEK293T cells to adjust for the presence of any common cell metabolites .", "When purified Wnt3a or AFM-Wnt3a complex were used , they were diluted in PBS from the stock solution and further diluted with a fresh DMEM containing 10% FCS .", "About 50-µl of the test reagents were added to the reporter cells and incubated for 6 prior to a brief washing and cell lysis .", "Reporter activities contained in the lysates were measured using a dual luciferase reporter assay system ( Promega ) .", "Each assay was performed in quadruplicate .", "For the Dvl2 phosphorylation assay , NIH3T3 cells were cultured in the presence of varying concentrations of Wnt5a proteins .", "As the stock solution of the purified untagged Wnt5a contained 1% CHAPS ( Kurayoshi et al . , 2007 ) , AFM-Wnt5a complex devoid of detergents were preincubated with 1% CHAPS for 1 hr at 4°C .", "Both samples were diluted with the fresh media containing 1% BSA , where the final concentration of CHAPS was below 0 . 01% .", "After the treatment with Wnt5a for 2 hr , the cells were lysed in lysis buffer ( 20 mM Tris-HCl [pH 8 . 0] , 1% Nonidet P-40 , 137 mM NaCl , 10% glycerol , 1 mM phenylmethylsulfonyl fluoride , 20 µg/ml aprotinin , 20 µg/ml leupeptin , 5 mM NaF , 5 mM Na3VO4 , and 50 mM β-glycerophosphate ) and cell lysates were subjected to the immunoblotting with anti-Dvl2 antibody ( Cell signaling Technology ) .", "The supporting effect of Wnt3a on the growth and expansion of human intestinal organoid cultures were evaluated using the method established previously ( Sato et al . , 2011 ) .", "Colonic samples were obtained at Keio University Hospital with written informed consent and consent to publish .", "Normal mucosa samples were derived from tissue biopsy specimen taken from patients with colonic tumor , which were located at least 5 cm away from the edge of tumors .", "Crypts were dissociated from the samples by a combination of EDTA treatment and physical pipetting , and isolated .", "They were suspended in Matrigel , placed at the center of each well of a 48-well plate , and overlaid with 125 µl of defined serum-free medium containing a cocktail of essential factors including R-spondin ( 500 ng/ml ) , EGF ( 50 ng/ml ) , Noggin ( 100 ng/ml ) , A83-01 ( TGF-β receptor 1 inhibitor , 500 nM ) , and SB 202190 ( p38 MAPK inhibitor , 10 µM ) .", "The culture was started by the addition of the same volume of Wnt3a-containing solution , either as a fresh CM of L-3a cells or concentration-adjusted Wnt3a or AFM-Wnt3a complex in PBS , and the crypt morphology and numbers were observed daily .", "Upon the outgrowth , the organoids were passaged every week by gentle pipetting at 1:5 ratio into the fresh medium ." ] ]
[ "Wnt plays important role during development and in various diseases .", "Because Wnts are lipidated and highly hydrophobic , they can only be purified in the presence of detergents , limiting their use in various in vitro and in vivo assays .", "We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media .", "Wnt3a forms a 1:1 complex with afamin , which remains soluble in aqueous buffer after isolation , and can induce signaling in various cellular systems including the intestical stem cell growth assay .", "By co-expressing with afamin , biologically active afamin-Wnt complex can be easily obtained in large quantity .", "As afamin can also solubilize Wnt5a , Wnt3 , and many more Wnt subtypes , afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years ." ]
[ "The Wnt signaling pathway helps animal cells to communicate with each other to coordinate the formation of tissues and organs .", "The pathway relies on a protein called Wnt that is released from cells and binds to a receptor protein called Frizzled on the surface of other cells to trigger changes in gene activation .", "Defects in the Wnt signaling pathway contribute to cancer and other diseases .", "Great progress has been made in understanding Wnt signaling , but certain types of experiments have been hindered because it has been difficult to isolate pure Wnt proteins .", "This is partly because Wnt proteins are attached to a fatty molecule that is important for their activity but also makes these proteins “hydrophobic , ” or repelled by water .", "Hydrophobic proteins have a strong tendency to clump or aggregate when they are isolated from cells , which reduces the biological activity of proteins .", "Adding detergents to the aggregates can break them apart , but can also hinder the proteins’ activities and cannot be used in all experiments .", "Previous research has shown that mammalian cells grown in the presence of blood serum can produce Wnt proteins that do not aggregate .", "Blood serum is a complex mixture of different molecules obtained from blood and is commonly added to cells grown in the laboratory .", "However , adding serum can have also undesirable effects and it is not understood why serum stops Wnt proteins forming aggregates .", "Using biochemical methods , Mihara et al . have now identified the component in blood serum that prevents Wnt proteins from aggregating .", "The experiments showed that a protein in the blood serum called afamin binds tightly to Wnt proteins .", "Furthermore , the complex between afamin and Wnt was biologically active , and could bind to the Frizzled receptor and trigger an appropriate response in cells .", "Mihara et al . then generated cells that produced both afamin and Wnt and used them to purify large amounts of biologically active Wnt/afamin complexes .", "This method avoids the potentially undesirable effects of using detergents or serum , and will therefore likely be useful for future experiments and therapeutic applications .", "Further work is also needed to understand why afamin binds to Wnt proteins and whether this is important for Wnt signaling ." ]
2016
[ "Introduction", "Results and discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics" ]
Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition
elife-13027-v2
[ [ "The eukaryotic 26S proteasome is a large multi-enzyme complex that functions as the primary degradation machinery for the selective turnover of aberrant or unneeded proteins within the cell .", "Proteins targeted for degradation are covalently labeled with polyubiquitin chains , which are recognized and removed by the proteasome ( Finley , 2009 ) .", "The barrel-shaped core peptidase complex of the proteasome , which sequesters the proteolytic active sites in an internal chamber ( Groll et al . , 1997 ) , is capped on one or both ends by a regulatory particle that acts as a discriminating gateway for targeted protein substrates ( Saeki and Tanaka , 2012 ) .", "The regulatory particle consists of two sub-complexes , known as the ‘base’ and the ‘lid’ ( Glickman et al . , 1998 ) .", "The base sub-complex contains the AAA+ ATPases Rpt1-Rpt6 , which form a heterohexameric ring that drives the mechanical substrate unfolding and translocation of the unstructured polypeptides into the degradation chamber of the core peptidase .", "Docked on one side of the base is the lid subcomplex , which contains the deubiquitinating enzyme ( DUB ) Rpn11 that cleaves polyubiquitin chains from targeted substrates as an essential step in proteasomal substrate processing ( Boehringer et al . , 2012 ) .", "The lid is an asymmetric , ~370 kDa complex that consists of 9 unique subunits ( Rpn3 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , Sem1 ) and exhibits a characteristic hand-shaped organization similar to that of the eukaryotic initiation factor 3 ( eIF3 ) and the COP9 signalosome ( CSN ) ( Lander et al . , 2012; Lingaraju et al . , 2014; des Georges et al . , 2015 ) .", "At the center of the lid , six Proteasome-CSN-eIF3 ( PCI ) -domain containing subunits ( Rpn3 , 5 , 6 , 7 , 9 , 12 ) interact via their winged-helix motifs to form a horseshoe-shaped scaffold .", "The amino-terminal domains of these 6 subunits extend radially like fingers from the central PCI horseshoe .", "The essential deubiquitinase Rpn11 is positioned in the ‘palm’ of the hand-shaped lid .", "Rpn11 is an Mpr1-Pad1 N-terminal ( MPN ) -domain containing metalloprotease of the JAB1/MPN/MOV34 ( JAMM ) family and forms a heterodimer with an enzymatically inactive MPN-subunit , Rpn8 .", "With the exception of Sem1 , a small 87-residue subunit located at the interface of the N-terminal domains of Rpn3 and Rpn7 ( Bohn et al . , 2013 ) , all lid subunits contain conserved C-terminal helices that assemble into a large bundle positioned next to the MPN heterodimer of Rpn11/Rpn8 in the palm of the complex ( Beck et al . , 2012 ) .", "Previous crystallographic and biochemical studies have focused on the mechanism of Rpn11 , which acts as a highly promiscuous DUB to remove ubiquitins from the wide variety of substrates during their translocation into the proteasome , likely by cleaving the isopeptide bond between the substrate lysine and the first ubiquitin moiety of the attached ubiquitin chain ( Worden et al . , 2014; Pathare et al . , 2014 ) .", "The Rpn11/Rpn8 heterodimer is active in isolation ( Worden et al . , 2014 ) , but is significantly inhibited in the context of the lid sub-complex and regains robust DUB activity in the assembled 26S proteasome ( Verma et al . , 2002; Yao and Cohen , 2002 ) .", "The isolated Rpn11/Rpn8 heterodimer is not present at considerable levels in the cell , whereas the presence of the lid and its assembly intermediates containing Rpn11/Rpn8 have been previously observed and characterized ( Tomko and Hochstrasser , 2011 ) .", "The inhibition of Rpn11 activity in the isolated lid and its assembly intermediates might therefore be important to prevent spurious deubiquitination of proteins in the cell , given the high promiscuity of this DUB .", "It has been suggested that interactions with Rpn5 are possibly involved in Rpn11 inhibition in the isolated lid ( Lander et al . , 2012 ) , but the specifics of this regulation and the mechanism by which Rpn11 is activated upon incorporation into the holoenzyme remain elusive ( Verma et al . , 2002; Lander et al . , 2012; Yao and Cohen , 2002 ) .", "Here , we present an atomic model of the isolated lid sub-complex of the yeast proteasome , as determined by cryo-electron microscopy ( cryoEM ) ( Figure 1A–C , Table 1 , Figure 1—figures supplements 1–4 ) , revealing the molecular mechanism for direct inhibition of the DUB active site , as well as Rpn11 activation through extensive conformational changes that occur during lid incorporation into the 26S holoenzyme . 10 . 7554/eLife . 13027 . 003Figure 1 . Architecture of the isolated proteasome lid sub-complex .", "( A ) The segmented 3 . 5 Å resolution cryo-EM density is shown at a low isocontour level , with each subunit colored differently .", "Rpn3 is shown in orange , Rpn5 in light blue , Rpn6 in yellow , Rpn7 in purple , Rpn8 in red , Rpn9 in magenta , Rpn11 in green , Rpn12 in light green , and Sem1 in tan .", "This coloring scheme is maintained throughout all figures .", "( B ) The unsegmented cryoEM density is shown at a higher isocontour level ( in gold ) to demonstrate the molecular details observable in the reconstruction ( ~3Å in certain regions ) .", "The lower isocontour level used for the segmented map is overlaid as a silhouette .", "( C ) The atomic model of the proteasome lid is depicted using a ribbon representation , with each subunit colored according to the segmentation shown in A . The central location of the six PCI domains is illustrated by a gray shadow underneath the structure .", "( D ) The PCI domains form a horseshoe , held together by an 18-stranded β-sheet .", "( E ) Close-up of the helical bundle and the MPN heterodimer . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00310 . 7554/eLife . 13027 . 004Figure 1—figure supplement 1 . CryoEM data collection .", "( A ) A representative cryo-electron micrograph ( scale bar = 100 nm ) after whole-frame alignment .", "( B ) Reference-free 2D class averages of frozen-hydrated particles , showing a variety of different projection views of the lid complex .", "( C ) A Fourier transform of the representative micrograph , with a simulated CTF estimated by CTFFind3 overlaid on the right half of the image .", "Thon rings are clearly observed beyond 4Å ( white circle ) .", "( D ) A 1-dimentional plot showing the correlation of the experimental CTF ( blue ) and the estimated CTF ( black ) .", "Images having a correlation of the of greater than 50% beyond 4 Å resolution were used for subsequent processing .", "( E ) Per-frame B-factors ( Bf , top ) and intercepts ( Cf , bottom ) .", "( F ) Per-frame weighting of spatial frequencies , based on the Bf and Cf estimates shown in e .", "First and last frames are colored in yellow , and the frame with the least negative relative Bf is colored in orange ( frame 5 ) .", "Frames 4–16 are the major contributors to the high-resolution spatial frequencies . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00410 . 7554/eLife . 13027 . 005Figure 1—figure supplement 2 . Single particle analysis of the lid complex . A total of 254 , 113 particles were extracted from micrographs , scaled by a factor of 0 . 25 , and subjected to 3D classification into 8 classes in RELION .", "The particle coordinates corresponding to the four highest resolution classes that showed density for all lid components were re-centered , based on the translations determined from 3D classification .", "These coordinates were used to extract 139 , 561 unbinned particles for 3D refinement with RELION , which yielded a reconstruction of 4 . 4 Å .", "After correcting for particle motion and electron beam damage ( particle polishing ) , the resolution was improved to 4 . 1 Å .", "A 3D mask surrounding the most structurally stable regions of the map ( the PCI domains , the MPN heterodimer , and the helical bundle ) was generated , and used in an alignment-free 3D classification of the data into 3 classes .", "The 109 , 396 particles contributing to the highest resolution 3D class were used for further 3D refinement in RELION without applying a mask , yielding a 3 . 6 Å structure .", "Continued refinement of these alignment parameters using the same 3D mask that was applied earlier improved the resolution of the central regions of the structure by 0 . 1 Å .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00510 . 7554/eLife . 13027 . 006Figure 1—figure supplement 3 . Resolution assessment of the reconstructions .", "( A ) 3D angular distribution plot , shown from three orthogonal angles of the reconstruction .", "The diameter and color saturation of the spheres increases with occupancy of particles at a given Euler angle .", "( B ) Fourier Shell Correlation plots of lid reconstructions at different stages of processing .", "( C ) The final map is shown at two contour levels , colored according to a local resolution estimation using Bsoft ( Heymann and Belnap , 2007 ) .", "A lower contour level ( left ) shows the more disordered regions , while the higher contour level ( right ) shows that regions of the map were resolved to 3 Å resolution .", "An outline of the lower contour is overlaid on top of the images on the right for reference . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00610 . 7554/eLife . 13027 . 007Figure 1—figure supplement 4 . Atomic modeling of the lid sub-complex .", "( A ) The 5 top-scoring models are shown , with each subunit colored differently .", "Close-up views of the cryo-EM density in the regions within the black boxes are shown in ( B–D ) .", "Side chains in the helical bundle ( B , C ) were well-resolved , and the 5 models were largely consistent in these regions .", "At the periphery of the complex , however , structural features were less well-resolved and the models are less consistent .", "( E ) The atomic model is colored according to B-values , which are largely consistent with the local resolution map shown in Figure 1—figure supplement 3 .", "Blue regions correspond to B-values of ~50 Å2 , while red regions correspond to B-factors of ~300 Å2 .", "( F ) The FSCs between the top-scoring atomic model refined against one of the half-maps and the final 3 . 5 Å map ( dashed line ) and the two independently refined half maps ( light and dark solid lines ) are shown .", "For comparison , the FSC between the independently refined half maps is also plotted ( dotted line ) .", "The overlap of the curves in the high spatial frequencies indicate that the model was not over-fitted . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00710 . 7554/eLife . 13027 . 008Figure 1—figure supplement 5 . Subunits of the yeast 26S proteasome lid sub-complex . The well-ordered cryo-EM densities for the 9 lid subunits are shown as transparent surfaces , with the atomic models shown as ribbons .", "Side-chain density is clearly visible for the C-terminal helices of the PCI-containing domains , and the MPN-containing subunits are well-resolved throughout .", "The cryoEM density for the PCI-containing subunits become less ordered near the N-terminus of these subunits , and is only visible at lower contour levels . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00810 . 7554/eLife . 13027 . 009Figure 1—figure supplement 6 . Comparison of PCI horseshoes in different complexes . Shown are ribbon representations of the PCI horseshoes of ( from left to right ) the unincorporated lid , the incorporated lid ( PDB ID: 4CR2 ) ( Unverdorben et al . , 2014 ) , the COP9 signalosome ( PDB ID: 4D18 ) ( Lingaraju et al . , 2014 ) , and eIF3 ( PDB ID: 5A5T ) ( des Georges et al . , 2015 ) .", "The PCI horseshoes all adopt a staircase arrangement , and the pseudo-helical pitch for each horseshoe was determined , as well as the radius of the helix .", "The PCI horseshoe in the unincorporated lid complex ( left ) is more open , and less planar than that of the incorporated lid .", "The helical parameters and diameter of the PCI horseshoe in the incorporated lid closely resembles that of the COP9 Signalosome .", "Of the four PCI horseshoes , the arrangement of eIF3 is most open and least helical in arrangement . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 00910 . 7554/eLife . 13027 . 010Figure 1—figure supplement 7 . Interactions of the helical bundle with surrounding lid components . The helical bundle ( which is emphasized by a semi-transparent Gaussian-filtered surface ) only makes significant interactions with the PCI domain of Rpn9 ( magenta-colored ribbon ) .", "There also appears to be a contact established between the C-terminus of the Rpn6 helix ( yellow ) and the N-terminal domain of Rpn3 ( orange ) .", "The cryoEM density is shown in the lower left panel , showing the putative Rpn3-Rpn6 interaction . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01010 . 7554/eLife . 13027 . 011Table 1 . CryoEM data collection , processing , and modelingDOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 011Data collectionMicroscopeFEI Titan KriosCameraGatan K2 SummitVoltage300 keVMagnification22 , 500Pixel size1 . 31 Å ( 0 . 655 Å , super-resolution ) Dose rate9 . 9 e-/pixel/sCumulative electron dose43 . 8 e-/Å2Exposure7 . 6 sNumber of frames38Defocus range1 . 5–3 . 5 µmMicrographs collected3 , 432Acquisition softwareLeginon ( Suloway et al . , 2005 ) Image ProcessingPreprocessing packageAppion ( Lander et al . , 2009 ) Frame alignment softwareMotionCorr ( whole image ) ( Li et al . , 2013 ) CTF estimation softwareCTFFind3 ( Mindell and Grigorieff , 2003 ) CTF cutoff criterion4 Å at 0 . 5 confidenceParticle picking softwareFindEM ( Roseman , 2004 ) Micrographs used3 , 365Particles selected254 , 112ReconstructionSoftwareRELION 1 . 3 ( Scheres , 2012 ) Particles contributed109 , 396Rotational accuracy1 . 392 degreesTranslational accuracy0 . 671 pixelsB-factor applied-75 . 9Final resolution @ FSC 0 . 1433 . 5 ÅModel building and RefinementNumber of residues2743 ( 86% ) Map CC ( whole unit cell ) 0 . 758 Map CC ( all atoms ) 0 . 853 R . M . S deviationsBond length ( Å ) 0 . 02Bond angle ( ˚° ) 1 . 15Ramachandran plot statsPreferred2646 ( 96 . 47% ) Allowed92 ( 3 . 35% ) Outlier5 ( 0 . 18% ) Rotamer outliers5 ( 0 . 20% ) C-beta deviations0 ( 0 . 00% )" ], [ "Our cryo-EM reconstruction of the isolated lid shows that the MPN heterodimer , PCI horseshoe , and helical bundle together comprise a rigid substructure that contains regions resolved to ~3 Å resolution ( Figure 1B , Figure 1—figure supplements 3–5 ) .", "The N-terminal portions of the PCI-domain containing subunits progressively decrease in resolution as they extend toward the periphery of the complex , likely due to intrinsic flexibility ( Figure 1—figure supplements 3C , 5 ) .", "The 3D reconstructions of the fully assembled lid and the lid lacking Rpn12 ( Figure 1—figure supplement 2 , top row , third reconstruction ) show that the N-terminal portions of Rpn6 and Rpn5 are fully extended , and the MPN heterodimer and helical bundle adopt identical orientations in both structures .", "These findings contradict a recent crosslinking study ( Tomko et al . , 2015 ) suggesting that incorporation of Rpn12 during lid assembly induces a large-scale rearrangement of the MPN dimer and the transition of the N-terminal portion of Rpn6 from an inward-folded state to an extended conformation that allows binding to the base .", "Further structural studies will therefore be required to better understand how Rpn12 incorporation affects lid binding to the base and core subcomplexes .", "We found that the six PCI winged-helix domains associate into a continuous 18-stranded β-sheet , forming an incomplete right-handed spiral at the center of the lid sub-complex ( Figure 1C , D , Figure 1—figure supplement 6 ) .", "This organization was also observed in the crystal structure of CSN , although the PCI horseshoe assembly of the isolated lid has a wider and steeper spiral ( Figure 1—figure supplement 6 ) .", "Recently , a similar succession of β-strands was shown for the PCI domains in eIF3 ( des Georges et al . , 2015 ) , but its domain organization is significantly more open and deviates from the spiral configuration observed in the proteasome lid and CSN ( Figure 1—figure supplement 6 ) .", "The significant conformational differences between the horseshoes of the lid , CSN , and eIF3 indicate that the PCI-domain assembly allows for substantial flexibility , while simultaneously serving as an organizational scaffold at the center of the complex .", "The C-terminal helices of all lid subunits ( except Sem1 ) assemble into a well-defined helical bundle that is centrally positioned within the lid sub-complex , adjacent to the PCI horseshoe and the MPN heterodimer ( Figure 1E , Figure 1—figure supplement 7 ) .", "Our cryoEM reconstruction contains sufficient structural detail to generate a complete atomic model of this helical bundle , providing an accurate depiction of the extensive inter-helical interactions .", "Furthermore , we were able to precisely assign the register of several helices that could not be unambiguously positioned in earlier lower-resolution models of this bundle ( Beck et al . , 2012; Unverdorben et al . , 2014; Estrin et al . , 2013 ) .", "Our structure shows that Rpn8 and Rpn11 are the only subunits that contribute multiple helices to the bundle and together contact all other subunits within the helical assembly .", "Notably , Rpn8 is the largest contributor to the bundle , which is consistent with previous biochemical work showing that the Rpn8 C-terminal helices are more critical for lid assembly than those of other subunits ( Estrin et al . , 2013 ) .", "The PCI horseshoe and MPN heterodimer are individually tethered to the bundle via short loops , but make only few direct surface contacts with the bundle .", "The cryoEM map also shows that the bundle’s position in the isolated lid is likely stabilized by interactions between α-helix 5 ( residues 186–215 ) of Rpn8 and the PCI domain of Rpn9 at one end of the bundle , and between the C-terminus of Rpn6 and the N-terminus of Rpn3 at the opposite end ( Figure 1E , Figure 1—figure supplement 7 ) .", "The cryoEM reconstruction of the isolated lid allowed us to examine the structural elements involved in regulating Rpn11 DUB activity .", "Notably , within the isolated lid , the Rpn11/Rpn8 heterodimer is positioned in a previously unobserved orientation relative to the other subunits , stably associated within the palm of the hand-shaped complex via two primary interfaces with the tetratricopeptide repeat ( TPR ) domain of Rpn5 and the α-solenoid of Rpn9 ( Figure 2A–C ) .", "The resulting organization produces the basis for Rpn11 DUB inhibition in the isolated lid . 10 . 7554/eLife . 13027 . 012Figure 2 . The MPN heterodimer interacts extensively with Rpn5 and Rpn9 . ( A ) Side view of the lid sub-complex shows that the MPN heterodimer ( Rpn8 in red , Rpn11 in green ) interacts closely with the Rpn5 ( blue ) and Rpn9 ( lavender ) subunits .", "Side-chain interactions likely responsible for maintaining the MPN heterodimer in this configuration are shown in detail in panels ( B ) and ( C ) .", "Residues that were mutated to alanine for deubiquitination assays are labeled in black .", "( D ) Measurements of fluorescence increase upon Rpn11-mediated cleavage of ubiquitin-AMC are shown for three lid mutants relative to the wild-type lid .", "( E ) Ubiquitin-AMC cleavage rates show activation of Rpn11 in the lid upon mutation of residues within Rpn5 and Rpn8 . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01210 . 7554/eLife . 13027 . 013Figure 2—figure supplement 1 . Buried surface area between the MPN heterodimer and Rpn9 and Rpn5 . An MSMS surface was generated from the atomic model of the lid sub-complex , and the buried surface area was calculated for the interfaces of Rpn8 and Rpn9 ( colored yellow ) , and Rpn11 and Rpn5 ( colored magenta ) .", "The Rpn11-Rpn5 interface is the largest site of interaction between the all of the PCI-containing subunits and the MPN-heterodimer , with a buried surface area of ~631 Å2 .", "The Rpn8-Rpn9 interface is the next largest , with a buried surface area of ~452 Å2 . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01310 . 7554/eLife . 13027 . 014Figure 2—figure supplement 2 . Interface mutations in Rpn5 and Rpn9 release the MPN dimer from its inhibited conformation . Negative stain EM analysis was performed on the lid mutants .", "On the left are 2D class averages depicting the canonical hand-shaped arrangement of the lid sub-complex from the front and top views .", "On the right are 3D reconstructions of the lid , showing the organization of the PCI horseshoe , as well as the position of the Rpn11/Rpn8 heterodimer ( red and green ) relative to Rpn9 ( pink ) and Rpn5 ( blue ) .", "The 2D and 3D analyses of the wild type particles show that the MPN heterodimer is closely associated with the Rpn5 and Rpn9 subunits ( top row ) .", "The mutants , however , show that the while overall organization of the PCI-containing subunits are preserved , the MPN dimer is released from its position in the palm of the lid sub-complex .", "In the 2D class averages , the detached MPN dimer can be identified as an additional density that is not observed in the WT class average ( indicated by a yellow arrow ) .", "In the 3D reconstructions , the WT structure shows the MPN dimer ( green and red ) are connected to Rpn5 ( blue ) and Rpn9 ( purple ) .", "The density corresponding to the dimer in all the mutants is clearly detached from Rpn5 and Rpn9 .", "The MPN dimer within the Rpn5 ( Y273A ) ( bottom row ) had such an increased level of flexibility , that the dimer itself is poorly resolved . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 014 We first probed the contacts between Rpn11 and Rpn5 for their contributions to Rpn11 inhibition in the isolated lid , as this interface is more extensive ( total buried surface area of ~630 Å2 ) than all other subunit interactions with the MPN heterodimer ( Figure 2—figure supplement 1 ) .", "Importantly , the N-terminal region of α-helix 13 in Rpn5 ( residues 275–285 ) is nestled against the end of Rpn11’s catalytic groove , with several residues from Rpn5 directly contacting loops that surround Rpn11’s catalytic Zn2+ ion ( Figure 2C ) .", "To test the functional importance of these contacts , we generated Rpn5-mutated lid variants and compared their ubiquitin-7-amino-4-methylcoumarin ( Ub-AMC ) cleavage rate with that of wild-type lid and the isolated Rpn11/Rpn8 dimer .", "Under our assay conditions , Rpn11 activity within the isolated lid is 5-fold lower compared to the free Rpn11/Rpn8 dimer .", "In the loop preceding α-helix 13 of Rpn5 , Tyr273 is in an orientation that enables hydrophobic interactions with Rpn11 Phe114 , located in a loop near the active site ( Figure 2C ) .", "Mutation of this Rpn5 residue ( Y273A ) increased Rpn11 activity to 61% of the isolated Rpn11/Rpn8 dimer ( Figure 2D–E ) , suggesting that Tyr273 aids in stabilizing Rpn11 in its inhibited conformation .", "Rpn5 residues His282 and Lys283 directly interact with the backbone atoms of two loops near the Rpn11 active site , and their substitution with alanine increased Rpn11 activity to 31% and 41% of the free MPN heterodimer , respectively ( Figure 2C , E ) .", "The effects of these mutations were additive , as the Rpn5 ( H282A , K283A ) double-mutant lid exhibited 51% DUB activity compared to the free MPN heterodimer ( Figure 2E ) .", "Structural analysis of the lid containing the Rpn5 ( Y273A ) or the Rpn5 ( H282A , K283A ) double-mutant by negative-stain EM shows that the Rpn11/Rpn8 heterodimer is released from its inhibitory conformation , while the overall organization of the PCI-containing subunits is identical to that of the isolated wild-type lid complex ( Figure 2—figure supplement 2 ) .", "Together , these activating mutations support a model wherein Tyr273 , His282 , and Lys283 of Rpn5 all stabilize the association of α-helix 13 with the Rpn11 active site , generating a structural barrier that blocks substrates from accessing the catalytic groove .", "In addition to preventing access to the Rpn11 active site by steric occlusion , the close proximity of Rpn5 in the isolated lid further blocks DUB activity through interaction with the catalytic Zn2+ ( Figure 3A ) .", "Two histidines ( His109 and His111 ) and an aspartate ( Asp112 ) coordinate the Zn2+ within the Rpn11 active site , a configuration that is preserved in all JAMM metalloenzymes ( Komander et al . , 2009 ) .", "This geometry allows for interaction with a fourth ligand , as Zn2+ is usually tetrahedrally coordinated in proteins ( Gerke and Moss , 2002 ) .", "Despite the close proximity of Rpn5’s α-helix 13 to the Rpn11 active site , intermolecular distances preclude direct interaction of any Rpn5 residues with the catalytic Zn2+ .", "The Rpn5 residue that is closest to the zinc is Asn275 , which is notably oriented with its carboxamide group directed towards the Rpn11 active site .", "Mutation of Asn275 to alanine increases Rpn11 DUB activity in the isolated lid to 51% of the isolated MPN heterodimer ( Figure 2E ) , and negative-stain EM of this lid mutant shows the Rpn11/Rpn8 heterodimer detached from its inhibited conformation ( Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 13027 . 015Figure 3 . The Rpn11 active site is inhibited in the isolated lid .", "( A ) The catalytic Zn2+ ( gray sphere ) within the Rpn11 active site ( green ribbon ) is coordinated by three residues from Rpn11 , and a water molecule acts as a fourth ligand , likely mediated by Asn275 from the neighboring Rpn5 subunit ( blue ) .", "The cryoEM density in this region is shown as a mesh .", "( B ) Comparison of the Rpn11 active sites from the isolated Rpn11/Rpn8 heterodimer crystal structure ( PDB ID: 4O8X , purple ) and isolated lid ( green ) shows that the two structures are nearly superimposable .", "( C ) A di-ubiquitin substrate ( tan ) was modeled into the active site and shown as a transparent surface rendering .", "The modeled substrate severely clashes with the locked Rpn11 Ins-1 loop and Rpn5 .", "( D ) In CSN , a Glu within the Ins-1 loop provides a fourth point of coordination for the Zn2+ ion .", "( E ) Similar to CSN , an AMSH mutant utilizes an Asp from the Ins-1 loop to establish tetrahedral coordination of the catalytic Zn2+ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01510 . 7554/eLife . 13027 . 016Figure 3—figure supplement 1 . Water-mediated tetrahedral coordination of active site Zn2+ in Rpn11 and AMSH orthologue Sst2 . The deubiquitinase active site of isolated lid’s Rpn11 subunit ( left ) and an Sst2 mutant ( PDB ID: 4MSM ) ( Shrestha et al . , 2014 ) ( right ) are shown , depicting the coordination of the catalytic Zn2+ ( gray sphere ) by three active site residues , and a catalytic water molecule ( red sphere ) .", "In both structures , the water molecule is hydrogen-bonded to a residue from an additional component .", "In the lid , the catalytic water in the Rpn11 active site is bound to N275 from Rpn5 , and in the Sst2 mutant , the water interacts with the carboxylate group of Gly76 from a cleaved Ubiquitin . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01610 . 7554/eLife . 13027 . 017Figure 3—figure supplement 2 . Relative B-values of the Rpn11 Ins-1 loop in the isolated Rpn11/Rpn8 heterodimer and in the isolated lid sub-complex . A ribbon representation of the Rpn11 Ins-1 loop in the crystal structure of the isolated Rpn11/Rpn8 heterodimer and within the isolated lid sub-complex is colored according to the B-factor values ( blue = low B-factor , red = high B-value ) .", "Notably , the Ins-1 loop in the isolated MPN heterodimer has significantly higher average B-values than the rest of Rpn11 , while the B-values of the Ins-1 loop in the cryo-EM structure of the lid sub-complex are slightly lower than the average B-factor value of the Rpn11 subunit .", "This suggests that the Ins-1 loop is stabilized in this conformation within the lid assembly . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 017 Although Rpn5 Asn275 is not within range to directly bind the catalytic Zn2+ ( ~5 Å from the Zn2+ to Nδ1 of Asn275 ) , the cryo-EM density in the Rpn11 active site shows connectivity between Asn275 and the catalytic Zn2+ ( Figure 3A ) , potentially corresponding to a coordinated water molecule .", "Indeed , a Zn-associated water molecule is known to play a key role in the peptide hydrolysis mechanism of Zn-dependent proteases and has been observed in the crystal structures of Rpn11 ( Worden et al . , 2014; Pathare et al . , 2014 ) and related DUBs of the JAMM family , such as AMSH ( Shrestha et al . , 2014; Davies et al . , 2011 ) .", "Furthermore , the co-crystal structure of the AMSH ortholog Sst2 bound to a post-cleavage ubiquitin fragment shows that the carboxylate of ubiquitin Gly76 forms a hydrogen bond with the catalytic water ( Shrestha et al . , 2014 ) in the same manner as Rpn5 Asn275 in the isolated lid ( Figure 3—figure supplement 1 ) .", "While the Sst2 structure presents a snapshot of the transient substrate cleavage product prior to its departure from the active site , the positioning of Rpn5 Asn275 establishes a stable tetrahedral coordination of the Zn2+ ion via this catalytic water molecule , inhibiting isopeptidase activity of Rpn11 in the isolated lid sub-complex .", "The other major interface involved in stabilizing the DUB-inhibited conformation of the isolated lid is found between Rpn8 and Rpn9 , and involves a 5-residue loop connecting α-helix 8 ( residues 143–159 ) and α-helix 9 ( residues 165–182 ) of Rpn9 .", "While the buried surface area of this interface ( ~450 Å2 ) is smaller than the Rpn5-Rpn11 interface ( Figure 2—figure supplement 1 ) , mutagenesis of the interface residues shows that these contacts also contribute significantly to maintaining the sequestered position of the MPN heterodimer within the palm of the isolated lid sub-complex .", "Our atomic model suggests that Rpn8 Gln115 interacts with the backbone atoms of Rpn9 Ile163 ( Figure 2B ) , and upon mutation of Gln115 to alanine , we observed elevated Rpn11 activity that was 33% of isolated MPN levels .", "Furthermore , two lysine residues in Rpn8 , Lys86 and Lys88 , are likely involved in electrostatic interactions with Rpn9 Asp158 , which is located at the C-terminal end of α-helix 8 ( Figure 2B ) .", "Mutation of Lys86 and Lys88 to alanine in Rpn8 of the isolated lid increases Rpn11 activity to 33% and 45% of the free MPN-heterodimer levels , respectively .", "The double mutant Rpn8 ( K86A , K88A ) was additive , stimulating Rpn11 activity to about 60% of the isolated MPN heterodimer .", "As with the Rpn5 ( H282A , K283A ) double mutant lid , negative-stain analysis of the Rpn8 ( K86A , K88A ) double mutant revealed that disruption of the Rpn8-Rpn9 interface releases the MPN dimer from its inhibited conformation ( Figure 2—figure supplement 2 ) .", "Combined with the structural data , our mutational analyses of the MPN-dimer contacts with Rpn5 and Rpn9 suggest that DUB inhibition requires establishment of a finely tuned network of interactions and perturbation of this system at any of the identified contact points disrupts the inhibitory conformation of the MPN dimer within the isolated lid sub-complex .", "Common structural motifs present in many members of the MPN family are the two insertion loops , Ins-1 and Ins-2 , which have been suggested to be involved in orienting ubiquitin chains for cleavage ( Sato et al . , 2008 ) .", "In Rpn11 , Ins-1 is required for catalysis and has been proposed to play a structural role in DUB activity by engaging and positioning the C-terminus of the ubiquitin substrate for hydrolysis ( Worden et al . , 2014 ) .", "Flexibility of this loop suggests that it may regulate access to the DUB active site by switching between different conformational states .", "Upon ubiquitin binding to Rpn11 , the Ins-1 loop may first open up to allow the ubiquitin C-terminus to enter the catalytic groove and then switch to a conformation that stabilizes the isopeptide bond for hydrolysis .", "Structures of the isolated Rpn11/Rpn8 dimer show the Ins-1 loop in a ‘closed’ conformation ( Worden et al . , 2014; Pathare et al . , 2014 ) , which is also observed in EM reconstructions of proteasomes that are actively processing a protein substrate ( Unverdorben et al . , 2014; Matyskiela et al . , 2013 ) .", "Interestingly , in the context of the isolated lid , the Ins-1 loop appears to be locked in this closed state through interactions with the neighboring Rpn5 subunit ( Figures 2B , 3B ) .", "In particular , the amino group of Rpn5 Lys283 interacts with the Ser74 carbonyl of Ins-1 ( Figure 2C ) , and introducing the Rpn5 K283A mutation in the isolated lid results in a significant increase in Rpn11 DUB activity , as indicated above .", "While the Ins-1 loop in the free Rpn11/Rpn8 heterodimer exhibited markedly elevated B-values ( Worden et al . , 2014; Pathare et al . , 2014 ) , the Rpn11 Ins-1 loop within the isolated lid has lower B-values than the average for all modeled Rpn11 atoms ( Figure 3—figure supplement 2 ) .", "These data suggest that the Ins-1 loop is locked in a closed conformation through contact with neighboring residues and is unable to switch to the ‘open’ state required for substrate access to the active site .", "The combined effects of the tetrahedral coordination of the catalytic Zn2+ by Asn275 ( Figure 3A ) , the steric hindrance imposed by Rpn5’s α-helix 13 in the Rpn11 catalytic groove ( Figure 3C ) , and the obstruction of the DUB active site by the Ins-1 loop result in robust DUB inhibition .", "Interestingly , the proposed mechanism for auto-inhibition of the catalytically active MPN subunit in CSN , Csn5 , also involves tetrahedral coordination of the active-site Zn2+ .", "However , the fourth ligand in Csn5 is not provided by a neighboring subunit , but intramolecularly by the Ins-1 loop that thereby gets stabilized in a closed conformation ( Lingaraju et al . , 2014 ) ( Figure 3D ) .", "A similar scenario is also observed for a mutant AMSH construct ( PDB ID: 3RZV ) that utilizes a nearby Asp within the Ins-1 loop to complete the tetrahedral geometry ( Shrestha et al . , 2014 ) ( Figure 3E ) .", "Upon incorporation into the 26S proteasome , the lid undergoes major conformational changes that involve the PCI-assembly , the helical bundle , and especially the MPN heterodimer .", "To visualize these rearrangements , we compared the atomic coordinates of the isolated lid sub-complex to the previously determined pseudo-atomic model of the lid in the context of the assembled proteasome ( PDB ID: 4CR2 ) ( Unverdorben et al . , 2014 ) ( Figure 4 and Video 1 ) .", "Lid binding to the base and core sub-complexes causes the PCI horseshoe to constrict , decreasing in radius by ~3 Å , and adopt a more planar conformation that closely resembles the reported architecture of the CSN ( Lingaraju et al . , 2014 ) ( Figure 4 , Figure 1—figure supplement 6 ) .", "As a result , Rpn3 , Rpn7 and Rpn12 , comprising one half of the PCI horseshoe , undergo considerable rotation toward the center of the regulatory particle , where Rpn3 and Rpn12 bind the scaffolding subunit Rpn2 , while Rpn7 contacts the AAA+ ATPase subunits Rpt3 and Rpt6 .", "By comparison , the other half of the PCI horseshoe , consisting of Rpn9 , Rpn5 and Rpn6 , goes through a much less pronounced conformational change .", "The N-terminal α-solenoid domain of Rpn9 extends toward the N-terminal coiled coil of Rpt4 and Rpt5 , generating the binding site for the ubiquitin receptor Rpn10 , and the highly flexible TPR segment of Rpn5 becomes stabilized through contact with the ATPase ring and the core peptidase .", "The N-terminal α-solenoid domain of Rpn6 also accommodates interactions with the ATPase ring and core peptidase by rotating ~34º° around its long axis ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 13027 . 018Figure 4 . The lid sub-complex undergoes a dramatic reorganization upon incorporation to the 26S holoenzyme . Motions associated with lid incorporation are shown from three orthogonal views .", "Top panels correspond to the isolated lid , while bottom panels represent the proteasome-incorporated lid .", "Atomic models of the lid subunits were used to generate semi-transparent Gaussian filtered surfaces for visualization .", "For clarity , the helical bundle , which moves as a rigid body , is shown as a single surface .", "Sem1 is not shown .", "The base and core peptidase components are depicted as shadows to not occlude details of the lid rearrangement .", "Notable rearrangements include: a 90° rotation of the MPN dimer away from the inhibited conformation , movement of Rpn3 , 7 , and 12 away from Rpn5 , 6 , and 9 , constriction of the PCI horseshoe , and an overall closure of the lid sub-complex around the regulatory particle . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01810 . 7554/eLife . 13027 . 019Figure 4—figure supplement 1 . The Rpn6 α-solenoid domain rotates during incorporation into the holoenzyme . The N-terminal α-solenoid domain rotates ~34° ( calculated using DynDom [Hayward and Berendsen , 1998] ) during incorporation of the lid into the holoenzyme .", "The PCI domain of the two conformations were aligned , and the overlay of these structures illustrates the solenoid movement , shown from the side and down the axis of rotation .", "The Rpn6 subunit in the isolated lid sub-complex is colored using a rainbow scheme , with residues at the N-terminus colored blue , and progressing through the color spectrum to red for C-terminal residues .", "The Rpn6 conformation in the incorporated lid is shown in transparent gray . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 01910 . 7554/eLife . 13027 . 020Figure 4—figure supplement 2 . Rpn11’s bundle helix binds to the N-ring of the holoenzyme ATPase . Upon incorporation into the holoenzyme , a short helix from Rpn11 within the helical bundle interacts with the N-ring of the ATPase .", "This interaction may aid in stabilizing the lid’s position at the side of the regulatory particle . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 02010 . 7554/eLife . 13027 . 021Figure 4—figure supplement 3 . Lid incorporation activates Rpn11 . ( A ) Rpn11 activation during proteasome incorporation monitored by ubiquitin-AMC hydrolysis .", "Fluorescence time courses for the isolated WT lid and Rpn11 ( AxA ) lid are shown in blue and red , respectively .", "Background DUB activity of proteasomes reconstituted with Rpn11 ( AxA ) lid is shown in green , and the activity of proteasomes reconstituted with WT lid is shown in orange .", "The difference between the time courses for proteasomes reconstituted with WT and Rpn11 ( AxA ) lid corresponds to Rpn11-dependent DUB activity .", "( B ) Quantification of the ubiquitin-AMC cleavage activities for WT and Rpn11 ( AxA ) proteasomes shown in a .", "( C ) Normalized Ubiquitin-AMC hydrolysis activity of Rpn11-containing complexes in isolation ( grey bars ) and background-corrected activity of Rpn11 in proteasomes reconstituted with different lid variants ( dark grey bars ) .", "Error bars in b and c correspond to 1 standard deviation of the data ( n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 02110 . 7554/eLife . 13027 . 022Video 1 . Lid subunit rearrangements that occur during incorporation into the 26S holoenzyme . The atomic model of the isolated lid is interpolated into the pseudo-atomic model of the 26S holoenzyme ( Unverdorben et al . , 2014 ) , showing the motions associated with lid incorporation . DOI: http://dx . doi . org/10 . 7554/eLife . 13027 . 022 The extensive rearrangements of the PCI-containing subunits upon interaction with base and core may also trigger movements of the helical bundle toward the ATPase ring of the base ( Figures 1A , B , 2A , and 4 ) .", "Because the bundle is connected to the PCI horseshoe through flexible loops , it can move as a single unit , ultimately adopting an orientation in the 26S holoenzyme that is more perpendicular to the hand-shaped arrangement of the PCI subunits .", "Both in the isolated and incorporated lid , the helical bundle contacts the N-terminal domain of Rpn3 , albeit through different interfaces .", "That the association between these two components is maintained during lid incorporation suggests that movement of the Rpn3/7/12 unit influences the positioning of the bundle ( Figure 4 and Video 1 ) .", "The final orientation of the helical bundle within the regulatory particle is likely also dictated by an extensive interaction between α-helix 6 of Rpn11 ( residues 263–272 ) and the N-terminal domain ring of the ATPase subunits in the base ( Figure 4—figure supplement 2 ) .", "The most pronounced conformational rearrangement of the lid involves the Rpn11/Rpn8 MPN-domain heterodimer .", "Upon lid incorporation , the Rpn11/Rpn8 dimer undergoes a dramatic 90º° rotation , moving from its inhibited state in the palm of the isolated lid to a highly extended conformation over the central substrate-translocation channel in the 26S holoenzyme ( Figure 4 ) .", "The inhibited conformation of the Rpn11/Rpn8 heterodimer in the isolated lid appears to be metastable , as mutations in either the Rpn5 or Rpn9 contact surfaces lead to release of the dimer .", "In fact , the extended conformation of the Rpn11/Rpn8 dimer in the proteasome is similar to its conformation in our DUB-activating lid mutants ( Figure 2—figure supplement 2 ) .", "During lid incorporation , it is likely that the conformational changes occurring in the PCI subunits upon their interactions with the core and base distort the Rpn11-Rpn5 and Rpn8-Rpn9 contact sites and release the Rpn11/Rpn8 dimer from its inhibited state .", "To assess the extent of Rpn11 activation upon lid incorporation , we compared Ub-AMC hydrolysis activity for Rpn11 in the isolated lid versus the assembled 26S proteasome .", "Incorporation of wild-type lid stimulated Rpn11 DUB activity to 150% of the isolated Rpn11/Rpn8 dimer levels .", "This hyperstimulation of Rpn11 in the proteasome may originate from an alternative Ins-1 loop conformation that is stabilized by the neighboring Rpt4/Rpt5 coiled coil .", "Rpn11 activity in lid sub-complexes that contain Rpn5 ( Y273A ) , Rpn5 ( N275A ) , or Rpn8 ( K86A , K88A ) mutations was also stimulated upon proteasome incorporation , although to a lower level than with the wild-type lid ( Figure 4—figure supplement 3 ) .", "None of the mutations are involved in interfaces between subunits in the proteasome holoenzyme , and we speculate that the slightly lower DUB activity of the reconstituted mutant proteasomes originates from an interference with normal lid incorporation due to a prematurely released MPN dimer .", "Interestingly , lid sub-complexes where Rpn11 lacked Ins-2 were completely deficient in Rpn11 stimulation upon incorporation into holoenzyme ( Figure 4—figure supplement 3C ) , even though Rpn11 activity was unaffected by Ins-2 deletion in the isolated lid ( Figure 2E ) .", "The Ins-2 region of Rpn11 is known to interact with the scaffolding subunit Rpn2 of the base and likely stabilizes the Rpn11/Rpn8 dimer in the extended conformation .", "In summary , our structural and functional data suggest that during lid incorporation , the MPN-domain heterodimer loses its stabilizing interactions with Rpn5 and Rpn9 , and extends out toward the center of the regulatory particle , leading to Rpn11 activation .", "This extended conformation enables the Rpn11 Ins-2 loop to interact with Rpn2 , which likely aids in positioning the DUB active site above the entrance to the AAA+ ATPase ring for highly regulated deubiquitination of protein substrates during translocation .", "The primary function of the lid sub-complex is to house the isopeptidase Rpn11 , an enzyme that is central to proteasomal substrate degradation .", "While earlier structural and biochemical work described the role of the lid scaffold in positioning Rpn11 and facilitating its activity in the context of the assembled proteasome holoenzyme , we illustrate here how interactions of the Rpn11/Rpn8 dimer with other lid subunits block premature DUB activity in the unincorporated lid assembly .", "Our atomic model of the isolated lid subcomplex showcases the dramatic conformational gymnastics undergone by this important component of the proteasome during incorporation into the regulatory particle and , while the molecular communication involved in promoting this massive reorganization is still an active area of structural and biochemical research , our work here has resolved an important mystery surrounding DUB inhibition and activation during proteasome assembly ." ], [ "Expression and purification of mutant and wild-type recombinant yeast proteasome lid complex was carried out in E . coli as described previously , with minor modifications ( Lander et al . , 2012 ) .", "Briefly , E . coli BL21-star ( DE3 ) cells containing the recombinant lid expression system ( pETDuet-1 Rpn5 , FLAG-Rpn6 , Rpn8 , Rpn9 , 6xHis-Rpn11] , pCOLADuet-1 [Rpn3 , Rpn7 , Rpn12] and pACYCDuet-1[Sem1 , Hsp90 ) were grown at 37°C in 6 liters of terrific broth ( Novagen ) supplemented with 150µM ZnCl2 . At OD600 = 1 . 0 , the temperature was reduced to 18°C and , at OD600 = 1 . 5 lid , expression was induced overnight with 1 mM isopropyl-β-D-thiogalactopyranoside . After centrifugation , cell pellets were re-suspended in lid buffer ( 60 mM HEPES , pH8 . 0 , 100 mM NaCl , 100 mM KCl , 10% Glycerol , 1 mM DTT ) supplemented with protease inhibitors ( Aprotinin , Pepstatin , Leupeptin , PMSF ) , 2mg/ml lysozyme , and bezonase . All purification steps were performed at 4°C . Cells were lysed by sonication and clarified by centrifugation at 16 , 000g for 30 min . Clarified lysate was incubated with anti-FLAG M2 resin ( Sigma-Aldrich ) , washed with lid buffer and eluted with lid buffer supplemented with 0 . 15mg/ml 3x-FLAG peptide . FLAG eluate was concentrated to ~500 μl in a 30 , 000 MWCO spin concentrator ( Amicon ) and further purified by size-exclusion chromatography on a Superose 6 column ( GE Healthcare ) that was pre-equilibrated in lid buffer . Peak fractions were concentrated and stored at -80°C . Purification of core particle , Rpn10 , Rpn11/Rpn8 MPN-domain dimer and recombinant base was performed as described previously ( Lander et al . , 2012; Worden et al . , 2014; Beckwith et al . , 2013 ) . All Ubiquitin-AMC cleavage experiments were performed at 30°C in lid buffer . Because Rpn11’s Km for various ubiquitin substrates ranges from ~20 to ~300 μM , we assayed our WT and mutant lid variants at a constant , sub-Km Ubiquitin-AMC concentration . For all lid variants and the Rpn11/Rpn8 MPN-domain dimer , 500 nM enzyme was incubated with 2 . 5 μM Ubiquitin-AMC ( Boston Biochem ) , and Rpn11-catalyzed ubiquitin cleavage was monitored by the increase in AMC fluorescence ( Ex: 360 nm , Em: 435 nm ) using a QuantaMaster spectrofluorometer ( PTI ) . The slopes of individual time traces were translated to initial cleavage rates using a standard curve for ubiquitin-AMC ( ranging from 0 . 5–2 . 5 μM ) that had been completely cleaved by the DUB Yuh1 . Ubiquitin-AMC cleavage rates for all variants were measured in triplicate except for WT lid , Rpn11/Rpn8 dimer , Rpn5 ( H282A , K283A ) and Rpn8 ( Q115A ) , where n = 11 , n = 6 , n = 4 , and n = 4 , respectively . Proteasomes were reconstituted in vitro with lid as the limiting component by mixing 250 nM lid , 375 nM core particle , 750 nM base and 1 μM Rpn10 in reconstitution buffer ( 60 mM HEPES , pH7 . 6 , 100 mM NaCl , 100 mM KCl , 10% glycerol , 10 mM MgCl2 , 1 mM DTT , 0 . 5 mM ATP ) that contained an ATP-regeneration system ( 5 mM ATP , 16 mM creatine phosphate , 6 μg/ml creatine phosphokinase ) . Deubiquitination reactions were initiated by the addition of 2 . 5 μM ubiquitin-AMC and monitored by the increase in AMC fluorescence ( Ex: 360 nm , Em: 435 nm ) using a QuantaMaster spectrofluorometer ( PTI ) . A low level background DUB activity co-purified with our yeast core particle . To subtract this background activity , we reconstituted proteasomes as described above , but with a lid variant containing Rpn11 active-site mutations that abolish zinc binding ( Rpn11 [AxA] ) .", "The background DUB activity of Rpn11 ( AxA ) proteasomes was subtracted from the DUB activity of proteasomes reconstituted with WT Rpn11 to get the DUB activity that was specifically contributed by Rpn11 .", "To directly compare the activity of proteasome-incorporated and unincorporated Rpn11 , we monitored the ubiquitin-AMC hydrolysis activity of 250 nM lid and Rpn11/Rpn8 MPN-domain dimers in reconstitution buffer containing the ATP regeneration system but with core particle , base , and Rpn10 omitted .", "For negative stain analysis , purified lid samples were diluted to ~50 nM in FLAG buffer ( 50 mM HEPES , pH7 . 6 , 100 mM NaCl , 100 mM KCl ) and directly applied to plasma-activated ( 20 s; 95% Ar , 5% O2 ) copper grids for staining with 2% uranyl formate .", "For analysis by cryoEM , samples were diluted to ~5 uM in FLAG buffer that contained 1 . 5 mM TCEP ( G Biosciences ) and 0 . 05% NP-40 ( Sigma ) .", "4 μl of each sample was then applied directly to holey carbon C-flat grids ( Protochips , 400 mesh , 1 . 2 μm holes ) that had been plasma-cleaned ( Gatan Solarus , 6 s; 95% Ar , 5% O2 ) for manual blotting and plunge-freezing in liquid ethane .", "All imaging data was collected using automated Leginon imaging software ( Suloway et al . , 2005 ) .", "Images of negatively stained samples of wild-type and mutant lid complexes were acquired on a Tecnai Spirit LaB6 electron microscope operating at 120 keV , with a random defocus range of -0 . 5 μm to -1 . 5 μm and an electron dose of 20 e-/Å2 .", "331 images were acquired for wild-type lid , 433 images for the Rpn5 ( H282A/K283A ) double-mutant , 412 images for the Rpn8 ( K86A/K88A ) double mutant , 181 for the Rpn5 ( N275A ) mutant , and 204 for the Rpn5 ( Y273A ) mutant .", "Images were collected at a nominal magnification of 52 , 000 X on an F416 CMOS 4K X 4K camera ( TVIPS ) with a pixel size of 2 . 05 Å/pixel at the sample level .", "Imaging of frozen hydrated samples was performed using a Titan Krios electron microscope operating at 300 keV , with a defocus range of -1 . 5 μm to -3 . 5 μm .", "A Gatan K2 Summit was used for counting individual electron events at a dose rate of 9 . 9e-/pixel/s , using an exposure of 7 . 6 s consisting of 38 frames at 200 ms/frame .", "This resulted in a total electron dose of 43 . 8 e-/Å2 , accounting for coincidence loss .", "A total of 3 , 432 images of wild-type lid were acquired at a nominal magnification of 22 , 500X , yielding a pixel size of 0 . 655 Å/pixel at the sample level when collected in super-resolution mode .", "All image preprocessing was performed using the Appion image-processing pipeline ( Lander et al . , 2009 ) .", "The contrast transfer function ( CTF ) was estimated using CTFFIND3 ( Mindell and Grigorieff , 2003 ) .", "For negative stain data , particles were selected using a difference of gaussians ( DoG ) picking algorithm ( Voss et al . , 2009 ) , and only micrographs having an overall CTF confidence of greater than 80% were used for subsequent processing .", "The phases of the micrograph images were corrected according to the estimated CTF , and the particles were extracted using a box size of 160 pixels , and pixel values were capped at 4 . 5 sigma above or below the mean .", "Boxed particles were binned by a factor of 2 for processing .", "Reference-free 2D class averages of the extracted particles were determined through five rounds of iterative multivariate statistical analysis and multi-reference alignment ( Ogura et al . , 2003 ) .", "The results of the 2D analysis were used to remove damaged , aggregated , or falsely selected particles from the dataset used for 3D analysis .", "All 3D analysis was performed with RELION v1 . 31 ( Scheres , 2012 ) .", "Using a previously determined reconstruction of the wild type yeast proteasome lid as an initial model ( EMD-1993 ) ( Lander et al . , 2012 ) , a 3D refinement of 17 , 680 particles wild-type lid complex provided a reconstruction at 19 . 6 Å resolution , according to a Fourier Shell Correlation at 0 . 143 of two independently determined half-maps .", "This volume was used as the initial model for all 3D analysis of the mutant lid datasets .", "3D classification was performed on each of the negative stain mutant lid datasets , and only 3D classes exhibiting well-ordered structural details were selected and combined within each dataset for 3D refinement .", "22 , 103 particles of the Rpn5 ( H282A/K283A ) mutant yielded a 25 . 2 Å reconstruction; 11 , 185 particles of the Rpn8 ( K86A/K88A ) mutant yielded a 27 . 3 Å reconstruction; 25 , 429 particles of the Rpn5 ( N275A ) mutant yielded a 23 . 4 Å reconstruction , and 44 , 272 particles of the Rpn5 ( Y273A ) mutant yielded a 21 . 8 Å reconstruction ( Figure 2—figure supplement 2 ) .", "UCSF Chimera ( Goddard et al . , 2007 ) was used to dock the atomic model model of the lid into the density .", "For cryo-EM image preprocessing , the super-resolution images were binned by a factor of two in reciprocal space , and motion-corrected using MotionCorr ( Li et al . , 2013 ) .", "The aligned frames were summed and used for all subsequent processing steps .", "The CTF was estimated using CTFFIND3 ( Mindell and Grigorieff , 2003 ) , and only micrographs having a CTF confidence value that was greater than 50% at 4Å resolution were used for further processing ( Figure 1—figure supplement 1C ) , resulting in a dataset of 3 , 365 micrographs .", "Particles were manually selected from the first 100 images , and the results of reference-free 2D analysis were used as templates for particle selection using FindEM ( Roseman , 2004 ) .", "A random subset of 50 , 000 particles were extracted from the micrographs with a box size of 256 and used for reference-free 2D analysis in order to rapidly assess the quality of particle selection ( Figure 1—figure supplement 1B ) .", "Very few classes corresponding to damaged or aggregated particles were observed; so all particles were used for single particle analysis in RELION .", "A total of 254 , 112 particles were extracted from the micrographs using a box size of 288 pixels , binned by a factor of 4 , and classified into 8 3D classes over the course of 22 iterations in RELION .", "The particles from the 4 classes that showed evidence of conformational and compositional stability were selected from this initial classification , providing a total of 139 , 561 particles .", "The x and y coordinates corresponding to these particles were adjusted according to the final translational alignments from the 3D classification , and the centered particle coordinates were used to extract an unbinned particle dataset for 3D refinement in RELION .", "3D refinement using the default RELION parameters yielded a 4 . 4 Å resolution structure after 22 iterations .", "These aligned particle parameters were used for the RELION ‘particle polishing’ method .", "Individual particle motion trajectories were estimated using a running average window of 7 frames and particle translations were limited using a prior with a standard deviation of 1 .", "Particle movements were fit to a linear trajectory using a running average window of 7 frames , with an inter-particle distance contribution value set to 300 pixels .", "Per-frame B-factors and intercepts were estimated by comparing the reconstructed half-maps from individual frames to the full-frame half maps , and the spatial frequency contribution from each frame weighted according ( Figure 1—figure supplement 1D , E ) .", "A new stack of particles was generated from the translationally aligned particles extracted from the weighted frames , which provided a reconstruction at 4 . 1 Å resolution .", "Due to the possibility that the flexible N-terminal domains of the PCI subunits were negatively influencing the particle alignment , a soft-edged 3D mask encompassing the PCI-domains , the helical bundle , and the MPN domains was generated ( blue mask shown in Figure 1—figure supplement", "2 ) and used for 3D classification of the particles into 3 classes .", "This 3D classification was performed using the alignments from the 3D refinement , without further alignment of particles .", "One of the 3D classes resulting from this analysis clearly exhibited higher resolution details than the other two , and the 109 , 396 particles contained in this class were further refined ( in the absence of a mask ) to achieve a 3 . 6 Å structure .", "The same soft-edged 3D mask that was used for the previous 3D classification was then used for continued 3D refinement , which improved the structural details of the region contained within this mask , and increased the resolution to 3 . 5 Å resolution .", "Modeling and visualization of the lid was performed in COOT ( Emsley and Cowtan , 2004 ) using mostly the cryo-EM map that had been generated using a soft mask encompassing the PCI domains and the C-terminal helical bundle ( deposited as EMD-6479 ) , as this is the highest resolved region , and cross-validated using the unmasked map .", "Available structures and homology models generated using Modeller v9 . 15 ( Eswar et al . , 2007 ) were initially fit into the unmasked cryo-EM map using Chimera ( Goddard et al . , 2007 ) .", "These included: 1 ) the crystal structure of Drosophila melanogaster Rpn6 ( residues 50–390 ) homolog ( PDB ID: 3TXN ) ( Pathare et al . , 2014 ) ;", "2 ) the crystal structure of the Saccharomyces cerevisiae Rpn11-Rpn8 heterodimer ( residues 24–220 and 10–280 , respectively; PDB ID: 4O8X ) ( Worden et al . , 2014 ) ;", "3 ) the NMR structures of the N-terminal ( residues 4–140 ( PDB ID: 2MQW ) and C-terminal ( residues 184–353 ( PDB ID: 2MRI ) ) domains of Saccharomyces cerevisiae Rpn9 ( Hu et al . , 2015 ) ; and", "4 ) the N-terminal domain of Schizosaccharomyces pombe Rpn12 homolog ( residues 6–200 , PDB ID: 4B0Z ) ( Boehringer et al . , 2012 ) .", "The most N-terminal helices of Rpn5 and Rpn6 were not modeled due to the limited resolution of these regions .", "Placement of the N-terminal helices of Rpn3 was possible , however the absolute sequence register could not be assigned and these helices were modeled as polyalanine .", "Following each round of real space refinement in Phenix v1 . 10 ( Adams et al . , 2010 ) , 100 models were generated in Rosetta ( DiMaio et al . , 2015 ) , clustered , and scored .", "The top scoring structures were then used for the next round of manual model building and an aggregate model was used for refinement in Phenix .", "For the final round of refinement , the SHAKE protocol in Phenix was used to displace all atoms of the top 5 scoring models by 0 . 5 Å before refinement against one of the unmasked half-maps .", "An ensemble of these 5 models have been deposited in the PDB under ID: 3JCK .", "To visualize conformational changes undergone by the lid complex upon incorporation into the 26S proteasome , we first rigid-body fit individual components of the atomic model of our isolated lid ( 6 PCI domains , 6 N-terminal extensions , the MPN heterodimer , and the helical bundle ) onto the pseudo-atomic model of the engaged lid ( PDB-ID: 4CR2 ) ( Unverdorben et al . , 2014 ) using the ‘MatchMaker’ tool in Chimera .", "These overlaid models were then docked into the EM density of the 26S holoenzyme in the S1 state ( Unverdorben et al . , 2014 ) .", "Overall , the secondary structure organization of the atomic models matched with high fidelity , although the register of the C-terminal helices of Rpn11 and the N-terminal helices of Rpn9 of the incorporated lid model were modified to correspond to the isolated lid model .", "The domain movements were visualized using the ‘morph conformations’ tool in UCSF Chimera .", "The motion of Rpn6 was evaluated using the software DynDom ( Hayward and Berendsen , 1998 ) .", "The EM density maps for the 26S proteasome lid sub-complex before and after masked refinement , as well as unsharpened maps and half-maps , have been deposited in the Electron Microscopy Data Bank under accession number EMD-6479 .", "The atomic coordinates of the five highest-scoring Rosetta models have been deposited under accession ID: 3JCK in the PDB ." ] ]
[ "The 26S proteasome is responsible for the selective , ATP-dependent degradation of polyubiquitinated cellular proteins .", "Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11 , a deubiquitinase within the ‘lid’ sub-complex .", "Prior to the lid’s incorporation into the proteasome , Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins .", "Here we present the atomic model of the isolated lid sub-complex , as determined by cryo-electron microscopy at 3 . 5 Å resolution , revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit , Rpn5 .", "Through mutagenesis of specific residues , we describe the network of interactions that are required to stabilize this inhibited state .", "These results provide significant insight into the intricate mechanisms of proteasome assembly , outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme , which ultimately activates the deubiquitinase for substrate degradation ." ]
[ "The proteins contained within cells are constantly under scrutiny by a sophisticated “quality control” system that tags damaged or malfunctioning proteins with chains made up of a protein called ubiquitin .", "These ubiquitin chains serve as markers that target these toxic proteins for destruction by a molecular complex called the proteasome .", "Removing ubiquitin chains from toxic proteins is a critical step in their degradation by the proteasome .", "This task is accomplished by an enzyme called a deubiquitinase , whose activity is tightly controlled .", "However , it was not clear how this enzyme is kept inactive before it is incorporated into the proteasome complex .", "The deubiquitinase is part of a sub-complex called the “lid” , which attaches to the side of the proteasome .", "Dambacher , Worden , Herzik et al . used electron microscopy to solve the structure of the lid complex in high detail – so that it was almost possible to view individual atoms .", "This revealed that the deubiquitinase was in a conformation that was very different from what had previously been observed in fully assembled proteasomes .", "The structures revealed that within the lid complex , a complicated network of interactions causes the deubiquitinase to be encompassed by neighboring subunits .", "This prevents the enzyme from interacting with ubiquitin chains .", "Importantly , this network of interactions appears to be set on a hair-trigger , as mutations that disrupt these interactions cause the deubiquitinase to be activated .", "As the lid complex integrates into the proteasome , the lid undergoes large-scale structural rearrangements; Dambacher , Worden , Herzik et al . expect that these disrupt the interactions that maintain the deubiquitinase in an inhibited conformation .", "Due to their ability to regulate the activity of the proteasome , deubiquitinases are becoming increasingly popular drug targets .", "Therefore , probing how they are activated in more detail will be of great importance to cell biologists and also contribute substantially to biomedical research ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "structural biology and molecular biophysics" ]
NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble
elife-18096-v2
[ [ "To control when and how fast genes are expressed , the activity of RNA polymerase ( RNAP ) is tightly regulated .", "Much of transcription regulation in all domains of life takes place at the initiation stage by modulating activities of promoters .", "The examples of on/off regulation at the transcript elongation stage , such as promoter-proximal pauses in metazoans ( Adelman and Lis , 2012 ) and antitermination in prokaryotes ( Santangelo and Artsimovitch , 2011 ) , are also known .", "In other cases , transcription elongation control is mediated by coupling of transcription to downstream processes , such as RNA translation , processing , and folding ( Proshkin et al . , 2010; Bubunenko et al . , 2013 ) .", "The multisubunit RNAPs evolved to elongate relatively inefficiently in the absence of proper coupling , thereby enabling the downstream processes to control the elongation rate .", "The ubiquitous family of NusG proteins ( SPT5/SPT4 in archaea and yeast , DSIF in mammals ) are the central components which mediate coupling between transcription and the downstream processes ( Werner , 2012 ) .", "The simplest member of the family , a bacterial NusG , consists of two domains connected by a flexible linker ( Figure 1 ) .", "The N-terminal domain ( NTD or NGN ) anchors NusG to the clamp helices of the RNAP β’ subunit , whereas the C-terminal domain ( CTD or KOW ) interacts with the components of the downstream processes ( reviewed in [Belogurov and Artsimovitch , 2015] ) .", "In E . coli , NusG CTD interacts with NusE as part of the ribosome on protein coding operons ( Burmann et al . , 2010 ) or as a part of a so-called antitermination complex ( NusABEG ) on ribosomal RNA operons ( Zellars and Squires , 1999; Shankar et al . , 2007; Singh et al . , 2016 ) ; in these contexts , NusG inhibits the function of a transcription termination factor Rho .", "If neither ribosome nor antitermination complex is engaged , which often implies that transcription is futile , NusG CTD binds to Rho and facilitates termination of transcription ( Cardinale et al . , 2008; Peters et al . , 2012 ) .", "tRNA and other non-coding RNA genes escape the premature termination by Rho possibly due to their extensive secondary structures and small size relative to the transcribed region required for the termination of transcription by Rho ( Mooney et al . , 2009a; Peters et al . , 2009 ) .", "NusG-mediated coupling of transcription with the pioneer round of translation is likely conserved in prokaryotes , whereas functioning of NusG CTD ( and additional KOW domains present in eukaryotic SPT5 and DSIF ) in RNA processing/maturation is likely conserved in all domains of life ( Belogurov and Artsimovitch , 2015 ) . 10 . 7554/eLife . 18096 . 003Figure 1 . An overview of the bacterial transcription elongation complex ( TEC ) with bound NusG . RNAP core subunits are depicted by simplified differentially colored molecular surfaces .", "β is depicted transparent to reveal the path of nucleic acids through the enzyme .", "The positions of NusG CTD and αCTD , connected via flexible linkers , were chosen arbitrary .", "The locations of RNAP cleft loops individually deleted in this study , β Gate Loop ( GL ) , β' Rudder Loop ( RL ) and β' Lid Loop ( LL ) are accentuated by ovals .", "The hypothetical path of the single stranded non-template DNA is depicted by a grey dashed line .", "The approximate location of GreA cleavage factor employed in backtracking experiments ( see results ) is depicted as a black dashed contour .", "The composite model was generated using the Thermus thermophilus TEC ( Vassylyev et al . , 2007 ) , NusG NTD from the NusG-RNAP model in Martinez-Rucobo et al . , 2011 and the elements from other structures ( see Materials and methods ) .", "The duplex DNA immediately upstream of the RNA:DNA hybrid was modeled de novo as described in the Results section . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 003 The regulation of gene expression by NusG-like proteins , which include several paralogs in some bacterial species , is complex .", "Even the housekeeping NusG may exhibit opposite effects on transcription in vivo depending on the protein partner ( s ) bound to its CTD domain .", "Furthermore , NusG from different bacteria display seemingly opposite effects on transcription by their cognate RNAPs in a purified in vitro system lacking the downstream components .", "The E . coli NusG has an intrinsic stimulatory effect on transcript elongation in vitro ( Bar-Nahum et al . , 2005; Burova et al . , 1995 ) , which persists when an isolated NusG NTD is used ( Mooney et al . , 2009b ) .", "It is hypothesized that this intrinsic stimulatory effect of NusG NTD may allow RNAP to transcribe more efficiently in vivo when coupled with the downstream processes and slower if the coupling is broken ( McGary and Nudler , 2013; Belogurov and Artsimovitch , 2015; Burmann and Rösch , 2011 ) .", "However , NusG from Thermus thermophilus slows down its cognate RNAP in vitro ( Sevostyanova and Artsimovitch , 2010 ) , whereas Bacillus subtilis NusG stimulates pausing by interacting with specific sequences in the non-template DNA ( Yakhnin et al . , 2008 , 2016 ) .", "We later suggest that these apparent incongruences result from the superimposition of several distinct consequences of the NusG NTD binding to the RNAP and considerable differences in the elongation properties of these RNAPs .", "But first we consider the mechanistic details of the elongation stimulation by E . coli NusG .", "The RNAP nucleotide addition cycle consists of", "( i ) the NTP substrate binding to a post-translocated transcription elongation complex ( TEC ) ;", "( ii ) a chemical step of the nucleotide incorporation; and", "( iii ) the post-catalytic relaxation of the resulting pre-translocated TEC , which involves the release of pyrophosphate and forward translocation ( reviewed in [Belogurov and Artsimovitch , 2015] ) .", "The processive repetition of this cycle is sometimes interrupted by off-pathway pause events .", "The latter can be classified into pauses involving backtracking of the RNAP on the DNA template by two or more registers , which occludes the active site with the nascent RNA ( Komissarova and Kashlev , 1997; Nudler et al . , 1997 ) , and diverse non-backtracked pauses ( Artsimovitch and Landick , 2000 ) .", "The non-backtracked pauses involve more complex and less understood rearrangement of the active site and the RNAP structure that impede the nucleotide addition ( Hein et al . , 2014; Zhang et al . , 2010; Kireeva and Kashlev , 2009 ) .", "Notably , many non-backtracked pauses likely involve partial opening of the β’clamp ( Hein et al . , 2014; Weixlbaumer et al . , 2013 ) , a large mobile domain that contributes most of the β’ subunit contacts with the nucleic acids .", "The elongation-enhancing effect of NusG may arise from", "( i ) accelerating the on-pathway elongation ,", "( ii ) diminishing some or all type of pauses , or both .", "Early studies suggested that NusG acts by reducing pausing ( Burova et al . , 1995 ) .", "Recent reports further specified that NusG homologues enhance elongation by restricting the conformational flexibility of the RNAP β’clamp ( Sevostyanova et al . , 2011; Hirtreiter et al . , 2010 ) , which is consistent with biophysical measurements ( Schulz et al . , 2016 ) and structural considerations ( Martinez-Rucobo et al . , 2011; Klein et al . , 2011 ) .", "On the other hand , E . coli NusG mainly reduces the frequency and duration of backtracked pauses ( Herbert et al . , 2010 ) that are not explicitly known to involve the β’ clamp opening ( Sekine et al . , 2015 ) and has only small stimulatory effect at non-backtracked pauses ( Artsimovitch and Landick , 2000; Belogurov et al . , 2010; Kolb et al . , 2014 ) .", "Here , we report a detailed analysis of NusG effects on the individual steps of the nucleotide addition cycle , backtracking , and the conformation of the upstream DNA .", "Our results suggest that NusG slows backtracking without affecting the on-pathway elongation in non-paused TECs .", "We also demonstrate that NusG inhibits backtracking by restricting the melting of the upstream DNA , independently of the NusG-RNAP contacts that are important for stabilization of the β’ clamp conformation .", "We further perform a comprehensive mapping of the upstream fork junction , determine the point of the upstream DNA reannealing , and provide a plausible mechanistic model of the anti-backtracking action of NusG ." ], [ "To determine the effect of NusG on the kinetics of nucleotide addition and translocation , we utilized a TEC design that was extensively validated in our previous studies ( Malinen et al . , 2012 , 2015 ) .", "The TEC was assembled on a synthetic nucleic acid scaffold and contained the fully complementary transcription bubble flanked by 20-nucleotide DNA duplexes upstream and downstream ( Figure 2—figure supplement 1 ) .", "The annealing region of a 16-nucleotide RNA primer was initially nine nucleotides , permitting the TEC extended by one nucleotide to adopt the post- and pre-translocated states , but disfavoring backtracking .", "The RNA primer was 5’ labeled with the infrared fluorophore ATTO680 to monitor the RNA extension by the denaturing PAGE .", "We performed parallel , time-resolved measurements of nucleotide incorporation by assembled TEC accompanied by forward translocation and , in a separate experiment , pyrophosphorolysis of extended TEC accompanied by backward translocation in the presence and absence of saturating concentration of NusG ( 2 µM , see later ) ( Figure 2A–C ) .", "RNA extension was monitored by a rapid chemical quench-flow method , whereas forward and backward translocation were monitored by measuring the fluorescence of the 6-methyl-isoxanthopterin base ( 6-MI ) incorporated in the template DNA strand in a stopped-flow instrument ( Malinen et al . , 2015 ) .", "NusG did not affect either of these on-pathway reactions .", "We then tested the effect of NusG on the TEC translocation bias in the equilibrium setup ( Figure 2D ) .", "We have previously demonstrated that the predominantly post-translocated TEC can be converted into the pre-translocated state by tagetitoxin ( TGT ) ( Malinen et al . , 2012 ) .", "TGT was equally potent in biasing the TEC towards the pre-translocated state in the presence and absence of NusG , suggesting that NusG does not affect the equilibrium between the post- and pre-translocated states .", "Overall , these experiments suggest that NusG does not measurably affect the on-pathway kinetics and thermodynamics of transcript elongation ( Table 1 ) in the non-paused TECs examined in this study . 10 . 7554/eLife . 18096 . 004Figure 2 . NusG does not affect on-pathway transcription elongation .", "( A ) The schematics of the system used for monitoring the forward and the backward kinetics of the nucleotide addition cycle .", "In addition , the translocation bias of the TEC was evaluated under equilibrium conditions by measuring the TEC response to tagetitoxin ( TGT ) .", "( B ) The effect of NusG on the rate of nucleotide addition ( discrete time-points and best-fit lines ) and forward translocation ( continuous time-traces ) .", "The lower and upper bounds of the reaction half-lives were calculated by combined analysis of data from several independent experiments ( Table 5 ) by FitSpace routine of Kintek Explorer software ( at a 10% increase in Chi2 ) .", "( C ) The effect of NusG on the pyrophosphorolysis-driven backward translocation .", "Inset: the gel control of the pyrophosphorolysis reaction .", "( D ) The effect of NusG on the potency of TGT to bias the TEC towards the pre-translocated state .", "Error bars indicate the range of duplicate measurements .", "Numerical values of the reaction rate constants and the median reaction times are presented in Table 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 00410 . 7554/eLife . 18096 . 005Figure 2—figure supplement 1 . TEC systems employed in the experiments presented in the main figures . X = 6-MI , Y = 2-AP , S = 6-TG , mismatches in the non-template DNA are colored blue . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 00510 . 7554/eLife . 18096 . 006Table 1 . Numerical values of the reaction rate constants and the median reaction times . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 006Figure2B−NusG+NusGlower boundbest fitupper boundlower boundbest fitupper boundnucleotide addition , s−1272830282830translocation , s−1606573627079Slow TEC fraction~8%~7%recovery rate , s−10 . 41 . 12 . 70 . 41 . 12 . 7inactivation rate , s−10 . 030 . 090 . 30 . 020 . 080 . 2The lower and upper bounds of rate constants were calculated by the combined analysis of data from several independent experiments ( Table 5 ) by FitSpace routine of Kintek Explorer software ( at a 10% increase in Chi2 ) . 2Cmedian pyrophosphorolysis time , s-NusG+NusG0 . 49 ± 0 . 080 . 51 ± 0 . 08Errors indicate the range of the bestfit estimates in duplicate experiments . 2DKD TGT , µM-NusG+NusG0 . 09–0 . 150 . 09–0 . 14The ranges represent 95% confidence interval for KD determined by the nonlinear regression analysis of data from two independent experiments . 3BMethodmedian reaction time , s−NusG+NusG6-MI13 . 2 ± 2 . 228 . 0 ± 0 . 7RNA1811 . 7 ± 1 . 130 . 4 ± 3 . 4RNA1611 . 6 ± 1 . 230 . 1 ± 3 . 62-AP12 . 4 ± 1 . 830 . 3 ± 2 . 2Errors indicate the range of the bestfit estimates in duplicate experiments . 3CRNADNARNAPmedian reaction time , s−NusG+NusGmatchedmatchedWT12 . 4 ± 1 . 830 . 3 ± 2 . 2matchedmatchedΔRL11 . 1 ± 1 . 925 . 7 ± 4 . 4matchedmatchedΔLL11 . 4 ± 1 . 320 . 6 ± 3 . 1matchedmatchedΔGL24 . 0 ± 2 . 354 . 1 ± 4 . 7matchedmm 1WT19 . 9 ± 2 . 242 . 9 ± 3 . 3matchedmm 1 and 2WT2 . 30 ± 0 . 043 . 11 ± 0 . 203’ mmmatchedWT0 . 34 ± 0 . 170 . 36 ± 0 . 103’ mm stands for mismatch against 3’ RNA nucleotide .", "Errors indicate the range of the bestfit estimates in duplicate experiments .", "NusG has been suggested to inhibit stochastic and sequence-specific backtracking during transcription through a long template in vitro ( Herbert et al . , 2010 ) .", "We developed a system where backtracking of the TEC is driven by the rapid cleavage of the nascent RNA by the RNAP active site .", "The reaction was initiated by adding 2–8 µM GreA protein that transforms the RNAP active site into a highly efficient RNAse .", "The TECs were assembled on a synthetic nucleic acid scaffold and contained the fully complementary transcription bubble ( Figure 2—figure supplement 1 ) .", "The annealing region of 18-nucleotide long RNA primer was 11 nucleotides , allowing the TEC to backtrack by one nucleotide .", "The RNA primer was 5’ labeled with ATTO680 to monitor the accumulation of a 16-nucleotide RNA cleavage product in a rapid chemical quench flow experiment .", "The RNA primer also contained 2-aminopurine ( 2-AP ) as the penultimate 3’ nucleotide ( Figure 3A ) , thereby permitting monitoring of 2-AP-p-C dinucleotide release by measuring an increase in 2-AP fluorescence in a stopped flow instrument .", "In a subset of experiments , the template DNA contained 6-MI nine registers upstream of the RNA 3’ end ( Figure 3A ) to directly monitor RNAP backtracking by measuring the decrease in 6-MI fluorescence in a stopped flow instrument .", "Importantly , the changes in 6-MI and 2-AP fluorescence were driven by the RNA cleavage and not GreA binding because the addition of the cleavage-deficient D41N GreA variant did not change either fluorescence ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 18096 . 007Figure 3 . NusG inhibits GreA assisted RNA cleavage by slowing backtracking .", "( A ) Three assays for monitoring the GreA-assisted RNA cleavage: TEC backtracking ( 6-MI fluorescence decrease ) , RNA cleavage ( PAGE ) , and the dinucleotide release ( 2-AP fluorescence increase ) .", "( B ) Left: the effect of NusG on the TEC backtracking ( continuous time-traces ) and RNA cleavage ( discrete time-points ) upon the addition of 8 µM GreA .", "Center: the effect of NusG on the release of the cleaved dinucleotide ( continuous time-traces ) and the RNA cleavage ( discrete time-points ) .", "Right: the median reaction times .", "( C ) The effect of NusG on the RNA cleavage by TECs with deletions of the RNAP domains , the mismatched upstream DNA and a 3' rC‑dA RNA:DNA mismatch .", "The median reaction times were determined by monitoring the increase in 2‑AP fluorescence at 8 µM GreA .", "The schematic on the right illustrates the location of DNA:DNA mismatches ( cyan bars ) .", "Numerical values of the median reaction times are presented in Table 1 .", "Error bars indicate the range of the bestfit estimates in duplicate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 00710 . 7554/eLife . 18096 . 008Figure 3—figure supplement 1 . Control experiment with the inactive GreA ( D41N variant ) .", "( A ) The decrease in 6-MI fluorescence is observed upon addition of the active GreA protein but not the inactive GreA D41N variant .", "( B ) The increase in 2-AP fluorescence is observed upon addition of the active GreA protein but not the inactive GreA D41N variant . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 00810 . 7554/eLife . 18096 . 009Figure 3—figure supplement 2 . Primary data for graphs in Figure 3B: The effect of NusG on GreA assisted RNA cleavage . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 00910 . 7554/eLife . 18096 . 010Figure 3—figure supplement 3 . The effect of NusG on RNA cleavage by wild-type TECs at 2 µM GreA . Error bars indicate the range of duplicate measurements . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 010 Parallel experiments demonstrated that within the limits of experimental uncertainty accumulation of the 16-nucleotide cleavage product , the release of 2AP-p-C dinucleotide , and RNAP backtracking occur with the same rate ( Figure 3B ) .", "Importantly , 3’ mismatched TECs that are more prone to backtracking were cleaved approximately thirty times faster than the matched TECs ( Figure 3C ) , suggesting that for the matched TECs the rates of RNA cleavage and the dinucleotide release were limited by the rate of backtracking .", "We therefore concluded that all three types of measurements can be used interchangeably to monitor backtracking in this system .", "Saturating concentrations of NusG ( 2 µM , see below ) slowed RNA cleavage , dinucleotide release ( increase in 2AP fluorescence ) , and backtracking ( decrease in 6-MI fluorescence ) approximately twofold ( Figure 3B ) .", "Similar inhibition was observed at 2 µM ( Figure 3—figure supplement 3 ) and 8 µM GreA ( Figure 3B ) , suggesting that NusG did not act by weakening the binding of GreA .", "Furthermore , NusG did not slow the dinucleotide release from the TEC that was biased towards the backtracked state by the RNA 3´end mismatch ( Figure 3C ) , suggesting that NusG did not directly inhibit RNA cleavage .", "NusG may slow backtracking of the TEC by affecting the conformation of RNAP and/or transcription bubble .", "To dissect the mechanism of anti-backtracking activity of NusG , we individually deleted β and β’ cleft loops near the NusG binding site ( Figure 1 ) .", "We evaluated the NusG effects on backtracking of TECs assembled with RNAPs lacking β Gate Loop ( ΔGL ) , β’ Lid Loop ( ΔLL ) and β’ Rudder Loop ( ΔRL ) .", "We also perturbed the base pairs of the upstream DNA that may affect backtracking .", "While the register of the upstream DNA reannealing is not exactly known , the bacterial TEC structures suggest that", "( i ) DNA may reanneal as early as ten nucleotides upstream of the RNA 3’ end in the post-translocated TEC and", "( ii ) DNA must be unpaired up to at least 11 nucleotides upstream of the RNA 3’ end in one-nucleotide backtracked TEC ( Figure 3C ) .", "Accordingly , we evaluated the anti-backtracking activity of NusG on the TECs where DNA reannealing ten and ten-eleven nucleotides upstream from the RNA 3´end was inhibited by mismatches .", "ΔRL and ΔLL did not affect backtracking rates in the absence of NusG , but the latter deletion reduced the TEC response to NusG twofold ( Figure 3C ) .", "In contrast , ΔGL and the DNA:DNA mismatch ten nucleotides upstream of the RNA 3’ slowed backtracking 1 . 5–2 fold but did not affect the TEC responses to NusG .", "Most notably , TEC with two DNA:DNA mismatches 10–11 nucleotides upstream of the RNA 3’ end backtracked approximately fivefold faster than the fully-matched TEC and was insensitive to NusG ( Figure 3C ) .", "At the same time , TEC with a 3’ RNA:DNA mismatch cleaved RNA further sevenfold faster than the TEC with two DNA:DNA mismatches ( Figure 3C ) , suggesting that backtracking limited the cleavage rate in the latter TEC .", "These results suggested that:", "( i ) NusG slows backtracking by inhibiting DNA melting eleven nucleotides upstream from the RNA 3’ end;", "( ii ) RL , GL and DNA:DNA pairing ten nucleotides upstream of the RNA 3’ end are dispensable for anti-backtracking activity of NusG;", "( iii ) LL may be involved in the anti-backtracking action of NusG , but is not critically important therein .", "To directly test the effect of NusG on the reannealing of the upstream DNA , we developed a system to probe the DNA reannealing by photocrosslinking with 8-methoxypsoralen ( 8-MP ) .", "8-MP specifically intercalates into the double-stranded 5´-TA-3´ sequence and introduces a T-T inter-strand crosslink upon illumination with UV light ( Figure 4—figure supplement 1 ) .", "We designed a fully complementary TEC containing a unique 5´-TA-3´ sequence motif positioned nine nucleotides upstream of the RNA 3’ end ( Figure 4A , Figure 2—figure supplement 1 ) .", "The template DNA and the RNA primer were 5’ labeled with ATTO680 to monitor DNA:DNA crosslinking and RNA extension by the denaturing PAGE .", "The system allowed us to probe DNA:DNA base pairing nine ( TEC16 ) , ten ( TEC17 ) and eleven ( TEC18 ) nucleotides upstream of the RNA 3’ end by the stepwise extension of the RNA with subsets of NTPs ( Figure 4A ) .", "The assembled TEC16 preparations produced only minute amounts of DNA:DNA crosslinked species as expected: the template DNA thymidine of TA site was anticipated to form the upstream-last base pair of the RNA:DNA hybrid .", "Upon formation of TEC17 , the efficiency of DNA:DNA crosslinking remained low ( <15% ) , despite the entire TA site being potentially available for pairing .", "In contrast , the crosslinking efficiency exceeded 40% in TEC18 , comparable to that observed in a protein-free DNA:DNA duplex ( ~60% ) .", "TGT reduced crosslinking efficiency in TEC18 at least twofold ( Figure 4A ) consistent with its ability to stabilize TECs in the pre-translocated state ( Malinen et al . , 2012 ) . 10 . 7554/eLife . 18096 . 011Figure 4 . Probing the effects of NusG and deletions of the RNAP domains on the structure of the upstream fork junction by DNA:DNA photocrosslinking with 8-methoxypsoralen ( 8-MP ) .", "( A ) TECs containing the unique 8-MP intercalation site were supplemented with 8-MP , walked by up to three nucleotides , supplemented with 5 µM TGT ( where indicated ) and illuminated with the UV light .", "( B ) DNA:DNA crosslinking in TECs formed by the wild-type and altered RNAPs in the absence ( grey bars ) and presence ( red bars ) of NusG .", "Error bars indicate the range of duplicate measurements or SDs of several measurements ( Table 5 ) .", "The gel panels were spliced from the same gel and the pixel counts were linearly scaled to span the full 8 bit grayscale range within each panel .", "Joined panels have the same scaling . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 01110 . 7554/eLife . 18096 . 012Figure 4—figure supplement 1 . Control experiment: the specificity of DNA:DNA photocrosslinking with 8-MP . Oligonucleotides were mixed in various combinations ( samples 1–7 ) , treated with indicated concentrations of 8‑MP and subjected , where indicated , to 30 min illumination with UV light .", "The vertically stacked panels were spliced from the same gel and the pixel counts were linearly scaled to span the full 8 bit grayscale range within each panel . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 012 NusG effects on DNA:DNA crosslinking in the wild-type TEC18 were within the margins of the experimental errors ( Figure 4B ) .", "However , NusG restored otherwise markedly reduced DNA crosslinking in ΔRL and ΔLL TEC18s .", "These observations suggest that NusG stabilizes the upstream DNA duplex 11 nucleotides upstream the RNA 3’ end , but does not affect crosslinking in the wild-type TEC18 because the TA site is double-stranded even in the absence of NusG .", "Interestingly , NusG marginally but measurably enhanced crosslinking in the wild-type and ΔLL TEC17s , suggesting that NusG may affect the DNA conformation immediately upstream of the RNA:DNA hybrid ( ten nucleotides upstream of the RNA 3’ end ) .", "In light of the effect of NusG on the upstream DNA reannealing , it was reasonable to test its effect on a related and spatially adjacent process of RNA:DNA separation .", "We used template DNA-RNA photo-crosslinking by a guanine analogue 6-thioguanine ( 6-TG ) , to probe the accessibility of RNA to DNA in the absence and presence of NusG .", "The TECs contained the fully complementary transcription bubble and the 5’ ATTO680-labeled 16-nucleotide RNA primer with the nine nucleotide annealing region .", "The initial TEC16 contained 6-TG in the template DNA eight base pairs upstream of the RNA 3’ end ( Figure 5A , Figure 2—figure supplement 1 ) .", "Upon exposure to UV light , the TEC16 produced crosslinked DNA-RNA species that migrated considerably slower than the un-crosslinked RNA primer in a denaturing gel ( Figure 5A ) .", "Two major crosslinked species ( a band and a smear ) were observed that likely originated from 6-TG crosslinks to different RNA bases within the crosslinking range .", "Walking the RNAP along the DNA revealed that 6-TG efficiently crosslinks to the RNA primer eight ( initial TEC16 ) and nine ( TEC17 ) nucleotides upstream of the RNA 3’ end .", "Crosslinking was largely abolished when the separation was increased to ten nucleotides in TEC18 , yet restored when TEC18 was stabilized in the pre-translocated state by TGT ( Figure 5A ) .", "Qualitatively similar results were obtained with the initial TEC16 containing 6-TG nine nucleotides upstream of the RNA 3’ end ( Figure 5—figure supplement 1 ) ; in this system , crosslinking was abolished upon an extension to form TEC17 . 10 . 7554/eLife . 18096 . 013Figure 5 . Probing the effects of NusG and deletions of the RNAP domains on the structure of the upstream fork junction by RNA:DNA photocrosslinking with 6-TG .", "( A ) TECs containing 6-TG in the template DNA were walked by up to three nucleotides , supplemented with 5 µM TGT ( where indicated ) and illuminated with the UV light .", "( B ) RNA:DNA crosslinking in TECs formed by the wild-type and altered RNAPs in the absence and presence of NusG .", "The gel panels were spliced from the same gel and the pixel counts were linearly scaled to span the full 8 bit grayscale range within each panel .", "The pixel intensity profiles for each gel lane are shown above the gels .", "The independent repeats are presented in Figure 5—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 01310 . 7554/eLife . 18096 . 014Figure 5—figure supplement 1 . RNA:DNA photocrosslinking with 6-TG reflects the physical accessibility of RNA to DNA . TECs containing 6-TG in the template DNA nine nucleotides upstream of the RNA 3’ end were walked by up to two nucleotides and illuminated with the UV light .", "The RNA:DNA complementarity was initially nine ( Left ) and eight ( Right ) base pairs .", "6-TG does not pair with RNA in the TEC16 on the right: the mismatch against 6‑TG is colored light blue .", "The vertically stacked panels were spliced from the same gel and the pixel counts were linearly scaled to span the full 8 bit grayscale range within each panel . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 01410 . 7554/eLife . 18096 . 015Figure 5—figure supplement 2 . The effects of NusG ( A ) , TGT ( B ) and ΔLL ( C ) on RNA:DNA photocrosslinking with 6-TG . This figure presents the independent repeats of the experiments in Figure 5 .", "The gel panels were spliced from the same gel and the pixel counts were linearly scaled to span the full 8 bit grayscale range within each panel .", "The pixel intensity profiles for each gel lane are shown above the gels . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 015 Deletion of the RL increased the overall intensity of crosslinks in TEC16 and TEC17 , presumably by eliminating the protein domain that competed with RNA for crosslinking .", "More importantly , DNA efficiently crosslinked to RNA in ΔLL TEC18 , but not in the ΔRL or the wild-type TEC18 ( Figure 5B ) .", "These observations suggest that ΔLL makes the RNA accessible to DNA ten nucleotides upstream the RNA 3’ end , but do not necessarily suggest that the RNA:DNA hybrid is longer in ΔLL TEC .", "An RNA with a mismatch against 6-TG efficiently crosslinked to DNA in the wild-type TEC17 , demonstrating that the crosslinks reflect the physical accessibility of RNA to DNA and do not require the RNA:DNA base pairing ( Figure 5—figure supplement 1 ) .", "NusG did not measurably affect the crosslinking efficiency eight , nine or ten nucleotides upstream of the RNA 3’ end in wild-type and altered TECs ( Figure 5B ) , indicating that NusG does not alter the accessibility of RNA to DNA at the upstream edge of the transcription bubble .", "We have previously reported that the base analogue fluorophore 6-MI positioned in template DNA strand within the 5’-TXG-3’ beacon sequence ( where X is 6-MI and G is a guanine functioning as a quencher ) displays 2–5 fold brighter fluorescence when positioned nine and ten nucleotides upstream the RNA 3’ end relative to fluorescence levels observed eight and eleven nucleotides upstream of the RNA 3’ end ( Figure 6A ) .", "This system was originally designed for the time-resolved studies of the RNAP translocation ( Malinen et al . , 2012 ) .", "Here we revisit this setup to complement the photocrosslinking techniques in assessing the effects of NusG on base pairing immediately upstream of the RNA:DNA hybrid and on the conformation of the upstream DNA . 10 . 7554/eLife . 18096 . 016Figure 6 . Probing the effects of NusG and deletions of the RNAP domains on the structure of the upstream fork junction by a fluorescent beacon in the template DNA . Error bars indicate the range of duplicate measurements or SDs of several measurements ( Table 5 ) .", "( A ) Walking 6-MI ( yellow dash ) through the upstream fork junction modulates 6-MI stacking with the upstream guanine ( black dash ) that functions as a strong quencher .", "( B ) The effects of ΔRL and ΔLL on the TEC fluorescence in the absence and the presence of NusG .", "( C ) The effects of NusG on 6-MI fluorescence of the wild-type and altered TECs .", "Monitoring the fluorescence of TEC17 upon mixing with NusG in a stopped flow instrument ( graph on the right , black curves ) allows for the estimation of the binding and the dissociation rate constants .", "The analysis scheme is depicted below the graph .", "The best fit curves ( red ) were simulated using k+1=9 . 2 µM−1s−1; k−1=1 . 1 s−1 .", "The lower and upper bounds of rate constants were calculated by combined analysis of data from two independent experiments by FitSpace routine of Kintek Explorer software ( at a 10% increase in Chi2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 01610 . 7554/eLife . 18096 . 017Figure 6—figure supplement 1 . Primary data for Figure 6: the effects of NusG and deletions of the RNAP domains on 6-MI fluorescence . Black vertical arrows depict the changes in fluorescence relative to the neighboring graph on the left .", "The wild-type TEC data ( faded graphs ) are presented for comparison .", "Error bars indicate the range of duplicate measurements or SDs of several measurements ( Table 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 017 The TECs contained the fully complementary transcription bubble and 16-nucleotide RNA primer with nine nucleotides annealing region ( Figure 6A , Figure 2—figure supplement 1 ) .", "The initial TEC16 contained 6-MI base in template DNA positioned eight base pairs upstream of the RNA 3’ end .", "The TEC was walked along the DNA by up to three positions by the addition of subsets of NTPs and the 6-MI fluorescence was monitored ( Figure 6A ) .", "We attribute the low fluorescence of 6-MI eight nucleotides upstream the RNA 3’ end ( the assembled TEC16 ) to the quenching effect of the upstream guanine that forms the upstream-most base pair of the RNA:DNA hybrid .", "The elevated 6-MI fluorescence nine nucleotides upstream of the RNA 3’ end is likely due to the unstacking of the upstream quenching guanine because it no longer belongs to the RNA:DNA hybrid .", "The elevated fluorescence persists ten nucleotides upstream of the RNA 3’ end , arguing that 6-MI does not reestablish the stacking interaction with the upstream guanine .", "Finally , the 6-MI fluorescence is reduced 11 nucleotides upstream from the RNA 3’ end , likely because 6-MI reestablishes the stacking interaction with the upstream guanine at this position .", "An experiment performed in the presence of TGT suggests that increased 6-MI fluorescence at the upstream edge of the RNA:DNA hybrid originates from a post-translocated TEC ( Figure 2D ) .", "The major effect of NusG on the wild-type TEC was the increase in the fluorescence of 6-MI at the upstream edge of the RNA:DNA hybrid ( in TEC17 ) .", "The effect was also observed in ΔLL and ΔRL TEC17s ( Figure 6C , Figure 6—figure supplement 1 ) .", "The rate of fluorescence increase in the wild-type TEC17 was dependent on NusG concentration with Kd ~120 nM ( Figure 6C ) .", "NusG binds about 30 Å from 6-MI at the upstream edge of the RNA:DNA hybrid and is therefore unlikely to affect the 6-MI fluorescence directly .", "Instead , we suggest that NusG increases 6-MI fluorescence by repositioning the quenching guanine immediately upstream of the RNA:DNA hybrid .", "In the absence of LL , NusG significantly changed 6-MI fluorescence also in TEC18 and TEC19 ( Figure 6B ) .", "Remarkably , ΔLL TECs deviated most from the wild-type TECs in the absence of NusG but displayed the identical fluorescence intensities in the presence of NusG ( Figure 6B ) .", "Overall , the effects of NusG on 6-MI fluorescence largely paralleled its effects on the DNA crosslinking with 8-MP leading to similar conclusions:", "( i ) NusG likely affects the DNA conformation immediately upstream of the RNA:DNA hybrid and", "( ii ) NusG reverses the alterations in the upstream DNA conformation introduced by deletion of the LL .", "However , in contrast to 8-MP crosslinking experiments , NusG did not compensate for and , instead , increased the differences in the fluorescence intensities between the wild-type and ΔRL TECs ( Figure 6B ) .", "NusG likely inhibits backtracking by acting on the upstream DNA that , at the time of writing , is universally absent from the crystal structures of bacterial TECs .", "At the same time , the conformation of the upstream DNA in published TEC models ( Opalka et al . , 2010; Martinez-Rucobo et al . , 2011; Andrecka et al . , 2009 ) as well as recent X-ray ( Barnes et al . , 2015 ) and CryoEM ( Bernecky et al . , 2016 ) structures of RNA polymerase II are incompatible with the structure of bacterial TEC ( in case of RNA polymerase II models ) , the upstream DNA mapping data presented here ( see below ) , and/or NusG binding .", "Similarly , the conformation of the upstream DNA resolved in the crystal structures of the bacterial initiation complexes ( Zuo and Steitz , 2015; Bae et al . , 2015; Liu et al . , 2016 ) is strongly influenced by the sigma factor and therefore is not suitable for modeling of the upstream fork junction in the TEC .", "To gain the mechanistic insights into the anti-backtracking action of NusG , we used our data to generate an accurate map of the upstream fork junction ( Figure 7B ) and further employed it to postulate a tentative structural model of a NusG-TEC complex with the upstream DNA ( Figures 1 , 8 ) . 10 . 7554/eLife . 18096 . 018Figure 7 . The effects of DNA mismatches suggest the minimal transcription bubble .", "( A ) DNA:DNA mismatches against quenching guanine ( top ) and 6-MI ( middle ) , or downstream of the TA site ( bottom ) alter the TEC properties when positioned ten nucleotides upstream of the RNA 3' end and further upstream .", "Error bars indicate the range of duplicate measurements or SDs of several measurements ( Table 5 ) .", "( B ) Mapping the upstream edge of the transcription bubble based on data in Figures 3C , 4A , 5A , 6A and 7A . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 01810 . 7554/eLife . 18096 . 019Figure 7—figure supplement 1 . Primary data for Figure 7A: the effects of DNA mismatches on 6-MI fluorescence . Black vertical arrows depict the changes in fluorescence relative to the graph on the left .", "Error bars indicate the range of duplicate measurements or SDs of several measurements ( Table 5 ) .", "( A ) The mismatch is against quenching guanine .", "( B ) The mismatch is against 6‑MI . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 01910 . 7554/eLife . 18096 . 020Figure 7—figure supplement 2 . Primary data for Figure 7A: the effect of a DNA mismatch downstream of the TA site on crosslinking with 8‑MP . Error bars indicate the range of duplicate measurements or SDs of several measurements ( Table 5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 02010 . 7554/eLife . 18096 . 021Figure 8 . A model of the upstream fork junction . DNA bases are numbered from the RNA 3' end in the post-translocated TEC .", "( A ) An overview: β’ LL and the structurally conserved five amino acid loop of NusG NTD form a channel accommodating the exiting upstream DNA .", "( B ) The template DNA nucleotide at position ten can be modeled to pair with the non-template DNA in a partially unstacked conformation ( top ) or interact with the cleft between the β’ RL and β’ LL ( bottom ) .", "DNA and RNA bases are shown as spheres , sugar-phosphate backbones as cartoons .", "The β subunit is omitted for clarity .", "( C ) The superimposition of Aquifex aeolicus NusG NTD ( PDB ID 1M1G ) and Pyrococcus furiosus Spt5 NTD ( PDB ID 3P8B ) .", "The cartoon color changes from magenta in well superimposed regions to cyan in poorly superimposed or unaligned regions .", "The β’ clamp helices ( yellow , from PDB ID 3QQC ) , the upstream DNA ( grey , from the model in A ) and Spt4 ( cyan cartoon , from PDB ID 3P8B ) are included to present the superimposition in the context of the TEC .", "( D ) Multiple sequence alignment of the structurally conserved five amino acid loop of NusG family proteins and the flanking secondary structure elements .", "Species names are abbreviated as follows: Eco , E . coli , Bsu , Bacillus subtilis , Mtu , Mycobacterium tuberculosis , Tma , Thermotoga maritima , Syn , Synechocystis sp .", "PCC 6803 , Tth , T . thermophilus , Aae , Aquifex aeolicus , Mja Methanocaldococcus jannaschii , Pfu , Pyrococcus furiosus , Sce , Saccharomyces cerevisiae , Hsa , Homo sapiens .", "Amino acid residues are shaded as follows: hydrophobic –green , polar –olive , Pro and Gly –yellow , Asp and Glu –red , Arg , Lys and His –cyan . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 02110 . 7554/eLife . 18096 . 022Figure 8—source data 1 . NusG-TEC model . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 022 Together , the 8-MP DNA:DNA photocrosslinking ( ± TGT ) , 6-TG DNA:RNA photocrosslinking ( ± TGT ) , 6-MI fluorescence pattern and crystal structures of bacterial TECs lacking the upstream DNA ( Vassylyev et al . , 2007 ) define the resting TECs as", "( i ) post-translocated ,", "( ii ) containing nine base pairs RNA:DNA hybrid , and", "( iii ) containing the upstream DNA duplex that starts eleven nucleotides upstream of the RNA 3’ end .", "The experiments with TECs containing mismatched non-template DNA strand additionally suggest that the upstream DNA base pairs ten nucleotides upstream of the RNA 3’ end ( Figure 7A ) .", "Specifically , the 8-MP crosslinks the TA site eleven nucleotides upstream of the RNA 3’ end only when DNA is matched ten nucleotides upstream of the RNA 3’ end ( Figure 7A , Figure 7—figure supplement 2 ) .", "Similarly , TECs containing a DNA:DNA mismatch either directly against or one nucleotide upstream of 6-MI start to differ in fluorescence levels from the matched TECs when the mismatch is ten nucleotides from the RNA 3’ end ( Figure 7A , Figure 7—figure supplement 1 ) .", "Therefore , DNA likely base pairs ten nucleotides from the RNA 3’ end , but the first upstream base pair deviates from the geometry of the conventional B-form DNA duplex and/or is highly dynamic ( Figure 8B ) .", "To generate a NusG-TEC model , we positioned NusG NTD over the β’ clamp helices in bacterial TEC lacking the upstream DNA ( Vassylyev et al . , 2007 ) guided by the model of NusG-RNAP complex in ( Martinez-Rucobo et al . , 2011 ) .", "We then modelled in the upstream duplex DNA following the overall direction suggested by the published models ( Opalka et al . , 2010; Andrecka et al . , 2009; Bernecky et al . , 2016 ) but avoiding clashes with the NusG NTD and maintaining a canonical B-duplex as far downstream as possible .", "Joining the upstream duplex with the RNA:DNA hybrid required altering the sugar-phosphate backbone of the downstream-most nucleotides of the upstream DNA but allowed maintaining the DNA base pairing .", "In the resulting model ( Figure 1 , 8 ) , the upstream DNA has an ample space to move away from the NusG NTD and the β’ clamp surfaces towards the cleft between the β1 and β flap domains by hinging around the downstream most base pair ( position ten in Figure 8 ) , reflecting the natural flexibility of the upstream DNA ( Coban et al . , 2006 ) .", "However , we suggest that the conformation where the upstream DNA lines β’ clamp and NusG NTD ( Figure 1 , 8 ) is the most relevant to the NusG effects on transcription elongation .", "In such a scenario , the two downstream- most base pairs of the upstream DNA ( positions ten and eleven in Figure 8 ) occupy a narrow channel walled by NusG NTD and LL , thereby explaining the functional interactions between the NusG , the LL and the upstream DNA in RNA cleavage , crosslinking and fluorescence assays ." ], [ "E . coli NusG was shown to enhance elongation in vitro over two decades ago , acting mainly by reducing pausing ( Burova et al . , 1995 ) .", "More specifically , NusG was found to reduce backtracked but not hairpin-stimulated pauses ( Artsimovitch and Landick , 2000; Pasman and von Hippel , 2000 ) .", "It was later established that NusG-family proteins bind to , and bridge , the β’ clamp with the β lobe across the RNAP cleft ( Belogurov et al . , 2007; Mooney et al . , 2009b; Klein et al . , 2011; Martinez-Rucobo et al . , 2011 ) .", "Biochemical studies further concluded that the archaeal NusG orthologue Spt5 ( Hirtreiter et al . , 2010 ) and the specialized NusG paralogue RfaH ( Sevostyanova et al . , 2011 ) enhance transcription elongation by stabilizing the β’ clamp in a closed conformation .", "However , E . coli RfaH accelerates RNAP at pause sites known to involve clamp opening , as well as at the backtracked pauses , whereas E . coli NusG has only marginal effect at the former sites ( Kolb et al . , 2014; Anthony et al . , 2000; Belogurov et al . , 2010 ) .", "Moreover , crystal structures of the backtracked TECs revealed the closed clamp ( Sekine et al . , 2015; Wang et al . , 2009 ) , whereas the specificity of E . coli NusG for backtracked pauses was reaffirmed in single molecule experiments ( Herbert et al . , 2010 ) .", "Together , these observations suggest that E . coli NusG enhances transcription elongation by means other than restricting the β’ clamp movement .", "Here , we show that NusG slows backtracking but does not affect the on-pathway elongation in the non-paused TEC used in our study .", "In contrast , Herbert et al ( Herbert et al . , 2010 ) concluded that NusG has a modest stimulatory effect ( 10–20% ) on the pause-free elongation rate , in addition to inhibiting backtracking .", "One possibility is that a subset of TECs backtracked by one nucleotide display the elongation rates within the pause-free range compiled by Herbert et al . Alternatively , NusG may have a marginal effect on the elongation rate in a subset of the non-paused on-pathway TECs with yet to be identified sequence determinants .", "In any case , the effect of NusG on the pause-free elongation rate estimated by Herbert et al is small comparing with the specific effect of NusG on the backtracking rate ( ~2 . 5 fold ) that we report here .", "We further demonstrate that two DNA mismatches immediately upstream of the RNA:DNA hybrid increase the backtracking rate and render the TEC insensitive to NusG .", "In contrast , NusG reduced backtracking normally in a TEC missing the GL , a contact point with the β subunit ( Figure 1 ) that is required for anti-pausing by E . coli RfaH ( Sevostyanova et al . , 2011 ) .", "β’ RL was similarly dispensable , whereas the β’ LL was slightly stimulatory for the anti-backtracking activity of NusG .", "Overall , our data suggest that the intrinsic action of NusG on the E . coli TEC is restricted to inhibiting backtracking and is exclusively mediated through the upstream fork junction .", "Remarkably , several other transcription factors , including Mfd ( Deaconescu et al . , 2006 ) and UvrD ( Epshtein et al . , 2014 ) that link transcription to DNA repair operate through the upstream fork junction .", "Interestingly , ΔGL TEC backtracked two-fold slower than the wild-type TEC both in the presence and in the absence of NusG ( Figure 3C ) .", "GL is located more than 20Å from the duplex DNAs and the RNA:DNA hybrid but may directly contact the single-stranded non-template DNA .", "Accordingly , we suggest that GL promotes backtracking by altering the conformation of the non-template DNA in a manner that increases the propensity of the TEC to backtrack , e . g . by facilitating the downstream DNA re-annealing .", "Indeed , GL restricts the downstream portion of the single-stranded non-template DNA within the main channel in the initiation complex ( Zhang et al . , 2012 ) and may therefore have a similar functionality in the TEC .", "The upstream DNA decisively emerged as the major determinant of NusG anti-backtracking effect .", "However , the upstream DNA is absent from the crystal structures of bacterial TECs , the published models are incompatible with NusG binding , and even the register of the upstream DNA reannealing is uncertain .", "To gain mechanistic insights into the anti-backtracking action of NusG , we performed a comprehensive mapping of the upstream fork junction using fluorescent base analogues and site-specific crosslinking .", "Our data are fully consistent with the nine base pairs RNA:DNA hybrid and the upstream DNA duplex that starts eleven base pairs upstream of the RNA 3’ end in the post-translocated TEC ( Figure 7B ) .", "The effects of DNA mismatches additionally suggest that DNA is paired immediately upstream of the RNA:DNA hybrid .", "In combination , the data obtained with the matched and mismatched TECs suggest that the first pair of the upstream DNA is unstacked from both the RNA:DNA hybrid and the rest of the upstream DNA .", "We then combined our mapping data with the model of the NusG-RNAP complex ( Martinez-Rucobo et al . , 2011 ) to sketch a NusG-TEC model with the upstream DNA .", "We found that in a subset of spatially feasible conformations of the upstream DNA ( see results ) , the two downstream-most base pairs are accommodated in a narrow 'exit' channel walled by the NusG and the LL ( Figure 1 , 8 ) .", "Such an arrangement plausibly explains the cooperation between NusG and the LL in stabilizing the upstream DNA pairing and inhibiting backtracking .", "Notably , NusG loop facing the upstream DNA is strictly conserved in size in bacteria and archaea ( five amino acids , Figure 8 ) , but the evidence for the conserved residue-specific contacts between the NusG and the upstream DNA is lacking .", "We propose that NusG provides a complementary molecular surface to the paired upstream DNA and possibly also affects the overall direction of the upstream DNA duplex .", "Finally , NusG likely stabilizes DNA pairing only in a subset of the upstream DNA conformations , yet influences the overall backtracking rate by targeting those conformations that are most favorable for backtracking , i . e . , the non-template DNA is optimally positioned for the strand exchange with the RNA .", "NusG inhibits backtracking by stabilizing the DNA base pair eleven nucleotides upstream of the RNA 3’ end ( Figure 3C ) .", "This pair corresponds to the second DNA pair upstream of the RNA:DNA hybrid in the post-translocated TEC ( pair eleven in Figure 8 ) .", "However , NusG does not affect the equilibrium between the pre- and post-translocated states ( Figure 2C–D ) and therefore likely acts on the pre-translocated TEC where the base pair eleven nucleotides upstream of the RNA 3’ end lies immediately upstream of the RNA:DNA hybrid ( pair ten in Figure 8 , see also schematics in Figure 3C ) .", "Accordingly , we propose that NusG facilitates DNA pairing immediately upstream of the RNA:DNA hybrid in the pre-translocated TEC , thereby reducing backtracking .", "It remains uncertain how NusG inhibits backtracking without affecting the equilibrium between the post- and pre- translocated states ( Figure 2C–D ) .", "One possible explanation is that backtracking and backward translocation are limited by different processes in our system .", "It has been hypothesized that , rather than moving in sync along the different nucleic acid chains of the TEC , RNAP moves forward by sequentially translocating the RNA:DNA hybrid and the downstream DNA ( Brueckner and Cramer , 2008 ) .", "The synchronous sliding of RNAP along the nucleic acids is only superficially plausible in the 2D schematics ( Figures 2–7 ) but is much less likely in the actual 3D structure of the TEC ( Figure 1 ) where the downstream DNA and the RNA:DNA hybrid are separated by a 90° bend .", "Analogously , the backward translocation may involve the sequential translocation of the downstream DNA and the RNA:DNA hybrid .", "The former process is NusG independent and may limit the rate of the backward translocation , whereas the latter process is modulated by NusG and may be thus rate limiting for backtracking .", "We further argue that the difficulty of reconciling the large effect of NusG on backtracking with its small ( Herbert et al . , 2010 ) or undetectable ( this work ) effect on the forward and backward translocation in the context of a single-step translocation model lends support to a two-step translocation mechanism .", "The available data suggest three independent and structurally plausible effects of NusG NTD on the TEC .", "First , NusG binds near the upstream fork junction and stabilizes the upstream DNA duplex , thereby inhibiting spontaneous backtracking at most template positions ( [Herbert et al . , 2010] and this work ) .", "Second , the NTD restricts the conformational flexibility of the β’ clamp , with different outcomes for the transcription elongation .", "Archaeal Spt5 ( Hirtreiter et al . , 2010; Schulz et al . , 2016 ) and E . coli RfaH ( Sevostyanova et al . , 2011 ) exert at least part of their elongation enhancing effects by stabilizing the clamp .", "In contrast , clamp stabilization by E . coli NusG only marginally contributes to anti-pausing ( Kolb et al . , 2014; Belogurov et al . , 2010 ) .", "Third , NusG binds to specific sequences in the single-stranded non-template DNA , thereby introducing infrequent yet physiologically relevant pauses ( Yakhnin et al . , 2016 , 2008 ) and facilitating intrinsic termination in some species ( Czyz et al . , 2014 ) .", "Similar effects are well documented for other dissociable factors and TEC components positioned near the single stranded non-template DNA ( Perdue and Roberts , 2011; Artsimovitch and Landick , 2002; Vvedenskaya et al . , 2014; Arimbasseri and Maraia , 2015 ) .", "Interestingly , T . thermophilus NusG slows down the cognate RNAPs at non-paused sites by an unknown mechanism ( Sevostyanova and Artsimovitch , 2010 ) .", "It remains to be determined if this unusual effect is mediated through the clamp or contacts with the non-template and upstream DNA .", "We argue that anti-backtracking represents the only conserved functionality of NusG family proteins .", "Backtracking is a universally conserved and functionally important feature of the multisubunit RNAPs that has been documented in vitro and in vivo in both bacteria and eukaryotes ( reviewed in [Nudler , 2012] ) .", "The stimulation of RNA chain elongation by NusG has been documented in vitro for bacterial ( Burova et al . , 1995 ) , archaeal ( Hirtreiter et al . , 2010 ) and eukaryotic ( Wada et al . , 1998 ) transcription systems .", "The NTD is sufficient for these effects on elongation , but the structural elements that superimpose well between the bacterial NusG NTD and the archaeal Spt5 NTD ( Figure 8C ) are limited to", "( i ) the beta sheet that comprises the RNAP-binding site ,", "( ii ) the conserved five amino acid loop that we implicate in the anti-backtracking action of E . coli NusG , and", "( iii ) the N-terminus of the α-helix that follows this loop and possibly interacts with the single-stranded non-template DNA ( Crickard et al . , 2016 ) .", "The lack of the strong conservation of the surface residues ( Figure 8D ) suggests that the anti-backtracking activity may be determined by the overall fold of the NusG NTD and is only weakly dependent on the nature of the individual amino acid side-chains , consistent with the mutational analysis of E . coli NusG ( Mooney et al . , 2009b ) .", "While the above considerations suggest that the stimulation of transcription elongation by NusG family proteins may be important for long-term survival and fitness , in the experimental systems studied to date , functional contacts established by the CTD , such as Rho and S10 in Bacteria , appear to be more critical .", "In E . coli , the essential function of NusG is to facilitate termination of transcription by Rho , thereby maintaining the operon borders ( Cardinale et al . , 2008 ) , suppressing pervasive antisense transcription ( Peters et al . , 2012 ) , and inhibiting R-loop formation ( Krishna Leela et al . , 2013 ) .", "Stimulating Rho-dependent termination is also likely the major , albeit a non-essential , function of B . subtilis NusG ( Ingham and Furneaux , 2000 ) .", "NusG and its paralog RfaH have also been proposed to mediate transcription-translation coupling via direct contacts with S10 ( Burmann et al . , 2010 , Burmann and Rösch , 2011 ) .", "However , the CTD contacts are not universally conserved: Rho is absent in eukaryotes and even some Bacteria ( D’Heygère et al . , 2013 ) , whereas S10 and RNAP are separated by a nuclear membrane in eukaryotes , where CTD interacts with proteins involved in splicing , polyadenylation , and other RNA processing pathways .", "In eukaryotes , the intrinsic stimulatory activity of Spt5 NTD on transcription elongation is non-essential , but abolishing it leads to the temperature sensitive phenotypes ( Crickard et al . , 2016 ) .", "In rare circumstances , the stimulatory effect of Spt4/5 may possibly be deleterious: Stp4/5 has been suggested to facilitate transcription through toxic repeat sequences in eukaryotes , thereby contributing to the progress of neurodegenerative disorders ( Kramer et al . , 2016 ) .", "Overall , we suggest that the universal conservation of the intrinsic stimulatory activity of NusG family proteins on transcription elongation underscores its importance , but the quantitative assessment of the in vivo role of this functionality in bacteria necessitates the analysis of transcription systems that natively lack Rho and Gre factors , e . g . , those of Cyanobacteria ." ], [ "DNA and RNA oligonucleotides were purchased from IBA Biotech ( Göttingen , Germany ) and Fidelity Systems ( Gaithersburg , MD , USA ) .", "DNA oligonucleotides and RNA primers are listed in Table", "2 . NTPs were from Jena Bioscience ( Jena , Germany ) .", "Tagetitoxin ( TGT ) was from Epicentre ( Madison , WI , USA ) , 8-methoxypsoralen ( 8-MP ) was from Sigma ( St . Louis , MO , USA ) .", "The following buffers were used for the TEC assembly and transcription assays: TB0 ( 40 mM HEPES-KOH pH 7 . 5 , 80 mM KCl , 5% glycerol , 0 . 1 mM EDTA , and 0 . 1 mM DTT ) , TB1 ( TB0 supplemented with 1 mM MgCl2 ) , TB2 ( TB0 supplemented with 2 mM MgCl2 and 300 mM KCl ) and TB10 ( TB0 supplemented with 10 mM MgCl2 ) . 10 . 7554/eLife . 18096 . 023Table 2 . DNA oligonucleotides and RNA primers used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 023NametypeSequence ( 5’→3’ ) EmploymentS041MtDNAGCTACTCTACTGACATGATGCCTCCTCTXGAACCTTAGATCGCTACAAGTFigures 2 , 6–7S154StDNAGCTACTCTACTGACATGATGCCTCCTCTGSAACCTTAGATCGCTACAAGTFigure 5—figure supplement 1S155StDNAGCTACTCTACTGACATGATGCCTCCTCTSGAACCTTAGATCGCTACAAGTFigure 5S042ntNAACTTGTAGCGATCTAAGGTTCCAGAGGAGGCATCATGTCAGTAGAGTAGCFigures 2 , 5–7S150ntDNAACTTGTAGCGATCTAAGGTTACAGAGGAGGCATCATGTCAGTAGAGTAGCFigure 7AS056MtDNAGCTACTCTACTGCAATGACGTCTCCTCTXGAACCTTAGATCGCTACAAGTFigures 2C , 3B , 7AS076tDNAGCTACTCTACTGCAATGACGTCTCCTCTGGAACCTTAGATCGCTACAAGTFigure 3S057ntDNAACTTGTAGCGATCTAAGGTTCCAGAGGAGACGTCATTGCAGTAGAGTAGCFigures 2C , 3 , 7AS152ntDNAACTTGTAGCGATCTAAGGTTCGAGAGGAGACGTCATTGCAGTAGAGTAGCFigures 3C , 7AS153ntDNAACTTGTAGCGATCTAAGGTTGGAGAGGAGACGTCATTGCAGTAGAGTAGCFigure 3CS173ntDNAACTTGTAGCGATCTAAGGTTAAAGAGGAGACGTCATTGCAGTAGAGTAGCFigure 3—figure supplement 3S114tDNACGTACTCTACTCGAATAGCATCTCCTCTGGAACCTTAGATCGTCACAAGTFigure 3CS115ntDNAACTTGTGACGATCTAAGGTTCCAGAGGAGATGCTATTCGAGTAGAGTACGFigure 3CS170tDNAAtto680- TGGTGTCTGCTGTCCGTCTGCCTCCTCTGTAGTCTGTGCTCGTGTCTGGTFigures 4 , 7AS171ntDNAACCAGACACGAGCACAGACTACAGAGGAGGCAGACGGACAGCAGACACCAFigures 4 , 7AS224ntDNAACCAGACACGAGCACAGACTAAAGAGGAGGCAGACGGACAGCAGACACCAFigure 7AR024RNAAtto680- CUCACAACCAGAGGAG Figures 2 , 5–7R052RNAAtto680- CUCACAACCAGAGGAGYC Figure 3R079RNAAtto680- CAACACAACAGAGGAG Figures 4 , 7 , Figure 5—figure supplement 1X = 6-methyl-isoxanthopterin; S = 6-thioguanine; Y = 2-aminopurineMismatches in ntDNA are marked in blue and underlined . R079 is a chimeric oligo: six 5’ nucleotides are DNA .", "All proteins were expressed in E . coli Xjb ( DE3 ) ( Zymo Research , Irvine , CA ) .", "The wild-type , ΔLL ( β´ΔP251-S263→GG ) , ΔRL ( β´ΔN309-K325 ) and ΔGL ( βΔR368-P376→GG ) RNAPs were purified by Ni- , heparin and Q-sepharose chromatography as described previously ( Svetlov and Artsimovitch , 2015 ) .", "E . coli NusG was captured from the lysate by Ni-sepharose , the N-terminal hexa-histidine tag was cleaved by TEV-protease , imidazole was removed by dialysis , and the un-cleaved NusG , the cleaved tag and the TEV-protease were absorbed by passing the NusG solution over the Ni-sepharose .", "E . coli GreA containing C-terminal hexa-histidine tag was captured from lysate by Ni-sepharose followed by gel filtration as described ( Perederina et al . , 2006 ) .", "All proteins were dialyzed against the storage buffer ( 50% glycerol , 20 mM Tris-HCl pH 7 . 9 , 150 mM NaCl ( 1M NaCl for GreA ) , 0 . 1 mM EDTA , 0 . 1 mM DTT ) and stored at −20°C .", "Plasmids are listed in Table", "3 . Sequences of the plasmids are provided as Supplementary file 5 ( plasmids . fas ) . 10 . 7554/eLife . 18096 . 024Table 3 . E .", "coli protein expression vectors used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 024NameDescriptionSource/referencepVS10wild-type RNAP ( T7p-α-β-β’_His6-T7p-ω ) ( Belogurov et al . , 2007 ) pTG011ΔβGL RNAP ( T7p-α-His6_β[ΔR368-P376→GG]-β’-ω ) this workpMT041Δβ’RL RNAP ( T7p-α-β-β’[ΔN309-K325]_TEV_His10-T7p-ω ) this workpHM001Δβ’LL RNAP ( T7p-α-β-β’[ΔP251-S263→GG]_TEV_His10-T7p-ω ) this workpIA578GreA ( T7p-GreA_His6 ) ( Perederina et al . , 2006 ) pGB043NusG ( T7p-His6_TEV_NusG ) made by GB in Artsimovitch lab . Sequences of the plasmids are provided as Supplementary file 5 ( plasmids . fas ) .", "TECs ( 1 µM ) were assembled by a procedure developed by Komissarova et al ( Komissarova et al . , 2003 ) .", "An RNA primer was annealed to the template DNA , incubated with RNAP for 10 min , and with the non-template DNA for 20 min at 25°C .", "RNA , template DNA , non-template DNA and RNAP were present at 1–2 µM during assembly .", "The exact ratios between the TEC components in different assays are listed in Table", "4 . The assembly was carried out in TB0 buffer for TECs used in backtracking , RNA cleavage and dinucleotide release experiment or in TB10 buffer for TECs used in all the other experiments .", "In the nucleotide addition experiments the assembled TEC16 were used .", "In the pyrophosphorolysis and NusG binding experiments the assembled TEC16s were pre-extended into TEC17s with 5 µM ATP or GTP , respectively .", "In the former case , the excess of ATP was further removed by passing through the desalting spin columns ( 40K cutoff ) with TB10 buffer .", "In the RNA cleavage experiments TEC18s were directly assembled in TB0 buffer . 10 . 7554/eLife . 18096 . 025Table 4 . TEC assembly ratios and reaction buffers . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 025Concentrations during assembly , µM Assembly bufferReaction buffer*RNAtDNAntDNARNAPTECadditiveNucleotide addition ( gel ) 11 . 421 . 5TB10TB10TB10Figure 2BRNA cleavage ( gel ) 11 . 421 . 5TB0TB0TB2Figure 3BForward translocation ( nucleotide addition ) 1 . 4121 . 5TB10TB10TB10Figure 2BBackward translocation ( pyrophosphorolysis ) 1 . 4121 . 5TB10TB10TB10Figure 2CBacktracking ( RNA cleavage ) 1 . 4121 . 5TB0TB0TB2Figure 3BNusG binding1 . 4121TB1TB1TB1Figure 6CEquilibrium 6-MI assays1 . 4121 . 5TB10TB10Figures 2D , 6–7Dinucleotide release ( RNA cleavage ) 11 . 421 . 5TB0TB0TB2Figure 38-MP crosslinking1 . 2111 . 5TB10TB10Figures 4 , 7A6-TG crosslinking1121 . 5TB10TB10Figure 5* In time resolved assays the equal volumes of the TEC and the additive solutions were mixed to initiate the reaction . 10 . 7554/eLife . 18096 . 026Table 5 . The number of repeats for each experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 18096 . 026FigureDataNumber of experimentswith independently assembled TECsincluding the experiments with the same TEC preparationwith independently assembled TECs not in the figurescontrol+NusGcontrol+NusGcontrol+NusG2BWT catalysis WT translocation4 33 28 >126 >82CWT pyrophosphorolysis22>8>82DWT TGT binding223BCWT RNA cleavage WT 6-MI WT 2-AP ΔRL 2-AP ΔLL 2-AP ΔGL 2-AP WT mm1 2-AP WT mm1-2 2-AP WT 3’mm 2-AP2 2 2 2 2 2 2 2 22 2 2 2 2 2 2 2 23 >8 >8 >8 >8 >8 >8 >8 >84 >8 >8 >8 >8 >8 >8 >8 >8623S3WT 6-MI WT 2-AP WT mm1-2 AA 2-AP WT mm1-2 GG 2-AP2 1 1 22 1 1 25 3 7 >85 3 8 >811The experiments reported in the figures were performed with the same batch of GreA .", "The older and the newer experiments cannot be directly combined with the reported experiments due to the variations in the specific activity of the GreA preparations .", "However , the relative effect of NusG on the reactions involving backtracking can be estimated from all available data .", "In the WT TEC NusG inhibits reactions that involve backtracking: 2 . 61 ± 0 . 28 fold ( n = 5 , 2 µM GreA ) ; 2 . 62 ± 0 . 22 fold ( n = 6 , 8 µM GreA ) 4ABWT 8-MP ΔRL 8-MP ΔLL 8-MP WT TEC18+TGT 8-MP7 3 3 23 2 2 5AB 5S2WT 6-TG ΔRL 6-TG ΔLL 6-TG WT TEC18+TGT 6-TG7 2 3 23 2 2 6AB , C ( Left ) WT 6-MI ΔRL 6-MI ΔLL 6-MI2 2 22 2 2>5 5* 5*>3 3* >3>7>7* Except TEC19 .", "SD of the fluorescence measurements with the same TEC preparation = 2 . 3 ± 1 . 3% ( n = 122 ) .", "SD of the fluorescence measurements with the independently assembled TECs = 16% ( n = 8 , WT TEC17 measured with different batches of the fluorescent oligonucleotides ) .", "Accordingly , the primary data from all fluorescent experiments cannot be directly combined with a figure but some NusG effects can be estimated with the highest accuracy and precision from all available data .", "NusG effects on the fluorescence intensity of the WT TECs: ( TEC17NusG–TEC16NusG ) / ( TEC17-TEC16 ) = 1 . 45 ± 0 . 09 ( n = 9 ) ( TEC18NusG–TEC16NusG ) / ( TEC18-TEC16 ) = 0 . 99 ± 0 . 02 ( n = 7 ) ( TEC19NusG–TEC16NusG ) / ( TEC19-TEC16 ) = 0 . 50 ± 0 . 35 ( n=3 ) 6C ( Right ) WT+NusG 6-MI2>67ATEC16-19 6-MI29 ( except TEC19 ) TEC16-19 6-MI24 ( except TEC19 ) TEC16-18 8-MP22 Time-resolved measurements of nucleotide addition were performed in an RQF 3 quench-flow instrument ( KinTek Corporation , Austin , TX ) .", "The reaction was initiated by rapid mixing of 14 µl of 0 . 4 µM TEC with 14 µl of 400 µM NTP .", "Both TEC and NTP solutions were prepared in TB10 buffer .", "The reaction was allowed to proceed for 0 . 004–10 s at 25°C , quenched with 86 µl of 0 . 5 M HCl and immediately neutralized by adding 171 µl of neutralizing-loading buffer ( 94% formamide , 290 mM Tris base , 13 mM Li4-EDTA , 0 . 2% Orange G ) .", "RNA extension was also followed in 6-MI fluorescence assays by withdrawing 8 µl aliquots from the fluorometer cuvette into 12 µl of gel loading buffer ( 94% formamide , 20 mM Li4-EDTA and 0 . 2% Orange G ) .", "RNA cleavage was monitored by manual mixing of 50 µl of 0 . 2 µM TEC in TB0 buffer with 50 µl of 16 µM GreA in TB2 buffer .", "The aliquots ( 8 µl ) were withdrawn at the indicated time points and quenched with 12 µl of the gel loading buffer .", "The TEC solutions were supplemented with 4 µM NusG where indicated .", "RNAs were separated on 16% denaturing polyacrylamide gels and visualized with Odyssey Infrared Imager ( Li-Cor Biosciences , Lincoln , NE ) ; band intensities were quantified using ImageJ software ( Abramoff et al . , 2004 ) .", "Measurements were performed in an Applied Photophysics ( Leatherhead , UK ) SX . 18", "MV stopped-flow instrument at 25°C .", "6-MI fluorophore was excited at 340 nm and emitted light was collected through 400 nm longpass filter .", "At least three individual traces were averaged for each reported curve .", "The nucleotide addition , pyrophosphorolysis and RNA cleavage reactions were initiated by mixing 60 µl of 0 . 2 µM TEC with 60 µl of 400 µM NTP , 1000 µM PPi and 16 ( or 4 ) µM of GreA , respectively .", "TEC solutions were supplemented with 4 µM NusG where indicated .", "The NusG binding reaction was initiated by mixing 60 µl of 0 . 4 µM TEC with 60 µl of 0 . 2–20 µM NusG .", "In the nucleotide addition and pyrophosphorolysis experiments reactant solutions were prepared in TB10 buffer , whereas in NusG binding experiments TB1 buffer was used .", "In the RNA cleavage experiments , TEC and GreA solutions were prepared in TB0 and TB2 buffers , respectively .", "Measurements were performed in an Applied Photophysics SX . 18", "MV stopped-flow instrument at 25°C .", "2-AP fluorophore was excited at 320 nm and emitted light was collected through 375 nm longpass filter .", "At least three individual traces were averaged for each reported curve .", "The RNA cleavage reactions were initiated by mixing 60 µl of 0 . 2 µM TEC with 60 µl of 16 ( or 4 ) µM of GreA .", "TEC and GreA solutions were prepared in TB0 and TB2 buffers , respectively .", "TEC solutions were supplemented with 4 µM NusG where indicated .", "Equilibrium levels of fluorescence were determined by continuously recording light emission at 420 nm ( excitation at 340 nm ) with an LS-55 spectrofluorometer ( PerkinElmer , Waltham , MA ) in a 16 . 160-F/Q/10 quartz cuvette ( Starna ) at 25°C .", "The assembled TECs were diluted at 100 nM into 200 µl of TB10 buffer and the NTP substrates ( 5 μM ) and/or the increasing concentrations of TGT ( where indicated ) were sequentially added into the cuvette .", "The reaction was allowed to proceed for up to two minutes between each successive addition to ensure that the fluorescence reached the equilibrium level .", "In 8-MP ( mono-adduct absorption maximum 342 nm [Tessman et al . , 1985] ) crosslinking experiments the reaction mixture contained 1 µM TEC , 0 . 92 mM 8-MP , 6 . 3% DMSO in TB10 buffer .", "In 6-TG ( absorption maximum 340 nm [Karran and Attard , 2008] ) crosslinking experiments the reaction mixture contained 1 µM TEC in TB10 buffer .", "NTPs ( 5 µM ) , TGT ( 5 µM ) and NusG ( 2 . 5 µM ) were added were indicated .", "TEC samples ( 5 µl ) were placed in an 18-well circular tray ( Ø=26 mm , all wells equidistant from the center ) in a closed thermally controlled chamber with the UV LED ( P8D1 365 nm , Seoul Viosys , Ansan , Korea ) in the top center ( height=17 mm ) .", "Samples were exposed to UV for 30 min at 25°C , 4 µl aliquots were quenched with 6 µl of loading buffer and separated on 14% denaturing PAGE gel .", "ATTO680 labeled species were visualized with Odyssey Infrared Imager ( Li-Cor Biosciences , Lincoln , NE ) ; band intensities were quantified using ImageJ software ( Abramoff et al . , 2004 ) .", "The composite model was generated using the structure of T . thermophilus TEC with the NTP analogue ( Vassylyev et al . , 2007 ) ( PDB ID 2O5J , the lineage specific domain ( β’132–456 ) omitted ) , NusG NTD from the model of T . thermophilus NusG-RNAP complex in Martinez-Rucobo et al . ( 2011 ) , NusG CTD ( G187-I248 ) from the crystal structure of Aquifex aeolicus NusG ( Steiner et al . , 2002 ) ( PDB ID 1M1G ) and αCTDs from the crystal structure of E . coli holoenzyme ( Murakami , 2013 ) ( PDB ID 4YG2 ) .", "The duplex DNA immediately upstream of the RNA:DNA hybrid was modeled de novo as described in results; the downstream DNA outside of the TEC was extended with the canonical DNA duplex .", "The positions of NusG CTD and αCTD were chosen arbitrary within the volume permitted by the length of the flexible linkers tethering those domains to the TEC .", "Parts of the linkers were modeled de novo using ModLoop RRID:SCR_008395 ( Fiser and Sali , 2003 ) .", "NusG CTD and αCTDs are highly conserved in bacteria but are likely irrelevant to the NusG effects in the present study .", "The above considerations justify the use of heterologous NusG CTD and αCTD in the composite model solely for the illustrative purposes .", "The model geometry was evaluated using MolProbity RRID:SCR_014226 ( Chen et al . , 2010 ) .", "The atomic coordinates of the TEC-NusG complex are provided as Figure 8—source data 1 ( NusG-TEC . pdb ) .", "To generate Figures 1 and 8A the simplified surfaces ( Gaussian resolution 6 , B-factor 50 ) were calculated and rendered in PyMOL Molecular Graphics System , RRID:SCR_000305 , ( Schrödinger , New York , NY ) , exported in VRML format , converted to OBJ format using Meshlab and further simplified using sculpting tools of Meshmixer ( Autodesk Inc . San Rafael , CA ) .", "The resulting meshes were imported into and rendered in Rhinoceros 4 . 0 RRID:SCR_014339 ( Robert McNeel & Associates , Seattle , WA ) .", "The superimposition of Aquifex aeolicus NusG NTD ( PDB ID 1M1G , residues A9-50 , A133-185 ) and Pyrococcus furiosus Spt5 NTD ( PDB ID 3P8B , residues B4-82 ) was performed using COLORBYRMSD PyMOL plugin ( by S . Shandilya , J . Vertrees , T . Holder ) .", "Time-resolved nucleotide incorporation and the forward translocation data were simultaneously fit to a three-step model using the numerical integration capabilities of KinTek Explorer software ( Johnson , 2009 ) ( KinTek Corporation , Austin , TX ) .", "The model postulated that the initial TEC16 slowly and reversibly interconverts between inactive and active states and , upon the addition of the NTP substrate , undergoes an irreversible transition to TEC17 , followed by irreversible translocation ( Supplementary file 1 ) ( Malinen et al . , 2014 ) .", "Pyrophosphorolysis , backtracking , RNA cleavage and dinucleotide release data were fit to the stretched exponential function and the median reaction times were used in place of half-lives to quantify the reaction progress ( Supplementary file 2 ) .", "Equilibrium titration data were fit to the dissociation equilibrium equations that accounted for changes in concentrations of all reactants upon complex formation using Scientist 2 . 01 software ( Micromath , Saint Louis , MO ) ( Supplementary file 3 ) .", "NusG binding data were fit to a one-step reversible binding model ( Supplementary file 4 ) .", "Numerical values of the reaction rate constants and the median reaction times are presented in Table 1 .", "The number of repeats for each experiment is indicated in Table 5 ." ] ]
[ "Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation , processing , and folding of the nascent RNA .", "Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism .", "Here we report that E . coli NusG slows Gre factor-stimulated cleavage of the nascent RNA , but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase .", "We demonstrate that NusG slows RNA cleavage by inhibiting backtracking .", "This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops , but is partially dependent on the lid loop .", "Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble ." ]
[ "Cells decode genes in two steps .", "First , they synthesize a molecule similar to DNA , called RNA , which is a complementary copy of the gene .", "This process , known as transcription , creates an intermediate RNA molecule that is turned into protein in the second step .", "RNA polymerase is an enzyme that carries out transcription; it separates the two strands of the DNA helix so that the RNA can be synthesized from the DNA template .", "By opening up the DNA downstream of where active copying is taking place , and re-annealing it upstream , RNA polymerase maintains a structure called a \"transcription bubble\" .", "RNA polymerases do not copy continuously but oscillate back and forth along the DNA .", "Sometimes larger backwards oscillations , known as backtracking , temporarily block the production of the RNA molecule and slow down the transcription process .", "A protein called NusG helps to couple transcription to the other related processes that happen at the same time .", "One end of the protein , the N-terminal domain , anchors it to RNA polymerase and stimulates transcription elongation .", "The other end , the C-terminal domain , interacts with other proteins involved in the related processes and can positively or negatively control transcription elongation .", "Nevertheless it was poorly understood how NusG carries out these roles .", "Turtola and Belogurov investigated how NusG from the bacterium Escherichia coli affects the individual steps of transcription elongation .", "A simple experimental system was used , consisting of short pieces of DNA and RNA , an RNA polymerase and NusG .", "A transcription bubble resembles an opening in a zipper with two sliders; and rather than affecting the synthesis of RNA , NusG affected the part that corresponds to the “slider” located at the rear edge of the bubble .", "NusG helped this slider-like element to bring the DNA strands at this edge of the bubble back together and modified it so that it behaved as a ratchet that inhibited RNA polymerase from backtracking .", "This did not affect the smaller backwards and forwards oscillations of RNA polymerase .", "Turtola and Belogurov suggest that these newly discovered effects play a key role in regulating transcription; NusG’s N-terminal domain makes the RNA polymerase more efficient , whilst the C-terminal domain makes it amenable to control by other proteins .", "Future studies will investigate whether these effects are seen in more complex experimental systems , which include proteins that interact with NusG ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "evolutionary biology", "epidemiology and global health" ]
COVID-19 CG enables SARS-CoV-2 mutation and lineage tracking by locations and dates of interest
elife-63409-v1
[ [ "Since the beginning of the pandemic , SARS-CoV-2 genomic data has been accumulating at an unprecedented rate ( 400 , 000+ virus genomes as of February 2020 on the GISAID database ) ( Elbe and Buckland-Merrett , 2017; Shu and McCauley , 2017 ) .", "Numerous countries have mobilized to sequence thousands of SARS-CoV-2 genomes upon the occurrence of local outbreaks , collectively and consistently contributing more than 20 , 000 genomes per month ( Figure 1—figure supplement 1A , B ) .", "It is important to note that , despite the slow accumulation of potentially functional ( nonsynonymous ) mutations , there has been a steady increase in the number of variants with more than six nonsynonymous mutations compared to the WIV04 reference , an early genome of SARS-CoV-2 that was sampled in Wuhan in December 2019 ( Figure 1—figure supplement 1C ) .", "To evaluate the outcomes of anti-COVID-19 measures and detect keystone events of virus evolution , it is important to track changes in SARS-CoV-2 mutation and population dynamics in a location and date-specific manner .", "Indeed , several countries and the National Institutes of Health ( NIH ) have recognized how critical it is to collect SARS-CoV-2 genomic data to support contact tracing efforts and to inform public health decisions – these are paramount to the re-opening of countries and inter-regional travel ( Collins , 2020; Gudbjartsson et al . , 2020; Oude Munnink et al . , 2020; Virological , 2020; Rockett et al . , 2020 ) .", "Yet , the quantity and complexity of SARS-CoV-2 genomic data ( and metadata ) make it challenging and costly for the majority of scientists to stay abreast of SARS-CoV-2 mutations in a way that is meaningful to their specific research goals .", "Currently , each group or organization has to independently expend labor , computing costs , and , most importantly , time to curate and analyze the genomic data from GISAID before they can generate specific hypotheses about SARS-CoV-2 lineages and mutations in their population ( s ) of interest ." ], [ "Analyzing SNVs by geography and time is critical as the frequency of each SNV may vary significantly across different regions over time .", "For instance , as of December 2020 , an S477N mutation in the receptor binding domain ( RBD ) has become dominant in Australia ( 69% of Australian SARS-CoV-2 genotypes , all time ) although it constitutes less than 6% of SARS-CoV-2 genotypes globally ( Figure 2A ) .", "SNV frequency in a given region can also shift over time , for example , an RBD N439K mutation not found in Ireland prior to July was present in 42% of the genomes collected mid-July through September , peaking in August and gradually fading after ( Figure 2B ) .", "Another rare RBD S477N mutation , which was found in only 1% of the Australian SARS-CoV-2 sequences before June , has constituted 84% of the sequenced June through December genomes ( Figure 2C ) .", "This geographical and temporal variation is important to incorporate into the design and testing of therapeutic antibodies ( such as those under development as therapeutics by Regeneron that specifically target the SARS-CoV-2 spike RBD ) , as well as mRNA or recombinant protein-based vaccines .", "This will help to assure developers of the efficacy of their therapeutics and vaccines against the SARS-CoV-2 variants that are present in the intended location of implementation .", "In addition , COVID-19 CG can be harnessed to track changes in SARS-CoV-2 evolution post-implementation of therapeutics and vaccines .", "It will be crucial to watch for rare escape variants that could resist drug- or immune-based interventions to eventually become the dominant SARS-CoV-2 variant in the community .", "This need was particularly emphasized by a Regeneron study that demonstrated that single amino acid variants could evolve rapidly in the SARS-CoV-2 spike to ablate binding to antibodies that had been previously selected for their ability to neutralize all known RBD variants; some of these RBD amino acid variations are already present at low frequency among human SARS-CoV-2 genomes globally ( Baum et al . , 2020 ) .", "The authors , Baum et al . , suggested that these rare escape variants could be selected under the pressure of single antibody treatment , and , therefore , advocated for the application of cocktails of antibodies that bind to different epitopes to minimize SARS-CoV-2 mutational escape .", "A recent study by Greaney et al . , 2021 generated high-resolution ‘escape maps’ delineating RBD mutations that could potentially result in virus escape from neutralization by 10 different human antibodies .", "Based on lessons learnt from the rise of multidrug resistant bacteria and cancer cells , it will be of the utmost importance to continue tracking SARS-CoV-2 evolution even when multiple vaccines and therapeutics are implemented in a given human population .", "Diagnostics developers can evaluate their probe , primer , or point-of-care diagnostic according to user-defined regional and temporal SARS-CoV-2 genomic variation .", "More than 665 established primers/probes are built into COVID-19 CG , and new diagnostics will be continually incorporated into the browser as they become publicly available .", "Users can also input custom coordinates or sequences to evaluate their own target sequences and design new diagnostics .", "A recent preprint alerted us to the finding that a common G29140T SNV , found in 22 . 3% of the study’s samples from Madera County , California , was adversely affecting SARS-CoV-2 detection by the NIID_2019-nCoV_N_F2 diagnostic primer used at their sequencing center; the single SNV caused a ~ 30-fold drop in the quantity of amplicon produced by the NIID_2019-nCov_N_F2/R2 primer pair ( Vanaerschot et al . , 2020 ) .", "We used COVID-19 CG to detect other SNVs that could impact the use of this primer pair , discovering that there are SARS-CoV-2 variants in several countries with a different C29144T mutation at the very 3’ end of the same NIID_2019-nCoV_N_F2 primer ( Figure 3A ) .", "The authors of the preprint , Vanaerschot et al . noted that SNVs could impact assay accuracy if diagnostic primers and probes are also being used to quantify viral loads in patients .", "We found that at least 10 other primer pairs could potentially be at risk in different geographical regions due to SNVs that appear proximal to the 3’ ends of primers ( Figure 3B–K ) : China-CDC-N-F and R; NIH , Thailand , WH-NIC N-F; US CDC 2019-nCoV-N1-R; US CDC 2019-nCoV-N2-F; ARTIC-V3_nCoV-2019_11_RIGHT; ARTIC-V3_nCoV-2019_13_LEFT; ARTIC-V3_nCoV-2019_34_LEFT; ARTIC-V3_nCoV-2019_39_LEFT ( note that the ARTIC primers are used for nanopore sequencing; Tyson et al . , 2020 ) ; WHO N_Sarbarco_R1; and Institut Pasteur , Paris 12759Rv .", "Labs and clinics can use COVID-19 CG ( https://covidcg . org ) to check their most commonly used primers and probes against the SARS-CoV-2 sequences that are prevalent in their geographical regions .", "We reiterate Vanaerschot et al . ’s exhortation that SARS-CoV-2 detection strategies should ideally target multiple viral genes and check for concordant Ct values .", "In addition , scientists have cautioned against lab-specific errors that exist among the available SARS-CoV-2 genomes , often co-localizing with commonly used primer binding sites ( Turakhia et al . , 2020 ) .", "These errors appear to be specific to particular labs and do not affect all of the genomes on the GISAID database .", "Therefore , we have added a caution to our site and a link to the evolving list of problematic sites ( Issues with SARS-CoV-2 sequencing data , 2020 ) so that users are made aware of these potential errors in some subsets of the genomes deposited on GISAID .", "Researchers and public health professionals can use COVID-19 CG to gain insights as to how the virus is evolving in a given population over time ( e . g . , in which genes are mutations occurring , and do these lead to structural or phenotypic changes ? ) .", "For example , users can track D614G distributions across any region of interest over time .", "Figure 4 shows a variety of different D614G population dynamics in different areas .", "Nonetheless , we caution against inferring ( 1 ) chains or directionality of transmission and ( 2 ) changes in the transmissibility of any SARS-CoV-2 SNV based on population dynamics alone .", "Inconsistent sampling , sampling biases , differences in founder host population traits ( even median patient age ) , superspreading events , regionally and temporally differential travel restrictions , and numerous other factors instead of virus biological differences can influence the global distribution of SNVs ( Grubaugh et al . , 2020 ) .", "Our site carries the following warning: ‘Inconsistent sampling in the underlying data can result in missing data and artefacts in this visualization . Please interpret this data with care . ’ In September 2020 , we observed that the SARS-CoV-2 spike S477N mutation had become more prevalent in Australia ( Figure 5A ) .", "Globally , the S477N mutation was first detected in a single sample of lineage B . 1 . 1 . 25 that was collected on March 19 , 2020 , in Victoria , Australia , and became the dominant SARS-CoV-2 variant in the region between June and September ( Figure 5B ) .", "In particular , the set of SNVs that co-occur with the S477N mutation in Australia ( all time , as well as prior to May 2020 before the most recent outbreak ) are different from the set of co-occurring SNVs in the United Kingdom ( Figure 5C ) — suggesting that the S477N mutation occurred separately in the Australian and the UK lineages .", "However , COVID-19 CG only reflects data contributed to GISAID .", "Variants of interest could be present in other countries , but not yet known to the public because the sequencing centers in those countries have not collected or deposited their data in GISAID .", "Furthermore , in instances where only a singular , sporadic variant is detected ( no sustained transmission ) , there is also the possibility of sequencing error resulting in incorrect lineage assignment .", "Due to these caveats , the genetic data must be used in combination with other types of data , such as from contact tracing efforts , before it is possible to draw conclusions about the international transmission of SARS-CoV-2 variants .", "In the case of the S477N variant that is now dominating in Australia , the sequencing data alone indicate that the local transmission of this variant in Australia since March 2020 or earlier cannot be ruled out ." ], [ "COVID-19 CG is one of a growing number of COVID-19 public browsers that analyze and visualize the SARS-CoV-2 genomes in the GISAID database , serving different and complementary user objectives .", "NextStrain ( Hadfield et al . , 2018 ) visualizes real-time tracking of SARS-CoV-2 evolution on a global and continental level , using phylogenetic trees , geographical maps , and entropy and frequency plots; CoV-GLUE ( Singer et al . , 2020 ) is a browsable database of amino acid replacements and indels; the UCSC SARS-CoV-2 Genome Browser is an extension of their widely used browser that enables layering of annotation tracks and new features such as conservation with similar viruses , immune epitopes , primers , and CRISPR guides ( Fernandes et al . , 2020 ) ; the COVID-19 Viral Genome Analysis Pipeline ( Korber et al . , 2020 ) allows for exploring mutations geographically and over time with a focus on the spike protein; COVIDep ( Ahmed et al . , 2020 ) is a browser for real-time reporting of vaccine target recommendations; Genomic Signature Analysis of Virus Causing COVID-19 ( Bauer et al . , 2020 ) visualizes the similarity between SARS-CoV-2 genomes in 2D space; SARS-CoV-2 Alignment Screen ( van Dorp et al . , 2020a; van Dorp et al . , 2020b ) is a visualization tool providing the distribution of SNPs and homoplasies; the WashU Virus Genome Browser ( Flynn et al . , 2020 ) provides different tools such as a phylogenetic tree and a genomic-coordinate track-based view of viral sequencing data .", "There are also websites aimed at helping users to check COVID-19 diagnostic primers and probes , for example , the SARS-CoV-2 target regions browser from the European Commission and the Status of SARS-CoV-2 detection systems ( RT-PCR ) browser from the University of Turin .", "GISAID also monitors and reports RBD mutations and assigns clades and lineages as well as genome quality .", "The current GISAID reports describe the geographical distribution of new mutations , total occurrence by country , mutations in commonly used diagnostics , and 3D structural maps of mutations in the four largest clades ( gisaid . org/spike ) .", "GISAID additionally provides CoVsurver ( gisaid . org/covsurver ) for users to screen for potential phenotypic changes based on curated literature annotations; BLAST searches and large-scale phylogenetic trees and alignments; and a new functionality for tracking emerging variants ( gisaid . org/variants ) .", "COVID-19 CG provides additional complementary functionalities to those that are available on the GISAID platform , as well as these other diverse COVID-19 public browsers .", "COVID-19 CG ( https://covidcg . org ) was built to help scientists and professionals worldwide , with varying levels of bioinformatics expertise , in their real-time analysis of SARS-CoV-2 genetic data .", "Mainly , COVID-19 CG enables users to track in real time , without sub-sampling , lineages , clades , and SNVs ( nucleotide or amino acid ) across the SARS-CoV-2 genome ( or any genomic region of choice ) while rapidly filtering on a user-friendly interface by geographical regions , date range , and other criteria of interest to the user .", "In this work , we explored several case studies that serve to highlight what users can quickly achieve using COVID-19 CG .", "As more detailed metadata is generated by COVID-19 studies and initiatives , we will update the application to enable filtering according to patient traits such as host species ( e . g . , human or mink ) , gender , age , ethnicity , and medical condition ( e . g . , symptoms , hospitalization ) .", "In addition , over the next year , as more mutations accumulate across the tens of millions of COVID cases worldwide , we plan to implement a server-client model for COVID-19 CG , where genomic data is filtered and processed on the server before being sent to the client for visualization .", "This change should significantly reduce the computational burden of COVID-19 CG on user’s computers , and allow our application to scale to a much larger number of genomes .", "Our team and colleagues continue to actively use the COVID-19 CG site to quickly generate hypotheses about COVID-19 before performing a deep analysis using the data on GISAID .", "We anticipate that novel questions will arise over the next year and intentionally designed COVID-19 CG ( https://covidcg . org ) to be modular in order to continually integrate different types of COVID-19 data and build in new features .", "Some of these functionalities exist on other platforms , and we recommend that scientists try each browser to find the one that best meets their needs .", "SARS-CoV-2 public browsers such as COVID-19 CG and others we have highlighted here help scientists around the world to parse through large quantities of SARS-CoV-2 genomic data and metadata for the purposes of informing vaccine , therapeutics , and policy development .", "We advocate for decision makers around the world to sustain or accelerate their sequencing of virus genomes in their geographical area .", "Collecting virus genomic data is particularly relevant to regions that are experiencing increases in COVID-19 cases .", "If only sparse genomic data are sampled , we risk the late detection of SARS-CoV-2 variants that exhibit enhanced transmissibility , virulence or resistance against therapeutics or vaccination programs in these pandemic hotspots .", "Furthermore , the widespread implementation of vaccines and antibody therapies could stimulate the emergence and selection of new escape variants ( Baum et al . , 2020 ) .", "To compound these risks , SARS-CoV-2 transmission from humans to minks ( and in some cases back into humans ) has already been detected at farms across at least 10 countries and one wild mink in the United States ( OIE - World Organisation for Animal Health , 2020 ) .", "This process of species crossing , if left unchecked , can risk the spread of SARS-CoV-2 into wild animal populations and the emergence of diverse SARS-CoV-2 variants .", "Coordinated sequencing and contact tracing efforts ( e . g . , in the United Kingdom , Singapore , the Netherlands , Italy , California , and Australia ) emphasize the urgency of establishing open access platforms to evaluate trends in virus introduction into each country or region in real time .", "Local policymakers , public health researchers , and scientists can use global SARS-CoV-2 genetic data , in complementation with contact tracing data , to better understand which lineages were imported into their region ( from which potential international locations ) , whether these were introduced multiple times , and if particular lineages are dying out or persisting .", "Labs in numerous countries are already making incredible efforts to sequence the SARS-CoV-2 variants circulating in their local populations ( Figure 6 ) .", "When each country actively contributes to the database of SARS-CoV-2 genomes , this protects against sampling biases that can impact the ability to perform phylogenetic analysis and interpret global SARS-CoV-2 data .", "Toward this goal that affects all of humanity , we advocate for the increased sequencing of SARS-CoV-2 isolates from patients ( and infected animals ) around the world , and for these data to be shared in as timely a manner as possible ." ], [ "Our data processing pipeline is written with the Snakemake scalable bioinformatics workflow engine ( Köster and Rahmann , 2012 ) , which modularizes our workflow and enables reproducibility and compatibility with cloud-computing .", "All code and relevant documentation are hosted on an open-source , publicly available GitHub repository ( https://github . com/vector-engineering/COVID19-CG; Chen , 2021; copy archived at swh:1:rev:e9558dc11b31b908f3af142e403d33e91d417b8a ) , providing example data for users to validate our pipeline .", "Data analysis is broken up into two Snakemake pipelines: ( 1 ) ingestion and ( 2 ) main .", "The ingestion pipeline downloads , chunks , and prepares metadata for the main analysis , and the main pipeline analyzes sequences , extracts SNVs , and compiles data for display in the web application .", "Configuration of the pipeline and the web application is defined by a single YAML file .", "At the time of publication , two ingestion workflows are available: workflow_genbank_ingest and workflow_gisaid_ingest .", "While the GISAID ingestion pipeline is provided as open source , it utilizes a custom GISAID endpoint and is intended only for internal use .", "Both ingestion pipelines are designed to be run daily and chunk sequence data in order to minimize expensive reprocessing/realignment in the downstream main analysis step .", "In addition , these ingest pipelines clean the provided sequence metadata by removing unwanted fields , renaming fields , and fixing typos ( see ‘Metadata Cleaning’ for details ) .", "We clean metadata with the aim of preserving the original intent of the authors and data submitters while presenting simpler and unified versions to end users .", "Sequencing metadata is cleaned to remove obvious typos , and to unify labels with the same meaning , for example , ‘MinION’ and ‘Nanopore MinION . ’ Location metadata is cleaned with the goal of simplifying the location selector in the sidebar .", "Locations with excessive children are collapsed to the nearest upper hierarchical grouping .", "For example , if a state has individual data for 200+ towns , these towns will be collapsed to the county level in order to facilitate easier data browsing .", "Typos and clear identities are also unified to prevent the display of duplicate locations in the application .", "Based on best practices , we filter out sequences meeting any of the following criteria: ( 1 ) present on the NextStrain’s exclusion list ( https://github . com/nextstrain/ncov/blob/master/defaults/exclude . txt; Hadfield et al . , 2018 ) , ( 2 ) genomes from non-humans ( animals , environmental samples , etc . ) , ( 3 ) genome length less than 29 , 700 nt , or ( 4 ) >5% ambiguous base calls .", "Sequences which pass all preprocessing filters are carried onto the next steps .", "SNVs and insertions/deletions ( indels ) at the nucleotide and amino acid level are determined using bowtie2 by aligning each sequence to the WIV04 reference sequence ( WIV04 is a high quality December 2019 genome that is 100% identical to the other commonly used publicly available SARS-CoV-2 genome reference Wuhan-Hu-1/NC_045512 . 2 , excepting the length of the poly ( A ) tail ) .", "Spurious SNVs and probable sequencing errors , defined as less than three global occurrences , are filtered out prior to downstream analysis .", "SNVs involving ambiguous base calls ( ‘N’ in the original sequences ) are ignored .", "Indels resulting in frameshifts are ignored , and SNVs/indels occurring in non-protein-coding regions are ignored when determining SNVs/indels on the AA level .", "Viral lineages , as defined by the pangolin tool ( Rambaut et al . , 2020 ) , and clades ( Tang et al . , 2020 ) are provided by GISAID .", "In accordance with pangolin , SNVs present in >90% of sequences within each lineage/clade will be assigned as lineage/clade-defining SNVs .", "The web application is written in Javascript and primarily uses the libraries React . js , MobX , and Vega .", "The code is compiled into Javascript bundles by webpack .", "Sequence data and metadata are combined and compressed into one file , which is downloaded by the web application on page load .", "This allows us to regularly update the data on https://covidcg . org .", "COVID CG ( https://covidcg . org ) is hosted by Google Cloud Run .", "The application code is assembled into a Docker image ( see Dockerfile ) , with a build environment ( node . js ) and deployment environment ( NGINX ) .", "All of the data shown in this manuscript and displayed on COVID CG ( https://covidcg . org ) are downloaded from the GISAID EpiCov database ( https://www . gisaid . org ) .", "All code and relevant documentation are hosted on an open-source , publicly available GitHub repository ( https://github . com/vector-engineering/COVID19-CG ) ." ] ]
[ "COVID-19 CG ( covidcg . org ) is an open resource for tracking SARS-CoV-2 single-nucleotide variations ( SNVs ) , lineages , and clades using the virus genomes on the GISAID database while filtering by location , date , gene , and mutation of interest .", "COVID-19 CG provides significant time , labor , and cost-saving utility to projects on SARS-CoV-2 transmission , evolution , diagnostics , therapeutics , vaccines , and intervention tracking .", "Here , we describe case studies in which users can interrogate ( 1 ) SNVs in the SARS-CoV-2 spike receptor binding domain ( RBD ) across different geographical regions to inform the design and testing of therapeutics , ( 2 ) SNVs that may impact the sensitivity of commonly used diagnostic primers , and ( 3 ) the emergence of a dominant lineage harboring an S477N RBD mutation in Australia in 2020 .", "To accelerate COVID-19 efforts , COVID-19 CG will be upgraded with new features for users to rapidly pinpoint mutations as the virus evolves throughout the pandemic and in response to therapeutic and public health interventions ." ]
[ "The discovery of faster spreading variants of the virus that causes coronavirus disease 2019 ( COVID-19 ) has raised alarm .", "These new variants are the result of changes ( called mutations ) in the virus’ genetic code .", "Random mutations can occur each time a virus multiplies .", "Although most mutations do not introduce any meaningful changes , some can alter the characteristics of the virus , for instance , helping the virus to spread more easily , reinfecting people who have had COVID-19 before , or reducing the sensitivity to treatments or vaccines .", "Scientists need to know about mutations in the virus that make treatments or vaccines less effective as soon as possible , so they can adjust their pandemic response .", "As a result , tracking these genetic changes is essential .", "But individual scientists or public health agencies may not have the staff , time or computer resources to extract usable information from the growing amount of genetic data available .", "A free online tool created by Chen et al . may help scientists and public health officials to track changes to the virus more easily .", "The COVID-19 CoV Genetics tool ( COVID-19 CG ) can quickly provide information on which virus mutations are present in an area during a specific period .", "It does this by processing data on mutations found in viral genetic material collected worldwide from hundreds of thousands of people with COVID-19 , which are hosted in an existing online database .", "The COVID-19 CG tool presents customizable , interactive visualizations of the data .", "Thousands of scientists , public health agencies , and COVID-19 vaccine and treatment developers in over 100 countries are already using the COVID-19 CG tool to find the most common mutations in their area and use it for research .", "They can use this information to develop more effective vaccines or treatments .", "Chen et al . plan to update and improve the tool as more information becomes available to help advance global efforts to end the COVID-19 pandemic ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "short report", "neuroscience" ]
DYT1 dystonia increases risk taking in humans
elife-14155-v3
[ [ "DYT1 dystonia is a rare , dominantly inherited form of dystonia , caused almost exclusively by a specific deletion of three base pairs in the TOR1A gene ( Ozelius et al . , 1997 ) .", "Clinically , DYT1 dystonia is characterized by variable severity of sustained or intermittent muscle contractions that produce abnormal movements .", "DYT1 dystonia patients have normal intelligence , and post-mortem examination of their brains does not reveal obvious abnormalities or evidence of neurodegeneration ( Paudel et al . , 2012 ) .", "Nevertheless , research in two different rodent models of DYT1 dystonia points to the existence of a fundamental deficit in synaptic plasticity .", "Specifically , brain slices of transgenic rodents expressing the human mutant TOR1A gene show abnormally strong long-term potentiation ( LTP; Martella et al . , 2009 ) and weak , or even absent , long-term depression ( LTD; Grundmann et al . , 2012; Martella et al . , 2009 ) in corticostriatal synapses , as compared to wild-type controls .", "Reinforcement learning theory ( Sutton and Barto , 1998 ) hypothesizes that dopamine-dependent synaptic plasticity in corticostriatal networks is the neuronal substrate for learning through trial and error ( Barnes et al . , 2005; Barto , 1995; Schultz et al . , 1997 ) .", "The core assumptions of this theory are that ( 1 ) dopamine release in the striatum signals errors in the prediction of reward , with dopamine levels increasing following successful actions ( to signal a positive prediction error ) and decreasing when actions fail to achieve the expected outcome ( a negative prediction error ) , ( 2 ) fluctuations in dopamine modulate downstream plasticity in recently active corticostriatal synapses such that synapses responsible for positive prediction errors are strengthened through long-term potentiation ( LTP ) , and those that led to disappointment are weakened through long-term depression ( LTD ) ( Reynolds et al . , 2001 ) , and ( 3 ) the efficacy of corticostriatal transmission affects voluntary action selection .", "Dopamine’s role as a reinforcing signal for trial-and-error learning is supported by numerous findings ( Pessiglione et al . , 2006; Schultz et al . , 1997; Steinberg et al . , 2013 ) , including in humans , where Parkinson’s disease serves as a human model for altered dopaminergic transmission ( Frank et al . , 2004 ) .", "However , the contribution of ( dopamine modulated ) corticostriatal plasticity to shaping action has remained unconfirmed in the behaving organism , as it is not clear that the behavioral effects of altered dopamine signaling in Parkinson’s disease ( and other conditions in which dopamine transmission is compromised ) indeed stem from the role of dopamine in modulating plasticity .", "Towards this end , here we test whether DYT1 dystonia , where corticostriatal plasticity is suggested to be altered despite preserved dopaminergic signaling , leads to the behavioral effects predicted by reinforcement learning with imbalanced plasticity .", "In particular , our predictions stem from considering the effects of intact prediction errors on an altered plasticity mechanism that amplifies the effect of positive prediction errors ( i . e . , responds to positive prediction errors with more LTP than would otherwise occur in controls ) and mutes the effects of negative prediction errors ( that is , responds with weakened LTD as compared to controls ) .", "We compared the behavior of DYT1 dystonia patients and healthy controls on an operant-learning paradigm with probabilistic rewards ( Niv et al . , 2012 ) .", "Participants learned from trial and error to associate four different visual cues with monetary rewards ( Figure 1a ) , optimizing their gain by selecting one of two cues in choice trials , and choosing the single available cue in forced trials .", "Three visual cues were associated with a payoff of 0¢ , 5¢ and 10¢ , respectively , while the fourth cue was associated with an unpredictable payoff of either 0¢ or 10¢ with equal probabilities ( henceforth the ‘risky 0/10¢’ cue ) .", "Based on the findings in rodents with the DYT1 mutation , we predicted that dystonia patients would learn preferentially from positive prediction errors ( putatively due to abnormally strong LTP ) and to a much lesser extent from negative prediction errors ( due to weak LTD ) ( Figure 1b ) .", "As a result , they should show a stronger tendency to choose the risky cue as compared to healthy controls . 10 . 7554/eLife . 14155 . 003Figure 1 . Behavioral task and hypothesis .", "( a ) In ‘choice trials’ two visual cues were simultaneously presented on a computer screen .", "The participant was required to make a choice within 1 . 5 s .", "The chosen option and the outcome then appeared for 1 s , followed by a variable inter-trial interval .", "( b ) Theoretical framework .", "Top: trials in which the risky cue is chosen and the obtained outcome is larger than expected ( trials with a 10¢ outcome ) should result in strengthening of corticostriatal connections ( LTP ) , thereby increasing the expected value of the cue and the tendency to choose it in the future .", "Conversely , outcomes that are smaller than expected ( 0¢ ) should cause synaptic weakening ( LTD ) and a resulting decrease in choice probability .", "Middle: In DYT1 dystonia patients ( red solid ) , increased LTP combined with decreased LTD are expected to result in an overall higher learned value for the risky cue , as compared to controls ( blue dashed ) .", "In the model , this is reflected in higher probability of choosing the risky cue when presented together with sure 5¢ cue ( Bottom ) .", "Simulations ( 1000 runs ) used the actual order of trials and mean model parameters of each group as fit to participants’ behavior .", "Gray shadow in the middle plot denotes trials in the initial training phase . DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 003" ], [ "We tested 13 patients with DYT1 dystonia ( 8 women , 5 men , age 20–47 , mean 28 . 6 years , henceforth DYT ) and 13 healthy controls ( CTL; 8 women , 5 men , age 19–46 , mean 28 . 8 years ) , matched on an individual basis for sex and age ( Mann-Whitney U test for age differences , z = −0 . 59 , df = 24 , P = 0 . 55 ) , all with at least 13 years of education .", "Patients had no previous neurosurgical interventions for dystonia ( including deep brain stimulation ) and were tested before their scheduled dose of medication when possible ( see Materials and methods ) .", "The number of aborted trials was similarly low in both groups ( DYT 2 . 3 ± 2 . 5 , CTL 1 . 1 ± 1 . 2 , Mann-Whitney z = −1 . 61 , df = 24 , P = 0 . 11 ) and reactions times were well below the 1 . 5s response deadline ( DYT 0 . 78s ± 0 . 11 , CTL 0 . 71s ± 0 . 10 , Mann-Whitney z = −1 . 49 , df = 24 , P = 0 . 14 ) , confirming that motor symptoms of dystonia did not interfere with the minimal motor demands of the task .", "Both groups quickly learned the task , and showed similarly high probabilities of choosing the best cue in trials in which a pair of sure cues ( sure 0¢ vs . sure 5¢ or sure ¢5 vs . sure 10¢ ) appeared together ( mean probability correct choice: DYT1 0 . 92 ± 0 . 08 , CTL 0 . 93 ± 0 . 05 , Mann-Whitney z = 0 . 08 , df = 24 , P = 0 . 94; Figure 2a ) , as well as in trials in which the risky cue appeared together with either the sure 0¢ or sure 10¢ cues ( mean probability correct: DYT1 0 . 84 ± 0 . 09 , CTL 0 . 89 ± 0 . 04 , Mann-Whitney z = −1 . 39 , df = 24 , P = 0 . 17; Figure 2b ) . 10 . 7554/eLife . 14155 . 004Figure 2 . Learning curves did not differ between the groups . Mean probabilities ( ± s . e . m ) of choosing the cue associated with the higher outcome , on average ,", "( a ) among pairs of two sure cues ( 15 trials per ‘block’ ) or", "( b ) when the risky 0/10¢ cue was paired with a sure cue of 0¢ or 10¢ value ( 20 trials per ‘block’ ) confirmed that both groups quickly learned to choose the best cue in trials in which one cue was explicitly better than the other .", "These results verify that both groups understood the task instructions and could perform the task similarly well ( in terms of choosing and executing their responses fast enough , etc . ) .", "Participants evidenced learning of values for deterministically-rewarded cues even in the first choice trials despite the fact that they were never informed verbally or otherwise of the monetary outcomes associated with each of the cues , and thus could only learn these from experience .", "However , for cues leading to deterministic outcomes , a little experience can go a long way ( Shteingart et al . , 2013 ) , and participants received 16 training trials prior to the test phase .", "Our data suggest that learning in this phase did not differ between the groups: in the first 5 choice trials in the test phase that involved a pair of sure cues , the probability of a correct response was 0 . 78 ± 0 . 18 in the DYT group and 0 . 81 ± 0 . 07 in the CTL group ( Mann-Whitney U test , df = 24 , P = 0 . 59 ) .", "We verified that that this level of performance could result from trial-and-error learning by simulating the behaviors of individuals using the best-fit learning rates ( see Materials and methods ) .", "The simulation confirmed that both groups should show similar rates of success on the first 5 choice trials ( DYT 0 . 81 ± 0 . 17 probability for correct choice , CTL 0 . 87 ± 0 . 13 , Mann-Whitney U test z = 0 . 88 , df = 24 , P = 0 . 38 ) despite differences in learning rates from positive and negative prediction errors ( see Results ) .", "Indeed the model , which started from initial values of 0 and learned only via reinforcement learning , performed on average better than participants . DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 004 On trials in which the risky 0/10¢ cue appeared together with the equal-mean 5¢ sure cue , control participants showed risk-averse behavior , as is typically observed in such tasks ( Kahneman and Tversky , 1979; Niv et al . , 2012 ) .", "In contrast , patients with DYT1 dystonia displayed significantly less risk aversion , choosing the risky stimulus more often than controls throughout the experiment ( Figure 3a , Mann Whitney one-sided test for each block separately , all z > 1 . 68 , df = 24 , P < 0 . 05; Friedman’s test for effect of group after correcting for the effect of time χ2 = 16 . 2 , df = 1 , P < 0 . 0001 ) .", "Overall , the probability of choosing the risky cue was significantly higher among patients with dystonia than among healthy controls ( Figure 3b , probability of choosing the risky cue over the sure cue DYT 0 . 44 ± 0 . 18 , CTL 0 . 25 ± 0 . 20 , Mann-Whitney z = 2 . 33 , df = 24 , P < 0 . 05 ) . 10 . 7554/eLife . 14155 . 005Figure 3 . Risk taking in DYT1 dystonia patients as compared to healthy sex- and age-matched controls .", "( a ) Mean proportion ( ± s . e . m ) of choosing the risky 0/10¢ cue over the sure 5¢ cue ( 15 trials per block ) in each of the groups .", "DYT1 dystonia patients ( red solid ) were less risk-averse than controls ( blue dashed ) .", "Results from several randomly-selected participants are plotted in the background to illustrate within-participant fluctuations in risk preference over the course of the experiment , presumably driven by ongoing trial-and-error learning .", "( b ) Overall percentage of choosing the risky 0/10¢ cue throughout the experiment .", "Horizontal lines denote group means; grey boxes contain the 25th to 75th percentiles .", "DYT1 dystonia patients showed significantly more risk-taking behavior than healthy controls .", "( c ) Proportion of choices of the risky 0/10¢ cue over the sure 5¢ cue , divided according to the outcome of the previous instance in which the risky cue was selected .", "Both controls and DYT1 dystonia patients chose the risky 0/10¢ cue significantly more often after a 10¢ ‘win’ than after a 0¢ ‘loss’ outcome , demonstrating the effect of previous outcomes on the current value of the risky 0/10¢ cue due to ongoing reinforcement learning .", "Error bars: s . e . m . The effect of recent outcomes on the propensity to choose the risky option was evident throughout the task , especially in the DYT group , and was seen after both free choice and forced trials ( Figure 3—figure supplement 1 ) , suggesting that participants continuously updated the value of the risky cue based on feedback , and used this learned value to determine their choices .", "( d ) Risk taking was correlated with clinical severity of dystonia ( Fahn-Marsden dystonia rating scale ) .", "The mean of the control group is denoted in blue for illustration purposes only .", "Interestingly , the regression line for DYT1 dystonia patients’ risk preference intersected the ordinate ( 0 severity of symptoms ) close to the mean risk preference of healthy controls . DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 00510 . 7554/eLife . 14155 . 006Figure 3—figure supplement 1 . Learning about the risky cue continued throughout the task .", "( a ) Our experimental design was aimed explicitly at focusing on learning about the risky cue so that we could analyze learning from positive and negative prediction errors decoupled from initial learning about deterministic cues .", "As shown in Figure 3c , participants’ tendency to choose the risky 0/10 cue over the same-mean 5¢ cue was dynamically adjusted according to experience: if the previous choice of the risky cue was rewarded with 10¢ , participants were significantly more likely to choose the risky cue again on the next time it was available , as compared to the case in which the previous choice of the risky cue resulted in 0¢ .", "To verify that the value of the risky cue was continuously updated , we calculated the proportion of choices of the risky cue over the sure 5¢ cue after different outcomes of the previous instance in which the risky cue was selected , for different time bins throughout the task ( 15 risky trials in each ) .", "A three way ANOVA ( group X outcome X time-bin ) revealed a significant effect for group ( P < 0 . 001 ) , outcome ( win or loss; P < 0 . 001 ) and no effect of time-bin or interactions .", "Post-hoc comparisons revealed that the differences between win and loss conditions were significant in all bins for the DYT group only ( all Ps < 0 . 05 , two tailed ) .", "The first two bins for the CTL group approached significance ( P = 0 . 054 , two-tailed ) .", "This analysis showed that DYT patients changed their behavior based on outcomes of the risky cue throughout training .", "Control participants , on the other hand , evidenced somewhat less learning as the task continued , with their behavior in the last quarter of training settling on a risk-averse policy that was not sensitive to local outcomes .", "In reinforcement learning , this could result from a gradual decrease of learning rates , which is optimal in a stationary environment .", "Indeed , the final risk-averse policy was predicted by our model , based on the ratio of positive and negative learning rates .", "In any case , these results suggest that participants learned to evaluate the risky cue based on experienced rewards , and that the locally fluctuating value of the risky cue affected choice behavior , at least in the first half of the experiment , and for the DYT group , throughout the experiment .", "( b ) Recent work on similar reinforcement learning tasks has shown that choice trials and forced trials may exert different effects on learning ( Cockburn et al . , 2014 ) .", "To test for this effect in our data , we examined separately the probability of choosing the risky cue over the sure cue following wins or losses , after either forced or choice trials .", "Our analysis revealed that choices were significantly dependent upon the previous outcome of the risky cue ( P < 0 . 01 , F = 7 . 45 , df = 1 for main effect of win versus loss; 3-way ANOVA with factors outcome , choice and group ) but not upon its context ( P = 0 . 38 , F = 0 . 93 , df = 1 for main effect of forced vs . choice trials ) .", "Similar to Cockburn et al . ( 2014 ) , we did observe a numerically smaller effect of the outcome of forced trials ( as compared to choice trials ) on future choices , however this was not significant ( interaction between outcome and choice P = 0 . 46 , F = 0 . 56 , df = 1 ) .", "P values in the figure reflect paired t-tests . DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 00610 . 7554/eLife . 14155 . 007Figure 3—figure supplement 2 . Effects of ongoing learning in the simulated data . Proportion of choices of the risky 0/10¢ cue over the sure 5¢ cue , divided according to the outcome of the previous instance in which the risky cue was selected , according to the asymmetric learning model with parameters fit to each participant’s behavior .", "The model captures the behavioral findings faithfully . DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 00710 . 7554/eLife . 14155 . 008Figure 3—figure supplement 3 . Sex of participants did not affect risk sensitivity in our task . To avoid any possible sex-dependent bias , we matched the sex of both groups when comparing control participants to DYT1 dystonia patients .", "Similar to Figure 3b , plotted are overall percentage of choices of the risky 0/10¢ cue over sure 5¢ cue throughout the experiment , for different participants ( Female N = 8 in each group , filled dots; Male , N = 5 in each group , open dots ) .", "A two-way ANOVA ( CTL/DYT x Male/Female ) did not reveal a significant main effect of sex ( P = 0 . 08 ) although this analysis is obviously underpowered .", "The difference between CTL and DYT remained significant in this analysis ( P = 0 . 01 for the main effect of group ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 00810 . 7554/eLife . 14155 . 009Figure 3—figure supplement 4 . Medication did not affect risk-sensitivity . To minimize the effect of medication on learning in our task , we tested patients before their scheduled dose of medication to the extent that this was possible .", "As in Figure 3b , plots show overall percentage of choices of the risky 0/10¢ cue over sure 5¢ cue throughout the experiment and its relation to medications and doses .", "( a ) Similar risk-taking ( choosing the risky 0/10¢ cue over the sure 5¢ cue ) behavior among untreated patients and those taking trihexyphenidyl or baclofen ,", "( b ) lack of correlation between risk-taking behavior and the daily dose of trihexyphenidyl ( Pearson’s r = 0 . 19 , df = 11 , P = 0 . 526 ) or", "( c ) baclofen ( Pearson’s r = −0 . 20 , df = 11 , P = 0 . 51 ) all suggested that medication did not contribute significantly to the observed results . DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 009 To rule out the possibility that DYT1 patients were simply making choices randomly , causing their behavior to seem indifferent to risk , we divided all 0/10¢ versus 5¢ choice trials according to the outcome of the previous trial in which the risky 0/10¢ cue was chosen .", "As shown in Figure 3c ( see also Figure 3—figure supplement 1 ) , both groups chose the risky 0/10¢ cue significantly more often after a 10¢ ‘win’ than after a 0¢ ‘loss’ outcome ( DYT P < 0 . 005 , CTL P < 0 . 05 , Wilcoxon signed-rank test ) , attesting to intact reinforcement learning in the DYT group ( see Figure 3—figure supplement 2 , for a reinforcement learning simulation of the same result ) .", "If anything , DYT1 dystonia patients showed a greater difference between trials following a win and those following a loss .", "We next tested for a correlation between risk-taking behavior and the clinical severity of dystonia , as rated on the day of the experiment ( see Materials and methods ) .", "The results showed that patients with more severe dystonia were more risk taking in our task ( Figure 3d , Pearson’s r = 0 . 62 , df = 11 , P < 0 . 05 ) .", "Risky behavior was not significantly affected by sex ( Figure 3—figure supplement 3 ) or the patient's regime of regular medication ( Figure 3—figure supplement 4 ) , and the relationship between risk taking and symptom severity held even when controlling for these factors ( p < 0 . 05 for symptom severity when regressing risk taking on symptom severity , age and either of the two medications; including both medications in the model lost significance for symptom severity , likely due to the large number of degrees of freedom for such a small sample size; age and medication did not achieve significance in any of the regressions ) .", "To test whether increased risk-taking in DYT1 dystonia could be explained by asymmetry in the effects of positive and negative prediction errors on corticostriatal plasticity , we modeled participants’ choice data using an asymmetric reinforcement-learning model ( see methods ) where the learning rate ( η ) is modulated by ( 1+κ ) when learning from positive prediction errors and by ( 1−κ ) when the prediction error is negative ( also called a 'risk-sensitive' reinforcement learning model; Mihatsch and Neuneier , 2002; Niv et al . , 2012 ) .", "Our model also included an inverse-temperature parameter ( β ) controlling the randomness of choices .", "This approach exploits fluctuations in each individual’s propensity for risk taking ( see Figure 3a ) as they update their policy based on the outcomes they experience , to recover the learning rate η and learning asymmetry κ that provide the best fit to each participant’s observed behavior .", "First , we tested whether the asymmetric-learning model is justified , that is , whether it explains participants’ data significantly better than the classical reinforcement-learning model with only learning-rate and inverse-temperature parameters .", "The results showed that the more complex model was justified for the majority of participants ( 16 out of 26 participants; DYT 6 , CTL 19 ) , and in particular , for participants who were risk seeking or risk taking ( but not risk-neutral; Figure 4a ) . 10 . 7554/eLife . 14155 . 010Figure 4 . Model comparison supports the asymmetric learning model . We compared three alternative models in terms of how well they fit the experimental data .", "( a ) To compare the asymmetric learning model with the classical ( symmetric ) reinforcement learning ( RL ) model , we used the likelihood ratio test , which is valid for nested models .", "Plotted are the log likelihood differences between the asymmetric learning model and the classical RL model .", "Black line: the minimal difference above which there is a P < 0 . 05 chance that the additional parameter improves the behavioral fit , as tested via a likelihood ratio test for nested models ( dots above this line support the asymmetric learning model ) .", "For the majority of participants ( 16 out of 26; DYT 6 , CTL 10 ) the more complex asymmetric model was justified ( chi square test with df = 1 , P < 0 . 05 ) .", "In particular , and as expected based on Niv et al . ( 2012 ) , the asymmetric learning model was justified for participants who were either risk-averse or risk-taking , but not those who were risk-neutral .", "( b ) The asymmetric learning model and the nonlinear utility model have the same number of free parameters and therefore could be compared directly using the likelihood of the data under each model .", "Plotted is the average probability per trial for the asymmetric learning model as compared to the nonlinear utility model , for healthy controls ( blue dots ) and patients with dystonia ( red ) .", "Dots above the black line support the asymmetric learning model .", "The asymmetric learning model fit the majority of participants better than the utility model ( 15 out of 26; DYT 6 , CTL 9 ) with large differences in likelihoods always in favor of the asymmetric model , and over the entire population the asymmetric learning model performed significantly better ( paired one-tailed t-test on the difference in model likelihoods , t = 1 . 92 , df = 25 , P < 0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14155 . 010 We then compared the individually fit parameters of the asymmetric model across the two groups .", "We found significant differences between the groups in the learning asymmetry parameter ( DYT −0 . 05 ± 0 . 27 , CTL −0 . 34 ± 0 . 27 , Mann-Whitney z = −2 . 51 , df = 24 , P < 0 . 05 ) , but no differences in the other two parameters ( learning rate DYT1 0 . 25 ± 0 . 19 , CTL 0 . 14 ± 0 . 11 , Mann-Whitney z = 1 . 33 , df = 24 , P = 0 . 18; inverse temperature DYT 0 . 68 ± 0 . 37 , CTL 0 . 93 ± 0 . 47 , Mann-Whitney z = −1 . 18 , df = 24 , P = 0 . 23 ) .", "Thus patients’ behavior was consistent with enhanced learning from positive prediction errors and reduced learning from negative prediction errors as compared to healthy controls , despite the overall rate of learning and the degree of noise in choices ( modeled by the inverse temperature parameter ) being similar across groups .", "A significant correlation was also observed between the learning asymmetry parameter and the severity of dystonia ( Pearson’s r = 0 . 64 , df = 11 , P < 0 . 05 ) .", "One alternative explanation for our results is that the nonlinearity of subjective utility functions ( Kahneman and Tversky , 1979 ) for small amounts of money is different between DYT1 dystonia patients and controls .", "However , replicating previous results from a healthy cohort ( Niv et al . , 2012 ) , formal model comparison suggested that choice behavior in our task is significantly better explained by the asymmetric-learning model above ( Figure 4b ) .", "Moreover , the impetus for our experiment was an a priori hypothesis regarding risk sensitivity as a consequence of asymmetric learning , based on findings from the mouse model of DYT1 dystonia , which has no straightforward equivalent interpretation in terms of nonlinear utilities .", "We note also that strongly nonlinear utilities in the domain of small payoffs such as those we used here are generally unlikely ( Rabin and Thaler , 2001 ) , again suggesting that risk sensitivity is more likely to arise in our experiment from asymmetric learning .", "Another alternative explanation for behavior in our task , is a win-stay lose-shift strategy that is perhaps utilized to different extent by DYT1 patients and controls .", "However , this model , equivalent to the classical reinforcement-learning model with a learning rate of 1 and only an inverse temperature parameter , fit 25 out of 26 participants’ data considerably worse than the asymmetric learning model , and therefore was not investigated further ." ], [ "We demonstrated that DYT1 dystonia patients and healthy controls have different profiles of risk sensitivity in a trial-and-error learning task .", "Our results support the dominant model of reinforcement learning in the basal ganglia , according to which prediction-error modulated LTP and LTD in corticostriatal synapses are responsible for changing the propensity to repeat actions that previously led to positive or negative prediction errors , respectively .", "Similar to Parkinson’s disease , at first considered a motor disorder but now recognized to also cause cognitive and learning abnormalities , it appears that DYT1 dystonia is not limited to motor symptoms ( Fiorio et al . , 2007; Heiman et al . , 2004; Molloy et al . , 2003; Stamelou et al . , 2012 ) , and specifically , that the suspected altered balance between LTP and LTD in this disorder has overt , readily measurable effects on behavior .", "DYT1 dystonia and Parkinson's disease can be viewed as complementary models for understanding the mechanisms of reinforcement learning in the human brain .", "In unmedicated Parkinson’s disease patients , learning from positive prediction errors is impaired due to reduced levels of striatal dopamine that presumably signal the prediction errors themselves , whereas learning from negative prediction errors is intact ( Frank et al . , 2004; Rutledge et al . , 2009 ) .", "This impairment , and the resulting asymmetry that favors learning from negative prediction errors , can be alleviated using dopaminergic medication ( Frank et al . , 2004; Shohamy et al . , 2004 ) .", "DYT1 dystonia patients , on the other hand , seem to have intact striatal dopamine signaling ( Balcioglu et al . , 2007; Dang et al . , 2006; Grundmann et al . , 2007; Zhao et al . , 2008 ) , but altered corticostriatal LTP/LTD that favors learning from positive prediction errors .", "Our a priori predictions were based on a simplified model of the role of corticostriatal LTP and LTD in reinforcement learning , and the entire picture is undoubtedly more complex .", "Controversies regarding the functional relationship between the direct and indirect pathways of the basal ganglia ( Calabresi et al . , 2014; Cui et al . , 2013; Kravitz et al . , 2012 ) and the large number of players taking part in shaping synaptic plasticity ( Calabresi et al . , 2014; Shen et al . , 2008 ) make it hard to pin down the precise mechanism behind reinforcement learning .", "Indeed , the DYT1 mouse model has also been linked to impaired plasticity in the indirect pathway due to D2 receptor dysfunction ( Beeler et al . , 2012; Napolitano et al . , 2010; Wiecki et al . , 2009 ) , which can lead to abnormal reinforcement ( Kravitz et al . , 2012 ) .", "In any case , our finding are compatible with the prominent 'Go'/'NoGo' model of learning and action selection in the basal ganglia ( Frank et al . , 2004 ) that incorporates opposing directions of plasticity in the direct and indirect pathways ( Collins and Frank , 2014 ) .", "In particular , current evidence suggests that corticostriatal LTP following positive prediction errors and LTD following negative prediction errors occur in D1 striatal neurons ( direct pathway ) , whereas plasticity in D2-expressing neurons ( indirect pathway ) is in the opposite direction ( Kravitz et al . , 2012; Shen et al . , 2008 ) .", "As the direct pathway supports choice ( ‘Go’ ) while the indirect pathway supports avoidance ( ‘NoGo’ ) , under this implementation of reinforcement learning both types of learning eventually lead to the same behavioral outcome: a positive prediction error increases the probability that the action/choice that led to the prediction error would be repeated in the future , and vice versa for negative prediction errors .", "As such , at the algorithmic level in which our asymmetric learning model was cast , the differences we have shown between dystonia patients and controls would still be expected to manifest behaviorally through diminished risk-aversion in dystonia patients .", "In particular , our results are compatible with several alternative abnormalities in corticostriatal plasticity in DYT1 dystonia:", "( a ) Abnormally strong LTP/weak LTD in D1-expressing striatal neurons only , with plasticity in the indirect pathway being intact; in this case , learning in the direct pathway would exhibit the abnormal asymmetries we argue for , whereas the indirect pathway would learn as normal .", "( b ) Abnormally strong LTP/weak LTD in D1-expressing striatal neurons and the opposite pattern , abnormally strong LTD and/or weak LTP in D2-expressing striatal neurons of the indirect pathway in DYT1 dystonia .", "As a result , a positive prediction error would generate extra strong positive learning in the Go pathway , and a similarly large decrease in the propensity to avoid this stimulus due to activity in the 'NoGo' pathway .", "Conversely , learning from negative prediction errors would generate relatively little decrease in the propensity to 'Go' to the stimulus and little increase in the propensity to 'NoGo' .", "In both cases , the effect on both pathways would be in the same direction as is seen in the behavioral asymmetry .", "( c ) Finally , abnormalities may exist in both pathways in the same direction ( stronger LTP and weaker LTD ) , but with a larger effect on LTP as compared to LTD .", "In this case , a positive prediction error would increase 'Go' activity considerably , but not decrease 'NoGo' activity to the same extent .", "Negative prediction errors , on the other hand , would increase 'NoGo' propensities while decreasing 'Go' propensities to a lesser extent .", "This type of asymmetry can explain why the rodent studies suggested almost absent ( not only weaker ) LTD , but nevertheless , patients did not behave as if they did not learn at all from negative prediction errors .", "Unfortunately , our model and behavioral results cannot differentiate between these three options .", "We hope that future data , especially from transgenic DYT1 rodents , will clarify this issue .", "Relative weighting of positive and negative outcomes shapes risk-sensitivity in tasks that involve learning from experience .", "Humans with preserved function of the basal ganglia have been shown to be risk-averse in such tasks .", "We showed that patients with DYT1 dystonia are more risk-neutral , a rational pattern of behavior given our reward statistics , and in such tasks in general .", "While this type of behavior may offer advantages under certain conditions , it may also contribute to impaired reinforcement learning of motor repertoire and fixation on actions that were once rewarded .", "In any case , these reinforcement-learning manifestations of what has been considered predominantly a motor disease provide support for linking corticostriatal synaptic plasticity and overt trial-and-error learning behavior in humans ." ], [ "Fourteen participants with genetically-proven ( c . 907_909delGAG ) ( Ozelius et al . , 1997 ) symptomatic DYT1 dystonia were recruited through the clinics for movement disorders in Columbia University and Beth Israel Medical Centers in New York and through publication in the website of the Dystonia Medical Research Foundation .", "Exclusion criteria included age younger than 18 or older than 50 years old and deep brain stimulation or other prior brain surgeries for dystonia .", "A single patient was excluded from further analysis due to choosing the left cue in 100% of trials .", "Thirteen age- and sex-matched healthy participants were recruited among acquaintances of the DYT1 patients and from the Princeton University community .", "Healthy control participants were not blood relatives of patients with dystonia and did not have clinical dystonia .", "All patients and healthy controls had at least 13 years of education .", "Nine DYT1 dystonia patients took baclofen ( n = 6 , daily dose 66 . 7 ± 28 . 0 mg , range 30–100 mg ) and/or trihexyphenidyl ( n = 7 , daily dose 30 . 9 ± 25 . 8 mg , range 12–80 mg ) for their motor symptoms .", "In order to reduce possible effects of medication , patients were tested before taking their scheduled dose .", "The median time interval between the last dose of medication and testing was 7 . 5 hr for baclofen ( range 1–20 hr ) and 13 hr for trihexyphenidyl ( range 1–15 hr ) .", "Given that the reported plasma half-life times of baclofen is 6 . 8 hr ( Wuis et al . , 1989 ) and of trihexyphenidyl is 3 . 7 hr ( Burke and Fahn , 1985 ) , three patients were tested within the plasma half life of the last dose of their medication .", "Finally , we could not find correlation between sex of participants ( Figure 3—figure supplement 1 ) or medication doses ( Figure 3—figure supplement 4 ) and relevant behavioral outcomes .", "All participants gave informed consent and the study was approved by the Institutional Review Boards of Columbia University , Beth Israel Medical Center , and Princeton University .", "Clinical scale of dystonia severity was scored immediately after consenting by a movement-disorders specialist ( DA ) , using the Fahn-Marsden dystonia rating scale ( Burke et al . , 1985 ) .", "This scale integrates the number of involved body parts , the range of actions that induce dystonia and the severity of observed dystonia .", "One patient was scored 0 since dystonia was not clinically observed on the day of her testing .", "Prior to , and after completing the reported task , all participants performed a short ( 8–9 min ) unrelated auditory discrimination task ( Baron , 1973 ) ( results not reported here ) that was not associated with any monetary reward .", "Participants were informed that the two tasks were not related .", "Four different pseudo-letters served as cues ( ‘slot machines’ ) and were randomly allocated to four payoff schedules: sure 0¢ , sure 5¢ , sure 10¢ , and one variable-payoff ‘risky’ stimulus associated with equal probabilities of 0¢ or 10¢ payoffs .", "Participants were not informed about the payoffs associated with the different cues and had to learn them from trial and error .", "Two types of trials were pseudo-randomly intermixed: In ‘choice trials’ , two cues were displayed ( left and right location randomized ) , and the participant was instructed to select one of the two cues by pressing either the left or right buttons on a keyboard .", "The cue that was not selected then disappeared and the payoff associated with the chosen cue was displayed for 1 s .", "After a variable ( uniformly distributed ) inter-trial interval of 1–2 s , the next trial began .", "In ‘forced trials’ , only one cue was displayed on either the left or right side of the screen , and the participant had to indicate its location using the keyboard to obtain its associated outcome .", "All button presses were timed out after 1 . 5 s , at which time the trial was aborted with a message indicating that the response was 'too slow' and the inter-trial interval commenced .", "Participants were instructed to try to maximize their winnings and were paid according to their actual payoffs in the task .", "On-screen instructions for the task also informed participants that payoffs depended only on the ‘slot machine’ chosen , not on its location or on their history of choices .", "Throughout the experiment , to minimize motor precision requirements , any of keys E , W , A , Z , X , D , and S ( on the left side of the keyboard ) served as allowable response buttons for choosing the left cue and any of keys I , O , L , < , M , J and K ( on the right side of the keyboard ) served as allowable response buttons for choosing the right cue .", "Each set of response keys was marked with stickers of different colors ( blue for left keys and red for right keys ) to aid in their visual identification .", "Participants were first familiarized with the task and provided with several observations of the cue–reward mapping in a training phase that included two subparts .", "The first part involved 16 pseudo-randomly ordered forced trials ( four per cue ) .", "The second part comprised 10 pseudo-randomly ordered choice trials ( two of each of five types of choice trials: 0¢ versus 5¢ , 5¢ versus 10¢ , 0¢ versus 0/10¢ , 5¢ versus 0/10¢ and 10¢ versus 0/10¢ ) .", "Before the experimental task began , on-screen instructions informed subjects that they would encounter the same cues as in the training phase .", "They were briefly reminded of the rules and encouraged to choose those ‘slot machines’ that yielded the highest payoffs , as they would be paid their earnings in this part .", "The task then consisted of 300 trials ( two blocks of 150 trials each , with short breaks after every 100 trials of the experiment ) , with choice and forced trials randomly intermixed .", "Each block comprised of 30 'risk' choice trials involving a choice between the 5¢ cue and the 0/10¢ cue , 20 choice trials involving each of the pairs 0¢ versus 0/10¢ and 10¢ versus 0/10¢ , 15 choice trials involving each of the pairs 0¢ versus 5¢ and 5¢ versus 10¢ , 14 forced trials involving the 0/10¢ cue and 12 forced trials involving each of the 0¢ , 5¢ and 10¢ cues .", "Trial order was pseudo-randomized in advance and was similar between patients and between blocks .", "Payoffs for the 0/10¢ cue were counterbalanced such that groups of eight consecutive choices of the risky cue included exactly four 0¢ payoffs and four 10¢ payoffs .", "All task events were controlled using MATLAB ( MathWorks , Natick , MA ) PsychToolbox ( Brainard , 1997 ) .", "Our modeling and quantification of the effects of abnormal learning from prediction errors rest solely on the risky cue , for which learning presumably continued throughout the experiment .", "However , one potential worry is that participants did not use trial-and-error learning to evaluate this cue , but rather ‘guessed’ its value using a cognitive system ( as in rule-based learning ) .", "To evaluate this possibility , we tested for a difference in the propensity to choose the risky cue after a previous win or a loss , throughout the task ( see Figure 3c and Figure 3—figure supplement 1 ) .", "To test the hypothesis that increased risk-taking in DYT1 dystonia was due to an enhanced effect of positive prediction errors and a weak effect of negative prediction errors , we modeled participants’ choice data using an asymmetric reinforcement learning model ( also called a risk-sensitive temporal difference reinforcement learning ( RSTD ) model ) ( Mihatsch and Neuneier , 2002; Niv et al . , 2012 ) .", "The learning rule in this model isVnew ( cue ) = Vold ( cue ) +η∙δ∙ ( 1±κ ) where V ( cue ) is the value of the chosen cue , δ = r – Vold ( cue ) is the prediction error , that is , the difference between the obtained reward r and the predicted reward Vold ( cue ) , η is a learning-rate parameter and κ is an asymmetry parameter that is applied as ( 1+κ ) if the prediction error is positive ( δ>0 ) and as ( 1−κ ) if the prediction error is negative ( δ<0 ) .", "This model is fully equivalent to a model with two learning rate parameters , one for learning when prediction errors are positive and another for learning when prediction errors are negative .", "Following common practice , we also assumed a softmax ( or sigmoid ) action selection function:p ( A ) = eβV ( A ) eβV ( A ) +eβV ( B ) where p ( A ) is the probability of choosing cue A over cue B , and β is an inverse-temperature parameter controlling the randomness of choices ( Niv et al . , 2012 ) .", "We fit the free parameters of the model ( η , κ , and β ) to behavioral data of individual participants , using data from both training and test trials ( total of 326 trials ) as participants learned to associate cues with their outcomes from the first training trial .", "Cue values were initialized to", "0 . We optimized model parameters by minimizing the negative log likelihood of the data given different settings of the model parameters using the Matlab function \"fminunc\" .", "The explored ranges of model parameters was [0 , 1] for the learning-rate parameter , [−10 , 10] for the learning-asymmetry parameters , and [0–30] for the inverse-temperature parameter .", "To avoid local minima , for each participant we repeated the optimization 5 times from randomly chosen starting points , keeping the best ( maximum likelihood ) result .", "This method is commonly used for temporal difference learning models and is known to be well-behaved ( Niv et al . , 2012 ) .", "Previous work has shown that the asymmetric learning model best explains participants’ behavior in our task ( Niv et al . , 2012 ) .", "To replicate those results in our sample population , we compared the asymmetric learning model to three other alternative models .", "The first was a classical reinforcement learning model with no learning asymmetry Vnew ( cue ) =Vold ( cue ) +η[R−Vold ( cue ) ] .", "The second alternative model was based on the classical nonlinear ( diminishing ) subjective utility of monetary rewards .", "The idea is that the 10¢ reward may not be subjectively equal to twice the 5¢ reward , therefore engendering risk-sensitive choices in our task .", "We thus defined learning in a nonlinear utility model as Vnew ( cue ) =Vold ( cue ) +η[ U ( R ) −Vold ( cue ) ] , where U ( R ) is the subjective utility of reward R . Without loss of generality , we parameterized the utility function over the three possible outcomes ( 0¢ , 5¢ or 10¢ ) by setting U ( 0 ) = 0 , U ( 5 ) = 5 and U ( 10 ) = a×10 , where the parameter a could be larger , equal to or smaller than 1 , and was fit to the data of each participant separately .", "If the effect of a loss is larger than that of a gain of the same magnitude , a should be smaller than", "1 . Finally , we tested a win-stay-lose-shift strategy model that is equivalent to the classic reinforcement learning model with a learning rate of", "1 . All models used the softmax choice function with an inverse temperature parameter β .", "The parameters of each of the models were fit to each participant’s data as was done for the asymmetric learning model .", "Because the relevant sets of data were not normally distributed ( tested using a Kolmogorov-Smirnov test , P < 0 . 05 ) , we analyzed the data using the nonparametric Mann-Whitney U test to compare two populations , Wilcoxon signed-rank test for repeated measures tests , and Friedman’s test for non-parametric one-way repeated measures analysis of variance by ranks .", "All statistical tests were two-sided unless otherwise specified ." ] ]
[ "It has been difficult to link synaptic modification to overt behavioral changes .", "Rodent models of DYT1 dystonia , a motor disorder caused by a single gene mutation , demonstrate increased long-term potentiation and decreased long-term depression in corticostriatal synapses .", "Computationally , such asymmetric learning predicts risk taking in probabilistic tasks .", "Here we demonstrate abnormal risk taking in DYT1 dystonia patients , which is correlated with disease severity , thereby supporting striatal plasticity in shaping choice behavior in humans ." ]
[ "We learn to choose better options and avoid worse ones through trial and error , but exactly how this happens is still unclear .", "One idea is that we learn 'values' for options: whenever we choose an option and get more reward than originally expected ( for example , if an unappetizing-looking food turns out to be very tasty ) , the value of that option increases .", "Likewise , if we get less reward than expected , the chosen option’s value decreases .", "This learning process is hypothesized to work via the strengthening and weakening of connections between neurons in two parts of the brain: the cortex and the striatum .", "In this model , the activity of the neurons in the cortex represents the options , and the value of these options is represented by the activity of neurons in the striatum .", "Strengthening the connections is thought to increase the value of the stimulus , but this theory has been difficult to test .", "In humans , a single genetic mutation causes a movement disorder called DYT1 dystonia , in which muscles contract involuntarily .", "In rodents , the same mutation causes the connections between the neurons in the cortex and the striatum to become too strong .", "If the theory about value learning is true , this strengthening should affect the decisions of patients that have DYT1 dystonia .", "Arkadir et al . got healthy people and people with DYT1 dystonia to play a game where they had to choose between a 'sure' option and a 'risky' option .", "Picking the sure option guaranteed the player would receive a small amount of money , whereas the risky option gave either double this amount or nothing .", "The theory predicts that the double rewards should cause the patients to learn abnormally high values , which would lure them into making risky choices .", "Indeed , Arkadir et al . found that players with DYT1 dystonia were more likely to choose the risky option , with the people who had more severe symptoms of dystonia having a greater tendency towards taking risks .", "Arkadir et al . showed that these results correspond with a model that suggests that people with DYT1 dystonia learn excessively from unexpected wins but show weakened learning after losses , causing them to over-estimate the value of risky choices .", "This imbalance mirrors the previous results that showed an inappropriate strengthening of the connections between neurons in rodents , and so suggests that similar changes occur in the brains of humans .", "Thus it appears that the changes in the strength of the connections between neurons translate into changes in behavior .", "This pattern of results might also mean that the movement problems seen in people with DYT1 dystonia may be because they over-learn movements that previously led to a desired outcome and cannot sufficiently suppress movements that are no longer useful .", "Testing this idea will require further experiments ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "plant biology", "cell biology" ]
Kinetochore protein depletion underlies cytokinesis failure and somatic polyploidization in the moss Physcomitrella patens
elife-43652-v2
[ [ "The kinetochore is a macromolecular complex that connects chromosomes to spindle microtubules and plays a central role in chromosome segregation .", "Kinetochore malfunction causes checkpoint-dependent mitotic arrest , apoptosis , and/or aneuploidy-inducing chromosome missegregation ( Potapova and Gorbsky , 2017 ) .", "Most of our knowledge on kinetochore function and impact on genome stability is derived from animal and yeast studies ( Musacchio and Desai , 2017 ) .", "Another major group of eukaryotes , plants , also possesses conserved kinetochore proteins ( Yu et al . , 2000; van Hooff et al . , 2017; Yamada and Goshima , 2017 ) .", "Although the localization and loss-of-function phenotype of some plant kinetochore proteins have been reported before ( Shin et al . , 2018; Zhang et al . , 2018; Wang et al . , 2012; Caillaud et al . , 2009; Komaki and Schnittger , 2017; Lermontova et al . , 2013; Sandmann et al . , 2017; Sato et al . , 2005; Du and Dawe , 2007; Ogura et al . , 2004 ) , the data are mostly obtained from fixed cells of specific tissues .", "No comprehensive picture of plant kinetochore protein dynamics and functions can be drawn as of yet .", "For example , 12 out of 16 components that form CCAN ( constitutive centromere associated network ) in animal and yeast cells cannot be identified by homology searches ( Musacchio and Desai , 2017; Yamada and Goshima , 2017 ) .", "How the residual four putative CCAN subunits act in plants is also unknown .", "The moss Physcomitrella patens is an emerging model system for plant cell biology .", "The majority of its tissues are in a haploid state , and , owing to an extremely high rate of homologous recombination , gene disruption and fluorescent protein tagging of endogenous genes are easy to obtain in the first generation ( Cove et al . , 2006 ) .", "The homology search indicated that all the P . patens proteins identified as the homologue of human kinetochore components are conserved in the most popular model plant species A . thaliana ( Yamada and Goshima , 2017 ) : therefore , the knowledge gained in P . patens would be largely applicable to flowering plants , including crop species .", "Another remarkable feature of P . patens is its regeneration ability; for example , differentiated gametophore leaf cells , when excised , are efficiently reprogrammed to become stem cells ( Sato et al . , 2017; Ishikawa et al . , 2011 ) .", "Thus , genome alteration even in a somatic cell can potentially spread through the population .", "In this study , we aimed to comprehensively characterize conserved kinetochore proteins in a single-cell type , the P . patens caulonemal apical cell .", "We observed that many proteins displayed localization patterns distinct from their animal counterparts .", "Furthermore , kinetochore malfunction led to chromosome missegregation and microtubule disorganization in the phragmoplast , eventually resulting in cytokinesis failure and polyploidy ." ], [ "To observe the endogenous localization of putative kinetochore components , we inserted a fluorescent tag in-frame at the N- and/or C-terminus of 18 selected proteins , which contain at least one subunit per sub-complex ( Figure 1—figure supplement 1 ) .", "Initially , we conducted C-terminal tagging since the success rate of homologous recombination is much higher than N-terminal tagging ( Yamada et al . , 2016 ) .", "For 10 proteins , function was unlikely perturbed by tagging , as the transgenic moss grew indistinguishably from wild-type , despite the single-copy protein being replaced with the tagged protein .", "For other proteins , the functionality of the tagged version could not be verified , since untagged paralogs are present in the genome .", "The C-terminal tagging line for CENP-S could not be obtained after two attempts , suggesting that tagging affected the protein’s function and thereby moss viability .", "The N-termini of CENP-S , CENP-O , and CENP-C were also tagged with Citrine .", "Among them , no paralogous protein could be identified for CENP-C; therefore , Citrine signals would precisely represent the endogenous localization .", "Exceptionally , histone H3-like CENP-A ( CenH3 ) localization was determined by ectopic Citrine-CENP-A expression , as tagging likely perturbs its function .", "Consistent with their sequence homology , many of the proteins were localized to the kinetochore at least transiently during the cell cycle .", "However , multiple proteins also showed unexpected localization ( or disappearance ) at certain cell cycle stages ( Figure 1—figure supplement 2–7; Videos 1–4 ) .", "Most surprising were CCAN protein dynamics: CENP-X , CENP-O and CENP-S did not show kinetochore enrichment at any stages ( Figure 1—figure supplement 3; Videos 1 and 3 ) , whereas CENP-C also dissociated from the kinetochore transiently in the post-mitotic phase ( Figure 1B−Figure 1—figure supplement 4; Videos 2 and 3 ) .", "Thus , we could not identify any ‘constitutive’ kinetochore proteins other than CENP-A .", "We failed to obtain knockout lines and/or induce frameshift mutation using CRISPR/Cas9 for the single-copy kinetochore proteins , except for the spindle checkpoint protein Mad2 , strongly suggesting that they are essential for moss viability .", "We therefore made conditional RNAi lines , targeting different proteins from both inner and outer kinetochores ( summarized in Figure 1—figure supplement 1 ) .", "In this RNAi system , knockdown of target genes was induced by the addition of β-estradiol to the culture medium 4–6 days prior to live-imaging ( Nakaoka et al . , 2012 ) .", "Since RNAi sometimes exhibits an off-target effect , we prepared two independent RNAi constructs for most target genes .", "Following the previously established protocol ( Nakaoka et al . , 2012; Miki et al . , 2016 ) , we screened for cell growth/division phenotypes in ≥10 transgenic lines for each construct by using long-term ( >10 hr ) fluorescent imaging .", "We observed mitotic defects in multiple RNAi lines , such as delay in mitotic progression , chromosome missegregation and/or multi-nuclei; these phenotypes were never observed in the control line ( Figure 2A , B; Video 5 ) .", "A full list of targeted genes and brief descriptions of the observed phenotypes are provided in Figure 1—figure supplement 1 .", "We first selected CENP-A for detailed analysis , the only constitutive centromeric protein identified in P . patens .", "As expected , we observed a significant mitotic delay and chromosome alignment/segregation defects in the CENP-A RNAi lines ( Figure 2—figure supplement 1; Video 6 ) .", "These phenotypes can be explained by a deficiency in proper kinetochore-microtubule attachment .", "Consequently , micronuclei were occasionally observed in the daughter cells , a hallmark of aneuploidy .", "We concluded that CENP-A , like in many organisms , is essential for equal chromosome segregation during mitosis in moss .", "Surprisingly , we also frequently observed cells with two large nuclei in both RNAi lines ( Figures 2B at 1 h 18 min ) , which is the typical outcome of cytokinesis failure in this cell type ( Kosetsu et al . , 2013; Miki et al . , 2014; Naito and Goshima , 2015 ) .", "To check if a similar phenotype is observed after the depletion of another kinetochore protein , we observed conditional RNAi line for SKA1 , an outermost kinetochore component that does not directly interact with CENP-A and that had not been functionally characterized in the plant cells yet .", "As expected , mitotic delay and chromosome missegregation were observed in the RNAi line ( Figure 2B−Figure 2—figure supplement 1; Video 5 ) .", "In addition , cytokinesis failure was also detected ( Figure 2B; Video 7 ) .", "To verify that the observed phenotype of SKA1 was not due to an off-target effect , we ectopically expressed RNAi-insensitive SKA1-Cerulean in the RNAi line and observed the rescue of all the above phenotypes ( Figure 2—figure supplement 2 ) .", "Furthermore , we observed a similar phenotype in RNAi lines targeting CENP-C ( CCAN ) , Nnf1 ( Mis12 complex ) , KNL1 and Nuf2 ( Ndc80 complex ) , suggesting that cytokinesis failure is a common outcome following kinetochore malfunction ( Figure 2; Video 5 ) .", "Although we could not detect any kinetochore enrichment of the CCAN subunit CENP-X , we analyzed its RNAi lines .", "Interestingly , we observed similar phenotypes to CENP-A and SKA1 , including cytokinesis failure ( Figure 2B−Figure 2—figure supplement 1; Video 6 ) .", "CENP-X RNAi phenotypes were rescued by the ectopic expression of CENP-X-Cerulean that was resistant to the RNAi construct ( Figure 2—figure supplement 2 ) .", "Thus , CENP-X has lost its kinetochore localization in moss , but is still essential for chromosome segregation and cell division .", "By analyzing a total of 44 cells from SKA1 ( 9 cells ) , CENP-X ( 18 cells ) and CENP-A RNAi ( 9 cells for one construct and 8 cells for the other ) lines that had lagging chromosomes , we noticed a correlation between cytokinesis failure and lagging chromosomes lingering for a relatively long time in the space between separated chromatids .", "We therefore quantified the duration of lagging chromosomes’ residence in the midzone between separating chromatids following anaphase onset .", "Interestingly , a minor delay of chromosomes in the midzone ( <4 min ) never perturbed cytokinesis ( 100% , n = 9 for CENP-A , n = 4 for CENP-X and n = 3 for SKA1 ) .", "By contrast , if we observed a longer delay of chromosome clearance from the midzone , even when only a single chromosome was detectable , cytokinesis defects occurred in 96% of the cells ( n = 9 , 14 and 5; Figure 2C , D−Figure 2—source data 1 ) .", "During plant cytokinesis , a bipolar microtubule-based structure known as the phragmoplast is assembled between segregating chromatids .", "The cell plate then forms in the phragmoplast midzone ( ~4 min after anaphase onset in P . patens caulonemal cells ) and gradually expands toward the cell cortex , guided by the phragmoplast ( Kosetsu et al . , 2013 ) .", "We observed that microtubules reorganized into phragmoplast-like structures upon chromosome segregation in every cell , regardless of the severity of chromosome missegregation ( e . g . 32 min in Figure 2B ) .", "However , high-resolution imaging showed that microtubule interdigitates at the phragmoplast midzone were abnormal in the kinetochore RNAi lines .", "In 5 out of 7 control cells , a sharp microtubule overlap indicated by bright GFP-tubulin signals was observed during cytokinesis , as expected from previous studies ( Kosetsu et al . , 2013; Hiwatashi et al . , 2008 ) ( yellow arrowhead in Figure 2E ) .", "In contrast , CENP-A and SKA1 RNAi lines that had lagging chromosomes and eventually failed cytokinesis never exhibited such focused overlaps ( 0 out of 12 cells ) ; instead , the overlap was broader and less distinguished ( Figure 2E ) .", "Finally , we checked if the cell plate was formed at any point in the cells that had cytokinesis defects , using the lipophilic FM4-64 dye .", "We could not observe vesicle fusion at the midzone following anaphase onset; thus , the cell plate did not form in the cells that had lagging chromosomes for a long time ( Figure 2B , E; Video 8 ) .", "From these results , we concluded that occupation of the midzone by lagging chromosomes for several minutes prevents proper phragmoplast assembly and cell plate formation , which subsequently causes cytokinesis failure .", "Lagging chromosomes are a major cause of aneuploidy in daughter cells , which is particularly deleterious for haploid cells .", "However , the above observation supports a different scenario , whereby cytokinesis failure induced by lagging chromosomes allows a cell to have a duplicated genome set in two or more nuclei .", "On the other hand , whether animal somatic cells that have failed cytokinesis can re-enter the cell cycle or not remains an ongoing debate ( Ganem and Pellman , 2007; Uetake and Sluder , 2004; Panopoulos et al . , 2014 ) .", "To address whether moss cells can recover from severe cell division defects and continue their cell cycle , we first analyzed the DNA content of cells in the CENP-A exon-targeting RNAi line , in which multi-nucleated cells were most prevalent .", "For comparison , we used the parental line: the nuclei of anaphase/telophase cells served as the 1N reference and randomly selected interphase nuclei as the 2N reference , as caulonemal cells are mostly in the G2 phase ( Ishikawa et al . , 2011; Schween et al . , 2003 ) .", "We observed that the majority of the multi-nucleated cells after CENP-A RNAi underwent DNA replication and became tetraploid or attained even higher ploidy ( Figure 3A; DNA was quantified at day 5 after β-estradiol treatment ) .", "Next , we checked if multi-nucleated cells continue cell cycling .", "We used SKA1 RNAi line for a long ( 46 hr ) time-lapse imaging; with this imaging , we expected to monitor the process of cytokinesis failure of a haploid cell and its fate .", "During the imaging period , we indeed observed cytokinesis failure and 10% or 25% multi-nucleated apical cells executed the next cell division by forming a single spindle ( n = 43 and 25 for experiments 1 and 2 , respectively , Figure 3B; Video 9 ) .", "The reason for the low frequency of this event is unclear; strong chromosome missegregation might result in a severe ‘aneuploid’ state for each nucleus , whereas the cell is overall polyploid , which might change the cell physiology .", "Nevertheless , this data strongly suggests that cells that have undergone cytokinesis failure can continue cell cycling as diploids at a certain probability .", "Diploid P . patens is known to develop protonema tissue with a few gametophores ( leafy shoots ) ( Schween et al . , 2005 ) ; therefore , a multi-nucleated cell produced by the cytokinesis failure of a caulonemal cell might proliferate and form a large protonema colony .", "To test this possibility , we isolated and cultured several cells ( Figure 3C ) that were seemingly multi-nuclear after SKA1 RNAi via laser dissection microscopy ( note that there is an unambiguity in identifying multi-nucleate cells; see Materials and methods for detailed explanation ) .", "After 6 weeks of culturing without β-estradiol ( i . e . RNAi was turned off ) , we obtained four moss colonies , two of which consisted mainly of protonemal cells with a few gametophores ( Figure 3D , colony 3 and 4 ) .", "DNA staining and quantification showed that the majority of the cells derived from those two colonies had DNA content approximately double of the control haploid cells , which were regenerated in an identical manner ( Figure 3E , colony 3 and 4 , regenerated from cell 3 and 4 , respectively ) .", "Thus , a polyploid plant was regenerated from a single multi-nucleated somatic cell ." ], [ "This study provides a comprehensive view of the dynamics of conserved kinetochore proteins in a single cell type of P . patens; furthermore , to the best of our knowledge , several proteins , including borealin , KNL1 and SKA subunits , have been characterized for the first time in plant cells .", "The tagged proteins were expressed under their native promoter at the original chromosome locus; thus , fluorescent signals of most , if not all , proteins would represent the endogenous localization .", "Overall , the behavior of outer subunits was largely consistent with their animal counterparts , suggesting that the mitotic function is also conserved .", "However , the timing of kinetochore enrichment differed from that of animal cells and even flowering plants ( e . g . Arabidopsis , maize ) ( Shin et al . , 2018; Du and Dawe , 2007; Hori et al . , 2003 ) : for example , P . patens Ndc80 complex gradually accumulated at the kinetochore after NEBD , unlike Arabidopsis and maize , where it showed kinetochore enrichment throughout the cell cycle ( Shin et al . , 2018; Du and Dawe , 2007 ) .", "More unexpected localizations were observed for inner CCAN subunits , namely CENP-C , CENP-O , CENP-S and CENP-X .", "For example , CENP-C disappeared from the centromeres shortly after mitotic exit .", "In animal cells , CENP-C has been suggested to act in cooperation with Mis18BP1/KNL2 to facilitate CENP-A deposition in late telophase and early G1 ( 2 ) .", "Hence , the mechanism of CENP-A incorporation might have been modified in plants .", "CENP-O , -S , or –X did not show kinetochore enrichment at any stage .", "CENP-X localization was unlikely an artifact of Citrine tagging , since the tagged protein rescued the RNAi phenotype .", "In human cells , 16 CCAN subunits , forming four sub-complexes , have been identified and shown to be critical for kinetochore assembly and function , not only in cells , but also in reconstitution systems ( Guse et al . , 2011; Weir et al . , 2016 ) .", "In plants , only four CCAN homologues have been identified through sequence homology search .", "It is therefore possible that less conserved CCAN subunits are present but could not be identified by the homology search .", "However , the complete lack of kinetochore localization for CENP-O , -S , -X suggests that plants have lost the entire kinetochore-enriched CCAN complex .", "Somewhat puzzlingly , CENP-X , despite its unusual localization , remained an essential factor for chromosome segregation in P . patens .", "In animals , it has been proposed that CENP-S and CENP-X form a complex and play an important role in outer kinetochore assembly ( Amano et al . , 2009 ) .", "It is an interesting target for further investigation if plant CENP-S/CENP-X preserves such a function .", "We observed lagging chromosomes as well as cytokinesis failure after knocking down kinetochore components .", "Failure in chromosome separation/segregation and cytokinesis can be caused by a single gene mutation , if the gene has multiple functions; for example , separase Rsw4 ( radially swollen4 ) in A . thaliana is involved in sister chromatid separation , cyclin B turnover and vesicle trafficking that is required for phragmoplast formation ( Chang et al . , 2003; Yang et al . , 2011; Moschou et al . , 2013; Wu et al . , 2010 ) .", "By contrast , in our study , both phenotypes were observed after RNAi treatment of CENP-A , a constitutive centromeric histone protein that is unlikely to play a direct role in cytokinesis .", "Furthermore , the cytokinesis phenotype frequently appeared in RNAi lines targeting other six kinetochore proteins , and only when lagging chromosomes were present .", "Based on these data , we propose that persistent lagging chromosomes cause cytokinesis failure .", "Lagging chromosomes might act as physical obstacles to perturb phragmoplast microtubule amplification and/or cell plate formation .", "Alternatively , persistent lagging chromosomes might produce an unknown signal or induce a certain cell state that inhibits phragmoplast expansion and/or cell plate formation in order to prevent chromosome damage , reminiscent of the NoCut pathway in animal cytokinesis ( Norden et al . , 2006; Amaral et al . , 2016 ) .", "We favor the latter model , as abnormal microtubule interdigitates were observed in the whole phragmoplast and not limited to the region proximal to the lagging chromosome ( Figure 2E ) .", "Notably , in a recent study , cytokinesis in moss protonema cells could be completed despite longer microtubule overlaps ( de Keijzer et al . , 2017 ) .", "It suggests that abnormal microtubule interdigitates represent the consequence of microtubule dynamics mis-regulation rather than the direct cause of cytokinesis failure .", "Our data further suggest that , in P . patens , chromosome missegregation in a single cell could lead to the generation of polyploid plants .", "Could lagging chromosomes cause polyploidization through somatic cell lineage in wild-type plants ?", "In our imaging of control moss cells , we could not find any lagging chromosome , since mitotic fidelity is very high in our culture conditions .", "Intriguingly , however , various mitotic abnormalities , including lagging chromosomes have been long observed in wild-type plants and crops , albeit at a low frequency and/or under harsh natural conditions ( Menéndez-Yuffá et al . , 2000; Nichols , 1941; Kvitko et al . , 2011 ) .", "Those studies did not analyze the relationship between lagging chromosomes and cytokinesis integrity; we expect the presence of lagging chromosomes for a certain duration to similarly perturb cytokinesis as observed in our study of moss , since the cytokinesis process is highly conserved between bryophytes and angiosperms ( Smertenko et al . , 2017 ) .", "Genome sequencing suggests that P . patens , like many other plant species , experienced whole genome duplication at least once during evolution ( Rensing et al . , 2008 ) .", "Polyploidization through spontaneous mitotic errors in somatic cells might have a greater impact on de novo formation of polyploid plants than previously anticipated ." ], [ "We generally followed protocols described by Yamada et al . ( 2016 ) .", "In brief , Physcomitrella patens culture was maintained on BCDAT medium at 25°C under continuous light .", "Transformation was performed with the polyethylene glycol-mediated method and successful endogenous tagging of the selected genes was confirmed by PCR ( Yamada et al . , 2016 ) .", "We used P . patens expressing mCherry-α-tubulin under the pEF1α promoter as a host line , except for Mis12-mCherry line where GFP-α-tubulin line was used as a host line .", "For knockout , CRISPR ( Lopez-Obando , 2016 ) and RNAi transformations , we used the GH line , expressing GFP-tubulin and HistoneH2B-mRFP .", "P . patens lines developed for this study are described in Supplementary file", "1 . Plasmids and primers used in this study are listed in Supplementary file", "2 . For the C-terminal tagging , we constructed integration plasmids , in which ∼800 bp C-terminus and ∼800 bp 3’-UTR sequences of the kinetochore gene were flanking the citrine gene , the nopaline synthase polyadenylation signal ( nos-ter ) , and the G418 resistance cassette .", "For the N-terminal tagging we constructed integration plasmids , in which ∼800 bp 5’-UTR and ∼800 bp N-terminus sequences of the kinetochore gene were flanking the citrine gene .", "CENP-A cDNA was amplified by PCR and sub-cloned into a vector containing the rice actin promoter , citrine gene , the rbcS terminator , the modified aph4 cassette , and flanked by the genomic fragment of the hb7 locus to facilitate integration .", "All plasmids were assembled with the In-Fusion enzyme according to manufacturer’s protocol ( Clontech ) .", "RNAi constructs were made by using the Gateway system ( Invitrogen ) with pGG624 as the destination vector ( Miki et al . , 2016 ) .", "We followed the protocol described by Vidali et al . ( 2007 ) with the following modifications: sonicated moss was cultured for 6–7 days on the BCDAT plate , containing 5 µM β-estradiol for RNAi induction and 20 µg/ml G418 to prevent contamination .", "Collected cells were preserved in a fixative solution ( 2% formaldehyde , 25 mM PIPES , pH 6 . 8 , 5 mM MgCl2 , 1 mM CaCl2 ) for 30 min and washed three times with PME buffer ( 25 mM PIPES , pH 6 . 8 , 5 mM MgCl2 , 5 mM EGTA ) .", "Following fixation , cells were mounted on 0 . 1%PEI ( polyethyleneimine ) -coated glass slides and subsequently incubated with 0 . 1% Triton X-100 in PME for 30 min and 0 . 2% driselase ( Sigma-Aldrich ) in PME for 30 min .", "Next , cells were washed twice in PME , twice in TBS-T buffer ( 125 mM NaCl , 25 mM Tris-HCl , pH 8 , and 0 . 05% Tween 20 ) and mounted in 10 µg/mL DAPI in TBS-T for observation .", "Images were acquired with the Olympus BX-51 fluorescence microscope equipped with ZEISS Axiocam 506 Color and controlled by ZEN software .", "Fluorescent intensity was measured with ImageJ .", "Cytoplasmic background was subtracted .", "A glass-bottom dish ( Mattek ) inoculated with moss was prepared as described in Yamada et al . ( 2016 ) and incubated at 25°C under continuous light for 4–7 days before live-imaging .", "To observe RNAi lines , we added 5 µM β-estradiol to culture medium ( Miki et al . , 2016 ) .", "For the high-magnification time-lapse microscopy , the Nikon Ti microscope ( 60 × 1 . 40 NA lens or 100 × 1 . 45 NA lens ) equipped with the spinning-disk confocal unit CSU-X1 ( Yokogawa ) and an electron-multiplying charge-coupled device camera ( ImagEM; Hamamatsu ) was used .", "Images were acquired every 30 s for localization analysis and every 2 min for RNAi analysis .", "The microscope was controlled by the Micro-Manager software and the data was analyzed with ImageJ .", "The rescue lines for RNAi were observed using a fluorescence microscope ( IX-83; Olympus ) equipped with a Nipkow disk confocal unit ( CSU-W1; Yokogawa Electric ) controlled by Metamorph software .", "Protonema tissue of P . patens was sonicated , diluted with BCD medium with 0 . 8% agar , and spread on cellophane-covered BCDAT plates that contain 5 µM estradiol to induce RNAi .", "After 5–6 days , small pieces of cellophane containing clusters of protonemal cells ( each containing 3–20 cells ) were cut with scissors and placed upside-down on a glass-bottom dish .", "Bi- or multi-nucleated cells were identified using Axio Zoom . v16 .", "Single bi-nucleated cell ( SKA1 RNAi line ) or random cell ( control GH line ) was selected for isolation , and all other cells were ablated with a solid-state ultraviolet laser ( 355 nm ) through a 20X objective lens ( LD Plan-NEOFLUAR , NA 0 . 40; Zeiss ) at a laser focus diameter of less than 1 µm using the laser pressure catapulting function of the PALM microdissection system ( Zeiss ) .", "Irradiation was targeted to a position distantly located from the cell selected for isolation to minimize the irradiation effect .", "Note that visual distinction of multi-nucleated cells from those with slightly deformed nuclei is not easy in P . patens , since in multi-nucleated cells , the nuclei maintain very close association with each other , so that nuclear boundaries often overlap .", "We interpret that two of four regenerated protonemata had haploid DNA content due to our unintentional isolation of a single cell with a deformed nucleus rather than multi-nuclei .", "Next , a piece of cellophane with single isolated cell was transferred from the glass-bottom dish to estradiol-free medium ( 20 µg/ml G418 was supplied to prevent bacterial/fungal contamination ) .", "DAPI staining was performed 5–6 weeks later as described above .", "Full-size amino acid sequences of the selected proteins were aligned using MAFFT ver . 7 . 043 and then revised manually with MacClade ver . 4 . 08 OSX .", "We used the Jones-Taylor-Thornton ( JTT ) model to construct maximum-likelihood trees in MEGA5 software .", "Statistical support for internal branches by bootstrap analyses was calculated using 1000 replications .", "Reference numbers correspond to Phytozome ( www . phytozome . net ) for Physcomitrella patens , the Arabidopsis Information Resource ( www . arabidopsis . org ) for Arabidopsis thaliana and Uniprot ( www . uniprot . org ) for Homo sapiens .", "Original protein alignments after MAFFT formatted with BoxShade ( https://embnet . vital-it . ch/software/BOX_form . html ) are shown in Supplementary file 3 ." ] ]
[ "Lagging chromosome is a hallmark of aneuploidy arising from errors in the kinetochore–spindle attachment in animal cells .", "However , kinetochore components and cellular phenotypes associated with kinetochore dysfunction are much less explored in plants .", "Here , we carried out a comprehensive characterization of conserved kinetochore components in the moss Physcomitrella patens and uncovered a distinct scenario in plant cells regarding both the localization and cellular impact of the kinetochore proteins .", "Most surprisingly , knock-down of several kinetochore proteins led to polyploidy , not aneuploidy , through cytokinesis failure in >90% of the cells that exhibited lagging chromosomes for several minutes or longer .", "The resultant cells , containing two or more nuclei , proceeded to the next cell cycle and eventually developed into polyploid plants .", "As lagging chromosomes have been observed in various plant species in the wild , our observation raised a possibility that they could be one of the natural pathways to polyploidy in plants ." ]
[ "Plants and animals , like all living things , are made of self-contained units called cells that are able to grow and multiply as required .", "Each cell contains structures called chromosomes that provide the genetic instructions needed to perform every task in the cell .", "When a cell is preparing to divide to make two identical daughter cells – a process called mitosis – it first needs to duplicate its chromosomes and separate them into two equal-sized sets .", "This process is carried out by complex cell machinery known as the spindle .", "Structures called kinetochores assemble on the chromosomes to attach them to the spindle .", "Previous studies in animal cells have shown that , if the kinetochores do not work properly , one or more chromosomes may be left behind when the spindle operates .", "These ‘lagging’ chromosomes may ultimately land up in the wrong daughter cell , resulting in one of the cells having more chromosomes than the other .", "This can lead to cancer or other serious diseases in animals .", "However , it was not known what happens in plant cells when kinetochores fail to work properly .", "To address this question , Kozgunova et al . used a technique called RNA interference ( or RNAi for short ) to temporarily interrupt the production of kinetochores in the cells of a moss called Physcomitrella patens .", "Unexpectedly , the experiments found that most of the moss cells with lagging chromosomes were unable to divide .", "Instead , they remained as single cells that had twice the number of chromosomes as normal , a condition known as polyploidy .", "After the effects of the RNAi wore off , these polyploid moss cells were able to divide normally and were successfully grown into moss plants with a polyploid number of chromosomes .", "Polyploidy is actually widespread in the plant kingdom , and it has major impacts on plant evolution .", "It is also known to increase the amount of food that crops produce .", "However , it is still unclear why polyploidy is so common in plants .", "By showing that errors in mitosis may also be able to double the number of chromosomes in plant cells , the findings of Kozgunova et al . provide new insights into plant evolution and , potentially , a method to increase polyploidy in crop plants in the future ." ]
2019
[ "Main Text", "Results", "Discussion", "Materials and methods" ]
[ "short report", "neuroscience" ]
Knockdown of hypothalamic RFRP3 prevents chronic stress-induced infertility and embryo resorption
elife-04316-v1
[ [ "High psychological stress inhibits reproductive function when both occur concomitantly ( Rivier and Rivest , 1991; Ferin , 1999; Tilbrook et al . , 2000; Louis et al . , 2011 ) .", "From an evolutionary perspective , inhibition of reproductive function by acute stress may be adaptive , delaying reproduction in times of duress or resource scarcity ( Wasser and Barash , 1983; Louis et al . , 2011 ) .", "Chronic stress , however can result in persistent , maladaptive sexual dysfunction and suppressed fertility ( Young et al . , 2006 ) .", "Little is understood about the lasting effects of stress exposure .", "For example , after its cessation , can a prior , persistent stressor have long-term negative after-effects on reproductive health ?", "In humans , a high-stress environment may be a significant barrier to sexual well-being and childbearing .", "In healthy couples under 30 years of age , 63–80% are unable to conceive within 3 months of attempting , and within 1 year of attempting pregnancy , 15% of couples remain unable to conceive ( Practice Committee of the American Society for Reproductive Medicine in collaboration with the Society for Reproductive Endocrinology and Infertility , 2008 ) .", "A molecular framework to understand the long-term effects of stress on female reproduction , and its implications for human health , is currently lacking .", "The present series of studies sought to answer two main questions:", "1 ) Do stressful events negatively impact female reproductive function even following recovery of the stressor , and , if so ,", "2 ) are the deficits observed mediated by stress-induced elevation of the inhibitory neuropeptide , RFamide-related peptide-3 ( RFRP3 ) ?", "To our knowledge , no study to date has elucidated the molecular mechanisms of stress-induced infertility nor has there been any investigation of long-lasting after-effects of pre-conception stress on reproductive success and pregnancy outcome .", "RFRP3 , the mammalian ortholog of gonadotropin-inhibitory hormone ( GnIH ) first identified in Japanese quail ( Tsutsui et al . , 2000 ) , is common across mammals , including rats and mice ( Ukena and Tsutsui , 2001; Ukena et al . , 2002; Kriegsfeld et al . , 2006 ) , hamsters ( Ubuka et al . , 2012 ) , non-human primates ( Ubuka et al . , 2009a ) , and humans ( Ubuka et al . , 2009b ) , and is a hypothalamic hormone that directly inhibits the firing of kisspeptin-sensitive gonadotropin-releasing hormone ( GnRH ) neurons in the hypothalamus in mice ( Ducret et al . , 2009; Wu et al . , 2009 ) .", "It also reduces downstream luteinizing hormone ( LH ) secretion in rats ( Johnson et al . , 2007; Murakami et al . , 2008 ) , mice ( Son et al . , 2012 ) , and hamsters ( Kriegsfeld et al . , 2006 ) .", "There is some debate as to whether RFRP3 is a hypophysiotropic hormone ( Murakami et al . , 2008; Kirby et al . , 2009; Pineda et al . , 2010a , 2010b ) or only centrally inhibits GnRH to elicit a response ( Rizwan et al . , 2009 , 2012 ) .", "Regardless of its mechanism of action , RFRP3 decreases the synthesis and release of pituitary gonadotropins , LH , and follicle stimulating hormone ( FSH ) , in many species , including rats and mice ( Ciccone et al . , 2004; Johnson et al . , 2007; Murakami et al . , 2008; Sari et al . , 2009; Kriegsfeld et al . , 2010; Ubuka et al . , 2011; Son et al . , 2012 ) .", "In females , RFRP was shown to be regulated throughout the ovulatory cycle in rats and hamsters , and it elicits a marked inhibitory effect on the pre-ovulatory LH surge through inhibition of GnRH activation in rats ( Anderson et al . , 2009 ) .", "In male rats , RFRP3 expression is elevated 24 hr after a chronic stressor , suggesting that RFRP3 may mediate enduring changes in reproductive function ( Kirby et al . , 2009 ) .", "Levels of the glucocorticoid stress hormone ( in rodents , corticosterone ) may mediate this effect; RFRP3 neurons in the rat hypothalamus were shown to express glucocorticoid receptor ( GR ) ( Kirby et al . , 2009 ) , as well as RFRP-expressing neuronal cell line in vitro ( Lee Son et al . , 2014 ) .", "Finally , the RFRP promoter region includes two glucocorticoid response elements ( GREs ) , all together supporting the hypothesis that RFRP may be directly regulated by circulating glucocorticoid levels ( Lee Son et al . , 2014 ) .", "Together , these findings provide support for the notion that stress-induced increases in RFRP3 might have long-lasting negative impact on female reproductive functioning .", "Despite knowledge of RFRP's responsiveness to stress and its role in regulating reproductive axis activity , no study to date has established a causal link between RFRP and fertility in any species .", "We set out to test the potential role of RFRP expression in stress-induced infertility in females ." ], [ "In sum , we found that chronic stress led to elevated RFRP3 at all stages of the ovulatory cycle .", "This elevated level of expression persisted after a full cycle of recovery from stress , indicating that the impact of stress on RFRP3 lasts well beyond removal of the stressor .", "Stressed females exhibited fewer successful copulation events , fewer pregnancies in those that did successfully mate , and increased frequency of embryo resorption in the achieved pregnancies .", "These marked effects of stress on fertility were completely blocked by knockdown of RFRP3 , even though RFRP3 function was restored following stress cessation .", "These findings indicate that stress has lingering negative consequences for female reproductive function that are mediated by a transient rise in RFRP3 .", "Female rats were subjected to an 18 day stress paradigm followed by quantification of hypothalamic markers of reproductive function either immediately after stress exposure or after one full estrous cycle ( 4 days ) of recovery ( Figure 1A ) .", "Serum levels of corticosterone ( CORT ) were measured on days 1 , 4 , 7 , 11 , and 18 of the 18 days immobilization stress paradigm , and on day 22 , 4 days after the cessation of stressor .", "Baseline levels at the onset of stress exposure sessions were unchanged throughout the 18 days .", "However , CORT levels were significantly elevated in samples drawn on days 1 , 4 , 7 , and 11 at the end of the 3-hr stress exposure . 10 . 7554/eLife . 04316 . 003Figure 1 . 18 days chronic stress leads to an upregulation of RFRP mRNA that persists for at least one estrous cycle in the rat .", "( A ) Experimental timeline .", "( B ) .", "Corticosterone was measured in serum samples from tail vein blood immediately before and after stress sessions on days 1 , 4 , 7 , 11 , and 18 , and on day 22 , 4 days post-stress cessation ( N = 36/group in 1 , 4 , 7 , 11 , 18 timepoints , N = 18/group on 22 ) .", "( C , E , G , I )", "Gene expression changes in the hypothalamus immediately after stress and ( D , F , H , J ) 4 days after stress .", "mRNA levels of all ( mean ± SEM , N = 6/group ) were determined using qRT–PCR relative to the ribosomal reference gene RPLP at day 0 and 4 post-stress cessation .", "Estrous cycle staging was determined by inspection of daily vaginal smears .", "*p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 .", "PCR statistics were done by a Kruskal–Wallis one-way ANOVA followed by Dunn's multiple comparison test for post-hoc analysis , CORT statistics analyzed by a repeated two-way ANOVA . DOI: http://dx . doi . org/10 . 7554/eLife . 04316 . 003 On day 22 , after 4 days of recovery from the stressor , the stressed rats exhibited serum CORT concentrations indistinguishable from baseline values ( Figure 1B ) .", "Rats exhibit a 4–5 days long estrous cycle , with rising estrogen concentrations triggering a surge of luteinizing hormone ( LH ) to initiate ovulation , and estrogen and progesterone driving sexual receptivity on the night of proestrus ( Blaustein , 2008 ) .", "Stress acutely inhibits the LH surge ( Du Ruisseau et al . , 1979 ) and subsequent sexual receptivity and fertility ( Sirinathsinghji et al . , 1983; Sato et al . , 1996 ) .", "However , it is unknown whether reproductive function continues to be negatively impacted even following recovery from stress ( defined as exhibiting baseline levels of CORT after 4 days of no stress exposure ) .", "Rats were monitored daily by vaginal smear to determine whether estrous cyclicity was affected during application of the stressor and to allow separation of animals into different cycle stages ( diestrus , proestrus , and estrus ) at the termination of the stressor .", "Stress did not affect estrous cyclicity , with all animals exhibiting normal vaginal cytology throughout the stressor , and all animals exhibited a normal 4- to 5-day estrous cycle after the cessation of stress .", "At all estrous cycle stages , RFRP3 mRNA expression in the hypothalamus was significantly elevated both 0 and 4 days after the stressor was terminated ( Figure 1C , D ) .", "Hypothalamic expression of the RFRP3 receptor , G-protein-coupled receptor-147 ( GPR147 ) , was also upregulated after stress during all stages of the cycle , and returned to baseline values after the cessation of stress ( Figure 1E , F ) .", "We did not find significant differences in either GnRH or kisspeptin ( KISS1 ) mRNA expression post-stress in any stage of the cycle ( Figure 1G–J ) .", "However , hypothalamic samples were taken from whole hypothalamus , precluding the ability to differentiate between rostral and caudal kisspeptin cell populations , potentially masking subtle differences .", "Notably , the persistent increases in the expression of both RFRP3 and its receptor specifically in proestrus coincide with the cyclical onset of sexual receptivity , suggesting that RFRP3 provides a mechanistic basis for long-lasting suppression of reproductive behavior after stress .", "To investigate whether the stress-induced increase in RFRP3 plays a causal role in prolonged sexual inhibition , we developed a conditional viral vector to knock down RFRP3 expression ( tet-OFF lentivirus RFRP3 shRNA ) in vivo during the strictly-defined time window of the chronic stressor .", "This lentiviral construct expresses RFRP3 shRNA from a constitutively active CMV promoter , driving both shRNA and blue fluorescent protein ( BFP ) marker expression .", "When exposed to doxycycline ( DOX , via drinking water ) ( Figure 2A ) , the tet-Off element is prevented from driving TRE-initiated transcription and both shRNA and BFP production cease ( location and extent of viral infection can be seen in Figure 2B , C ) .", "Stereotaxic infusion of RFRP3 shRNA lentivirus into the hypothalamus led to an 87% down-regulation of RFRP3 mRNA expression within 7 days relative to a control-scrambled shRNA ( Figure 2D ) .", "Immunohistological labeling verified that the peptide level in the hypothalamus was similarly knocked down by 85% compared to scrambled control virus , measured 2 weeks following viral injection ( Figure 2E , representative images of RFRP labeling with either scramble or RFRP-shRNA virus and pre- and post-DOX administration Figure 2F–I ) .", "Critically , administration of doxycycline in the drinking water restored RFRP3 mRNA to normal levels within 4 days ( Figure 2D ) .", "This viral vector system permitted knocking down of RFRP3 expression during chronic stress and restoration of RFRP3 during the later stages of copulation , mating , and birthing , which may rely on RFRP3 function in unknown ways . 10 . 7554/eLife . 04316 . 004Figure 2 . RFRP-shRNA successfully knocks down RFRP expression in the dorsal medial hypothalamus , and expression is recovered upon DOX induction .", "( A ) Map of RFRP-shRNA viral plasmid .", "( B ) Brain sectioned and stained with an anti-BFP antibody to label virus infection ( green ) and anti-RFRP 2 weeks post-injection ( WPI ) to show injection location and ( C ) spread .", "Scale bar indicates 100 µm .", "( D ) mRNA levels of RFRP following injection of RFRP-shRNA viral vector were determined using qRT-PCR ( WPI = weeks post-injection , mean ± SEM , N = 4 ) .", "( E ) RFRP3-ir cells/section counts in the DMH after 2 weeks post-injection with either scramble or RFRP-shRNA virus .", "( F–I )", "Brain sectioned stained with anti-RFRP3 antibody 2 weeks post-injection with scramble or RFRP-shRNA virus and before and after DOX administration .", "Scale bar indicates 100 µm .", "*p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 .", "Statistics were done by one-way analysis of variance ( ANOVA ) and Bonferroni post-hoc tests .", "For mRNA data , PCR statistics were done by a Kruskal–Wallis one-way ANOVA followed by Dunn's multiple comparison test for post-hoc analysis and statistics for protein counts were a student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 04316 . 004 A second group of female rats received dorsomedial hypothalamic injections of either RFRP3 shRNA or a scrambled control shRNA lentivirus 3 weeks before the 18 days of immobilization .", "Estrous cycles were monitored for each rat with immobilization timed to coincide with the onset of estrus , leaving most rats in proestrus 4 days after the end of stress .", "All rats were administered DOX on the final day of stress so that restoration of RFRP3 expression coincided with the onset of proestrus after the 4-day recovery period .", "( Figure 3A ) .", "After one full estrous cycle of recovery from stress , rats underwent a timed mating test on the night of proestrus and were monitored through gestation and birth , to assess the long-term effects of stress on reproductive success including successful copulation and pregnancy outcome . 10 . 7554/eLife . 04316 . 005Figure 3 . Knocking down RFRP during stress completely prevents stress-induced reproductive dysfunction .", "( A ) Experimental time line .", "( B ) Corticosterone concentrations were measured in serum samples from tail vein blood immediately before and after stress sessions on days 1 , 11 , and 18 .", "( C ) Total reproductive success was measured as percentage of females that successfully brought a litter to full term ( Scramble/control N = 17 , Scramble/Stress N = 14 , shRNA/control N = 20 , shRNA/stress N = 14 , g-statistics: G = 5 . 836 , df = 1 p = 0 . 016 , fisher's exact test p = 0 . 0031 ) .", "Breaking down total reproductive success , ( D ) copulation success was measured as percentage of females that exhibited lordosis and allowed a male to achieve intromission within 15 min ( g-statistics: G = 2 . 405 , df = 1 p = 0 . 028 , fisher's exact test p = 0 . 0062 ) , and ( E ) pregnancy success refers to the percentage of females that got pregnant out of the subgroup that successfully copulated ( Scramble/control N = 15 , Scramble/Stress N = 6 , RFRP-shRNA/control N = 18 , RFRP-shRNA/stress N = 11 ) .", "( F ) Litter sizes measured as number of pups born alive immediately after birth ( dams-Scramble/control N = 13 , Scramble/Stress N = 3 , RFRP-shRNA/control N = 16 , RFRP-shRNA/stress N = 9 ) .", "( G ) Embryos implanted measured as number of placental scars identified in the dam's uterine horns after birth .", "( H ) Embryo survival was calculated as the number of birthed pups divided by number of maternal placental scars and shown as a percentage ( indicative of initial implantation , mean ± SEM ) *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 .", "Reproductive success statistics were done by G-statistics tests followed by Fisher's Exact test , statistics for litter size , placental scars , and embryo resorption were done by a two-way analysis of variance ( ANOVA ) followed by Bonferroni post-hoc tests and CORT statistics analyzed by a repeated two-way ANOVA . DOI: http://dx . doi . org/10 . 7554/eLife . 04316 . 00510 . 7554/eLife . 04316 . 006Figure 3—figure supplement 1 . RFRP-shRNA animals had increased plasma estradiol on proestrus during stress and RFRP-shRNA animals that mated had higher circulating estradiol than scrambled animals .", "( A ) Estradiol levels as measured from tail bleed samples over the cycles of all stressed rats with either scrambled or RFRP-shRNA virus ( n = 14 ) .", "( B ) Within proestrus , estradiol measurements were separated by mating success and virus ( n = 10 , 21 , 11 , 25 samples for each successive group ) .", "( C ) Lordosis intensity , or quality of the lordosis pose , scored between 0 and 3 as published in Hardy and Debold ( 1971 ) .", "We found a significant main effect of stress ( F ( 1 , 61 ) = 5 . 15 , p = 0 . 0268 ) , however , no significant differences within groups .", "( D ) Lordosis quotient was calculated as the ratio of male mounts to female lordosis poses of a score of 2 or 3 .", "We found a significant main effect of stress ( F ( 1 , 61 ) = 11 . 66 , p = 0 . 0011 ) , as well a significant decrease in the scramble stress group .", "p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 .", "Estradiol , lordosis intensity and quotient statistics were a two-way analysis of variance ( ANOVA ) followed by Bonferroni post-hoc tests . DOI: http://dx . doi . org/10 . 7554/eLife . 04316 . 006 Tail vein serum samples taken at the onset and end of stress sessions on days 1 , 11 , and 18 revealed that post-stress circulating levels of CORT were elevated on days 1 and 11 of the immobilization period ( Figure 3B ) .", "Moreover , RFRP3 knockdown during stress did not significantly alter CORT response during stress , indicating an intact hormonal stress response ( Figure 3B ) .", "Stress exposure led to a profound decrease in total reproductive success in females that received the control virus: only 21% of stressed females became pregnant and carried to live birth , as compared to 76% of non-stressed females with control virus ( Figure 3C ) .", "80% of the females that received the RFRP-shRNA virus became pregnant and carried to live birth ( Figure 3C ) .", "The stress-induced decline in reproductive success resulted from a cumulative decrease in mating success ( from 88 and 90% in non-stressed groups to 43% in the stress-scrambled group , Figure 3D ) and pregnancy rates in the females that mated ( from 87 and 89% in non-stressed groups to 50% in the stress-scrambled group , Figure 3E ) .", "Interestingly , knockdown of RFRP3 expression in the hypothalamus during stress exposure prevented the stress-induced suppression of reproduction , leading to 79% copulation success , 82% pregnancy success , and overall reproductive success to 64% , a rate statistically equivalent to control ( non-stress ) levels ( 76% , Figure 3C ) .", "Exposure to acute stress on the evening of the third day of pregnancy was reported to lead to reduced litter size , via inhibition of implantation which occurs normally 5 days after mating ( Zhao et al . , 2013 ) .", "Therefore , we next assessed whether pre-copulation stress exposure affects pregnancy outcome .", "Stressed females that received control-scrambled shRNA had significantly smaller litter sizes ( Figure 3F , 12 . 77 ± 0 . 91 vs 7 . 667 ± 2 . 60 pups/litter , p < 0 . 05 ) with no difference in placental scars ( Figure 3G , 13 . 0 ± 0 . 91 vs10 . 667 ± 3 . 93 , p > 0 . 05 ) .", "Embryo survival was analyzed in the females that were successfully impregnated by determining the ratio of placental scars ( indicative of successful implantation ) to the number of live pups in the litter .", "These were first pregnancies for all females , so the number of placental scars is indicative of implantation events during this pregnancy .", "Embryo survival in stressed females that received scrambled shRNA was 78 . 8 ± 11 . 7% of fetuses , compared to 98 . 1 ± 0 . 95% , and 97 . 8 ± 1 . 5% survival in the control scrambled and control RFRP-shRNA groups , respectively ( Figure 3F , p < 0 . 05 ) .", "Most remarkably , RFRP3 shRNA administration suppressed stress-induced fetal resorption , showing a 93 . 4 ± 3 . 2% fetal survival rate ( Figure 3H ) .", "These results demonstrate that stress-induced increases in RFRP3 expression has long-term detrimental effects on female reproductive fitness that persist long after the stressor has been removed and CORT levels have returned to baseline .", "In addition , knocking-down RFRP3 expression during stress eliminated the stress-induced decrease in sexual motivation , decrease in pregnancy success , and subsequent increase in embryo resorption .", "We next examined plasma estradiol concentration in animals throughout the stress period in both scrambled and RFRP-shRNA groups .", "We found that animals with RFRP knocked down had significantly higher circulating estradiol in proestrus during the stress exposure than animals that received the scrambled virus ( Figure 3—figure supplement 1A , p < 0 . 01 ) , indicating that RFRP knockdown reverses the stress-induced blockade of the E2 rise that occurs during proestrus .", "Examining animals more closely during proestrus , we found that the RFRP-shRNA animals that successfully mated after the stressor had significantly higher circulating estradiol in their proestrus periods over the course of the stressor than both scrambled groups ( Figure 3—figure supplement 1B , p < 0 . 05 ) .", "Finally , we investigated behavioral measures of female receptivity to test its potential contribution to the stress-induced reproductive deficits observed .", "Lordosis intensity is a rating ( from 0-3 ) of the quality of all lordosis poses the female exhibits during the mating session , when 0 marks no lordosis and 3 is a fully mounted spinal flexion pose .", "In rats , a common index of relative sexual receptivity of a female in the presence of males , is the lordosis quotient ( LQ ) , calculated by the number of times the female adopts a lordosis posture scored 2 or higher , divided by the number of times a male mounts her .", "All females included in the study exhibited lordosis when introduced to a male ( indicating that they were in the correct stage of their cycle ) .", "Lordosis intensity did not differ within groups ( Figure 3—figure supplement 1C ) but a significant main effect of stress revealed that lordosis intensity was significantly suppressed by stress ( F ( 1 , 61 ) = 5 . 15 , p = 0 . 0268 ) .", "Furthermore , lordosis quotient measures revealed significantly lower ratio in the scrambled stress group compared to the non-stressed groups that received scrambled or RFRP-shRNA ( 0 . 30 ± 0 . 10 vs 0 . 73 ± 0 . 07 and 0 . 68 ± 0 . 07 ) , indicating that stress exposure decreased the relative sexual receptivity of the females ( Figure 3—figure supplement 1D ) , congruent with the stress-induced drop in mating success we found .", "Interestingly , LQ ratios in stressed females that received RFRP-shRNA were not significantly different from controls ratios ( 0 . 53 ± 0 . 10 , Figure 3—figure supplement 1D ) , demonstrating that knock-down of RFRP reversed the stress-induced decrease in sexual receptivity , and congruent with the reversal of mating success found in this group ." ], [ "In humans , high anxiety and psychological stress can lead to long-term impaired fertility , ranging from reduced libido , delayed pregnancy success to the extreme of complete reproductive axis suppression as in the case of hypothalamic amenorrhea ( Ferin , 1999 ) .", "In our studies , selective knock-down of hypothalamic RFRP3 during stress exposure preserved all aspects of reproductive function that were otherwise reduced in stress-exposed animals ( summary schematic , Figure 4 ) .", "The stress-induced spike in RFRP3 initiates a long-lasting suppression of reproduction , well after the removal of the stressor , perhaps via positive feedback that maintains elevated RFRP3 levels or engages downstream suppressive targets .", "These findings reveal a single molecular target that persistently underlies a range of different reproductive dysfunctions that may provide a novel translational framework for clinical study of human reproductive health . 10 . 7554/eLife . 04316 . 007Figure 4 . Schematic illustration of experiments . Female rats ( in pink at top ) were injected with either an inducible RFRP-shRNA or a scrambled control virus .", "Each group was furthered separated into a no stress control or subjected to 18 days of immobilization stress .", "Stressed females exhibited fewer successful copulation events , fewer pregnancies in those that did successfully mate , and increased frequency of embryo resorption .", "These marked effects of stress on fertility were completely blocked by knockdown of RFRP3 . DOI: http://dx . doi . org/10 . 7554/eLife . 04316 . 007 The stress-induced rise in RFRP may be acting on neural circuits influencing mating and pregnancy , potentially independently of sex steroids .", "RFRP projects to multiple brain regions responsible for successful reproduction and mating behavior , including the medial pre-optic area ( mPOA ) ( where it is known to affect GnRH release ) as well as the BNST , medial amygdala , anterior hypothalamus , and arcuate nucleus ( Kriegsfeld et al . , 2006 ) .", "Piekarski et al . found that administering RFRP3 to hamsters reduced sexual motivation ( as measured by percent of time spent with castrated vs intact males ) and vaginal scent marking without effect on lordosis behavior , similar to our present findings .", "Additionally , RFRP3 administration altered cellular activation in regions of the brain implicated in female sexual motivation , including the mPOA , medial amygdala , and BNST—all regions that receive RFRP projections .", "These effects were independent of gonadal steroids and kisspeptin cellular activation ( Piekarski et al . , 2013 ) .", "While we were unable to measure progesterone or prolactin in this study , it is possible that RFRP projections to the arcuate nucleus affect dopaminergic signaling required for prolactin release and maintenance of progesterone levels and pregnancy success .", "Future studies aimed at systematically examining each step in these processes is required to gain a full understanding of the neural circuits underlying the deleterious effects of stress on reproduction .", "In humans , RFRP 1 and 3 , and their cognate receptor are expressed in the hypothalamus ( Ubuka et al . , 2009b ) .", "It is possible that manipulation of RFRP3 signaling in humans may relieve stress-related reproductive dysfunction , including decreased sex drive , impaired fertility , and increased miscarriages .", "Likewise , if similar mechanisms of stress-induced reproductive suppression are common across species , such strategies may be similarly relevant to species bred in captivity that are susceptible to stress-induced infertility , in particular , endangered species whose preservation depends on captive breeding programs ." ], [ "Adult female Sprague–Dawley rats were triple-housed on a 12/12-hr light–dark cycle with lights on at 0700 hr and ad libitum food and water .", "For all studies , rats were acclimated for a week and then vaginal smears were obtained daily to verify normal cyclicity for 12 days before the studies commenced .", "Rats that did not cycle normally were removed from the study .", "For the chronic stress experiment , rats were immobilized daily from 9 am to 12 pm ( N = 6 for each cycle time point ) or left undisturbed in their home cages ( N = 6 for each cycle time point ) until terminal samples were collected ( stress paradigm described below ) .", "In the RFRP knockdown study , animals received stereotaxic injections of either RFRP-shRNA ( N = 30 ) or scrambled control ( N = 28 ) , then allowed to recover for 3 weeks .", "After recovery , rats were exposed to the same stress paradigm as the previous experiment .", "After cessation of stress all animals were left undisturbed in their home cage for 4 days , and on the night of the fourth day observed during timed mating ( see below ) .", "Rats that successfully mated were left in their home cages for the duration of gestation and then perfused within 24 hr after parturition .", "( stress/shRNA , N = 14 , control/shRNA , N = 20 , stress/scrambled , N = 14 , control/scrambled , N = 17 ) .", "One cage of control/scrambled animals were removed from analysis due to fighting .", "All animal care and procedures were approved by the University of California–Berkeley Animal Care and Use Committee ( Protocol R303-0313BC ) .", "Rats were immobilized in Decapicone bags ( Braintree scientific ) and placed in individual cages in a fume hood for 3 hr/day for 18 days from 9am-12pm .", "Blood samples were collected for corticosterone measurement on days 1 , 4 , 7 , 11 , and 18 at the onset of the stressor and again at the end of the 3 hr .", "All blood samples were collected from tail vein and centrifuged at 2000×g for 15 min .", "Plasma was extracted and stored at −20°C until assayed .", "Corticosterone was measured using a Corticosterone EIA kit ( Enzo Life Sciences , Farmingdale , NY ) with individual samples used for analysis .", "Sample values below the detection level of the assay were included as the lowest detectible value .", "Samples were assayed in duplicate and groups were balanced across different plates .", "Inter-assay coefficients were <3% and intra-assay coefficients were <5% .", "Estradiol assays were run at the University of Virginia Center for Research in Reproduction and were measured by CalBiotech EIA ( Spring Valley , CA ) in singlet with individual samples used for analysis .", "Again , sample values below the detection level of the assay were included as the lowest detectible value .", "Inter-assay coefficients were <2% and intra-assay coefficients were <8% .", "Females verified to be in proestrus were paired with a novel male in a large rectangular cage under red light illumination during the lights off phase .", "The male was permitted to mate with the female for up to two ejaculations after which the male was removed from the cage .", "The interactions were videotaped and both male and female behaviors were blindly scored post-hoc .", "Females that never exhibited lordosis posture during the test were removed from analysis .", "Females that exhibit at least one lordosis posture but that did not allow the male to achieve intromission were termed an ‘unsuccessful maters’ and after 15 min removed from the cage .", "All mating tests were videotaped in real-time for subsequent behavioral scoring .", "Videos were scored by two individuals blind to the experimental conditions .", "Behaviors of male and female animals were scored .", "The lordosis intensity was scored on a 4-point scale ( 0–3 ) as described by Hardy and DeBold ( Hardy and Debold , 1971 ) where 0 indicates no lordosis response and 3 indicates a pronounced spinal flexion , and averaged over the number of lordotic poses presented .", "The lordosis quotient ( LQ ) was determined as the number of lordosis responses ( scores of 2 or 3 ) divided by the total number of mounts during the scored session .", "The number of proceptive behaviors was calculated as number of ear wiggles/minute during duration of test , as well as the number of darts and hops through duration of test .", "Males were scored for total number of mounts and intromissions .", "Post-partum mothers were sacrificed 1-day post-partum .", "The abdominal cavity was opened and both uterine horns gently removed .", "Placental scars were identified as distinctive dark brown spots , counted , and logged ( Conaway and Conaway , 1955 ) .", "The viral vector pLenZs-tetOFF-BFP-shRNAmir-HygR was redesigned based on the backbone of pGIPZ vector originally from Open Biosystems to implement the new features and better single restriction enzyme cutting sites for molecular cloning .", "Briefly , PCR products for tetOFF and its response elements ( TetOff Gene Expression System from Clontech , Mountain View , CA ) , tagBFP ( pTagBFP-H2B vector from Evrogen , Farmingdale , NY ) , and a hygromycin-resistant gene ( pSilencer-hygro vector from Ambion , Grand Island , NY ) were inserted in to the original pGIPZ vector to replace the unwanted components ( e . g . , original fluorescent protein and the puromycin resistant gene ) .", "The constructed vector map is shown in Figure 2D .", "To construct the shRNA against RFRP , a 22 nucleotide-mer oligo against RFRP gene was designed using the online program maintained by Dr Ravi Sachidanandam's Lab ( http://katahdin . mssm . edu/siRNA/RNAi . cgi ? type=shRNA ) .", "The oligo was inserted into the linearized pLenZs-tetOFF-BFP-shRNAmir-HygR vector using KpnI and EcoRI enzymes after adding enzyme arms on both sides of the oligo using PCR .", "Lentiviral particles were prepared by PEG-2000 purification of transfected Hek-293 cells and concentrated to titers of 109–1010 infectious particles per ml .", "The control virus was a non-silence vector commercially available from Open Biosystems ( Lafayette , CO ) , with similar GC content and BLASTed to verify non-specificity .", "RFRP sequence: CACAGCAAAGAAGGTGACGGAA .", "Control sequence: CTCTCGCTTGGGCGAGAGTAAG .", "Stereotaxic microinjections of the RFRP-shRNA and scrambled control viral particles were injected in the hypothalamus as described previously ( Goosens and Maren , 2001 ) .", "Coordinates for viral injection into the dorsal medial hypothalamus were: −3 . 3 mm anterior/posterior , ±0 . 5 mm medial/lateral relative to bregma , −8 . 4 mm relative to dura with skull level between bregma and lambda .", "Virus was infused at a rate of 0 . 2 μl/min for 5 min ( 1 μl total ) .", "At 6–8 hr after surgery , all rats received an injection of meloxicam ( 2 mg/kg , s . c . ) .", "One series of free-floating sections were rinsed in 0 . 1M PBS then incubated in 0 . 3% H202 in PBS for 10 min .", "After rinsing , tissue was blocked with 2% normal donkey serum , 0 . 3% Triton-X 100 in PBS , then transferred into primary antibody against GnIH ( PAC123/124 , Bentley ) 1:5000 in PBS plus 0 . 3% Triton-100 [PBS-T] and sections were incubated in antibody overnight , on rotation , at 4°C .", "The next day , sections were rinsed in PBS and incubated in secondary for 1 hr at room temperature ( Biotin donkey anti-rabbit 1:500 , Jackson ImmunoResearch , West Grove , PA ) .", "Following rinsing , sections were incubated in ABC reagent ( Vector ) and then amplified by incubating in biotinylated tyramide for 30 min .", "Tertiary incubation for 1 hr at room temperature followed with streptavidin-Alexa594 ( 1:1000 in PBS , Jackson Immunoresearch ) .", "Following tertiary incubation , sections were incubated in an antibody against blue-fluorescent protein ( anti-BFP; 1:5000 , Abcam , Cambridge , MA ) on a rotating stage , overnight , at 4°C .", "The next day , sections were rinsed in PBS then incubated in secondary antibody for 2 hr at room temperature ( donkey anti-rabbit cy5 , Jackson Immunoresearch ) .", "After rinsing in PBS-T , slides were coverslipped using DABCO antifading medium and stored in the dark at 4°C .", "Real-time reverse transcriptase PCR was run on TRIzol-extracted RNA further purified with DNase ( DNA-free , Ambion ) .", "Rat primers were designed using NCBI Primer BLAST software , which verifies specificity .", "The Ct values were determined using PCR miner ( Zhao and Fernald , 2005 ) and normalized to the ribosomal reference gene , ribosomal protein L16P ( RPLP ) .", "There were no significant differences in RPLP values across any groups .", "For all studies , two-step PCR was used , following the manufacturer's instructions for iScript cDNA synthesis kit ( BioRad , Hercules , CA ) and then the manufacturer's instructions for SsoAdvanced SYBR supermix ( BioRad ) .", "Samples were run in a BioRad CFX96 real-time PCR system .", "After the PCR was complete , specificity of each primer pair was confirmed using melt curve analysis , and all samples run on a 2% ethidium bromide agarose gel with a 50-bp DNA ladder ( Invitrogen , Carlsbad , CA ) to verify correct product size .", "Primer sequences:PrimerForwardReverseTempProduct SizeRPLPATCTACTCCGCCCTCATCCTGCAGATGAGGCTTCCAATGT55159RFRPCCAAAGGTTTGGGAGAACAAGGGTCATGGCATAGAGCAAT55110GPR147GGTCAGAACGGGAGTGATGTAGGAAGATGAGCACGTAGGC55119LHβGCAAAAGCCAGGTCAGGGATAGAGGCCCACACCACACTTGG5592FSHβTTCAGCTTTCCCCAGGAGAGATAGATCTTATGGTCTCGTACACCAGCT55305TSHβTCGTTCTCTTTTCCGTGCTTCGGTATTTCCACCGTTCTGT55245glycoprotein alpha subunitCTATCAGTGTATGGGCTGTTGCTTGTGGTAGTAACAAGTGC55199KISS1TGGCACCTGTGGTGAACCCTGATCAGGCGACTGCGGGTGGCA61 . 4202GnRHGCAGATCCCTAAGAGGTGAACCGCTGTTGTTCTGTTGACT55201 In the chronic stress and reproductive success experiments , group differences in reproductive success , mating success , and pregnancy success were examined using G-statistics and Fisher's exact tests on raw numbers , not percentages .", "Litter size , placental scar , embryo survival , estradiol measurements , lordosis quotient , and intensity differences were assessed using two-way analysis of variance ( ANOVA ) followed by Bonferroni post-hoc tests .", "Differences in genes examined via RT-PCR were analyzed by a Kruskal–Wallis one-way ANOVA followed by Dunn's multiple comparison test for post-hoc analysis .", "Differences in corticosterone concentrations were subjected to repeated two-way ANOVAs followed by Bonferroni post-hoc test to determine statistical differences .", "*p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 .", "Statistics were performed using R ( for G-statistics and Fisher's exact test ) and Prism software ." ] ]
[ "Whereas it is well established that chronic stress induces female reproductive dysfunction , whether stress negatively impacts fertility and fecundity when applied prior to mating and pregnancy has not been explored .", "In this study , we show that stress that concludes 4 days prior to mating results in persistent and marked reproductive dysfunction , with fewer successful copulation events , fewer pregnancies in those that successfully mated , and increased embryo resorption .", "Chronic stress exposure led to elevated expression of the hypothalamic inhibitory peptide , RFamide-related peptide-3 ( RFRP3 ) , in regularly cycling females .", "Remarkably , genetic silencing of RFRP3 during stress using an inducible-targeted shRNA completely alleviates stress-induced infertility in female rats , resulting in mating and pregnancy success rates indistinguishable from non-stress controls .", "We show that chronic stress has long-term effects on pregnancy success , even post-stressor , that are mediated by RFRP3 .", "This points to RFRP3 as a potential clinically relevant single target for stress-induced infertility ." ]
[ "Infertility has become alarmingly common in otherwise healthy women and around 15% of healthy couples younger than 30 years old are unable to conceive within the first year of trying .", "High-stress levels are known to decrease short-term fertility in humans and other animals , which may serve to prevent pregnancy during times when food or other resources are in short supply .", "However , it is not clear if exposure to stress has lasting effects on fertility .", "Previous studies have found that when male rats experience stress , they release a protein called RFRP3 .", "This protein inhibits brain activity , leading to a reduction in the release of reproductive hormones .", "Geraghty et al . took a closer look at how stress may cause lasting fertility problems in female rats .", "The researchers exposed female rats to stress by restricting their movements for 3 hr each day over the course of 18 days , which increased the levels of stress hormones in the animals .", "They allowed the rats to recover for one full reproductive cycle—equivalent to a month in humans—and found that while their stress hormone levels returned to normal , RFRP3 levels in the brain remained high .", "Even after the recovery period , the females were less likely to mate .", "Also , the females that did mate were less likely to become pregnant , and the ones that did were more likely to lose some of the embryos .", "Overall , the level of reproductive success in these rats was only 21% , down from 76% in the control group ( who were not exposed to the stress ) .", "Next , Geraghty et al . injected a genetically engineered virus into the brain of the stressed rats to switch off the gene that makes RFRP3 during the stress period .", "This reduced the levels of the RFRP3 protein and restored the mating , pregnancy , and embryo survival rates to the normal levels seen in unstressed rats .", "These results suggest that increased levels of RFRP3 during stress can have lasting negative effects on fertility .", "In the future , developing therapies that lower RFRP3 levels may help individuals who experience fertility problems ." ]
2015
[ "Introduction", "Results and discussion", "Materials and methods" ]
[ "evolutionary biology", "developmental biology", "short report" ]
A shift in anterior–posterior positional information underlies the fin-to-limb evolution
elife-07048-v1
[ [ "Regulatory interactions between transcriptional factors play important roles for interpreting a morphogen gradient as positional information ( Balaskas et al . , 2012 ) .", "Changes in these regulatory interactions may therefore be key players for patterning changes during morphological evolution .", "The fin-to-limb transformation is a prominent but still unsolved example of morphological evolution .", "150 years ago Carl Gegenbaur subdivided the skeletal elements of shark pectoral fins into three segments along the anterior–posterior ( AP ) axis: propterygium , mesopterygium , and metapterygium ( Gegenbaur , 1865 ) ( Figure 1A ) , which are also found in the majority of chondrichthyans , none-teleost actinopterygians , placoderms , and acanthodians ( Orvig , 1962; Coates , 1994 , 2003 ) .", "Therefore , possession of propterygium , mesopterygium , and metapterygium is considered to be a plesiomorphic state for gnathostomes .", "In the sarcopterygians ( lobe-finned fishes including tetrapods ) , the propterygium and mesopterygium have been lost ( Coates , 2003 ) , thus , suggesting that anterior positional values have been lost or reduced during tetrapod evolution . 10 . 7554/eLife . 07048 . 003Figure 1 . Anterior–posterior patterning in Scyliorhinus canicula pectoral fin buds .", "( A ) Skeletal patterns of S . canicula pectoral fin and mouse limb .", "Blue colours , homologous elements .", "( B–G )", "In situ hybridisation for Alx4 ( B ) , Pax9 ( C ) , Hand1 ( D ) , Zic3 ( E ) , Hand2 ( F ) , and Tbx2 ( G ) in S . canicula pectoral fin buds at stage 30 and mouse limb bud at E11 . 5 ( left panel in G ) .", "Arrowheads in G , anterior boundary of posterior Tbx2 expression .", "Dorsal view; anterior is to the top .", "Scale bars , 100 μm .", "( H ) Schematic of the gene expression patterns .", "Arrowheads , Hand2 expression boundary .", "Expressions of mouse limb buds at E11 . 5 are after EMBRYS database ( Yokoyama et al . , 2009; Fernandez-Teran et al . , 2003 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 00310 . 7554/eLife . 07048 . 004Figure 1—figure supplement 1 . Molecular phylogenetic trees of relevant S . canicula genes .", "( A–E ) , Trees for Alx4 ( A ) , Pax9 ( B ) , Hand1 ( C ) , Zic3 ( D ) , Tbx2 ( E ) , and Ptch1 ( F ) were generated from amino acid sequences of the homeodomain and neighbouring sequences ( A ) , the paired box and C-terminal sequences ( B ) , Helix-loop-helix domain and C-terminal sequences ( C ) , N-terminal sequences ( D ) , and C-terminal sequences ( E and F ) .", "The neighbour-joining method was used for constructing the trees .", "The numbers at nodes indicate bootstrap probabilities with 1000 replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 00410 . 7554/eLife . 07048 . 005Figure 1—figure supplement 2 . Temporal expression analysis of Alx4 , Pax9 , Hand2 , and Hoxa13 in S . canicula pectoral fins .", "( A–D )", "Expression of Alx4 ( A ) , Pax9 ( B ) , Hand2 ( C ) , and Hoxa13 ( D ) at the indicated stages .", "Dorsal view of right pectoral fin buds of S . canicula embryos ( anterior to the top ) .", "( A ) Alx4 was initially expressed throughout the fin buds at stage 25 , but the posterior expression of Alx4 was reduced by stage 27 .", "Alx4 expression was then restricted to the anterior two-thirds of the fin bud at stage 29 and to the anterior half of the fin bud at stage 31 .", "( B ) Pax9 expression was not detected at stage 27 but was present in anterior distal fin buds at stage 29 .", "This expression persisted until at least stage 31 .", "( C ) Hand2 was initially expressed throughout the fin bud , although slightly more-robust expression was observed at the posterior side .", "Subsequently , Hand2 expression became posteriorly restricted by stage 27 .", "( D ) Hoxa13 , which is a marker for a late developmental stage in tetrapod limb buds , was detected since stage 29 in a distal domain .", "( E ) OPT scans of Sox9 expressions ( red ) stained with propidium iodide ( green ) at stage 28 ( left ) and late stage 29 ( right ) .", "pg , pectoral girdle; ms , mesopterygium; mt , metapterygium .", "( F ) Comparison of Alx4 ( black ) , Pax9 ( grey ) , Hand2 ( blue ) , and Hoxa13 ( light green ) expression patterns between S . canicula fin buds and chick forelimb and hindlimb buds ( see Figure 1—figure supplement 3 for detailed gene expression in chick limb buds ) .", "st , stage .", "Scale bars , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 00510 . 7554/eLife . 07048 . 006Figure 1—figure supplement 3 . Temporal expression analysis of Alx4 , Pax9 , Hand2 , and Hoxa13 in chick limb buds .", "( A–H )", "Expression of Alx4 ( A , B ) , Pax9 ( C , D ) , Hand2 ( E , F ) , and Hoxa13 ( G , H ) at the indicated stages .", "Dorsal views of chick forelimb ( A , C , E , G ) and hindlimb ( B , D , F , H ) buds ( anterior to the top; distal to the right ) .", "( A , B )", "At stage 19 , Alx4 expression was broad but was slightly weaker in the posterior side of forelimb ( A ) and hindlimb ( B ) buds .", "At later stages , its expression became restricted to the anterior one-third of the limb buds , with subsequent further restriction to the anterior proximal region both in forelimb ( A ) and hindlimb ( B ) buds .", "Alx4 expression also appeared in the posterior distinct region of chick forelimb ( A ) and hindlimb ( B ) buds at stage 25 .", "( C , D )", "Pax9 expression was first present in the anterior region at stage 24 in forelimb buds ( C ) and stage 23 in hindlimb buds ( D ) but was more distally restricted than Alx4 expression .", "At stage 25 , expression of Pax9 was robust , but the extent of its overlap with Alx4 expression was limited both in forelimb ( C ) and hindlimb ( D ) buds .", "( E , F )", "Hand2 expression was initially complementary with Alx4 and subsequently with Pax9 expression in forelimb ( E ) and hindlimb ( F ) buds .", "( G , H )", "At stage 21 , Hoxa13 expression fully overlapped with the distal Hand2 expression domain in forelimb ( G ) and hindlimb ( H ) buds .", "At later stages , co-expression of Pax9 and Hoxa13 was observed in the anterior distal portion of the limb buds , but this overlap was not as extensive as that between the Hand2 and Hoxa13 expression domains .", "st , stage .", "Scale bars , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 006 In mouse limb buds , Hand2 , Gli3 , and Shh are key genes for controlling AP patterning ( Riddle et al . , 1993; Te Welscher et al . , 2002 ) .", "One of the earliest patterning events is the mutual transcriptional repression between Gli3 in the anterior tissue and Hand2 in the posterior ( Te Welscher et al . , 2002; Osterwalder et al . , 2014 ) .", "This early polarity in expression contributes to the subsequent posterior localized expression of Shh , and this morphogen in turn reinforces the anteriorly restricted Gli3 protein activity ( Shh inhibits the default processing of the Gli3 protein to its repressor form ( Gli3R ) , thus , creating a gradient of Gli3R along the AP axis ) ( Wang et al . , 2000 ) .", "Several studies on fin development of actinopterygians and chondrichthyans have revealed that posterior Shh expression is conserved among gnathostomes ( Dahn et al . , 2007; Davis et al . , 2007; Yonei-Tamura et al . , 2008; Sakamoto et al . , 2009a ) .", "However , in fish fin development , the detailed roles of Shh signalling for AP patterning are not well studied and the role of the Hand2-Gli3 mutual interaction remains to be elucidated ." ], [ "To investigate changes in AP patterning during the fin-to-limb transition , we first cloned a number of AP patterning genes from the non-model species Scyliorhinus canicula ( Figure 1B–H and Figure 1—figure supplement 1 for phylogentic analyses ) .", "In the mouse limb bud , Alx4 , Pax9 , Hand1 , and Zic3 are positively regulated by Gli3R ( Te Welscher et al . , 2002; Fernandez-Teran et al . , 2003; McGlinn et al . , 2005; Vokes et al . , 2008 ) and thus are expressed in a localized anterior domain ( one-third of the axis ) , while Hand2 and Tbx2 show broad posterior expression domains ( two-thirds and one-half of the axis , respectively ) .", "In stage 30 S . canicula embryos ( staged according to Ballard et al . , 1993 ) , we found instead that the anterior genes Alx4 , Pax9 , Hand1 , and Zic3 were expressed in broad domains , which extend more posteriorly than in the mouse ( half the fin bud for Alx4 , two-thirds for Pax9 and Hand1 , and the whole axis for Zic3 , Figure 1B–E ) .", "By contrast , the Hand2 and Tbx2 domains were more posteriorly restricted in S . canicula fin buds than in mouse limb buds ( Figure 1F , G ) .", "All of these 6 AP patterning genes show the same trend—their expression boundaries are more posterior in S . canicula fin buds ( Figure 1H ) , apparently reflecting a gross shift in the AP coordinate system .", "We chose 3 of these genes to test at multiple time-points to determine whether this was a transient gene expression state ( Figure 1—figure supplements 2 , 3 ) , but in all cases these shifts were observed from stage 29 to stage 31 ( which covers ∼30 days of S . canicula development ) .", "In particular , stage 29 is a stage where Sox9 expression ( a prechondrogenic marker ) starts in the proximal part of the pectoral fin buds ( Figure 1—figure supplement 2E ) , which suggest that the observed shift of AP values would affect proximal skeletal elements as well as distal .", "Since the above AP patterning genes are regulated by Shh–Gli3 pathway ( Te Welscher et al . , 2002; Fernandez-Teran et al . , 2003; McGlinn et al . , 2005; Galli et al . , 2010 ) , we cloned Gli3 from S . canicula fin buds ( Figure 2A and Figure 2—figure supplement 1A for phylogenetic tree ) and analysed its expression in pectoral fin buds .", "In striking contrast to tetrapod limb buds ( Büscher et al . , 1997; Schweitzer et al . , 2000 ) , Gli3 expression is not restricted to the anterior region—thus again indicating a general posterior shift of AP positional values in the S . canicula .", "To address whether this situation is conserved in other chondrichthyans , we also cloned and analysed the expression of Gli3 in pectoral fin buds of a holocephalian , Callorhinchus milii , which has propterygium ( Figure 2B ) , and again found expression in the posterior part of pectoral fin bud at stage 31 ( staged according to Didier et al . , 1998; Figure 2B ) .", "Since Hand2 is expressed posteriorly and thus now overlaps with Gli3 , this strongly suggests that the Hand2–Gli3 mutual inhibition seen in tetrapods is weak or non-existent in chondrichthyans . 10 . 7554/eLife . 07048 . 007Figure 2 . Expression and processing of Gli3 and Gli2 in S . canicula embryos .", "( A ) Expression of Gli3 in S . canicula pectoral fins .", "( B ) Alcian blue staining of C . milii pectoral fin at stage 35 ( top , the ventral view of a right fin flipped horizontally ) and Gli3 expression at stage 31 ( bottom , a left pectoral fin flipped horizontally ) .", "pro , propterygium .", "( C ) Expression of Gli2 in S . canicula pectoral fin buds .", "Scale bars , 100 μm .", "( D ) The Gli3 chimera constructs .", "hGli3 PDD , full-length human Gli3 ( grey box ) with Myc tags .", "hGli2 , ScGli2 and ScGli3 PDD , chimeric Gli3 genes recombined at the processing determinant domain ( PDD ) with human Gli2 , S . canicula Gli2 and Gli3 , respectively .", "( E ) Protein processing of the chimeric constructs in cell cultures treated with either FSK ( + ) or DMSO ( − ) .", "Truncated Gli3 is detected only in hGli3 PDD ( lane 4 ) and ScGli3 PDD ( lane 8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 00710 . 7554/eLife . 07048 . 008Figure 2—figure supplement 1 . Phylogenetic tree of Gli2 and Gli3 , and PDD amino acid sequences .", "( A ) Trees for Gli2 and Gli3 were generated from amino acid sequences of the zinc finger domain and PDD .", "The neighbour-joining method was used for constructing the trees .", "The numbers at nodes indicate bootstrap probabilities with 1000 replicates .", "( B ) Human Gli3 ( the upper diagram ) is composed of an N-terminal repressor domain ( Repressor ) , a DNA-binding domain ( Zinc finger ) , and a C-terminal activator domain ( Activator ) .", "The alignment shows the homologous PDD domains from both human and S . canicula Gli2 and Gli3 . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 008 In chick and mouse limb buds , Gli2 does not play a major role in AP patterning because of its weak processing efficiency to produce its repressor form ( Wang et al . , 2000 ) .", "However , in zebrafish , Gli2 does indeed act as a repressor ( Maurya et al . , 2013 ) , so we checked whether Gli2 could be playing the repressor role in S . canicula fin buds .", "First , we analysed Gli2 expression in S . canicula embryos and found it to be uniform until stage 29 ( Figure 2C ) and then subsequently restricted to the posterior region ( Figure 2C ) .", "Second , we checked whether Gli3 and Gli2 of S . canicula have the repressor function , by measuring their processing efficiencies .", "We analysed the processing determinant domain ( PDD ) , which determines the differential processing efficiencies of Gli3 and Gli2 in mice and humans ( Pan and Wang , 2007 ) .", "We inserted the PDDs from human Gli2 and S . canicula Gli2 or Gli3 into the human Gli3 PDD region ( Figure 2D and Figure 2—figure supplement 1 for the amino acid sequences ) , transfected these constructs into HEK293 cells , and treated the cells with forskolin ( FSK ) to induce Gli processing .", "Human and S . canicula Gli2 PDD did not induce Gli3R , whereas their Gli3 PDDs did ( Figure 2E ) .", "Thus , in S . canicula ( as in chick and mouse ) , Gli3 , but not Gli2 , plays the major role in repressor production .", "We next wished to explore if a genetic explanation could be found for the lack of Gli3 repression in the posterior part of pectoral fin buds of S . canicula and C . milii .", "To compare Gli3 enhancers in chondrichthyans and tetrapods , we used the VISTA enhancer browser ( Visel et al . , 2007 ) and found a limb-specific Gli3 enhancer , element 1586 , which replicates anterior Gli3 expression in mouse limb buds .", "We identified the homologues of element 1586 in S . canicula and C . milii and compared them with those from other vertebrates .", "Consistent with the slow evolutionary rate of chondrichthyans and coelacanth ( Amemiya et al . , 2013; Renz et al . , 2013; Venkatesh et al . , 2014 ) , element 1586 is conserved in tetrapods , coelacanth , and chondrichthyans , but not in gar , medaka , and zebrafish ( Figure 3A ) .", "To assess whether the element 1586 in different species has different functionalities , we cloned this element from chick , S . canicula , and C . milii in front of a basal promoter followed by a GFP reporter ( Ochi et al . , 2012; Figure 3B ) .", "These constructs were electroporated into chick forelimb buds with a constitutively active RFP vector ( to determine the spatial efficiency of electroporation ) .", "As with endogenous Gli3 expression ( Büscher et al . , 1997 ) , the chick element 1586 drove GFP expression specifically in anterior tissue and was repressed in the posterior region , even though RFP was expressed throughout the buds ( Figure 3C ) .", "The element 1586 from both S . canicula and C . milii also drove GFP expression in the chick limb buds , confirming that its general activity is conserved from sharks to tetrapods .", "However , in both cases , the specific posterior repression observed in the chick element was absent ( Figure 3D , E ) .", "Thus , the differential activity of this enhancer ( with tetrapods showing posterior repression , and chondrichthyans not ) recapitulates the differences in Gli3 expression within these groups .", "Furthermore , by recombining S . canicula and chick enhancers , we identified a sequence that can exert the posterior repression when inserted into the S . canicula enhancer ( Figure 3F , G and Figure 3—figure supplement 1 ) .", "This sequence contains tetrapod or sarcopterygian-specific sequences , suggesting that the posterior repressive activity would have been acquired in a stepwise fashion . 10 . 7554/eLife . 07048 . 009Figure 3 . The Gli3 limb-specific enhancer of S . canicula and C . milii .", "( A ) VISTA plots of Gli3 intron 3 from indicated animals .", "Blue vertical bars , exons of human Gli3; black rectangle , element 1586 .", "Regions with >70% identity are indicated: blue , exon; pink , non-coding sequences .", "( B ) The enhancer construct .", "( C ) GFP expression in chick forelimb buds driven by chick element 1586 at stage 19 ( top , n = 3/3 ) , stage 23 ( middle , n = 14/14 ) , and empty vector ( bottom , n = 0/7 ) .", "pCAGGS-RFP ( right ) .", "( D–F )", "GFP expression driven by element 1586 of S . canicula ( D , n = 11/11 ) , C . milii ( E , n = 10/10 ) and Sc1586mt ( F , n = 4/4 ) .", "Scale bars , 100 μm .", "( G ) Scheme of Sc1586mt , S . canicula enhancer ( blue ) partially replaced by chick sequence ( green ) and alignment .", "Boxes indicate tetrapod", "( i ) and sarcopterygian", "( ii ) specific sequences . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 00910 . 7554/eLife . 07048 . 010Figure 3—figure supplement 1 . Detailed functional analyses of element 1586 . ( A ) Alignment of element 1586 sequences .", "Points where chick and S . cacnicula enhancers are recombined in ( B ) are indicated by arrows a and b .", "Box i and Box ii are sequences that are replaced with S . canicula sequence in ( C ) , activities of element 1586 recombined at the indicated points .", "Note that the posterior repressive activity is not altered at arrow a , but partially destroyed at arrow b , suggesting that the repressive sequences are located between arrow a and b and after arrow b .", "( C ) Activities of chick element 1586 that are partially recombined with S . canicula sequences at Box i and Box ii in ( A ) .", "The sequences were chosen because of specific conservation among tetrapods ( Box", "i ) and sarcopterygians ( tetrapod and coelacanth , Box", "ii ) .", "Each of replacement can slightly alter the posterior repression of GFP ( n = 3/3 ) .", "When the chick enhancer is replaced by both sequence ( the most right panel ) , the distal GFP expression is significantly shifted to the posterior limb buds ( n = 4/4 , white arrow ) .", "And weak expressions are also detected in the posterior proximal limb buds ( n = 2/4 . arrow head in the bottom panel , magnified view of the above white box with enhanced contrast ) , suggesting that the sequences at Box i and Box ii are partially responsible for the posterior repression .", "Scale bars , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 010 Finally , we wished to address whether changes to AP positional information could modify skeletal arrangement of the propterygium and mesopterygium in catshark .", "For this purpose , we explored methodologies for performing manipulative experiments on this very slow-developing non-model fish ( see ‘Materials and methods’ ) .", "We treated S . canicula embryos with retinoic acid ( RA ) to increase Shh-signalling activity ( at stage 29 with 1 μg/ml of RA for 4 days ) .", "Activation of Shh signalling by RA is known to be conserved among vertebrate limbs/fins ( Riddle et al . , 1993; Hoffman et al . , 2002; Dahn et al . , 2007 ) , and as expected , the most reliable Shh target gene , Ptch1 expression ( Marigo et al . , 1996; Vokes et al . , 2008; and see Figure 1—figure supplement 1F for phylogenetic analysis ) was increased and expanded anteriorly ( Figure 4A ) .", "Consistent with this , Hand2 expression also extended anteriorly ( Figure 4B ) —probably due to inhibition of Gli3 repressor formation by ectopic activation of Shh signalling ( as revealed by the extended Ptch1 expression ) .", "On the other hand , Pax9 expression ( an anterior marker ) was significantly downregulated and showed only weak expression in the anterior part of the fin buds ( Figure 4C ) .", "The most anterior regions may not be sensitive to this treatment , as expression of Alx4 was not significantly shifted ( Figure 4D ) , and this may be due to the lack of inhibitory regulation from Hand2 to Gli3 described above .", "To test whether the results of RA treatment were due to specific effects on AP patterning or instead due to a more general interference with limb development , we examined a marker for proximal-distal ( PD ) patterning in mouse and chick limb buds—Hoxa13 ( Tamura et al . , 1997; Mercader et al . , 2000; Yashiro et al . , 2004 ) .", "In RA-treated pectoral fin buds , Hoxa13 expression was weaker than in control , but a shift in its expression domain was not seen ( Figure 4E ) , showing that the impact of RA in these experiments is primarily on the AP patterning ( the shifts of Ptch1 , Hand2 , and Pax9 , Figure 4A–C ) , rather than on PD patterning or a general impact on development .", "Most intriguingly , we examined skeletal patterns of S . canicula pectoral fins in these partially ‘posteriorised’ fin buds ( Figure 4F ) .", "Phenotypes varied from mild to severe but in all cases the appearance of distinct anterior elements ( propterygium and mesopterygium ) was lost .", "In the mild cases , a proximal element anterior to the metapterygium is attached to the pectoral girdle ( single asterisk in Figure 4F ) .", "This proximal element may result from a fusion of the proximal parts of propterygium and mesopterygium .", "Whereas , the severe cases still have a fused element anterior to the metapterygial axis ( double asterisk in Figure 4F ) , but this element is not directly attached to the pectoral girdle , indicating that the pectoral fin of the severe phenotype has lost the anterior proximal elements .", "By contrast , the posterior metapterygium itself was larger than normal , but retained its strong identity as the primary axis from which radial branching was observed .", "Although RA treatment potentially cause non-specific effects , given the clear affect on AP patterning ( the shifts of Ptch1 , Hand2 , and Pax9 , Figure 4A–C ) , while causing no obvious effects on PD patterning , the main cause of the phenotype is likely caused by the AP pattern change . 10 . 7554/eLife . 07048 . 011Figure 4 . RA treatment causes ectopic activation of Shh signalling and loss of anterior skeletal elements .", "( A–E )", "In situ hybridisation of S . canicula pectoral fin buds for Ptch1 ( A; left fins flipped horizontally ) , Hand2 ( B ) , Pax9 ( C ) , Alx4 ( D ) , and Hoxa13 ( E ) treated with 1% DMSO or 1 μg/ml retinoic acid ( RA ) ( n = 2/2 for each for each except n = 4/4 for Hand2 ) .", "Arrowheads in C , a weak expression of Pax9 .", "White brackets in E , width of Hoxa13 expression domain along the proximal-distal ( PD ) axis .", "( F ) Pectoral fin skeletal patterns of 1% DMSO control ( n = 4 ) and 1–2 μg/ml RA ( n = 4 ) .", "Right panels , schematics of interpretive skeletal patterns .", "* , an anterior proximal radial; ** , a fused radial attached to the metapterygium; pg , pectoral girdle; ppr , pms and pmt , proximal propterygium , mesopterygium and metapterygium .", "Scale bars , 100 μm .", "( G ) Comparison between S . canicula fin and mouse limb .", "Green and blue colours represent anterior–posterior ( AP ) positional information .", "ppr , pms , and pmt denote proximal propterygium , mesopterygium , and metapterygium , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 07048 . 011 In the present study , we have found that S . canicula pectoral fin buds have a gross posterior shift in the AP coordinate system compared to mouse limb buds .", "We show that S . canicula and C . milii lack a specific enhancer activity for Gli3 , which in tetrapods mediates the posterior repression , and that this genetic difference likely contributes to the shift of AP positional information .", "Finally , RA treatment analyses suggest that a partial posteriorisation of S . canicula fin buds leads to a loss of anterior proximal elements ( propterygium and mesopterygium ) .", "Thus , while the loss of the anterior proximal elements during evolution was associated with cis-regulatory changes of Gli3 in the RA experiments , it was driven by a Shh-mediated affect on the Gli3 protein itself , but in both cases achieving similar phenotypic changes by anterior shift in AP pattern .", "In support of our observations , a recent study also showed that anterior extension of Shh signalling accompanied with an anterior shift of Gli3 expression resulted in a loss of anterior skeletal elements in mouse limbs ( Li et al . , 2014 ) .", "Considering all these data together , we therefore propose that one of the key events during the fin-to-limb transition was an anterior shift of AP positional information ( a posteriorisation ) , which caused the loss of anterior proximal elements ( Figure 4G ) .", "In the RA treatment experiments , we also observed that the anterior distal radials reduced and only metapterygial radials retained , suggesting that anterior shift in AP positional information may also have had an impact on the distal radials during the fin-to-limb transformation .", "Interestingly , nearly 30 years ago , a classic study proposed that the distal end of the metapterygial axis ( which has a uniformly posterior position in chondrychthians ) bent anteriorly during acquisition of digits—the so-called digital arch model ( Shubin and Alberch , 1986; Oster et al . , 1988 ) .", "Although the detailed validity of this model is unclear ( Wagner and Larsson , 2007 ) , there is a possibility that an AP shift in molecular patterning was involved in the acquisition of digits .", "In addition to our RA treatment analysis , knockdown analyses of actinotrichia proteins , which are components of fin rays and lost in tetrapod , show an anterior shift in several gene expressions in zebrafish pectoral fin buds ( Zhang et al . , 2010 ) .", "Therefore , it is interesting to speculate that AP positional information may have shifted several times until the acquisition of digits .", "We have shown that the Gli3 regulatory region of S . canicula and C . milii lacks the tetrapod-specific repressive element , which is likely needed for the Gli3–Hand2 interaction in mouse limb buds .", "In mice , Gli3−/−; Hand2−/c limbs show a severe dysplastic humerus ( some of them have ectopic protrusion in humerus; Osterwalder et al . , 2014 ) , suggesting that the Gli3–Hand2 interaction has an important role for patterning the proximal elements .", "However , how Gli3 regulates the proximal skeletal pattern is not well understood even in mice .", "Although Gli3 is involved in the stylopod ( humerus/femur ) formation in mice , the phenotype in stylopod always appears with combination of other gene knockouts .", "For example , Gli3−/−; Plzf−/− mice lack a femur , and Gli3−/−; Alx4−/− mice exhibit humerus malformation ( Barna et al . , 2005; Panman et al . , 2005 ) .", "These facts suggest that evolutionary modification of Gli3 regulation is likely necessary , but additional regulatory modifications are required for the loss of the anterior elements .", "Since Alx4 and Hand2 are expressed in S . canicula pectoral fin bud , and Plzf is involved only in hindlimb development , currently there is no obvious candidate that would be involved in the loss of propterygium and mesopterygium .", "Although S . cacnicula genome has not been sequenced , systematic studies at genome-wide level such as ChIP-seq in S . cacnicula pectoral fin buds would be invaluable to provide a more complete picture of evolutionary mechanism of the loss of the anterior elements in the future .", "In conclusion , by taking advantage of the slow evolutionary rates of chondrichthyian genomes , we were able to precisely compare the gene expression , function and regulation between pectoral fin and limb development , and discover a key difference between them .", "In particular , our study suggest that changes in morphogen interpretation by gene regulatory network mutations may have a major impact on morphological evolution ." ], [ "Experiments were performed in accordance with guidelines for animal experiments of Tokyo Tech and CRG , and experiments involved in mice were approved by animal ethics committees of CRG ( JMC-07-1001P3-JS ) .", "Catshark ( S . canicula ) eggs were incubated at 12–16°C in seawater and staged according to ( Ballard et al . , 1993 ) .", "C . milii eggs and embryos were collected as described ( Takagi et al . , 2012 ) and staged according to ( Didier et al . , 1998 ) .", "C52BL/6 ( Charles River Laboratories , Wilmington , MA ) mouse timed-pregnant females were sacrificed at different days after gestation E11 . 5 .", "Chicken ( Gallus gallus ) eggs were incubated at 38°C in a humidified incubator until the desired Hamburger–Hamilton ( HH ) stage ( Hamburger and Hamilton , 1951 ) was reached .", "For in situ hybridisation , embryos were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline , dehydrated in a graded methanol series , and stored in 100% methanol at −20°C .", "Total RNA was extracted from stage 24 to 29 S . canicula embryos , stage 28 chick embryos and E11 . 5 mouse embryos using an RNeasy kit ( Qiagen , Netherlands ) .", "cDNA was synthesised by reverse transcription and used as a template for PCR .", "Extraction of total RNA and cDNA synthesis from C . milii embryos were carried out as described ( Takagi et al . , 2012 ) .", "To clone S . canicula and C . milii genes , we used primers that were based on the nucleotide sequences of putative C . milii orthologues found in the Elephant Shark Genome Project database ( http://esharkgenome . imcb . a-star . edu . sg/ ) ( Venkatesh et al . , 2007 ) for Pax9 , Alx4 , and Gli3; SkateBase ( http://skatebase . org/ ) ( Wang et al . , 2012 ) for Hand1 , Zic3 , Tbx2 , and Ptch1; and GenBank for Gli2 ( EU196410 ) and Sox9 ( EU241880 ) : S . canicula Alx4 , 5′-AGGAATGAACGGCGAGACTTG-3′ and 5′-TCATGTTGCCCAAGATATAGC-3′; S . canicula Pax9 , 5′-GCTGTGTCAGCAAGATACTGG-3′ and 5′-CCGCACTGTATGTCATGTAGG-3′; S . canicula Gli3 , 5′-CAGCCCAGCAGAATACTACC-3′ and 5′-GAGATCTCAGCGCCATTGATG-3′; S . canicula Gli2 , 5′-GTAAAGCTTACTCACGACTCG-3′ and 5′-CGTAAGAGTCAGCCGAGCTGATG-3′; S . canicula Sox9 , 5′-CCCAGGTGCTGAAGGGATAC-3′ and 5′-GGCAGGTACTGGTCGAACTC-3′; S . canicula Hand1 , 5′-GAGAGCATCAACAGCGCATTCGC-3′ and 5′-TTCCTGGTCCTCAACCTGGTCAG-3′; S . canicula Zic3 , 5′-GTGGCCATGGCGATGTTACTGGATGGTG-3′ and 5′-GTTTCTCGCCGGTGTGCACTCGGATGTG-3′; S . canicula Tbx2 , 5′-GACACAGAAACCAGCTTCAGTCACAGTC-3′ and 5′-GAAAGTCGCGATACCCAATGTGGATCAG-3′; S . canicula Ptch1 , 5′-GAGGTTTCACCTCTCGATGGGAGAACC-3′ and 5′-CCATACTAATGTGTTCTGTTCCCACTG-3′; C . milii Gli3 , 5′-GAGATCTCAGCGCCATTGATG-3′ and 5′-GAGATCTCAGCGCCATTGATG-3′ .", "To clone chick and mouse genes , we used primers that were based on the nucleotide sequences of Pax9 ( NM_204912 ) ( Muller et al . , 1996 ) , Hoxa13 ( NM_204139 ) , and Tbx2 ( NM_009324 ) ( Bollag et al . , 1994a ) : chick Pax9 , 5′-TGAGCGACACCTCGTCGTACC-3′ and 5′-GGTTATGCGATCCACTGCTA-3′; chick Hoxa13 , 5′-GTCATGTTCCTCTACGACAAC-3′ and 5′-GGTGGACTTCCAGAGGTGAGG-3′; mouse Tbx2 , 5′-ATCCTGAACTCCATGCACAAGTACC-3′ and 5′-GAACTGCTGCCCATGCAGGTGGCTG-3′ .", "The gene fragments were cloned into pBluescript SK− or pCR4 ( Invitrogen , Thermo Fisher Scientific Inc . , Waltham , MA ) .", "The partial coding sequences for Alx4 ( 1112 bp ) , Pax9 ( 729 bp ) , Gli3 ( 1937 bp ) , Hand1 ( 465 bp ) , Tbx2 ( 926 bp ) , Zic3 ( 919 bp ) and Ptch1 ( 791 bp ) of S . canicula and Gli3 ( 428 bp ) of C . milii have been submitted to GenBank under accession numbers KC507187–9 , KF748129 , and KP055651-KP055653 , KF297620 , respectively .", "Phylogenetic analysis was used to confirm the orthology of newly identified S . canicula and C . milii genes .", "Amino acid sequences were aligned using ClustalX ( Thompson et al . , 1997 ) .", "Regions that could not be aligned were excluded from the analysis .", "Neighbour-joining phylogenetic trees of amino acid sequence data sets were constructed with MEGA5 ( Tamura et al . , 2011 ) .", "Bootstrapping was carried out with 1000 replicates .", "Chick Alx4 ( NM_204162 ) was kindly provided by Dr Toshihiko Ogura .", "Riboprobes for Hand2 ( AY057890 ) and Hoxa13 ( EU005550 ) of S . canicula and for chick Alx4 were synthesised as described ( Takahashi et al . , 1998; Tanaka et al . , 2002a; Sakamoto et al . , 2009a ) .", "The cloned genes described above were used as templates for riboprobe synthesis .", "Whole-mount in situ hybridisation was carried out as described ( Tanaka et al . , 2002a ) .", "Sox9 expressions were scanned with Optical Projection Tomography ( OPT ) as described ( Sharpe et al . , 2002 ) and analysed with Volviewer ( Lee et al . , 2006 ) .", "Human Gli3 ( clone name: pFN21AE1055 ) and Gli2 ( Roessler et al . , 2005 ) were obtained from the Kazusa DNA Research Institute ( Nagase et al . , 2008 ) and Addgene , respectively .", "pCAGGS was kindly provided by Dr Toshihiko Ogura and originated from Dr Jun-ichi Miyazaki ( Niwa et al . , 1991 ) .", "For Western blotting analysis , the N-terminal HaloTag in the human Gli3 construct was replaced with a 6×Myc tag ( Myc-hsGli3 ) .", "Then , the human Gli3 PDD ( amino acids 644–842 ) ( Pan and Wang , 2007 ) was replaced with the homologous domain from human Gli2 and S . canicula Gli2 and Gli3 by a combination of PCR ( Wurch et al . , 1998 ) and restriction enzyme digestions .", "The HEK293 cell line was kindly provided by Dr Masayuki Komada .", "HEK293 cells were grown in Dulbecco's modified Eagle medium ( Sigma–Aldrich , St . Louis , MO ) supplemented with 10% foetal bovine serum ( Gibco , Thermo Fisher Scientific Inc . , Waltham , MA ) and penicillin/streptomycin ( Sigma–Aldrich ) at 37°C .", "For Western blot analysis , cells were plated in 6-well plates without penicillin/streptomycin and transfected with 4 μg of constructs using polyethylenimine ( GE Healthcare , England ) for 3 hr .", "After the transfection , the medium was changed , and cells were cultured for 24 hr and then treated with 50 μM forskolin ( FSK; Sigma ) in Dimethyl sulfoxide ( DMSO ) DMSO or with DMSO alone for 24 hr .", "Whole-cell extracts were prepared by solubilisation in lysis buffer containing 50 mM Tris-HCl , pH 7 . 5; 150 mM NaCl; 1 mM ethylenediaminetetraacetic acid ( EDTA ) ; 1% Triton X-100; 0 . 1% Sodium Dodecyl Sulfate ( SDS ) S; 1% sodium deoxycholate; and protease inhibitor cocktail ( Roche , Switzerland ) .", "Whole-cell lysates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and analysed by Western blotting and anti-c-Myc ( Sigma–Aldrich ) , anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase ( Jackson ImmunoResearch , West Grove , PA ) , and enhanced chemiluminescence detection ( GE Healthcare ) .", "The limb-specific Gli3 enhancer was found with VISTA enhancer browser ( http://enhancer . lbl . gov/ ) ( Visel et al . , 2007 ) .", "The enhancer ID is hs1586 , which is located in Gli3 intron 3 in the human genome ( hg19 ) .", "For alignment , Gli3 intron 3 sequences from mouse ( Mus musculus ) , chick ( G . gallus ) , frog ( Xenopus tropicalis ) , coelacanth ( Latimeria chalumnae ) , gar ( Lepisosteus oculatus ) , medaka ( Oryzias latipes ) , and zebrafish ( Danio rerio ) from the Ensembl and Pre Ensembl genome browsers ( http://www . ensembl . org/ , http://pre . ensembl . org/ ) were collected .", "The element 1586 homologue from elephant shark ( C . milii ) was retrieved from the genome assembly ( http://esharkgenome . imcb . a-star . edu . sg/ ) ( Venkatesh et al . , 2007 ) by using human element 1586 sequence as the query .", "The GenBank accession number of the C . milii element 1586 is AAVX01295166 .", "The S . canicula counterpart of element 1586 was amplified by PCR with primers designed from conserved sequences of the upstream exon and the distal part of element 1586: 5′-AGTGGACCCCCGAAATGGCTACATGGACC-3′ and 5′-GAACATCTTCTAATTTACTGGAATCCCAG-3 .", "The amplified fragment was then cloned into pBluescript SK− .", "The sequence of S . canicula element 1586 was deposited in GenBank under accession number KF297619 .", "The alignment was carried out with the SLAGAN method , and overall sequence similarities in the alignment were visualised with mVISTA ( Mayor et al . , 2000; Brudno et al . , 2003; Frazer et al . , 2004 ) .", "For functional analysis , the element 1586 homologues were isolated from chick and C . milii genomes by PCR .", "The following forward and reverse primers were used: chick element 1586 , 5′-CGAGCTCCCTCCTCAGTCATTCAGTTCTGC-3′ and 5′-TGTGTGAGACATACTTTGATC-3′; C . milii element 1586 , 5′-GAGCTCGTACAGTGATGACTGAAATGGTG-3′ and 5′-GAGATTTCGAGTCTCTTTGATC-3′ .", "The amplified DNA fragments were cloned into pBluescript SK− .", "To subclone the S . canicula 1586 fragment , we used the following primers: 5′-CCGCTCTAGAACTAGCATCAATATGATTTGCTGAG-3′ and 5′-CGGGGGATCCACTAG GCTTCACGAGCATCAGGAAC-3′ .", "The element 1586 sequence from each species was subcloned in front of a chicken β-actin basal promoter that is followed by a GFP reporter ( Ochi et al . , 2012 ) .", "Recombined enhancers were created by PCR .", "In ovo , electroporation was carried out as described ( Suzuki and Ogura , 2008 ) .", "A DNA solution was prepared with Maxi Prep ( Qiagen ) .", "pCAGGS-RFP was kindly provided by Dr Cheryll Tickle .", "Gli3 limb enhancers and empty β-actin basal promoter–GFP at ∼6 μg/μl , coloured with ∼3% fast green , and co-electroporated with pCAGGS-RFP ( ∼2 μg/μl ) into the presumptive forelimb field of stage 13–14 embryos .", "A CUY21EDIT II electroporator ( BEX Co . , Ltd . , Japan ) was used .", "Electric pulses consisted of one short pulse ( 25 V , 0 . 05 ms ) and a 0 . 1-ms interval , followed by five long pulses ( 8 V , 10 ms ) with 1-ms intervals .", "The electric pulses were applied during injection of the DNA solution .", "S . canicula embryos were removed from their egg shells , then placed into 6-well plates .", "4–6 ml of artificial seawater containing penicillin/streptomycin was used for culturing embryos .", "RA was dissolved in DMSO to 2 mg/ml as a stock solution and diluted in the artificial seawater to 1–2 μg/ml .", "1% DMSO in the artificial seawater was used as negative controls .", "Embryos at stage 28–29 were cultured with RA for 4 days for gene expression analyses .", "For alcian blue staining , embryos at stage 28–29 were cultured with 1–2 μg/ml RA for 20 days and additional 10–18 days after removing RA .", "Note that effect of RA is highly dependent on individual embryos .", "Some batches of embryos were lethal at 2 μg/ml of RA , probably due to season or parents' condition .", "In this case , embryos were treated with 1 μg/ml of RA ." ] ]
[ "The pectoral fins of ancestral fishes had multiple proximal elements connected to their pectoral girdles .", "During the fin-to-limb transition , anterior proximal elements were lost and only the most posterior one remained as the humerus .", "Thus , we hypothesised that an evolutionary alteration occurred in the anterior–posterior ( AP ) patterning system of limb buds .", "In this study , we examined the pectoral fin development of catshark ( Scyliorhinus canicula ) and revealed that the AP positional values in fin buds are shifted more posteriorly than mouse limb buds .", "Furthermore , examination of Gli3 function and regulation shows that catshark fins lack a specific AP patterning mechanism , which restricts its expression to an anterior domain in tetrapods .", "Finally , experimental perturbation of AP patterning in catshark fin buds results in an expansion of posterior values and loss of anterior skeletal elements .", "Together , these results suggest that a key genetic event of the fin-to-limb transformation was alteration of the AP patterning network ." ]
[ "Humans , mice , and other animals with four limbs belong to a group of land-dwelling animals known as the tetrapods .", "This group of animals evolved from ancient fish and one crucial adaptation to life on land involved the modification of fins to form limbs .", "The front pair of limbs ( the ‘arms’ ) evolved from the ‘pectoral’ fins of the ancient fish .", "These fins contain numerous bones that fan out from a set of bones called the pectoral girdle .", "However , most of the bones nearer the front side ( the thumb side in the human limb ) were lost in the ancestors of tetrapods as they moved onto land .", "Only the bone nearest the back remained as the ‘humerus’ , which forms the upper part of the limb ( i . e . , the upper arm of humans ) .", "In the embryos of mice and other animals , the limbs develop from structures called limb buds .", "For the limb to develop properly , the cells in the limb bud need to receive specific instructions that depend on their position in the bud .", "A protein called Gli3R provides cells with information about their position along the ‘anterior–posterior’ ( or thumb-to-little finger ) axis of the bud .", "This protein regulates several genes that are involved in limb development , and this results in different genes being expressed in cells along the anterior–posterior axis .", "For example , Alx4 is only expressed in a small area at the anterior end of the bud , while Hand2 expression is found in a large area towards the posterior part .", "Gli3R is also found in a fish called the catshark , but it is not clear how it controls the formation of fins .", "Onimaru et al . show that the pattern of gene expression in the catshark fin bud is different to that of the mouse limb bud .", "For example , Alx4 is expressed in a larger area of the fin bud that extends further towards the posterior , while Hand2 is only found in a much smaller area at the posterior end of the bud .", "The experiments also suggest that Gli3R is active in a much larger area of the fin bud than in the limb bud .", "Next , Onimaru et al . used a drug on the catshark embryos to increase the activity of another protein that can inhibit Gli3R .", "The fin buds of these shark had anterior shift in several gene expression domains , and the fins that formed were missing several anterior bones and had only a single bone connected to the pectoral girdle .", "Onimaru et al . 's findings suggest that during the evolution of the tetrapods , there may have been a shift in the anterior–posterior patterning of the fin bud to form a limb .", "An important area for future work will be to use genome-wide studies to study the fin/limb buds of other species ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "physics of living systems", "tools and resources", "microbiology and infectious disease" ]
A lung-on-chip model of early Mycobacterium tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth
elife-59961-v3
[ [ "Early tuberculosis ( TB ) , a respiratory infection caused by Mycobacterium tuberculosis ( Mtb ) is strongly influenced by host physiology; due to the small diameter of respiratory bronchioles only the smallest aerosol droplets containing one to two bacilli are successfully transported to the alveolar space ( Ma and Darquenne , 2011 ) , and the ‘first contact’ with a naive host is by default a single-cell interaction between an Mtb bacillus and a host cell .", "There is some evidence that pulmonary surfactant plays host-protective role in these early interactions ( Torrelles and Schlesinger , 2017 ) , but a complete understanding of the role of surfactant is difficult to obtain from animal infection models owing to the lethality of surfactant deficiency .", "In addition , experiments in animal models ( Collins and Orme , 1994 ) cannot provide information about the dynamics of host-Mtb interactions at this early stage with sufficient spatiotemporal resolution ( Westphalen et al . , 2014; Looney et al . , 2011 ) .", "A commonly used in vitro model , infection of macrophages with Mtb ( Lerner et al . , 2017 ) , has been used to probe the role of certain surfactant components ( Arcos et al . , 2011 ) , but these studies cannot address the role of native surfactant secreted by alveolar epithelial cells ( ATs ) at an air-liquid interface ( ALI ) , a condition that has been reported to alter Mtb physiology ( Ojha et al . , 2008 ) .", "Organ-on-chip systems recreate tissue-level complexity in a modular fashion , allowing the number of cellular components , their identity , and environmental complexity to be tailored to mimic key aspects of the relevant physiology , such as an ALI in a lung-on-chip ( LoC ) ( Huh et al . , 2010 ) .", "These systems have emerged as crucial tools for the replacement of animal models in drug development , toxicity testing , and personalized medicine ( Ghaemmaghami et al . , 2012; Ronaldson-Bouchard and Vunjak-Novakovic , 2018 ) .", "A far less-explored line of enquiry has been to use them as models to study the dynamics of host-pathogen interactions in a realistic physiological setting ( Grassart et al . , 2019 ) , where they can combine key advantages of both simpler in vitro models and animal models ( Torrelles and Schlesinger , 2017 ) .", "Here , we develop an LoC model of early TB infection and use time-lapse microscopy to study the infection dynamics for ATs and macrophages as independent sites of first contact , and the impact of surfactant on infection of ATs and macrophages under ALI conditions that mimic the alveolar environment in vivo ." ], [ "Freshly isolated mouse ATs comprise a mix of type I cells ( Figure 1A ) and type II cells that produce normal surfactant ( NS ) levels ( Figure 1B ) .", "Prolonged in vitro passage causes ATs to adopt a phenotype with deficient surfactant ( DS ) levels ( Figure 1C ) .", "DS cells had reduced expression of type II markers such as Abca3 ( required for surfactant export [Beers and Mulugeta , 2017; Besnard et al . , 2010] ) and all four surfactant proteins Sftpa , Sftpb , Sftpc , and Sftpd ( Figure 1—figure supplement 1A ) as measured by RT-PCR .", "These cells also showed reduced expression of some type I markers such as the membrane proteins Aquaporin ( Aqp5 ) and Podoplanin ( Pdpn ) but had elevated expression of other type I markers such as Caveolin-1 ( Cav1 ) and Insulin Growth Factor Binding Protein 2 ( Igfbp2 ) .", "These observations are consistent with a majority of the cells in this populations having a terminal type I phenotype ( Wang et al . , 2018 ) .", "DS ATs also have fewer and smaller lamellar bodies ( Figure 1—figure supplement 1B , C ) .", "We reconstituted LoC ( schematic in Figure 1D ) with confluent monolayers of NS or DS ATs ( Figure 1E ) and endothelial cells ( Figure 1F ) on opposite faces of a porous membrane ( Figure 1G ) and an ALI mimicking the alveolar environment .", "Macrophages added to the epithelial face may remain there or transmigrate across the membrane to the endothelial face ( Figure 1E–G ) .", "Typical numbers of ATs , endothelial cells , and macrophages are given in Table 1 , the low macrophage: AT ratio is consistent with alveolar physiology ( Weibel , 2015 ) .", "The macrophages are obtained from a mouse line that constitutively expresses GFP ( false-colored magenta in all Figures unless otherwise specified ) to enable unambiguous identification of these cells during live-cell microscopy .", "A maximum intensity projection of a field of view on the epithelial face of an LoC reconstituted with NS ATs ( Figure 1H , additional examples in Figure 1—figure supplement 2A–C ) and maintained for 24 hr at the ALI shows more intense pro-SPC signal than a corresponding maximum intensity projection for an LoC reconstituted with DS ATs ( Figure 1I , additional examples in Figure 1—figure supplement 2D–F ) .", "LoCs reconstituted with DS ATs retain deficient surfactant expression on-chip at the ALI with fewer ( Figure 1J ) and smaller ( Figure 1K ) lamellar bodies detected across multiple fields of view .", "This phenotype is also reflected in the reduced expression of some type II markers , notably Abca3 , in DS LoCs as compared to NS LoCs ( Figure 1L , Figure 1—figure supplement 2G ) .", "In contrast , expression of type I markers including Aqp5 and Pdpn as well as the lung chemokine Cxcl15 was not significantly different between NS and DS LoCs at the ALI ( Figure 1L , Figure 1—figure supplement 2H ) .", "This established LoCs reconstituted with DS ATs as a tool for the direct study of the role of AT-secreted pulmonary surfactant in early infection without significantly altering other aspects of AT biology .", "DS LoCs retained surfactant deficiency for up to 6 days at the ALI ( Figure 1—figure supplement 2I , J vs . Figure 1—figure supplement 2K , L ) .", "Inoculation of the LoC with between 200 and 800 Mtb bacilli led to infection of both macrophages ( white boxes in Figure 1M , P , zooms in Figure 1O , R ) and ATs ( yellow boxes in Figure 1M , P , zooms in Figure 1N , Q ) under both NS ( Figure 1M–O ) and DS ( Figure 1P–R ) conditions .", "The current paradigm in TB focuses on macrophages as sites of first infection; we therefore examined all the Mtb-infected cells isolated from the lungs of a mouse at 8 days post-infection ( dpi ) in an unbiased manner to ascertain if ATs also served as a site of first contact .", "This revealed that 7 . 3% of infected cells ( n = 163 ) were CD45- pro-SPC+ type II ATs ( Figure 1—figure supplement 3A–F , Video 1 ) .", "We did not find instances of type I AT infection , but this likely reflects the challenges in isolating this cell type .", "Thus , the LoC model faithfully reproduces AT infection that also occurs in vivo ( Figure 1—figure supplement 3G ) albeit at a higher frequency ( Figure 1—figure supplement 3H ) .", "This in turn enables us to study the infection dynamics in ATs and macrophages simultaneously .", "We used time-lapse microscopy to quantify the intracellular growth of Mtb in the host cells of first contact by measuring the total fluorescence intensity of bacteria within single infected cells over time ( Figure 2 ) .", "The refractive index differences at the ALI significantly degrades axial resolution and signal-to-noise ratios; nonetheless , we are able to identify and track individual infected cells over time .", "Between 3 and 5 dpi under NS ( Figure 2 , A-D , Figure 2—video", "1 ) or DS ( Figure 2 , E-H , Figure 2—video", "2 ) conditions , intracellular growth of Mtb is highly variable in both ATs ( Figure 2I , K ) and macrophages ( Figure 2J , L ) .", "We used high-resolution confocal imaging of the epithelial face of an infected LoC fixed at 4 dpi to obtain direct verification that growth in both ATs and macrophages is intracellular ( Figure 2—figure supplement 1 and Videos 2 and 3 ) .", "Plots of the logarithm of total bacterial fluorescence intensity within individual infected cells ( representing the spread in growth rates ) indicate that bacterial growth is exponential in ATs ( Figure 2I , K ) and macrophages ( Figure 2J , L ) .", "However , under NS conditions , we identified substantial fraction of bacteria that show very slow growth ( doubling time >168 hr ) or even a decrease in fluorescence intensity over time .", "In the absence of a reliable live/dead marker for Mtb , we identify this as a ‘non-growing fraction’ ( NGF ) .", "Intracellular bacterial growth is slower in macrophages compared to ATs under NS conditions but growth rates in both cell types are equivalent under DS conditions ( Figure 2M ) .", "Compared to bacterial growth in axenic 7H9 cultures , growth in both cell types is slower under NS conditions ( Figure 2—figure supplement 2A ) but significantly faster under DS conditions ( Figure 2—figure supplement 2B ) .", "We also found that Mtb grows very poorly when cultured axenically in the ALI medium ( Figure 2—figure supplement 3 vs Figure 2—figure supplement 4B ) , which provides indirect evidence that Mtb growth on-chip is likely to be intracellular .", "Interestingly , there is a much larger spread in growth rates and a small fraction of bacteria continue to grow rapidly even under NS conditions .", "We observed no spatial pattern of intracellular Mtb growth rates within the LoC in both NS and DS conditions , confirming that the observed distributions of growth rates are not due to spatial heterogeneity within the device ( Figure 2—figure supplement 5A , B ) .", "Taken together , these results suggest that surfactant deficiency shifts the host-pathogen equilibrium in favor of Mtb , resulting in uncontrolled bacterial growth even in macrophages .", "We compared the growth rate measurements from the LoC with those obtained from Gill et al . , 2009 which uses of a plasmid-loss assay in the mouse model of infection and represents the state-of-the-art in quantitative measurements of Mtb growth in vivo .", "Table 1 ( Gill et al . , 2009 ) in lists a mean growth rate r = 0 . 78 and a mean death rate δ = 0 . 41 for Mtb replication for the period of days 1–14 post-infection in the mouse model of infection .", "Thus , the net growth rate equals r-δ=0 . 37 , which corresponds to a doubling time ( or generation time ) of td=ln⁡ ( 2 ) r-δ∙24 hr = 45 hr .", "In the notation of the current manuscript , this converts to a growth rate ( h−1 ) of 0 . 022 , which is in good agreement with the mean or median growth rate that we report for macrophage infections with wild-type Mtb in NS conditions ( Table 2 ) .", "Notably , the LoC model provides the entire population distribution of growth rates with an approximately 200-fold higher temporal resolution .", "Although reduced surfactant secretion in DS LoCs correlates with increased Mtb replication , this shift could reflect other physiological changes that occur during in vitro passage of ATs .", "We therefore asked whether uncontrolled intracellular replication of Mtb in LoCs reconstituted with DS ATs could be rescued by exogenous addition of surfactant .", "A 1% solution of Curosurf , a pulmonary surfactant formulation comprising dipalmitoylphospatidylcholine ( DPPC ) and the hydrophobic surfactant proteins SP-B and SP-C , was used to treat either the Mtb or the DS LoC prior to infection .", "Both procedures attenuated intracellular Mtb growth and generated a non-growing fraction similar in magnitude to infected NS LoCs ( Figure 2N , Table 2 ) .", "Curosurf treatment affects neither Mtb viability ( Figure 2—figure supplement 4A ) nor replication in vitro in the absence of host cells ( Figure 2—figure supplement 4B ) suggesting that surfactant protects by altering the interaction of Mtb with host cells rather than by any direct effect on bacterial physiology .", "We conclude that uncontrolled intracellular growth of Mtb in DS LoCs is largely attributable to reduced surfactant secretion .", "We examined whether Mtb mutants that were previously shown to be attenuated in the mouse model of TB are also attenuated in the LoC model and whether surfactant plays a role in attenuation .", "Mtb lacking both the isocitrate lyase genes icl1 and icl2 grows normally under standard conditions in vitro but is incapable of growth in the lungs of mice and is rapidly cleared ( Muñoz-Elías and McKinney , 2005 ) .", "In the LoC model , we found that the Δicl1Δicl2 strain is unable to grow in either ATs or macrophages even under the more-permissive DS conditions ( Figure 3C vs . A , Figure 3—figure supplement 1A–C for widefield images and Figure 3—figure supplement 1C–F for confocal images ) , indicating that attenuation of this mutant is similar in the LoC and mouse models and independent of surfactant secretion .", "These results once again are suggestive of intracellular Mtb growth in the LoC model because the Δicl1Δicl2 strain has a growth defect relative to wild-type only when intracellular in a host cell due to the accumulation of metabolic intermediates ( Upton and McKinney , 2007 ) , whereas there are no differences in growth between the axenic cultures of Δicl1Δicl2 and wild-type strains in 7H9 medium ( Muñoz-Elías and McKinney , 2005 ) or in the ALI media used in the LoC experiments ( Figure 2—figure supplement 3 ) .", "The activity of the ESX-1 Type VII secretion system , a major Mtb virulence factor that is required for escape from the phagosome into the cytosol ( van der Wel et al . , 2007 ) , is upregulated during AT infection ( Ryndak et al . , 2015 ) .", "In comparison to wild-type Mtb , whose intracellular growth rate is strongly dependent on surfactant levels ( Figure 3D–G ) , intracellular growth of the 5’Tn::pe35 strain ( Chen et al . , 2013 ) that is deficient in ESX-1 secretion but retains PDIM secretion ( Figure 4—figure supplement", "1 ) is largely independent of surfactant levels ( Figure 3H–K ) .", "Under NS conditions , a greater fraction of ESX-1-deficient bacteria are ‘non-growing’ in ATs ( Figure 3I ) .", "Under DS conditions , the ESX-1-deficient strain is unable to grow as rapidly as wild-type in both macrophages and ATs ( Figure 4B , D ) and a fraction ( ca . 12% ) of ESX-1-deficient bacteria are non-growing in macrophages ( Figure 3K ) .", "This attenuation in DS conditions is evident by visual inspection at 6 dpi ( Figure 3B vs 3A ) .", "Macrophages are less permissive than ATs for intracellular growth of ESX-1-deficient Mtb under both NS and DS conditions ( Figure 3—figure supplement 2C , D ) .", "In contrast , wild-type Mtb , grows more slowly in macrophages than in ATs only under NS but not DS conditions ( Figure 3—figure supplement 2A , B ) .", "Overall , attenuation of ESX-1-deficient bacteria relative to wild-type is not rescued by surfactant deficiency .", "This demonstrates that ESX-1 secretion is necessary for rapid intracellular growth in the absence of surfactant , consistent with the hypothesis that surfactant may attenuate Mtb growth by depleting ESX-1 components on the bacterial cell surface ( Raffetseder et al . , 2019 ) .", "Although median values of growth rate per hour are similar for wild-type and strains of Mtb under NS conditions ( Figure 4A , C vs . Figure 4B , D ) , subtle differences in the probability density functions for each distribution ( reflected in the 1–99 percentile interval in Figure 4A , C ) could nevertheless generate significant differences in population sizes over a few days .", "This is particularly true for TB where growth is exponential in early infection , and bacterial numbers are enumerated over weeks or months of infection in the mouse model .", "To determine if this could account for the attenuation of the esx-1 strain observed in vivo , we simulated the progression of a low-dose mouse infection ( infectious dose = 50 CFU at 1 dpi ) using intracellular bacterial growth rates randomly chosen from the growth rate distributions of each strain in macrophages in the LoC model ( Figure 3F , J ) .", "At 2 dpi , population sizes for the ESX-1-deficient strains ( Figure 4E , n = 100 , p=2 . 3×10−6 ) are already significantly smaller than for wild-type Mtb .", "The levels of attenuation predicted by this simple model using values from NS ( Figure 4F ) but not DS ( Figure 4G ) LoC conditions are in good agreement with the experimental data for both mutants from the mouse model in the acute phase of infection ( Chen et al . , 2013; Rao et al . , 2005 ) .", "These results provide a strong validation that surfactant secretion by freshly isolated ATs in NS conditions in the LoC model provide a better mimic of the native lung environment than DS conditions and serve to benchmark the LoC model .", "Although attenuation of the ESX-1-deficient strain is completely independent of surfactant in ATs ( Figure 3H , I ) , surfactant still has a small but significant impact on growth of ESX-1-deficient Mtb in macrophages ( Figure 3J , K ) .", "We therefore hypothesized that an additional mechanism of surfactant-dependent protection could be the removal of virulence-associated lipids from the Mtb cell surface .", "Consistent with this idea , microscopic examination of Mtb exposed to fluorescently labeled Curosurf with a fluorescent analogue of DPPC ( Figure 5—figure supplement 1A ) revealed that surfactant readily coated the bacteria ( Figure 5A , B , Figure 5—figure supplement 1B–E ) , albeit heterogeneously ( compare Figure 5—figure supplement 1B , C with Figure 5—figure supplement 1D , E ) .", "We also examined the effect of surfactant on the composition of the Mtb cell surface by comparing the total cell-associated lipids and the free ( released ) lipids prepared from untreated and Curosurf-treated Mtb .", "We found that Curosurf partially strips the Mtb cell surface of sulfoglycolipids ( SGL ) ( Figure 5C , E ) and trehalose dimycolate ( TDM ) ( Figure 5D , E ) , but not phthiocerol dimycocerosates ( PDIM ) ( Figure 5—figure supplement 2 ) .", "The surfactant-mediated removal of these virulence-associated lipids ( Dulberger et al . , 2020 ) suggests an additional mechanism for the attenuation of intracellular growth of Mtb in macrophages ." ], [ "ATs are the major cellular component of the distal lung , yet despite sporadic reports of AT infection in human TB ( Barrios-Payán et al . , 2012; Hernández-Pando et al . , 2000 ) , the role of ATs remains controversial .", "Previous work has revealed the specific roles of hydrophilic SP-A and SP-D proteins ( Ferguson et al . , 2006; Pasula et al . , 1997 ) in altering the uptake and intracellular processing of Mtb in macrophages .", "Surfactant hydrolase enzymes within the alveolar lining fluid Arcos et al . , 2011; Scordo et al . , 2017 have also been shown to release Mtb cell wall fragments as well as alter Mtb growth in ATs .", "Together , these studies show that , for the most part , specific components of surfactant can suppress intracellular Mtb growth .", "However , surfactant lipids have also been reported to upregulate Mtb growth via increased expression of CD36 ( Dodd et al . , 2016 ) , and the alveolar lining fluid from some patients has also been shown to increase intracellular replication in epithelial cells and exacerbate infection ( Scordo et al . , 2019 ) .", "However , all these studies focused on the role of specific components of surfactant and none of these studies probed the role of endogenous surfactant expression in co-cultures maintained at the ALI .", "In contrast , in the LoC model of early TB presented here , secretion of native surfactant by ATs at an ALI can be modulated .", "This physiological perturbation , which cannot be achieved in animal models due to the lethality of surfactant deficiency , provides a comprehensive view of the role of ATs in early TB and is an important advance over previous co-culture models for TB ( Bermudez et al . , 2002; Bielecka et al . , 2017; Parasa et al . , 2014 ) .", "The unexpectedly rapid and uncontrolled intracellular growth of Mtb at the ALI in the absence of surfactant has not been reported previously in simpler in vitro models of host cell infection .", "Under NS conditions , we identified a substantial non-growing fraction of intracellular Mtb in both ATs and macrophages , which may be equivalent to the ‘non-growing but metabolically active’ ( NGMA ) populations previously observed in the lungs of mice ( Manina et al . , 2015 ) .", "These examples underscore some of the advantages of the LoC model , which provides a more faithful mimesis of the complex in vivo environment compared to conventional in vitro models .", "Taken together , our findings indicate that pulmonary surfactant plays an important role in host innate immunity during early Mtb infection , which may partly explain why individuals with defective surfactant function ( Finley and Ladman , 1972; Subramaniam et al . , 1995; Moliva et al . , 2019 ) also show an increased risk of developing active TB .", "We also report that ATs are more permissive to Mtb growth than macrophages under NS ( but not DS ) conditions .", "Infection of ATs is not an artefact of the LoC model , as we also identified infected ATs in the lungs of aerosol-infected mice using a sensitive microscopy-based approach , which might be more discriminating than FACS-based approaches used previously ( Cohen et al . , 2018 ) .", "Alveolar macrophages in the lung have been shown to be sessile ( Westphalen et al . , 2014 ) , and so the higher incidence of AT infection in the LoC model is probably due to differences in the method of Mtb inoculation .", "This in turn suggests a deeper link between the airway and alveolar geometries that determine aerosol deposition in the lungs ( which are not captured in the LoC model but could be addressed with 3D bioprinted models [Grigoryan et al . , 2019] ) and resident lung immunity .", "Given the very low inoculum size in human Mtb ( most infected individuals harbor just one primary lesion originating from a single bacillus Mckinney et al . , 1998 ) , we speculate that first contact with an AT , albeit much rarer in vivo could potentially lead to a more aggressive infection .", "This could provide one explanation for the observation that the proportion of exposed individuals who develop clinical TB is low .", "Time-lapse imaging at an ALI in the LoC infection model directly quantifies bacterial growth rates with a spatiotemporal resolution that is unachievable using indirect measurements of bacterial growth rates in mouse models of TB ( e . g . plating tissue homogenates to measure colony-forming units [CFU] ) .", "The deliberate choice to use murine over human cells allows us to add macrophages to the chip from a mouse line that constitutively expresses GFP , and provides unambiguous identification of these cells over multiple days which would not be possible with CellTracker and other fluorescent dyes .", "It also enables us to benchmark the model with previous reports from the mouse model for TB .", "For example , average growth rates for wild-type Mtb under NS conditions are in good agreement with net Mtb growth rates measured using a plasmid-loss assay in mice ( Gill et al . , 2009 ) .", "However , our microscopy-based approach also reveals the population distribution of growth rates , which highlights that even under NS conditions , a small proportion of cells show robust intracellular Mtb growth , and that robust and attenuated Mtb growth can occur in close proximity to each other on-chip .", "These observations reinforce an important role for cell-to-cell heterogeneity in host-Mtb outcomes ( Lin et al . , 2014 ) .", "The growth characteristics of ESX-1-deficient Mtb in our LoC model are also in agreement with experiments from animal models , further validating the LoC model .", "Exogenous addition of Curosurf , a surfactant replacement formulation of phospholipids and hydrophobic proteins , rescues the effects of surfactant deficiency on mycobacterial growth to a large extent .", "Phospholipids are the main component of native surfactant , and lipid recycling is a key function of type II ATs and alveolar macrophages ( Olmeda et al . , 2017 ) .", "The two-way interaction of surfactant with the bacterial cell surface ( surfactant removes virulence-associated Mtb surface lipids and proteins , and surfactant lipids coat the Mtb surface ) may alter how host cells take up these bacteria , or how they are processed after uptake .", "More generally , surfactant phospholipids have been shown to have antiviral properties and can serve as a potent adjuvant for antiviral vaccines either on their own ( Kimoto et al . , 2013 ) or as a medium for delivery of immune-stimulating molecules to ATs ( Wang et al . , 2020 ) .", "Our findings suggest a potential role for pulmonary surfactant replacement formulations in host-directed therapies against TB .", "These insights were made possible by use of an organ-on-chip system that reproduces host physiology in a modular and tuneable fashion , which is frequently impossible to achieve in vivo ." ], [ "Primary C57BL/6 alveolar epithelial cells ( ATs ) and lung microvascular endothelial cells were obtained from Cell Biologics , USA , and were certified mycoplasma negative by the supplier .", "Each vial of ATs consisted of a mix of Type I and Type II ATs , which was verified by both immunostaining and qRT-PCR for type I and type II markers ( Figure 1A–C , Figure 1—figure supplement 1 ) .", "Both cell types were cultured in vitro in complete medium comprising base medium and supplements ( Cell Biologics , USA ) in 5% CO2 at 37°C .", "NS ATs were seeded directly on the LoC ( see below ) , without prior in vitro culture .", "DS ATs were passaged 6–11 times before use and were verified to be free of mycoplasma contamination prior to use .", "Bone marrow was obtained from 6- to 8-week-old Tg ( CAG-EGFP ) 131Osb/LeySopJ ( also known as Tg ( act-EGFP ) Y01Osb ) mice ( Jackson Laboratories , USA , Stock Number 006567 ) and cryopreserved .", "This transgenic line constitutively expresses enhanced GFP under the control of the chicken beta-actin promoter and the cytomegalovirus enhancer .", "Mice were housed in a specific pathogen-free facility .", "Animal protocols were reviewed and approved by EPFL's Chief Veterinarian , by the Service de la Consommation et des Affaires Vétérinaires of the Canton of Vaud , and by the Swiss Office Vétérinaire Fédéral .", "Bone marrow was cultured in Dulbecco’s Modified Eagle Medium ( DMEM ) ( Gibco ) supplemented with 10% fetal bovine serum ( FBS , Gibco ) and differentiated for 7 days with 20 ng/ml recombinant murine Macrophage-Colony Stimulating Factor protein ( M-CSF ) ( Thermo Fisher Scientific ) .", "Bone marrow was cultured in plastic petri dishes without pre-sterilization ( Greiner Bio-one ) so that differentiated macrophages could be detached .", "No antibiotics were used in the cell culture media for all cell types to avoid activation of macrophages or inhibition of Mtb growth , and the frozen marrow was verified to be free of mycoplasma contamination prior to use .", "Freshly isolated ATs ( NS ) were grown overnight in cell-culture microdishes ( Ibidi ) or T-25 cell culture flask ( TPP , Switzerland ) .", "Passaged ATs ( DS ) were grown to confluency in a T-75 cell culture flask ( TPP ) .", "Growth media was removed from the flask , and the cells were incubated with the appropriate volume of TRIzol ( Ambion ) as per the manufacturer’s instruction .", "TRIzol-treated cell lysates were stored at −20°C before further processing .", "RNA was precipitated with isopropanol , washed in 75% ethanol , resuspended in 50 μl of DEPC-treated water , treated with Turbo DNase ( Ambion ) , and stored at −80°C until use .", "DNase-treated RNA was used to generate cDNA using the SuperScriptII First-Strand Synthesis System with random hexamers ( Invitrogen ) , and was stored at −20°C .", "For RNA isolation from LoCs , one NS or DS LoC each were established as per the protocols described and maintained for 24 hr at the ALI .", "RNA from the apical and vascular channels was isolated separately in approximately 350 μl of the RLT Plus buffer of the Qiagen Micro Plus RNA Isolation Kit , and subsequently processed as per the manufacturer’s instructions .", "cDNA was generated using the SuperScript IV First-Strand Synthesis System with random hexamers ( Invitrogen ) , and subsequently stored at −20°C .", "Specific primers used are listed in Table", "3 . Sequences for the primers for Pdpn , Cav1 , and Igfbp2 were obtained from Wang et al . , 2018 and the sequences for the remaining primers were obtained from Origene and primers were obtained from a commercial supplier ( Microsynth , Switzerland ) .", "qRT-PCR reactions were prepared with SYBRGreen PCR Master Mix ( Applied Biosystems ) with 1 μM primers , and 1 or 2 μl cDNA .", "Reactions were run as absolute quantification on ABI PRISM7900HT Sequence Detection System ( Applied Biosystems ) .", "Amplicon specificity was confirmed by melting-curve analysis .", "Freshly isolated ATs ( NS ) or passaged ATs ( DS ) were grown overnight in 35-mm cell-culture microdishes ( Ibidi GmbH , Germany ) .", "The confluent layer of cells was subsequently fixed with 2% paraformaldehyde ( Thermo Fisher Scientific ) in phosphate-buffered saline ( PBS , Gibco ) at room temperature for 30 min , washed with PBS , and incubated with a blocking solution of 2% bovine serum albumin ( BSA ) in PBS for 1 hr at room temperature .", "The blocking solution was removed , and the cells were incubated with the primary antibody ( 1:100 dilution in 2% BSA solution in PBS ) overnight at 4°C .", "Antibodies used were anti-Podoplanin Monoclonal Antibody ( eBio8 . 1 . 1 ( 8 . 1 . 1 ) ) , Alexa Fluor 488 , eBioscience ( ThermoFisher Scientific ) , and anti-pro-SPC antibody ( ab40879 , Abcam ) .", "The cell-culture microdishes were washed 3x in PBS , incubated with a fluorescent secondary antibody ( Donkey anti-rabbit Alexa Fluor 568 ( A10042 Thermo Fisher ) ) in a solution of 2% BSA in PBS for 1 hr at room temperature , then thoroughly washed with PBS and incubated with Hoechst 33342 nuclear staining dye ( 1:1000 dilution , Thermo Fisher Scientific ) for 15–20 min for nuclear staining .", "Confocal images were obtained on a Leica SP8 microscope in the inverted optical configuration at the EPFL BIOP core facility .", "All bacterial strains were derived from Mtb strain Erdman and cultured at 37°C .", "Liquid medium: Middlebrook 7H9 ( Difco ) supplemented with 0 . 5% albumin , 0 . 2% glucose , 0 . 085% NaCl , 0 . 5% glycerol , and 0 . 02% Tyloxapol .", "Solid medium: Middlebrook 7H11 ( Difco ) supplemented with 10% OADC enrichment ( Becton Dickinson ) and 0 . 5% glycerol .", "Aliquots were stored in 15% glycerol at −80°C and used once .", "All strains were transformed with a plasmid integrated at the chromosomal attB site to allow constitutive expression of the fluorescent protein tdTomato under the control of the hsp60 promoter .", "Wild-type ( WT ) refers to the Erdman strain constitutively expressing tdTomato .", "The 5’Tn::pe35 ( ESX-1 deficient ) strain was generated using transposon mutagenesis ( Chen et al . , 2013 ) .", "Female C57BL/6 mice ( Charles River Laboratories ) were housed in a specific pathogen-free facility .", "Animal protocols were reviewed and approved by EPFL's Chief Veterinarian , by the Service de la Consommation et des Affaires Vétérinaires of the Canton of Vaud , and by the Swiss Office Vétérinaire Fédéral .", "Mice were infected by the aerosol route using a custom-built aerosol machine , as described ( MacMicking et al . , 2003 ) .", "Bacteria were grown to exponential phase , corresponding to an optical density at 600 nm ( OD600 ) of 0 . 5 , collected by centrifugation at 2850 g for 10 min , and resuspended in PBS supplemented with 0 . 05% Tween 80 ( PBS-T ) .", "The bacterial suspension was subjected to low-speed centrifugation ( 700 g ) for 5 min to remove bacterial aggregates .", "The cell suspension was adjusted to OD600 0 . 1 with PBS-T in a final volume of 20 ml , which was used to infect mice by aerosol .", "At 1 dpi , a group of four mice were euthanized by CO2 overdose; the lungs were removed aseptically and homogenized in 3 ml of 7H9 medium .", "Serial dilutions were plated on 7H11 plates containing 100 μg/ml cycloheximide ( Sigma ) , and colonies were counted after 4–5 weeks of incubation at 37°C .", "The aerosol infection corresponded to a bacterial load of between 60 and 100 CFU per mouse at 1 dpi .", "At 8 dpi , a group of five mice were euthanized by an overdose of ketamine/xylazine anesthetic , and the lungs were washed with PBS delivered via injection through the right ventricle of the heart to remove excess red blood cells .", "Lungs from each mouse were removed aseptically , minced into small pieces with scissors , and added to 2 . 5 ml of lung dissociation media reconstituted as per the manufacturer’s instructions ( Lung Dissociation Kit – Mouse , Miltenyi Biotec ) .", "The lungs were then dissociated using a gentleMACS Octo Dissociator ( Miltenyi Biotec ) .", "The resulting homogenate was filtered through a 40-μm cell filter , centrifuged for 10 min at 300 g , and resuspended in alveolar epithelial cell media supplemented with 10% FBS .", "The homogenate was then plated in 50-mm glass-bottom cell-culture dishes ( Ibidi ) and incubated for 36–48 hr to allow for epithelial cell attachment .", "Additional medium was added to each cell-culture dish at 24 hr .", "Adherent cells from the single-cell suspension were subsequently fixed with paraformaldehyde and stained for immunofluorescence as already described .", "Antibodies: anti-Podoplanin Monoclonal Antibody ( eBio8 . 1 . 1 ( 8 . 1 . 1 ) ) , Alexa Fluor 488 , eBioscience ( ThermoFisher Scientific ) to label Type I ATs , anti-pro-SPC antibody ( ab40879 , Abcam ) followed by secondary antibody staining ( Donkey anti rabbit Alexa Fluor 488 ( A21206 Thermo Fisher ) ) to label Type II ATs , and Alexa 647 anti-CD45 antibody ( 103124 , BioLegend ) to label immune cells .", "LoCs made of polydimethylsiloxane ( PDMS ) were obtained from Emulate ( Boston , USA ) .", "Extracellular matrix ( ECM ) coating was performed as per the manufacturer’s instructions .", "Chips were activated using ER-1 solution ( Emulate ) dissolved in ER-2 solution at 0 . 5 mg/ml ( Emulate ) and exposed for 20 min under UV light .", "The chip was then rinsed with coating solution and exposed again to UV light for a further 20 min .", "Chips were then washed thoroughly with PBS before incubating with an ECM solution of 150 μg/ml bovine collagen type I ( AteloCell , Japan ) and 30 μg/ml fibronectin from human plasma ( Sigma-Aldrich ) in PBS buffered with 15 mM HEPES solution ( Gibco ) for 1–2 hr at 37°C .", "If not used directly , coated chips were stored at 4°C and pre-activated before use by incubation for 30 min with the same ECM solution at 37°C .", "Endothelial cells were cultured overnight at 37°C and 5% CO2 in T-75 cell culture flasks , detached with 0 . 05% Trypsin , concentrated to 5–10 million cells/ml , and seeded on the bottom face of the PDMS membrane .", "The chip was then incubated for a short period at 37°C to allow the endothelial cells to spread and subsequently seeded with ATs .", "Freshly isolated ATs were seeded directly from cryopreserved vials received from the supplier , whereas DS LoCs were seeded from cells cultured overnight at 37°C and 5% CO2 , in both cases at a concentration of 1–2 million cells/ml .", "The chip was incubated overnight with complete epithelial and endothelial media in the epithelial and endothelial channels , respectively , under static conditions .", "The next day , the chip was washed and a reduced medium for the ALI was flowed through the vascular channel using syringe pumps ( Aladdin-220 , Word Precision Instruments ) at 60 μl/hr as described ( Hassell et al . , 2017 ) .", "The composition of the ALI media used was as described in Hassell et al . , 2017 but with an FBS concentration of 5% .", "The epithelial face was incubated with epithelial base medium with 200 nM dexamethasone ( Sigma Aldrich ) without FBS supplementation to promote tight junction formation and surfactant expression as reported in previous LoC studies ( Huh et al . , 2010; Hassell et al . , 2017 ) .", "Flow was maintained over 2–3 days with daily replacement of the medium on the epithelial face ( with dexamethasone supplementation ) .", "At the end of this period , GFP-expressing macrophages differentiated for 7 days in M-CSF ( described above ) were detached from the petri dish using 2 mM ethylenediaminetetraacetic acid ( EDTA , Sigma Aldrich ) in PBS at 4°C , centrifuged at 300 g for 5 min , and resuspended in a small volume of epithelial cell media without dexamethasone .", "This solution containing macrophages was introduced onto the epithelial face and incubated for 30 min at 37°C and 5% CO2 to allow macrophages to attach to the epithelial cells .", "Medium on the epithelial face was then removed and the chip was maintained overnight at the ALI .", "Chips that successfully maintained the ALI overnight were transferred to the biosafety level 3 ( BSL-3 ) facility for Mtb infection .", "No antibiotics were used in any of the cell culture media for setting up the LoC model .", "Uninfected LoCs were maintained at an ALI for up to 7 days after addition of macrophages , during which time ALI medium was flowed through the endothelial channel at 60 μl/hr .", "After 7 days at the ALI , the chip was fixed for immunostaining as described above; a permeabilization step with a solution containing 2% w/v saponin ( Sigma Aldrich ) and 0 . 1% Triton X-100 ( Sigma Aldrich ) was performed before incubation with the secondary antibody .", "F-actin on both the epithelial and endothelial face was stained using Sir-Actin dye ( Spherochrome ) at 1 μM for 30 min concurrently with Hoechst staining , as described above .", "Confocal images were obtained on a Leica SP8 microscope in the inverted optical configuration at the EPFL BIOP core facility .", "The chip was assembled into a stage top incubator ( Okolab , Italy ) prior to infection and flow of medium through the vascular channel was maintained throughout the course of the experiment by use of a syringe pump .", "A 1 ml aliquot of a culture of Mtb grown to exponential phase ( OD6000 . 3–0 . 5 ) was centrifuged at 5000 g for 5 min at room temperature , the supernatant was removed , and the cell pellet was resuspended in 200 μl of epithelial cell media without FBS .", "A single-cell suspension was generated via filtration through a 5-μm syringe filter ( Millipore ) .", "The single-cell suspension was diluted 100-fold in epithelial media and 30 μl was added to the epithelial channel of the LoC .", "The infectious dose was measured by plating serial dilutions of the single-cell suspension on 7H11 plates and counting CFU after 3–4 weeks of incubation at 37°C and varied between 200 and 800 Mtb bacilli .", "The chip was incubated for 2–3 hr at 37°C and 5% CO2 to allow Mtb infection of cells on the epithelial face , after which the solution on the epithelial face was withdrawn .", "The proportion of bacteria that remained on the chip was estimated by plating serial dilutions of the withdrawn solution on 7H11 plates and counting CFU after 3–4 weeks of incubation at 37°C .", "The epithelial face was returned to ALI and the inlets of the infected chip were sealed with solid pins as a safety precaution for time-lapse microscopy imaging in the BSL-3 facility .", "The LoC was placed in a microscope stage-top incubator and mounted on the stage of a widefield Nikon Ti-2 microscope .", "The stage-top incubator was connected to a gas mixer ( Okolab ) to maintain 5% CO2 throughout the imaging period .", "Flow of medium through the vascular channel was maintained throughout this period via the use of a syringe pump .", "The chip was imaged using a long working distance 20x phase-contrast objective ( NA = 0 . 75 , Ph2 , Nikon ) at 1 . 5 hr or 2 hr imaging intervals .", "The epithelial face of the chip ( where the refractive index differences were highest due to the ALI ) was maintained in focus using the Nikon Perfect Focus System .", "At each timepoint , a Z-stack of 9–10 images with an axial spacing of 10 μm was taken series for a series of fields of view along the length of the chip to account for the dynamic 3D movement of macrophages between both faces , as well as drift in focus over time .", "Each field of view was ~660 × 600 μm2 .", "Using a Sola SE II light source ( Lumencor , USA ) , macrophages and Mtb were identified through fluorescence emission in the green ( macrophages ) and red ( Mtb ) channels using GFPHQ and mcherryHQ 32 mm dichroic filters , respectively .", "Phase-contrast images were also captured; the poor quality of these images due to the refractive index differences at the ALI serves as a continuous verification that ALI is maintained .", "All images were captured with an EMCCD camera ( iXON Ultra 888 , Andor ) cooled to −65°C , with an EM gain setting of 300 to allow the sample to be illuminated with a low intensity of incident light in all fluorescent channels with reduced photodamage .", "Co-localization of the green and red fluorescence signals over a time course was identified as consistent with macrophage infection .", "Bacteria that did not co-localize with macrophages over time were assumed to infect ATs , which was verified by subsequent immunostaining .", "Images were visualized using FIJI .", "Macrophage and AT infections from each field of view were visually curated by assessing the co-localization of fluorescent signals over time .", "Smaller stacks of one to two microcolonies were assembled .", "Custom-written software in MATLAB was used to measure the total fluorescence intensity of each intracellular bacterial microcolony which used the nestedSortStruct algorithm for MATLAB ( Hughley , 2018; https://github . com/hugheylab/nestedSortStruct ) written by the Hughey lab .", "Briefly , at each timepoint , the Z-stack with the highest intensity in the fluorescence channel was identified; this image was then segmented to identify the bacterial microcolony; total fluorescence was measured by summing the intensity of all the pixels in this region after subtracting a value for each pixel that represented the average background fluorescence .", "We chose to measure the total fluorescence intensity because it accounts for both bacterial growth and dilution of the fluorescent protein due to growth ( which is slow in a slow-growing bacterium like Mtb ) .", "We were unable to measure microcolony volumes accurately using widefield imaging due to poor axial resolution caused by large refractive index differences at the ALI; therefore , we obtained this value from only the Z stack with the highest intensity .", "Statistical analysis was performed using Origin 9 . 2 ( OriginLabs ) , and p-values were calculated using the Kruskal-Wallis one-way ANOVA test , with the null hypothesis that the medians of each population were equal .", "Z-stacks from confocal images were visualized using ImageJ , 3D projection views and Videos 1 , 2 and 3 were made using the ClearVolume plugin in ImageJ ( Royer et al . , 2015 ) .", "Custom written software in MATLAB was used to segment lamellar bodies in each slice of the Z-stack , measure area and intensity , and collate the mean intensity and volume over multiple slices , along the lines of the analysis of intracellular bacterial growth described above .", "Growth rate datasets for wild-type and ESX-1-deficient strains of Mtb in NS and DS LoC conditions were fitted with a non-parametric Kernel Smoothed distribution .", "We simulated a low-dose aerosol infection of 50 bacteria in the alveolar space of n = 100 or n = 1000 mice , and conservatively assumed that every bacterium interacted with a macrophage upon first contact .", "Each bacterium was assigned a growth rate picked at random from the Kernel Smoothed distributions and assumed to grow exponentially with these growth rates to generate an intracellular microcolony .", "The total bacterial numbers in each mouse at 2 , 3 , 5 , 7 , and 14 dpi were obtained by summing the bacterial counts from each microcolony for each mouse and are shown in Figure 4E–G .", "Total bacterial numbers for n = 100 mice of WT and ESX-1-deficient strains are shown in Figure", "4 . Curosurf ( Chiesi Pharmaceuticals , Italy ) was used as a 1% solution in epithelial medium for all LoC experiments .", "In the case where Curosurf was added to a DS LoC , a 1% solution was introduced to the epithelial face after the macrophages were added but before ALI was introduced for 2 min , and then removed .", "The following morning , this procedure was repeated just prior to the addition of the single-cell suspension of Mtb in the manner described above .", "Alternatively , a 1 ml aliquot of Mtb in exponential phase in 7H9 media was centrifuged at 5000 g for 5 min , resuspended in 1 ml of cell culture media containing 1% Curosurf , and incubated for 10–15 min at room temperature .", "This solution was then centrifuged again at 5000 g for 5 min and a single-cell suspension of Mtb was generated as described above .", "Fluorescent labeling of surfactant was achieved by adding TopFluor phosphatidylcholine ( 10% v/v , Avanti Polar Lipids ) to Curosurf before dilution in cell culture medium .", "Mtb cultures ( 10 ml each ) were grown to stationary phase in 7H9 with 10 μCi of 14C-propionate added during exponential phase .", "Total free lipid extraction from the bacterial pellet , supernatant , and supernatant from bacteria pre-treated with 3% Curosurf for 15 min at 37°C were extracted as described ( Parish and Roberts , 2015 ) .", "Extracted free lipids were air-dried , resuspended in 2:1 v/v solution of chloroform: methanol , and aliquots were spotted on 5 × 10 cm TLC silica gel 60 F254 ( Merck ) .", "Running solvent was 90:10:1 chloroform: methanol: water for the analysis of sulfoglycolipids ( SGL ) , 80:20:2 chloroform: methanol: ammonium hydroxide for the analysis of TDM , and 9:1 petroleum ether: diethyl ether for the analysis of pthiocerol dimycocerosates ( PDIM ) and triacyclglycerols ( TAG ) .", "The developed TLC plate was exposed to an Amersham Hyperfilm ECl ( GE Healthcare ) for phosphorescence imaging and visualized with a Typhoon scanner ( GE Healthsciences ) .", "Intensities of the bands observed were quantified using ImageJ .", "For characterization of PDIM secretion by the WT and ESX-1-deficient strains , lipid extraction was performed via a protocol optimized for extraction of apolar lipids as described ( Ojha et al . , 2008 ) .", "Extracted lipids were subsequently processed as described above ." ] ]
[ "We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host-Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection .", "Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells .", "In contrast , under normal surfactant levels , a significant fraction of intracellular bacteria are non-growing .", "The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations , which have no effect on bacterial viability in the absence of host cells .", "Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface .", "Consistent with this mechanism , the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels .", "These findings may partly explain why smokers and elderly persons with compromised surfactant function are at increased risk of developing active tuberculosis ." ]
[ "Tuberculosis is a contagious respiratory disease caused by the bacterium Mycobacterium tuberculosis .", "Droplets in the air carry these bacteria deep into the lungs , where they cling onto and infect lung cells .", "Only small droplets , holding one or two bacteria , can reach the right cells , which means that just a couple of bacterial cells can trigger an infection .", "But people respond differently to the bacteria: some develop active and fatal forms of tuberculosis , while many show no signs of infection .", "With no effective tuberculosis vaccine for adults , understanding why individuals respond differently to Mycobacterium tuberculosis may help develop treatments .", "Different responses to Mycobacterium tuberculosis may stem from the earliest stages of infection , but these stages are difficult to study .", "For one thing , tracking the movements of the few bacterial cells that initiate infection is tricky .", "For another , studying the molecules , called ‘surfactants’ , that the lungs produce to protect themselves from tuberculosis can prove difficult because these molecules are necessary for the lungs to inflate and deflate normally .", "Normally , the role of a molecule can be studied by genetically modifying an animal so it does not produce the molecule in question , which provides information as to its potential roles .", "Unfortunately , due to the role of surfactants in normal breathing , animals lacking them die .", "Therefore , to reveal the role of some of surfactants in tuberculosis , Thacker et al . used ‘lung-on-chip’ technology .", "The ‘chip’ ( a transparent device made of a polymer compatible with biological tissues ) is coated with layers of cells and has channels to simulate air and blood flow .", "To see what effects surfactants have on M . tuberculosis bacteria , Thacker et al . altered the levels of surfactants produced by the cells on the lung-on-chip device .", "Two types of mouse cells were grown on the chip: lung cells and immune cells .", "When cells lacked surfactants , bacteria grew rapidly on both lung and immune cells , but when surfactants were present bacteria grew much slower on both cell types , or did not grow at all .", "Further probing showed that the surfactants pulled out proteins and fats on the surface of M . tuberculosis that help the bacteria to infect their host , highlighting the protective role of surfactants in tuberculosis .", "These findings lay the foundations for a system to study respiratory infections without using animals .", "This will allow scientists to study the early stages of Mycobacterium tuberculosis infection , which is crucial for finding ways to manage tuberculosis ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "cell biology" ]
Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans
elife-04591-v2
[ [ "RNA granules are ubiquitous cytoplasmic organelles that contain RNA and RNA-binding proteins ( Kedersha et al . , 2013; Buchan , 2014 ) .", "Several types of RNA granules have been described , including germ granules in germ cells , neuronal granules in neurons , and stress granules and P bodies in somatic cells .", "Their functions include mRNA transport and storage and the regulation of mRNA degradation and translation .", "Unlike other organelles , RNA granules are not bound by limiting membranes and their internal components are in constant flux with the surrounding cytoplasm .", "RNA granules assemble and disassemble in response to developmental or environmental cues ( Kedersha et al . , 2013; Buchan , 2014 ) .", "Live imaging studies in Caenorhabditis elegans zygotes have suggested that RNA granules behave like liquid droplets that undergo phase transitions ( Brangwynne et al . , 2009 ) .", "Granule components exist in a condensed liquid or gel-like phase in the granule and a dispersed phase in the cytoplasm ( Weber and Brangwynne , 2012; Toretsky and Wright , 2014 ) .", "In vitro studies have lent support to this hypothesis by demonstrating that purified proteins can undergo phase transitions in aqueous solutions .", "For example , proteins that contain weak , multivalent protein-binding domains undergo liquid–liquid demixing in concentrated solutions to form micron-sized droplets ( Li et al . , 2012 ) .", "Similarly , RNA-binding proteins that contain low sequence-complexity domains assemble into ordered fibers that coalesce into hydrogels when maintained at low temperature ( Frey et al . , 2006; Kato et al . , 2012 ) .", "The proteins that drive the phase transitions in vivo , however , are not known .", "Several proteins and RNAs are required for granule integrity ( Buchan , 2014 ) , and recently the kinase DYRK3 has been implicated in the regulation of stress granule dissolution and condensation ( Wippich et al . , 2013 ) .", "In the present study , we identify the substrates of the C . elegans DYRK3 homolog MBK-2 and demonstrate that phosphorylation and dephosphorylation of these substrates drive the dissolution and condensation of P granules in embryos .", "P granules are the germ granules of C . elegans ( Strome and Wood , 1982; Updike and Strome , 2010 ) .", "P granules are perinuclear during most of the germline development .", "During the oocyte-to-embryo transition , P granules detach from nuclei and disperse in the cytoplasm for asymmetric segregation to the nascent embryonic germline .", "Preferential dissolution of the granules in the anterior and condensation in the posterior of the zygote cause the granules to accumulate in the cytoplasm destined for the germline blastomere P1 ( Figure 1 ) ( Brangwynne et al . , 2009; Gallo et al . , 2010 ) .", "P granules segregate asymmetrically for three more divisions until the P granules are uniquely concentrated in the germline founder cell P4 ( Strome and Wood , 1983 ) .", "Two groups of RNA-binding proteins form the core components of P granules: the RGG domain proteins PGL-1 and PGL-3 and the Vasa-related helicases GLH-1 , 2 , 3 , and 4 ( Updike and Strome , 2010 ) .", "A self-association domain in the PGL family is essential for granule nucleation ( Hanazawa et al . , 2011 ) , and FG repeats in GLH-1 are required for P granules to associate with nuclei ( Updike et al . , 2011 ) . 10 . 7554/eLife . 04591 . 003Figure 1 . MBK-2 and PP2APPTR−1/2 are an opposing kinase/phosphatase pair .", "( A ) P granule dynamics during the oocyte to embryo transition .", "Green puncta are P granules , pale green color represents P granule components that diffuse into the cytoplasm .", "Orange represents pronuclei .", "P1 is the germline blastomere .", "( B ) Zygotes of the indicated genotypes expressing GFP::PGL-1 and mCherry::Histone 2B during pronuclear migration .", "Full genotypes are pptr-1 ( tm3103 ) , pptr-1 ( tm3103 ) pptr-2 ( RNAi ) , mbk-2 ( RNAi ) , and mbk-2 ( RNAi ) ;pptr-1 ( tm3103 ) pptr-2 ( RNAi ) .", "Right: working model .", "Phosphorylation by MBK-2 disassembles granules .", "Dephosphorylation by PP2APPTR−1/2 assembles granules .", "Each embryo is 50 μm in length .", "Anterior is to the left , posterior is to the right . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 00310 . 7554/eLife . 04591 . 004Figure 1—figure supplement 1 . Oocyte P granule disassembly during the oocyte-to-zygote transition . First four columns: time points from movies of eggs ( highlighted by dashed lines ) undergoing the oocyte-to-embryo transition ( top to bottom ) and expressing GFP::PGL-1 ( green ) and mCherry::H2B ( red ) .", "At time point zero , the oocytes are still in oviduct and not yet fertilized .", "At 24 min , fertilization has occurred and the zygotes are undergoing the first meiotic division .", "The oocyte granules have disassembled , except in meg-3 meg-4 zygotes .", "At 31 min , the zygotes are finishing the second meiotic division , and the zygote granules are forming .", "Zygote granule assembly is impaired in pptr-1 pptr-2 and meg-3 meg-4 zygotes .", "Full genotypes are: pptr-1 ( tm3103 ) pptr-2 ( RNAi ) , mbk-2 ( RNAi ) , and meg-3 ( RNAi ) meg-4 ( RNAi ) .", "Fifth column: time points from movies of eggs expressing mCherry::PGL-3 .", "Note that the top image was taken at 4:00 .", "All other images are time-matched to those taken in GFP::PGL-1 mCherry::H2B strains . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 004 In previous studies , we identified two potential regulators of P granule dynamics: MBK-2 , the C . elegans DYRK3 homolog ( Aranda et al . , 2011; Wippich et al . , 2013 ) and PPTR-1 , a regulatory subunit of the heterotrimeric phosphatase PP2A ( Padmanabhan et al . , 2009 ) .", "Loss of mbk-2 and pptr-1 has opposite effects on P granule dynamics .", "All P granules remain condensed in mbk-2 zygotes and all P granules disperse in pptr-1 zygotes ( Pellettieri et al . , 2003; Quintin et al . , 2003; Gallo et al . , 2010 ) .", "These phenotypes suggest that MBK-2 and PPTR-1 proteins may share substrates whose phosphorylation regulates the phase of P granules .", "To investigate this possibility , we searched for MBK-2 and PPTR-1 substrates and identified a group of intrinsically disordered proteins that regulate P granule dynamics ." ], [ "To determine the earliest time point at which P granules become dynamic during the oocyte-to-zygote transition , we monitored P granules in the oviduct and uteri of live hermaphrodites from ovulation through the first embryonic divisions .", "We used a GFP::PGL-1 transgene to mark P granules and an mCherry::histone transgene to mark chromosomes .", "Shortly after ovulation , P granules that were present in the oocyte cytoplasm ( oocyte granules ) disassemble and new granules ( zygote granules ) form throughout the cytoplasm as the maternal pronucleus completes the second meiotic division ( Video 1 , Figure 1A , Figure 1—figure supplement 1 ) .", "After meiosis , as the zygote becomes polarized along the anterior/posterior axis , granules in the anterior cytoplasm are quickly disassembled and granules in the posterior cytoplasm continue to grow and fuse to form large ( ≥1 micron ) granules ( Figure 1 , Video 2 ) .", "We observed the same dynamics with a mCherry fusion to a second P granule component ( PGL-3 ) ( Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 04591 . 005Video 1 . P granule dynamics during the oocyte-to-zygote transition . Time-lapse of eggs undergoing the oocyte-to-embryo transition in the gonad of a hermaphrodite expressing GFP::PGL-1 and mCherry::H2B .", "An oocyte in the oviduct , several sperm in the spermatheca , and a zygote in the uterus are highlighted in the first frame of the movie .", "Key stages in the P granule disassembly and assembly process are also highlighted in later frames .", "Images are maximum intensity projections of 11 z stacks separated by 1 µm steps .", "Stacks were taken every 30 s , total movie time is 30 min 30 s , movie is played back in 60× real time . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 00510 . 7554/eLife . 04591 . 006Video 2 . P granule dynamics in wild-type and meg-3 meg-4 zygotes . Time-lapse of embryos expressing GFP::PGL-1 and mCherry::H2B .", "Images are maximum intensity projections of 8 z planes separated by 1 μm steps .", "Stacks were taken every 8 s , total movie time is 17 min 52 s , movie is played back in 80× real time .", "Full genotype of mutant is meg-3 ( tm4259 ) meg-4 ( ax2026 ) .", "Very small granules form transiently in the posterior of the meg-3 meg-4 embryo at 7:20 .", "The bright red puncta in the anterior ( left ) side of the embryos are polar bodies .", "In the wild-type movie , there are also red puncta above the embryo , these are sperm outside of the egg shell . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 006 The kinase MBK-2 is required for P granule asymmetry in zygotes ( Pellettieri et al . , 2003; Quintin et al . , 2003 ) .", "We found that in zygotes derived from mothers lacking mbk-2 ( hereafter referred to as mbk-2 zygotes ) , oocyte granule disassembly and zygote granule assembly proceeded as in wild-type during meiosis ( Figure 1—figure supplement 1 ) .", "The zygote granules , however , failed to disassemble in the anterior cytoplasm during zygote polarization ( Figure 1B ) .", "PPTR-1 is a B′/B56 regulatory subunit of PP2A that is required for P granule maintenance during mitosis ( Gallo et al . , 2010 ) .", "In pptr-1 mutant zygotes , disassembly of oocyte granules and assembly of zygotic granules proceeded normally during meiosis .", "After meiosis , however , granules in the posterior did not grow ( Figure 1B ) and all granules eventually disassembled by the onset of mitosis , as previously reported ( Gallo et al . , 2010 ) .", "PPTR-1 is 58% identical at the amino acid level to PPTR-2 , another predicted B′/B56 regulatory subunit of PP2A ( WormBase; [Harris et al . , 2014] ) .", "To determine whether PPTR-2 might also regulate P granule dynamics , we compared zygotes derived from pptr-1 mothers and pptr-1 pptr-2 mothers .", "In pptr-1 pptr-2 , zygote granule assembly during meiosis was less robust and disassembly began even earlier during pronuclear migration ( Figure 1B and Figure 1—figure supplement 1 ) .", "pptr-2 single mutants appeared as wild-type ( data not shown ) .", "We conclude that pptr-1 and pptr-2 contribute redundantly to the assembly/stabilization of P granules in zygotes .", "Intriguingly , when we depleted mbk-2 in pptr-1 pptr-2 , we observed the same phenotype as in mbk-2: new P granules assembled in the zygote and remained stable through the first division ( Figure 1B ) .", "mbk-2 therefore is epistatic to pptr-1 pptr-2 , as might be expected in a ‘no phosphorylation’ scenario .", "These observations suggest that MBK-2 and PP2APPTR−1/2 work in opposition to promote P granule disassembly ( MBK-2 ) and re-assembly ( PP2APPTR−1/2 ) .", "One possibility is that MBK-2 and PP2APPTR−1/2 share substrates that , when phosphorylated , destabilize P granules in zygotes ( working model in Figure 1B ) .", "To identify potential substrates of PP2APPTR−1/PPTR−2 , we screened a LexA-PPTR-1 fusion against cDNAs from a mixed-stage C . elegans library fused to the GAL-4 activation domain .", "From 1 . 2 × 107 transformants , we obtained ∼4500 positive colonies .", "We sequenced plasmids from 111 colonies and identified 32 unique genes ( Figure 2—source data 1 ) .", "We tested each gene by RNAi ( 1 ) for defects in P granule distribution in wild-type embryos , or ( 2 ) for suppression of the pptr-1 hyper-granule disassembly phenotype and identified three genes: meg-1 , F52D2 . 4 , and C36C9 . 1 ( Figure 2—source data 1 ) .", "meg-1 ( and its paralog meg-2 ) was identified previously as a P granule component specific to early embryos ( Leacock and Reinke , 2008 ) .", "F52D2 . 4 was identified previously in a yeast two-hybrid screen as a gex-3 interactor and named gei-12 ( Tsuboi et al . , 2002 ) .", "Based on our findings ( see below ) , we have renamed this gene meg-3 .", "C36C9 . 1 is 71% identical to F52D2 . 4/meg-3 at the amino acid level , and we have named it meg-4 ( Figure 2 ) .", "meg-3 and meg-4 were identified previously as pptr-2 interactors in a genome-wide yeast two-hybrid screen ( Simonis et al . , 2009 ) .", "All four meg genes are X-linked and code for 70–95 . 8 kDa proteins that contain 11 . 8–14 . 3% serine and 7 . 2–9 . 6% asparagine residues and >60% residues predicted to be in disordered regions ( Figure 2 ) .", "meg-1/2 , however , are not homologous to meg-3/4 ( WormBase; [Harris et al . , 2014] ) . 10 . 7554/eLife . 04591 . 007Figure 2 . The MEG proteins contain sequences predicted to be intrinsically disordered .", "( A ) Graphs showing the disorder tendency of sequences along each protein calculated using IUPRED ( http://iupred . enzim . hu/ ) using ‘long disorder’ parameters ( Dosztányi et al . , 2005 ) .", "Scores above 0 . 5 indicate disorder .", "Number of predicted disordered residues: MEG-1: 420 of 636 aa ( 66% ) , MEG-2: 715 of 819 aa ( 87% ) , MEG-3: 542 of 862 aa ( 63% ) , MEG-4: 570 of 832 aa ( 69% ) , PPTR-1: 97 of 542 aa ( 18% ) .", "PPTR-1 is included here as an example of a mostly ordered protein .", "( B ) Lines below graph: regions of low complexity sequence as defined using SEG ( http://mendel . imp . ac . at/METHODS/seg . server . html ) ( Wootton , 1994 ) .", "Using the default SEG parameters ( SEG 12 2 . 2 2 . 5 ) as used in Kato et al . ( 2012 ) , there are no low complexity sequences greater than 36 residues in the MEG proteins .", "Using a larger window size ( SEG 45 3 . 4 3 . 75 ) , all MEGs have predicted low complexity regions as shown , but PPTR-1 does not .", "( C ) Protein sequence alignment of MEG-3 and MEG-4 , which are 71% identical to each other .", "Yellow highlight: serine and threonine residues .", "Red: predicted MBK-2 phosphorylation site mutated in Figure 3 kinase assay .", "Blue: antigen sequence of MEG-3 antibody .", "Orange highlight: [GS]Y[GS] tripeptide motif ( Kato et al . , 2012 ) .", "There is only 1 instance of this sequence in MEG-4 and none in MEG-3 .", "Red star: position of the frameshift in the meg-3 ( tm4259 ) and meg-4 ( ax2026 ) mutants .", "meg-3 ( tm4259 ) is a deletion of 623 nucleotides starting at nucleotide 543 ( amino acid 165 ) , followed by an insertion of ‘TACGA’ .", "The frameshift inserts the sequence ‘KQGRH’ at amino acid 165 followed by a premature stop .", "meg-4 ( ax2026 ) is a deletion of 7 nucleotides starting at nucleotide 37 ( amino acid 13 ) .", "The frameshift inserts the amino acids ‘EETEKVTALIVEAIWKVHRKIWDTTLVLKNCFIRS’ at amino acid 13 , followed by a premature stop .", "See Table 2 for full allele descriptions .", "( D ) Amino acid composition of indicated proteins .", "Basic residues are highlighted in yellow , acidic in blue .", "Red residues are overrepresented in MEG proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 00710 . 7554/eLife . 04591 . 008Figure 2—source data 1 . Candidates from yeast two-hybrid screen . DNA was extracted and sequenced from 111 colonies grown on –Trp–Leu–Ura–His plates .", "RNAi feeding vectors for each candidate were obtained from the Ahringer or OpenBiosystems RNAi banks , or if unavailable were PCR amplified from genomic DNA and cloned into pL4440 .", "*meg-3 ( RNAi ) also knocks out meg-4 and vice-versa .", "+ in the pptr-1 suppression column means that the RNAi treatment restores P granules in P blastomeres .", "Pleiotropic indicates a P granule phenotype accompanied by additional cellular defects .", "Entries in red are the MEG proteins , which uniquely affect P granules in the zygote stage ( meg-3 and meg-4 ) or suppress the pptr-1 phenotype ( meg-1 ) without inducing other phenotypes .", "Other phenotypes were as follows: emb = embryonic lethal , dpy = dumpy , egl = egg laying defect . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 008 To test that the MEG-1 , MEG-3 , and MEG-4 proteins interact directly with PPTR-1 and PPTR-2 , we performed GST pull down assays ( Figure 3A ) .", "We found that all three bound to PPTR-1 and/or PPTR-2 but not to maltose-binding protein ( MBP ) or to PAA-1 , the scaffolding subunit of PP2A .", "MEG-1 , MEG-3 , and MEG-4 also interacted with the P granule component PGL-1 ( Figure 3A ) . 10 . 7554/eLife . 04591 . 009Figure 3 . MEG-1 and MEG-3 are substrates of MBK-2 and PP2APPTR−1/2 . ( A ) Westerns of E . coli lysates expressing the indicated MBP fusions before ( input ) and after immobilization on columns containing the indicated GST fusions .", "MBP and PAA-1 are negative controls .", "PAA-1 is the scaffolding subunit of PP2A .", "Dashed lines separate individual gels .", "( B ) In vitro kinase assay .", "MBP fused to wild-type or kinase-dead ( KD ) MBK-2 was incubated with gamma32P-ATP and MBP-fused substrates: MEI-1 ( 94 kDa ) ; MEI-1 ( S92A ) ( 94 kDa ) ; MEG-3 ( 138 kDa with degradation band at ∼100 kDa ) ; MEG-3 ( T541A S582A T605A ) ( 138 kDa with degradation band at ∼100 kDa ) ; MEG-1 ( 112 kDa ) ; MEG-1 ( S574A ) ( 112 kDa ) .", "MBP::MBK-2 is 99 kDa and autophosphorylates .", "MEI-1 is a previously known substrate of MBK-2 ( Stitzel et al . , 2006 ) .", "Coomassie staining to control for loading is shown below .", "Phosphorylation is diminished in MEG-3 ( T541A S582A T605A ) ( 64% of wild-type phosphorylation ) and MEG-1 ( S574A ) ( 88% of wild-type phosphorylation ) .", "( C ) Anti-GFP westerns of C . elegans embryonic lysates run on SDS-PAGE gels with ( top ) or without Phos-tag ( bottom ) .", "Wild-type , pptr-1 ( tm3103 ) , and mbk-2 ( RNAi ) embryonic lysates expressing GFP::MEG-3 or wild-type , pptr-1 ( RNAi ) , and mbk-2 ( RNAi ) embryonic lysates expressing GFP::MEG-1 were treated with or without alkaline phosphatase ( AP ) and equal amounts were loaded on gels with or without Phos-tag .", "Dashed lines separate individual gels .", "Loading control was protein run on SDS-PAGE without Phos-tag .", "( D ) Graphs showing % unphosphorylated protein relative to wild-type in four Phos-tag experimental replicates .", "% unphosphorylated was calculated as the ratio of the intensity of the band corresponding to unphosphorylated protein on a Phos-tag gel over the intensity of the band on a non-Phos-tag gel ( total protein ) and normalized to wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 00910 . 7554/eLife . 04591 . 010Figure 3—figure supplement 1 . MEG-1 and MEG-3 are phosphorylated in vivo . Anti-GFP western of embryonic lysates expressing GFP::MEG-1 or GFP::MEG-3 treated with increasing amounts of alkaline phosphatase and run on Phos-tag SDS-PAGE ( top ) and SDS-PAGE without Phos-tag ( bottom ) .", "Equivalent amounts of total cell lysates were loaded in each lane .", "Top arrow indicates position of phosphorylated band , bottom arrow indicates position of unphosphorylated band .", "Asterisk indicates alkaline phosphatase , which is detected to varying extents using these Western blotting conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 010 To test whether the MEGs are also MBK-2 substrates , we expressed MEG-1 and MEG-3 as recombinant MBP fusions in Escherichia coli and performed in vitro kinase assays with recombinant MBK-2 as in Cheng et al . ( 2009 ) .", "MBK-2 could phosphorylate MBP::MEG-1 and MBP::MEG-3 , but not MBP alone ( Figure 3B ) .", "Using the DYRK consensus site K/R X1–3 S/T P ( Himpel et al . , 2000; Campbell and Proud , 2002 ) , we identified putative consensus sites in MEG-1 ( S574 ) and MEG-3 ( T541 , S582 , T605 ) .", "S574 in MEG-1 is reported as a phosphorylated site in Phosida ( Gnad et al . , 2007 ) .", "We mutated each consensus site to alanine and repeated the kinase assays .", "Phosphorylation was reduced but not eliminated in the alanine mutants ( Figure 3B ) .", "We conclude that recombinant MBK-2 phosphorylates MEG-1 and MEG-3 on consensus sites in vitro , as well as other sites that remain to be determined .", "To examine whether MEG-1 and MEG-3 are phosphoproteins in vivo , we ran lysates from embryos expressing GFP::MEG-1 or GFP::MEG-3 on Phos-tag SDS-PAGE .", "Phos-tag retards the migration of phosphorylated proteins ( Kinoshita et al . , 2006 ) .", "We detected one primary species for both GFP::MEG-1 and GFP::MEG-3 and several higher molecular weight species that did not resolve well into specific bands .", "Treatment with alkaline phosphatase eliminated the higher molecular weight species and increased the intensity of the lower species ( unphosphorylated isoform , Figure 3C , Figure 3—figure supplement 1 ) .", "The relative abundance of the unphosphorylated isoform increased in lysates depleted of mbk-2 and decreased in lysates depleted of pptr-1 ( Figure 3C , D ) .", "We conclude that MEG-1 and MEG-3 are phosphoproteins in vivo and that MBK-2 and PPTR-1 promote and antagonize , respectively , their phosphorylation .", "Under all conditions , GFP::MEG-1 and GFP::MEG-3 still migrated as a mixture of phosphorylated and unphosphorylated isoforms .", "Because our assays were done in lysates that were depleted for MBK-2 by RNAi , and lysates that lacked PPTR-1 but retained PPTR-2 , we do not know whether MBK-2 and PP2APPTR−1/2 are the only regulators of MEG-1 and MEG-3 phosphorylation or whether other kinases and phosphatases are also involved .", "To determine the loss of function phenotype of meg-3 , we obtained a deletion allele of meg-3 from the National Bioresource Project ( Mitani , 2009 ) .", "tm4259 is an out-of-frame , 623-nucleotide deletion that creates a frameshift at amino acid 165 followed by premature stop at amino acid 170 and therefore is a likely null ( Figure 2 ) .", "Since RNA-mediated interference experiments suggested that meg-3 and meg-4 function redundantly ( Figure 4—figure supplement 1 ) , we generated a meg-4 allele in the meg-3 ( tm4259 ) background by non-homologous end joining repair of CRISPR/Cas9-induced cuts in meg-4 ( Paix et al . , 2014 ) .", "meg-4 ( ax2026 ) is a 7 bp deletion which causes a frameshift at amino acid 13 , followed by a premature stop at amino acid 48 ( Figure 2 ) .", "We also generated a second meg-4 allele in the wild-type background by non-homologous end joining repair of two CRISPR/Cas9 cuts flanking the meg-4 ORF ( Paix et al . , 2014 ) .", "meg-4 ( ax2081 ) is a 3 . 2 kb deletion that removes 733 bases upstream of the meg-4 ATG and 2565 bases of the meg-4 coding sequence ( total length: 2589 bases ) .", "To determine whether meg-3 and meg-4 are required for P granule dynamics , we crossed in GFP::PGL-1 and mCherry::histone transgenes to analyze P granule dynamics in the meg-3 , meg-4 single and double mutants ( Figure 4 ) .", "We observed the strongest phenotype in the double mutant .", "First , we noticed that disassembly of oocyte granules was incomplete in meg-3 meg-4 zygotes ( Video 2 , Figure 1—figure supplement 1 ) , causing a few oocyte granules to persist through the first mitotic division ( arrows in Figure 4A ) .", "During mitosis , a few new granules appeared in the posterior cytoplasm ( Figure 4A ) , but these remained small ( <1 micron ) and most were not maintained during the division ( Video 2 ) .", "As a result , the total number of granules ( including oocyte granules ) in meg-3 meg-4 zygotes in mitosis was ∼11% that of wild-type ( Figure 4B ) .", "In the 2-cell stage , new small granules formed again transiently in P1 , but again these were fewer and smaller than in wild-type ( Figure 4A ) .", "This pattern was repeated at each division such that by the 28-cell stage , only a few scattered granules were observed throughout the embryo , with no detectable enrichment in the germline founder cell P4 ( Figure 4A ) .", "We observed the same P granule dynamics when staining meg-3 meg-4 zygotes with an antibody against another P granule component , the Vasa-homolog GLH-2 ( Figure 4—figure supplement 1 ) .", "meg-3 meg-4 zygotes showed similar levels of overall GFP:PGL-1 fluorescence compared to wild-type and similar levels of PGL-3 by western blot analysis ( Figure 4—figure supplement 5 ) .", "We conclude that the loss of meg-3 and meg-4 causes a dramatic reduction in the number of P granules but does not affect PGL-1 and PGL-3 levels significantly . 10 . 7554/eLife . 04591 . 011Figure 4 . meg-1 , meg-3 , and meg-4 regulate P granule dynamics .", "( A ) Live embryos of the indicated genotypes and stages expressing GFP::PGL-1 ( green ) and mCherry::H2B ( red ) or GFP::PGL-1 only ( last two ) .", "Arrows in meg-3 meg-4 point to oocyte granules .", "For pptr-1 zygotes , the number of zygotes with visible posterior P granules is indicated .", "P0 is 1-cell stage , P1 is 2-cell stage , P4 is 28-cell stage .", "( B ) Number of GFP::PGL-1 granules in zygotes in anaphase of the first mitosis .", "Each dot represents a different zygote , and mean and standard deviation are shown .", "Asterisks indicate data that are statistically significantly different from wild-type ( one asterisk: p < 0 . 005 , three asterisks: p < 0 . 0001 ) .", "( C ) Percent sterility of adult hermaphrodites of the indicated genotypes grown at 20°C .", "Number of hermaphrodites scored ( n ) is written above the x axis .", "( D ) Primordial germ cells in the L1 larval stage expressing GFP::PGL-1 .", "By this stage , PGCs express P granule components ( Kawasaki et al . , 2004 ) .", "Perinuclear P granules form in all three genotypes .", "GFP::PGL-levels are lower in the meg mutants due to the lack of P granules inherited from the early embryonic stages .", "Full genotypes are meg-1 ( vr10 ) , meg-1 ( vr10 ) meg-2 ( RNAi ) , meg-3 ( tm4259 ) , meg-1 ( vr10 ) meg-3 ( tm4259 ) , meg-4 ( ax2081 ) , meg-3 ( tm4259 ) meg-4 ( ax2026 ) , pptr-1 ( tm3103 ) , pptr-1 ( tm3103 ) ;meg-1 ( vr10 ) , pptr-1 ( tm3103 ) ;meg-3 ( tm4259 ) , mbk-2 ( pk1427 ) and mbk-2 ( pk1427 ) ; meg-3 ( RNAi ) meg-4 ( RNAi ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01110 . 7554/eLife . 04591 . 012Figure 4—figure supplement 1 . meg-3 and meg-4 are required redundantly for P granule assembly in zygote . 2-cell embryos stained for the P granule component GLH-2 ( Gruidl et al . , 1996 ) .", "meg-3 ( tm4259 ) hermaphrodites were treated with meg-4 or F52D2 . 12 RNAi .", "The RNAi constructs were designed to target regions in meg-4 and F52D2 . 12 corresponding to the region deleted in meg-3 ( tm4259 ) to avoid any possible cross-silencing .", "Genotypes are meg-3 ( tm4259 ) , meg-3 ( tm4259 ) meg-4 ( RNAi ) , and meg-3 ( tm4259 ) F52D2 . 12 ( RNAi ) .", "F52D2 . 12 is a more distantly related homolog of meg-3 and meg-4 ( 48% amino acid identity between meg-3 and F52D2 . 12 , compared to 71% amino acid identity between meg-3 and meg-4 ) .", "No phenotype was observed in meg-3 ( tm4259 ) F52D2 . 12 ( RNAi ) nor in wild-type animals treated with meg-4 ( RNAi ) or F52D2 . 12 ( RNAi ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01210 . 7554/eLife . 04591 . 013Figure 4—figure supplement 2 . Comparison of wild-type and meg-3 meg-4 mutants .", "( A ) 28-cell stage embryos hybridized to a probe for nos-2 RNA .", "In wild-type embryos , nos-2 is enriched in the P blastomere ( white outline ) .", "In meg3 meg-4 , nos-2 RNA is segregated equally to the P blastomere ( white outline ) and its sister somatic blastomere ( blue outline ) .", "Older somatic blastomeres have already turned over nos-2 RNA .", "( B ) Live zygotes expressing GFP::PATR-1 , a marker for P bodies .", "Assembly of P bodies is not affected in meg-3 ( RNAi ) meg-4 ( RNAi ) zygotes .", "( C ) Live zygotes expressing GFP::PIE-1 and mCherry::MEX-5 .", "Localizations are indistinguishable , except for the lack of PIE-1 on granules in meg-3 ( RNAi ) meg-4 ( RNAi ) zygotes .", "( D ) L4 larvae expressing GFP::PGL-1 and mCherry::H2B .", "The germline is highlighted in white dashed lines .", "9 of 10 meg-3 meg-4 L4 larvae developed a full germline , as in wild-type .", "The remaining one meg-3 meg-4 L4 larva only contained 4 germ cells . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01310 . 7554/eLife . 04591 . 014Figure 4—figure supplement 3 . meg-1 meg-2 embryos exhibit defects in P granule disassembly in 2-cell and later embryos leading to missegregation of P granules to somatic blastomeres .", "( A ) Time points from movies of a wild-type and a meg-1 ( RNAi ) meg-2 ( RNAi ) embryo expressing GFP::PGL-1 and mCherry::H2B .", "The embryos are precisely aged-matched .", "White dashed lines outline the P blastomeres P1 ( 2-cell stage ) and P2 ( 3-cell and later stages ) , blue dashed lines outline the somatic blastomere EMS .", "In wild-type , P granules are disassembled in the anterior cytoplasm of P1 and segregate only to P2 .", "In meg-1 meg-2 , P granule disassembly is defective in P1 , resulting in some P granules segregating into EMS .", "These ectopic P granules are disassembled by the late 4-cell stage .", "( B ) Table showing the number of 4-cell and later stage embryos with missegregated P granules in meg-1 ( vr10 ) and meg-1 ( vr10 ) meg-2 ( RNAi ) .", "( C ) Live meg-3 ( tm4259 ) meg-4 ( ax2026 ) and meg-1 ( vr10 ) meg-2 ( RNAi ) meg-3 ( tm4259 ) meg-4 ( RNAi ) zygotes expressing GFP::PGL-1 and mCherry::H2B .", "Phenotypes are indistinguishable . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01410 . 7554/eLife . 04591 . 015Figure 4—figure supplement 4 . MEG proteins are required for germ cell development .", "( A ) Nomarski images of live L3 larvae .", "Worms were staged according to vulval development .", "Germline is outlined .", "10 of 13 meg-1 meg-3 animals had wild-type germlines as shown , and 3 of 13 had underdeveloped germlines similar to meg-1 meg-3 meg-4 .", "Full genotypes are: meg-3 ( tm4259 ) meg-4 ( ax2026 ) and meg-1 ( vr10 ) meg-3 ( tm4259 ) meg-4 ( RNAi ) .", "( B ) Graph showing the total number of germ cells in wild-type and meg-1 ( vr10 ) meg-3 ( tm4259 ) meg-4 ( RNAi ) L4 larvae .", "Each dot represents a single larva .", "Mean and standard deviation are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01510 . 7554/eLife . 04591 . 016Figure 4—figure supplement 5 . meg-3 meg-4 do not affect PGL-1 and PGL-3 levels significantly .", "( A ) Western blot for endogenous PGL-3 ( detected using KT3 antibody ) and tubulin in wild-type ( N2 ) embryos and meg-3 ( tm4259 ) meg-4 ( RNAi ) embryos .", "( B ) Total GFP::PGL-1 fluorescence in wildtype and meg-3 ( tm4259 ) meg-4 ( ax2026 ) zygotes .", "Difference between wildtype and meg-3 meg-4 is not statistically significant ( p = 0 . 3 ) .", "Mean and standard deviation are shown .", "( C ) Percent GFP::PGL-1 fluorescence present in granules in wild-type and meg-3 ( tm4259 ) meg-4 ( ax2026 ) zygotes at mitosis .", "Mean and standard deviation are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 016 meg-3 single mutants exhibited the same phenotypes as the meg-3 meg-4 double mutant but with lower expressivity .", "In meg-3 zygotes , oocyte granule disassembly was delayed but was eventually completed before the first division ( Video 3 ) .", "Zygote granule assembly was also impaired , but less than in meg-3 meg-4 zygotes: we observed a 30% reduction in the number of P granules in meg-3 zygotes compared to the 89% reduction for meg-3 meg-4 zygotes ( Figure 4B ) .", "We also observed only a slight reduction in P granule number in meg-4 zygotes ( Figure 4B ) .", "We conclude that meg-3 and meg-4 contribute redundantly to the assembly of zygote granules , with meg-3 providing the greater contribution . 10 . 7554/eLife . 04591 . 017Video 3 . P granule dynamics in wild-type and meg-3 . Time-lapse of embryos expressing GFP::PGL-1 and mCherry::H2B .", "Wild-type embryo is same one as shown in Video 2 .", "Images are maximum intensity projections of 8 z planes separated by 1 μm steps .", "Stacks were taken every 8 s , total movie time is 17 min 20 s , movie is played back in 80× real time .", "Full genotype of mutant is meg-3 ( tm4259 ) .", "The bright red puncta in the anterior ( left ) side of the embryos are polar bodies .", "In the wild-type movie , there are also red puncta above the embryo , these are sperm outside of the egg shell . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 017 We next tested whether RNA components of P granules are affected in meg-3 meg-4 embryos .", "In wild-type , during each asymmetric division of the germline P blastomeres , the P granule-associated RNA nos-2 segregates preferentially with the P granules ( asymmetric segregation ) ( Subramaniam and Seydoux , 1999 ) .", "The lower levels inherited by the somatic daughter are turned over after division ( degradation ) .", "In meg-3 meg-4 zygotes , we found that nos-2 RNA was distributed equally to P blastomeres and their somatic sisters , consistent with the lack of stable P granules .", "nos-2 RNA , however , was still quickly degraded in the somatic blastomeres after division as in wild-type ( Figure 4—figure supplement 2A ) .", "Thus , as previously reported for pptr-1 mutants ( Gallo et al . , 2010 ) , despite symmetric segregation of nos-2 RNA , an inherent asymmetry between somatic and germline blastomeres in meg-3 meg-4 embryos causes nos-2 RNA to be maintained only in the germline blastomeres .", "Loss of meg-3 and meg-4 also did not affect the formation of P bodies ( Figure 4—figure supplement 2B ) nor the distribution of asymmetric proteins that segregate independently of P granules ( PIE-1 and MEX-5 ) ( Figure 4—figure supplement 2C ) .", "By the L1 larval stage , when new P granule components are synthesized in the PGCs ( Kawasaki et al . , 2004 ) , low levels of perinuclear P granules became visible in the primordial germ cells of meg-3 meg-4 double mutants ( Figure 4D ) .", "By the L4 larval stage , P granule levels were indistinguishable from wild-type in fertile meg-3 meg-4 mutants ( Figure 4—figure supplement 2D ) .", "We conclude that MEG-3 and MEG-4 are only required for P granule assembly in pre-gastrulation embryos and are not required for other soma/germline asymmetries , or for the assembly of P bodies or perinuclear P granules .", "To determine the role of meg-1 and meg-2 in P granule assembly and disassembly , we again used the GFP::PGL-1 and mCherry::H2B transgenes to follow granule dynamics live .", "Leacock and Reinke ( 2008 ) reported that meg-1 and meg-1 meg-2 mutants assemble P granules , but occasionally missegregate P granules to somatic blastomeres in 4-cell and later stage embryos .", "Consistent with those findings , we observed only a modest reduction in the number of P granules in meg-1 meg-2 zygotes ( Figure 4A , B ) .", "In 2-cell stage meg-1 mutants , P granules failed to disassemble in the anterior cytoplasm of the P1 blastomere ( Figure 4A ) , causing a few P granules to be inherited by the EMS blastomere ( Figure 4—figure supplement 3A ) .", "meg-2 ( RNAi ) on meg-1 mutants enhanced the percent of P granule missegregation to somatic blastomeres ( Figure 4—figure supplement 3B ) , as reported previously ( Leacock and Reinke , 2008 ) .", "We conclude that , unlike meg-3 and meg-4 , meg-1 and meg-2 contribute only weakly to granule assembly and contribute primarily to granule disassembly .", "To determine whether the meg genes act redundantly in granule assembly , we compared double , triple , and quadruple loss-of-function zygotes , combining mutations and RNAi ( see Figure 4 legend for complete genotypes ) .", "We found that the P granule assembly defects observed in meg-3 and meg-3 meg-4 zygotes were not affected by the additional loss of meg-1 or meg-1 and meg-2 ( Figure 4A and Figure 4—figure supplement 3C ) .", "In the L1 stage , we observed low levels of perinuclear P granules in the PGCs of meg-1 meg-3 meg-4 triple mutants as seen in meg-3 meg-4 double mutants ( Figure 4D ) .", "We conclude that meg-3 and meg-4 are the primary contributors to granule assembly in early embryos , and that none of the megs are required for the assembly of perinuclear granules in PGCs later in development .", "meg-1 meg-2 double mutants are 100% sterile ( Leacock and Reinke , 2008 ) .", "To examine whether other meg mutant combinations are also sterile , we determined the percent of adult animals with empty uteri ( sterile animals ) .", "We found that 27% of meg-3 meg-4 mutants and 100% of meg-1 meg-3 meg-4 mutants were sterile ( Figure 4C ) .", "In wild-type animals , the two PGCs begin to divide in the first larval stage ( L1 ) to generate >1000 germ cells by adulthood .", "In contrast , we observed <10 germ cells in meg-1 meg-3 meg-4 larvae and no further growth by the adult stage , despite the presence of a somatic gonad ( Figure 4—figure supplement 4 ) .", "This phenotype is identical to that reported for meg-1 meg-2 mutants ( Leacock and Reinke , 2008 ) .", "We conclude that meg-1 , meg-2 , meg-3 , and meg-4 contribute redundantly to germ cell proliferation during larval development .", "To test whether the meg genes are epistatic to pptr-1 and mbk-2 , we examined the effect of loss of meg-1 and meg-3 in pptr-1 and mbk-2 zygotes .", "In pptr-1 mutants , all P granules disassemble during mitosis .", "If this defect were due to hyper-phosphorylation of a PPTR-1 target , then elimination of that target might suppress the pptr-1 phenotype and restore some P granules .", "Consistent with this possibility , we found that the loss of meg-1 suppressed the granule hyper-disassembly phenotype of pptr-1 zygotes ( Figure 4A ) .", "Interestingly , the loss of meg-3 also partially suppressed pptr-1 ( Figure 4A ) , indicating that meg-3 also contributes to disassembly .", "In mbk-2 mutants , P granule disassembly does not occur during mitosis .", "We found that in mbk-2 , meg-3 meg-4 zygotes , P granules failed to assemble as is observed in meg-3 meg-4 zygotes ( Figure 4A ) .", "This finding indicates that meg-3 and meg-4 are essential for granule assembly even when disassembly is inhibited .", "Together with the finding that mbk-2 is epistatic to pptr-1/2 ( Figure 1 ) , these genetic interactions are consistent with the meg genes functioning downstream of mbk-2 and pptr-1/2 , to regulate the balance of granule assembly and disassembly in embryos .", "The experiments above suggest a direct role for the MEGs in disassembling and assembling P granules .", "MEG-1 was previously reported to localize to embryonic P granules from the 4-cell stage to the 100-cell stage ( Leacock and Reinke , 2008 ) .", "To determine the localization of MEG-3 , we generated a rescuing GFP-tagged transgene and a polyclonal serum raised against a MEG-3 peptide ( ‘Materials and methods’ and Figure 5—figure supplement 1 ) .", "For MEG-4 , we inserted a 3× FLAG tag at the carboxy terminus of the meg-4 open reading frame by homology dependent repair of a CRISPR/Cas9-induced cut ( Paix et al . , 2014 ) .", "We found that both MEG-3 and MEG-4 localize to P granules from the 1-cell stage to ∼100-cell stage ( Figure 5 and Figure 5—figure supplement 2A ) .", "After the birth of the primordial germ cells Z2/Z3 ( 100-cell stage ) , when P granules are fully perinuclear and no longer dynamic , MEG-3 and MEG-4 levels quickly faded ( Figure 5 , Figure 5—figure supplement 2 and data not shown ) . 10 . 7554/eLife . 04591 . 018Figure 5 . MEG-3 localizes to a dynamic domain that surrounds and penetrates the P granules .", "( A ) Gonad of an adult hermaphrodite expressing the GFP::MEG-3 transgene under the control of the meg-3 promoter and 3′ UTR .", "GFP::MEG-3 associates strongly with P granules only in embryos .", "( B ) Fixed wild-type zygote stained with anti:MEG-3 and anti-PGL-1 sera .", "Inset: magnification of merged image .", "Arrow points to a MEG-3-positive/PGL-1-negative granule .", "( C ) Live wild-type and pgl-1 ( RNAi ) ;pgl-3 ( bn104 ) zygotes expressing GFP::MEG-3 .", "GFP::MEG-3 localizes to a cytoplasmic gradient and P granules .", "P granule localization is lost in pgl-1 ( RNAi ) ;pgl-3 ( bn104 ) zygotes , but the cytoplasmic gradient remains .", "( D ) Still images from a movie acquired using lattice light sheet microscopy ( Video 5 ) .", "Max intensity projection of a Z stack through a pair of fusing granules .", "Time in seconds ( Video", "5 ) is indicated above each panel .", "GFP::MEG-3 and mCherry:PGL-3 domains are not completely co-localized .", "Also see Figure 5—figure supplement 3B .", "Resolution is 238 nm × 238 nm × 500 nm .", "Scale bars: upper left = 500 nm .", "( E ) Lattice light sheet 3D-SIM mode reconstruction of GFP::MEG-3 in P granules in a living zygote ( also see Video 6 ) .", "Scale bars: upper left = 500 nm .", "First granule on the left: acquisition time is 1 s ( Video 6 ) .", "Subsequent granules: acquisition time is 1 . 7 s ( Video 7 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01810 . 7554/eLife . 04591 . 019Figure 5—figure supplement 1 . GFP::MEG-3 rescues meg-3 mutant and the MEG-3 antibody is specific .", "( A ) Fixed zygotes stained for P granules ( K76 antibody , red ) .", "Note the oocyte granules ( arrows ) in meg-3 ( tm4259 ) which are absent in the rescued strain meg-3 ( tm4259 ) ;GFP::MEG-3 .", "At least 20 mothers were stained for each genotype and these images are representative .", "We could not test whether GFP::MEG-3 also rescues the P granule assembly defect of meg-3 ( tm4259 ) mutants , since this defect can only be scored using GFP::PGL-1 ( antibody staining of P granules is not reliable enough for accurate counts at this stage ) .", "( B ) Fixed meg-3 ( tm4259 ) zygote stained for P granules ( K76 antibody , red ) and with anti-MEG-3 serum .", "Anti-MEG-3 serum does not stain granules in meg-3 ( tm4259 ) embryos , but does so in wild-type ( Figure 5B ) , demonstrating specificity of the serum . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 01910 . 7554/eLife . 04591 . 020Figure 5—figure supplement 2 . MEG-4 localizes to P granules and MEG-3 and MEG-4 assemblies persist longer in disassembling granules than PGL-1 . ( A ) Embryos expressing MEG-4::FLAG ( endogenous locus ) and stained with anti-FLAG antibody .", "( B ) Top row: maximum projection image of embryo expressing MEG-4::FLAG ( endogenous locus ) and stained with anti-FLAG antibody and anti-PGL-1 ( K76 ) antibody .", "Note the MEG-4:FLAG-positive/PGL-1 negative granules in the anterior part of the cell ( where P granules disassemble ) .", "Bottom row: maximum projection of image of wild-type embryo ( no MEG-4::FLAG ) stained with anti-FLAG antibody and anti-PGL-1 ( K76 ) antibody .", "Note the lack of FLAG staining confirming the specificity of the FLAG antibody .", "( C ) Time-lapse of a disassembling P granule from an embryo expressing GFP::MEG-3 and mCherry::PGL-3 granule .", "During granule disassembly , GFP::MEG-3 persists for longer than mCherry::PGL-3 .", "Also see Video 4 .", "( D ) Zygotes expressing GFP::MEG-3 imaged at mitosis .", "Genotypes are: pptr-1 ( RNAi ) pptr-2 ( RNAi ) and mbk-2 ( RNAi ) .", "Note the perduring GFP::MEG-3 clusters in the pptr-1pptr-2 zygotes which disassemble all PGL clusters by this stage ( Figure 4A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 02010 . 7554/eLife . 04591 . 021Figure 5—figure supplement 3 . MEG-3 and PGL-1/3 overlap only partially in P granules .", "( A ) Confocal images of two P granules in a fixed zygote expressing GFP::MEG-3 .", "Endogenous PGL-1 is visualized using the K76 antibody .", "In all fixed preparations , GFP::MEG-3 never co-localized perfectly with PGL-1 , but the appearance of the PGL-1 signal varied between experimental replicates .", "( B ) Single plane images of GFP::MEG-3 and mCherry::PGL-3 in fused granule is also shown in Figure 5D .", "Time points are indicated above in seconds .", "Four consecutive z planes are shown with 0 . 3 μm z steps .", "( C ) Graph plotting area of mCherry::PGL-3 signal ( x axis ) vs area of GFP::MEG-3 signal ( y axis ) for 37 granules from two embryos visualized by lattice light sheet microscopy ( dithered mode ) .", "Each dot represents an individual granule .", "Diagonal lines indicate 1× and 1 . 5× fold increase of GFP::MEG-3 area over mCherry::PGL-3 area .", "In 34 of 37 granules , the GFP::MEG-3 signal occupied a larger area than the mCherry::PGL-3 signal ( 1 . 57× increase on average ) .", "The area occupied by the mCherry::PGL-3 signal was always entirely contained within the area occupied by the GFP::MEG-3 signal ( 37/37 granules analyzed ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 02110 . 7554/eLife . 04591 . 022Figure 5—figure supplement 4 . MEG-1 only partially co-localizes with PGL-1 . Fixed embryos imaged using confocal spinning disk microscopy .", "Top: endogenous MEG-1 antibody ( green ) , K76 ( anti-PGL-1 , red ) .", "Middle: K76 ( anti-PGL-1 , green ) , GLH-2 ( red ) .", "Bottom: GFP::MEG-3 ( green ) , endogenous MEG-1 antibody ( red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 02210 . 7554/eLife . 04591 . 023Figure 5—figure supplement 5 . Lattice light sheet microscopy identifies sub-granular MEG-3 domain .", "( A and B )", "Single plane images of granules from live embryos expressing GFP::MEG-3 imaged by lattice light sheet 3D-SIM .", "Granule in B is the same as in Video 6 and Figure 5E .", "Top row: each structured illumination phase is shown .", "Bottom row , left: maximum intensity image of the raw phase data .", "Bottom row , right: image after reconstruction .", "Note that the structure seen in the reconstructed images is present in the raw maximum images .", "( C ) Top: maximum projections of lattice light sheet images of GFP::MEG-3 and mCherry::PGL-3 granules .", "MEG-3 sub-granular structure is preserved over successive frames .", "Bottom: maximum projections of lattice light sheet images of cytoplasmic GFP::MEG-3 and mCherry::PGL-3 .", "Image contrast was enhanced to the same extent for both channels .", "mCherry::PGL-3 is not visible in the cytoplasm under these conditions .", "In contrast , GFP::MEG-3 is easily detected and is distributed non-uniformly .", "The partial pattern repetition seen in the three successive time points and the high signal-to-noise ratio ( D ) suggest that the cytoplasmic MEG-3 pattern represents true structure and not noise .", "( D ) Photon counts and signal to noise for each movie . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 023 We did not detect MEG-3 or MEG-4 in the perinuclear P granules of adult gonads ( Figure 5A for MEG-3 and data not shown ) .", "We detected GFP::MEG-3 in the cytoplasm of immature germ cells and oocytes ( Figure 5A ) .", "In zygotes , cytoplasmic GFP::MEG-3 formed an anterior-to-posterior gradient with highest levels in the posterior ( Figure 5C ) .", "In embryos depleted for pgl-1 and pgl-3 , which do not form P granules ( Hanazawa et al . , 2011 ) , GFP::MEG-3 was still present in an anterior-to-posterior gradient but no longer localized to large granules ( Figure 5C ) .", "We conclude that MEG-3 and MEG-4 are maternally provided proteins that segregate with the P lineage and associate with P granules during the embryonic period where P granules are most dynamic .", "Live-imaging of zygotes expressing GFP::MEG-3 and mCherry::PGL-3 revealed that , during granule disassembly , GFP::MEG-3 perdures longer in the granules than mCherry::PGL-3 ( Video 4 and Figure 5—figure supplement 2C ) .", "Consistent with that observation , in fixed zygotes , we observed MEG-4-positive/PGL-1-negative and MEG-3-positive/PGL-1-negative granules at the anterior most edge of the P granule domain ( Figure 5B and Figure 5—figure supplement 2B ) .", "We also observed GFP::MEG-3-positive/PGL-1-negative granules in pptr-1 zygotes where disassembly is enhanced ( Figure 5—figure supplement 2D ) .", "We conclude that MEG-3/4 and PGL-1 exhibit different dynamics during disassembly . 10 . 7554/eLife . 04591 . 024Video 4 . MEG-3 and PGL-3 dynamics in the anterior of P1 cell . Time-lapse of embryos expressing GFP::MEG-3 and mCherry::PGL-3 .", "Images are maximum intensity projections of 8 z planes separated by 1 μm steps .", "Stacks were taken every 8 s , total movie time is 9 min 12 s , movie is played back in 80× real time . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 024 Consistent with the different MEG and PGL dynamics , immunostaining experiments in fixed embryos suggested that MEG-1 and MEG-3 do not co-localize precisely with PGL-1 in P granules ( Figure 5—figure supplement 3A and Figure 5—figure supplement 4 ) .", "To avoid potential artifacts from fixation , we turned to lattice light sheet microscopy , a new method that uses ultra-thin light sheets derived from optical lattices to image 3D volumes with high temporal and spatial resolution ( Chen et al . , 2014 ) .", "Imaging of live zygotes co-expressing GFP::MEG-3 and mCherry:PGL-3 revealed that both were non-uniformly distributed and constantly rearranged within the granules ( Figure 5D , Video 5 ) .", "The GFP::MEG-3 and mCherry::PGL-3 signals overlapped but were never perfectly coincident , even during granule fusion ( Figure 5D , Figure 5—figure supplement 3B ) .", "In 34 of 37 granules analyzed , the GFP::MEG-3 signal extended over a larger area than the mCherry::PGL-3 signal ( Figure 5—figure supplement 3C ) , with GFP::MEG-3 extending further out at the periphery of each granule compared to mCherry::PGL-3 . 10 . 7554/eLife . 04591 . 025Video 5 . Lattice light sheet movie of an embryo expressing GFP::MEG-3 and mCherry::PGL-3 . Time-lapse of the posterior cytoplasm of a zygote ( anterior to the left , posterior to the right ) acquired using lattice light sheet microscopy in dithered mode .", "Two fusing granules are highlighted ( also shown in Figure 5D ) .", "Images are maximum intensity projections of 11 z planes separated by 0 . 3 μm steps , capturing the entire depth of the fusing pair .", "Stacks were taken every 580 ms , total movie time is 34 s , movie is played back in 5× real time . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 025 Images acquired every 580 ms revealed changes in the distribution of GFP::MEG-3 and GFP::PGL-3 at every time point ( Figure 5—figure supplement 3B ) .", "Overall granule shape was also dynamic , with none of the granules maintaining a perfectly spherical shape ( Figure 5D , Video 5 ) .", "Fusion between granules was comparatively slow , on the order of tens of seconds as documented previously ( Brangwynne et al . , 2009 ) .", "Granules remained close to each other for several seconds before initiating fusion .", "In the typical example shown in Figure 5D and Video 5 , the fusing granules took 25 s to return to a quasi-spherical shape .", "We also detected smaller , dynamic GFP::MEG-3 assemblies in the cytoplasm away from the granules ( Video 5 , Figure 5—figure supplement 5 ) .", "To examine the distribution of GFP::MEG-3 at higher resolution , we collected single-color , single time point light sheet images in the structured illumination ( SIM ) mode .", "In this mode , individual images are collected as the lattice light sheet is stepped along the x axis and reconstructed into a 3D image with resolution beyond the diffraction limit in the x and z directions ( 194 nm × 238 nm in xz; [Chen et al . , 2014] ) .", "The single time point SIM images confirmed that GFP::MEG-3 is not uniformly distributed in the granules .", "The non-uniform pattern of GFP::MEG-3 was also visible in raw , unprocessed images ( Figure 5—figure supplement 5 ) .", "In 5 out of 5 granules reconstructed in SIM mode , GFP::MEG-3 localized to a discontinuous ribbon-like domain that surrounded and penetrated the granule ( Figure 5E and Videos 6 and 7 ) .", "We conclude that P granules are not homogeneous assemblies and that MEG-3 localizes to dynamic sub-domains within each granule that only partially overlap with PGL-3 . 10 . 7554/eLife . 04591 . 026Video 6 . 3D reconstruction of GFP::MEG-3 granule . Single time point SI images acquired with lattice light sheet microscopy of an individual P granule in an embryo expressing GFP::MEG-3 .", "Acquisition time was 1 s .", "Resolution is 194 nm × 238 nm × 419 nm .", "Anterior is to the right , posterior to the left . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 02610 . 7554/eLife . 04591 . 027Video 7 . 3D reconstructions of GFP::MEG-3 granules . Single time point SI images acquired with lattice light sheet microscopy of individual P granules in an embryo expressing GFP::MEG-3 .", "Four examples are shown .", "Acquisition time was 1 . 75 s .", "Anterior is to the right , posterior to the left . DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 027" ], [ "During the oocyte-to-zygote transition , oocyte granules disassemble and are replaced by smaller and much more dynamic granules ( zygote granules ) .", "We suggest that this transition depends on scaffolding of new granules by MEG assemblies throughout the cytoplasm .", "We propose that , shortly after fertilization , MEG assemblies promote PGL condensation , depleting the cytoplasmic PGL-1 pool available to maintain oocyte granules and causing PGL-1 to redistribute from oocyte granules to MEG-scaffolded zygote granules .", "In meg mutants , this competition does not take place , allowing oocyte granules to persist longer .", "The alternative possibility that the primary role of the MEGs is to disassemble oocyte granules , which indirectly is required for zygote granule assembly , is unlikely since ( 1 ) the meg-3 single mutant disassembles oocyte granules , yet is still partially defective in zygote granule assembly ( Figure 4 ) , ( 2 ) mbk-2 , which is required for the disassembly of zygote granules , is not required for the disassembly of oocyte granules ( Figure 1—figure supplement 1 ) , and ( 3 ) oocyte granules represent less than 1% of the total PGL-1 pool available in meg-3 meg-4 zygotes , so their mere presence is unlikely to interfere with the assembly of new granules ( Figure 4—figure supplement 5 ) .", "We detected GFP::MEG-3 in the cytoplasm of oocytes before fertilization , yet loss of meg-3 does not affect oocyte granules until after fertilization .", "We do not know what cue activates the scaffolding function of MEG proteins specifically in zygotes .", "How do MEG proteins stimulate PGL-1 condensation ?", "Unlike the RNA-binding proteins that associate with hydrogels in vitro ( Frey et al . , 2006; Kato et al . , 2012 ) , the MEGs do not contain a recognizable RNA-binding domain or short repeated motifs ( Figure 2 ) .", "The MEGs contain , however , extended regions with predicted high disorder ( Figure 2 ) .", "Intrinsically disordered proteins have been shown to oligomerize and organize scaffolds that template supramolecular structures ( Turoverov et al . , 2010; Toretsky and Wright , 2014 ) .", "For example , ameloblastin self-assembles in vitro into ribbons that can extend to hundreds of nanometers in length ( Wald et al . , 2013 ) .", "In vivo , ameloblastin is essential for the organization of enamel matrix proteins during teeth biomineralization .", "MEG-1 and MEG-3 bind directly to PGL-1 in vitro , consistent with a role in organizing PGL-1 assemblies .", "When over-expressed in mammalian cells , PGL-1 can assemble granules on its own ( Hanazawa et al . , 2011 ) .", "In meg-3 meg-4 zygotes , a few small PGL-1 granules form transiently in the posterior cytoplasm but are not maintained .", "We suggest that , in zygotes , the self-assembly properties of PGL-1 are only sufficient to form small , unstable granules , which must be stabilized by a MEG-dependent scaffold .", "Our observations using light sheet microscopy indicate that MEG-3 localizes to small assemblies in the cytoplasm and larger assemblies surrounding a PGL core in the P granules .", "During granule disassembly , MEG-3 and MEG-4 assemblies persist longer than the PGL-1 core , consistent with occupying a distinct domain .", "Together these observations suggest the following working model: the MEGs promote granule assembly by forming dynamic scaffolds in the cytoplasm that stabilize and wrap around PGL condensates .", "By electron microscopy , embryonic P granules were reported to appear as homogenous , fibrillar granular bodies ( Wolf et al . , 1983 ) .", "Our observations suggest that embryonic P granules in fact contain distinct surface and internal zones , as also reported for post-embryonic , perinuclear P granules ( Pitt et al . , 2000; Sheth et al . , 2010 ) .", "Nucleoli , which also behave like liquid droplets , also contain a distinct surface shell ( Brangwynne et al . , 2011 ) .", "Nucleoli , however , are perfect spheres ( Brangwynne et al . , 2011 ) , whereas P granules are imperfect spheres with irregular , asymmetric contours that are constantly in flux .", "We suggest that this difference is due to the fact that P granules are undergoing continuous exchange with smaller assemblies in the surrounding cytoplasm .", "Consistent with this possibility , photobleaching experiments have demonstrated rapid exchange of GFP::PGL-1 and GFP::MEG-3 between granules and cytoplasm ( Brangwynne et al . , 2009 and data not shown ) .", "We conclude that P granules in embryos are not stable , homogeneous droplets , but dynamic assemblies of two ( or more ) condensates that mix only partially and are constantly in flux with smaller assemblies in the cytoplasm .", "MEG-1 and MEG-3 are direct targets of MBK-2/DYRK and the PP2A phosphatase .", "The MEGs are rich in serines ( 75 serines in MEG-1 and 119 serines in MEG-3 ) , and their migration on Phos-tag gels suggests a complex pattern of phosphorylation with many phosphoisoforms .", "Initial attempts to mutate individual serines in MEG-1 and MEG-3 have not yielded detectable phenotypes , suggesting that the serines may function cumulatively ( data not shown ) .", "Accumulation of negatively charged phosphates could interfere with the interactions that stabilize the MEG scaffold or its interactions with PGL-1 .", "Consistent with this possibility , phosphorylation of the low complexity domain of the FUS stress granule protein interferes with its incorporation into hydrogels in vitro ( Han et al . , 2012 ) .", "Our genetic analyses suggest that all MEGs contribute to assembly and disassembly but to different extents , with MEG-3 and MEG-4 together making the most important contribution to assembly .", "The predicted pI ( isoelectric point ) of MEG-1 and MEG-2 in the unphosphorylated state is 6 . 63 and 6 . 04 , compared to 9 . 74 and 9 . 33 for MEG-3 and MEG-4 .", "We speculate that the intrinsic negative charge of MEG-1 and MEG-2 , which is increased further by phosphorylation , favors a role for these proteins in disassembly .", "In contrast , the intrinsic weak positive charge of MEG-3 and MEG-4 allows them to contribute robustly to both disassembly and assembly , as they switch to negative and back to positive upon phosphorylation and dephosphorylation .", "How are disassembly and assembly spatially segregated ?", "At the onset of disassembly , MBK-2/DYRK is enriched in the anterior half of the zygote ( Pellettieri et al . , 2003 ) .", "If dephosphorylation by PP2A continuously antagonizes phosphorylation by MBK-2 , even small changes in the level of MBK-2 levels along the anterior/posterior axis could bias MEG phosphorylation and P granule disassembly .", "Paradoxically , MBK-2/DYRK also accumulates in P granules , especially late in the first cell cycle as the granules continue to grow in the posterior cytoplasm ( Pellettieri et al . , 2003 ) .", "Interestingly , DYRK3 , the vertebrate homolog of MBK-2 that regulates stress granule dynamics , also localizes in the granules ( Wippich et al . , 2013 ) .", "Drug inhibition studies suggest that DYRK3 enhances granule condensation when its kinase activity is inhibited and promotes granule dissolution when activated ( Wippich et al . , 2013 ) .", "An attractive possibility is that , as P granules increase in size , MBK-2 activity decreases within the granule , perhaps due to the accumulation of an inhibitor .", "Interestingly , GFP::PPTR-1 also becomes enriched in P granules in zygotes ( data not shown ) .", "There are also other asymmetries in the zygote cytoplasm that could bias P granule dynamics .", "GFP::MEG-3 forms an anterior-low/posterior-high gradient along the length of the zygote , and the RNA-binding protein MEX-5 , which is required for full disassembly , forms an opposing gradient with higher levels in the anterior ( Schubert et al . , 2000 ) .", "Modeling studies have already demonstrated that , in principle , even weak gradients of phase transition regulators are sufficient to segregate P granules ( Lee et al . , 2013 ) .", "It will be important to determine which asymmetry in the zygote cytoplasm is directly responsible for patterning P granule dynamics .", "Analysis of single , double , and triple meg combinations indicate that the megs display synthetic sterility .", "For example , meg-1 and meg-3 meg-4 mutants are ∼4% and ∼30% sterile , respectively , whereas meg-1 meg-3 meg-4 mutants are 100% sterile ( this work and Leacock and Reinke , 2008 ) .", "In the sterile meg-1 meg-3 meg-4 worms , the primordial germ cells stop dividing soon after initiating divisions in the gonad , a phenotype previously reported for meg-1 meg-2 mutants ( Meg phenotype: maternal-effect germ cell defective ) ( Leacock and Reinke , 2008 ) .", "We conclude that the MEGs contribute redundantly to an activity essential for germ cell viability and/or proliferation .", "The MEGs are components of the C . elegans germ plasm , the specialized maternal cytoplasm that segregates with the embryonic germline and specifies germ cell fate .", "Germ plasm is thought to have evolved independently several times during metazoan evolution ( Extavour and Akam , 2003 ) , and the molecules that initiate germ plasm and/or germ granule assembly in eggs are not conserved between different species , although some contain intrinsically disordered regions ( e . g . , Bucky Ball/Xvelo in vertebrates , MEGs in C . elegans ) ( Marlow and Mullins , 2008; Bontems et al . , 2009; Nijjar and Woodland , 2013 ) .", "In all species examined , the germ plasm contains microscopically visible P granule-like structures that are enriched for conserved mRNAs and RNA-binding proteins required for germ cell development ( e . g . , nanos RNA and VASA-related helicases ) ( Voronina et al . , 2011 ) .", "Despite this conservation , our genetic analyses suggest that the MEGs' contribution to fertility is not linked to their effects on P granules , as also suggested by Leacock and Reinke ( 2008 ) .", "meg-1 meg-2 embryos assemble embryonic P granules and meg-1 meg-3 meg-4 embryos do not , yet both display the same fully penetrant Meg sterility phenotype ( Figure 4 and Leacock and Reinke , 2008 ) .", "Furthermore , meg-3 meg-4 embryos show the same dramatic defect in P granule assembly as meg-1 meg-3 meg-4 embryos , yet most meg-3 meg-4 animals ( 70% ) are fertile ( Figure 4 ) .", "We conclude that P granule assembly in the germ plasm of early embryos is neither required nor sufficient for fertility in C . elegans .", "In contrast , the perinuclear P granules that form in primordial germ cells and their descendents are required for fertility ( Updike and Strome , 2010 ) .", "Consistent with that view , fertile meg mutants that do not assemble P granules in the germ plasm assemble perinuclear P granules de novo later in development .", "De novo assembly of perinuclear germ granules is also observed in animals that do not inherit germ plasm and specify their primordial germ cells through inductive mechanisms later in development ( Voronina et al . , 2011 ) .", "Previously , our analyses of pptr-1 mutants led us to conclude that asymmetric segregation of core P granule components is not essential to distinguish germline from soma in embryos ( Gallo et al . , 2010 ) .", "Our results here support this view and suggest that the essential activity of the germ plasm resides not in the assembly of P granules per se but in the MEG proteins that regulate granule assembly .", "How the maternally provided MEGs contribute to the health and proliferating potential of the PGCs will be an interesting area for future exploration ." ], [ "C . elegans was cultured according to standard methods ( Brenner , 1974 ) .", "Transgene plasmids were generated by InFusion cloning ( Clontech , Mountain View , CA ) and introduced into unc-119 worms by microparticle bombardment ( Praitis et al . , 2001 ) .", "GFP::MEG-3 is driven by its endogenous promoter and 3′UTR and consist of 1128 bp of genomic DNA sequence upstream of the MEG-3 ATG ( up to but not including the ATG of the next gene Y40A1A . 2 ) , GFP from pCM1 . 53 ( Merritt et al . , 2008 ) and 1872 bp of genomic DNA downstream of the stop codon , past both polyA sites annotated in WormBase ( WBsf257432 and WBsf216670 ) .", "GFP::MEG-1 is described in Leacock and Reinke ( 2008 ) and is driven by the pie-1 promoter and pie-1 3′UTR .", "CRISPR/Cas9 experiments to generate meg-3 and meg-4 alleles were performed as described in Paix et al . ( 2014 ) .", "See Tables 1 and 2 for list of strains , allele descriptions , and sgRNA and repair templates sequences . 10 . 7554/eLife . 04591 . 028Table 1 . List of strains used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 028NameDescriptionGenotypeReferenceJH2842pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRunc-119 ( ed3 ) III; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVGallo et al . , 2010JH2843pptr-1 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRpptr-1 ( tm3103 ) V; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVGallo et al . , 2010JH3055meg-3 mutantmeg-3 ( tm4259 ) XThis studyJH3147meg-3 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRmeg-3 ( tm4259 ) X; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVThis studyJH3225meg-3 meg-4 mutant—see Table 2meg-3 ( tm4259 ) meg-4 ( ax2026 ) XThis studyJH3149meg-3 meg-4 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRmeg-3 ( tm4259 ) meg-4 ( ax2026 ) X; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVThis studyJH3148meg-1 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRmeg-1 ( vr10 ) X; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVThis studyJH3229meg-1 meg-3 mutantmeg-1 ( vr10 ) meg-3 ( tm4259 ) XThis studyJH3150meg-1 meg-3 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRmeg-1 ( vr10 ) meg-3 ( tm4259 ) X; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVThis studyJH3156pptr-1; meg-1 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRpptr-1 ( tm3103 ) V; meg-1 ( vr10 ) X; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVThis studyJH3155pptr-1; meg-3 mutant; pie-1 prom::GFP::PGL-1-pgl-1 3′UTR; pie-1 prom::mCherry::H2B::pie-1 3′UTRpptr-1 ( tm3103 ) V; meg-3 ( tm4259 ) X; axIs1522[pCM4 . 11]; ltIs37 [pAA64] IVThis studyJH2932mbk-2 null mutant; pgl-1::TY1::eGFP::3xFLAG992C12unc-24 ( e1172 ) mbk-2 ( pk1427 ) IV / nT1[let- ? ( m435 ) ] ( IV;V ) ; ddEX16This studyYL183meg-1 mutant; pie-1 prom::GFP::MEG-1::pie-1 3′UTRmeg-1 ( vr10 ) X; GFP::MEG-1Leacock and Reinke , 2007JH3016meg-3 prom::GFP::MEG-3::meg-3 3′UTRunc-119 ( ed3 ) ; axIS2076[pJW6 . 01]This studyJH3064pptr-1 mutant; meg-3 prom::GFP::MEG-3::meg-3 3′UTRpptr-1 ( tm3103 ) V; axIS2076[pJW6 . 01]This studyJH3019meg-3 prom::GFP::MEG-3::meg-3 3'UTR; pie-1 prom::GFP::PGL-3::pie-1 3′UTRunc-119 ( ed3 ) III; axIS2076[pJW6 . 01]; axIS2077[pJW2 . 03]This studyJH3230pgl-3 mutant; meg-3 prom::GFP::MEG-3::meg-3 3′UTRpgl-3 ( bn104 ) V; axIS2076[pJW6 . 01]This studyJH3153meg-3 mutant; meg-3 prom::GFP::MEG-3::meg-3 3′UTRmeg-3 ( tm4259 ) X; axIS2076[pJW6 . 01]This studyJH3247C-terminal FLAG insertion in genomic meg-4 locus—see Table 2meg-4 ( ax2080 ) This studyJH3248meg-4 mutant—see Table 2meg-4 ( ax2081 ) This studyAbbreviations: prom–Promoter , 3′ UTR–3′ untranslated region , ALL CAPs–coding regions of indicated genes . All transgenes also contain a wild-type copy of unc-119 ( transformation marker ) . 10 . 7554/eLife . 04591 . 029Table 2 . Strains generated by CRISPR/Cas9DOI: http://dx . doi . org/10 . 7554/eLife . 04591 . 029StrainAlleleEditMethodsgRNA ( PAM sequence ) Genomic site of editJH3247meg-4 ( ax2080 ) C-terminal 3xFLAG insertion in meg-4Homology directed repair with ssODN .", "Sequence: ( homology arms ) ( cctgtcagatacttgaatgcaaaacgagaatggctgga atctatttttgacccaccgagagatcaa ) gactacaaaga ccatgacggtgattataaagatcatgatatcgattacaa ggatgacgatgacaag ( tgattgtactgatatatatctatt tcatgtcgagtattttgtattttattcttgttcattgacc ) caatcattgatctctgggt ( ggg ) X:1686208JH3248meg-4 ( ax2081 ) Deletion removing 733 base pairs upstream of the meg-4 start and the first 2565 bases of the geneNHEJgagcgcgaaatagtgtgtg ( ggg ) tgggaccaaaaagcaagaa ( tgg ) atttatttatggtctgccc ( agg ) ctgcccaggaacttgtaac ( ggg ) X:1682223 . . 1685521JH3325meg-3 ( tm4259 ) meg-4 ( ax2026 ) Deletion of 7 nucleotides starting at nucleotide 37 of meg-4 ( amino acid 13 ) .", "The frameshift inserts the amino acids ‘EETEKVTALIVEAIWKVHRKIWDTTLVLKNCFIRS’ at amino acid 13 followed by a premature stopNHEJ for meg-4 ( ax2026 ) in meg-3 ( tm4259 ) tctctgtttcctctggagtt ( tgg ) caagttccgttgattccagc ( tgg ) X:1682992 RNAi was performed by feeding ( Timmons and Fire , 1998 ) .", "Feeding constructs were obtained from the Ahringer or Openbiosystems libraries and sequenced or newly cloned from C . elegans cDNA .", "HT115 bacteria transformed with feeding vectors were grown at 37°C in LB + ampicillin ( 100 µg/ml ) for 5 hr , induced with 5 mM IPTG for 45 min , plated on NNGM ( nematode nutritional growth media ) + ampicillin ( 100 µg/ml ) + IPTG ( 1 mM ) , and grown overnight at room temperature before adding L4 worms at 24°C for 24–30 hr .", "For Phos-tag gel experiments , worms were fed starting in the L1 stage and scored for embryonic lethality ( mbk-2 ( RNAi ) ) or P granule phenotype ( pptr-1 ( RNAi ) ) .", "For sterility counts , at least eight gravid adult worms were allowed to lay embryos for 1 to 2 hr .", "Adult progeny were scored for empty uteri ( ‘white sterile’ phenotype ) on a dissecting microscope .", "To determine the number of germ cells in meg larvae , we counted the number of nuclei in GFP::PGL-1-expressing cells , using vulval morphology to stage the larvae .", "Yeast two-hybrid assays were performed using DUALhybrid kit ( P01004 , Dualsystems Biotech , Switzerland ) .", "All plasmids were converted to Gateway-compatible vectors ( Invitrogen , Carlsbad , CA ) and Gateway recombination was used to create N-terminally tagged pLexA-PPTR-1 bait vector , pY3H-PAA-1 bridge vector , and candidate prey vectors .", "For library screening , yeast transformed with PPTR-1 bait and PAA-1 bridge were used with prey library from Dualsystems consisting of polyA+ cDNA from mixed stage C . elegans with 5 . 7 × 106 independent clones ( 28 μg of prey library ) .", "Total transformation efficiency was 4 . 3 × 105 clones per μg DNA , 1 . 2 × 107 total transformants , with 2 . 1× library coverage .", "GST fusion proteins were cloned into pGEX6p1 ( GE Healthcare , Pittsburgh , PA ) .", "MBP fusion proteins were cloned into pJP1 . 09 , a Gateway-compatible pMAL-c2x ( Pellettieri et al . , 2003 ) .", "Proteins were expressed in E . coli BL21 cells overnight at 16°C , following induction with 0 . 4 mM IPTG .", "200 mg of bacterial pellet of GST fusion proteins was resuspended in 10 mM EGTA , 10 mM EDTA , 500 mM NaCl , 0 . 1% Tween , PBS pH 7 . 4 with protease and phosphatase inhibitors , lysed by sonication , and bound to GST beads .", "Beads were washed and incubated with MBP fusion proteins at 4°C for 1 hr in 50 mM Hepes pH 7 . 4 , 1 mM EGTA , 1 mM MgCl2 , 500 mM KCl , 10% glycerol , 0 . 05% NP-40 , pH 7 . 4 with protease and phosphatase inhibitors .", "After washing , beads were eluted with 10 mM reduced glutathione and eluates were loaded on SDS-PAGE .", "MBP fusion proteins were cloned into pJP1 . 09 , expressed and partially purified as previously described ( Griffin et al . , 2011 ) .", "In vitro kinase assays were performed as described ( Cheng et al . , 2009 ) .", "Embryos were harvested from synchronized young adult worms and sonicated in 2% SDS , 65 mM Tris pH 7 , 10% glycerol with protease and phosphatase inhibitors .", "Lysates were spun at 14 , 000 rpm for 30 min and cleared supernatants treated with 100 U alkaline phosphatase ( Roche , Indianapolis , IN ) .", "Samples were run in parallel on Phos-tag gels ( 7% SDS-PAGE with 25 μM Phos-tag and 50 μM MnCl2 , Phos-tag from Wako Chemicals , Japan ) and 7% SDS-PAGE at 30 mA for 2 . 5 hr .", "Gels were washed in transfer buffer with 1 mM EDTA twice for 10 min each and washed in transfer buffer without EDTA twice for 10 min each .", "Western blot transfer was performed for 1 hr at 4°C onto nitrocellulose membranes .", "Membranes were blocked and washed in 5% milk , 0 . 1% Tween-20 in PBS and probed with JL-8 antibody ( 1:240 dilutions , Clontech ) .", "Peptide antibodies against MEG-3 were made by Covance ( Princeton , NJ ) using KLH conjugation in rabbits against the sequence Ac-HAFKKGHKDNKNASC-amide .", "In situ hybridization of nos-2 mRNA was performed using fluorescent oligonucleotide probes as described previously ( Voronina et al . , 2012 ) .", "Gravid adult hermaphrodites were laid on a slide coated with 0 . 01% poly-L-lysine , and embryos were extruded by squashing with a coverslip .", "Embryos were frozen on pre-chilled aluminum blocks .", "Coverslips were removed and slides were incubated in −20°C methanol for 15 min , followed by −20°C acetone for 10 min .", "Slides were pre-blocked in PBS/0 . 1% Tween/0 . 1% BSA ( PBT ) for 30 min and incubated with primary antibody overnight at 4°C .", "Primary antibodies were diluted in PBT as follows: K76 ( 1:10 , DSHB , Iowa City , IA ) , rabbit anti-MEG-3 ( 1:250 , Covance ) , rabbit anti-MEG-1 ( 1:100 , gift from Valerie Reinke ) , chicken anti-GLH-2 ( 1:200 , gift from Karen Bennett ) , anti-FLAG ( 1:500 , Sigma , St . Louis , MO ) .", "Secondary antibodies were applied for 2 hr at room temperature .", "Images were acquired using a Zeiss Axio Imager fitted with a Yokogawa spinning disc confocal scanner with Slidebook software ( Intelligent Imaging Innovations , Denver , CO ) using a 63× objective ( embryos ) or 40× objective ( in utero movies ) .", "Embryos were dissected from gravid mothers and mounted on 3% agarose pads in M9 solution at room temperature .", "For single time point images , 10 z planes with a z step size of 1 μm , spanning 9 μm , were acquired .", "Exposure time was 100 ms per plane per color with one exposure acquired in each color before proceeding to the next plane .", "Granule counts were performed with Slidebook using Mask Segmentation tools and manually verified for each embryo .", "For time-lapse movies of embryos , z-stacks ( 8 z planes , step size 1 μm ) were acquired every 8 s .", "Exposure time was 100 ms per plane per color with one exposure acquired in each color before proceeding to the next plane .", "For in utero movies , young adult mothers were anesthetized in 0 . 3 mM levamisole for 15 min prior to mounting on 3% agarose pads .", "Z-stacks ( 11 z planes with a z step size of 1 μm ) were acquired every 30 s .", "Exposure time was 100 ms per plane per color with one exposure acquired in each color before proceeding to the next plane .", "Embryos were dissected from gravid mothers and mounted on 5-mm diameter coverslips in M9 solution at room temperature .", "Coverslips were pre-cleaned and coated with 2 μl of BD Cell-Tak ( BD Biosciences , San Jose , CA ) and 2 μl of poly-D-lysine .", "In the time-lapse dithered mode , 21 z planes with a z step size 0 . 3 μm , spanning 6 μm , were acquired .", "Exposure time was 10 ms per plane per color with one exposure acquired in each color before proceeding to the next plane , for a total exposure time of 21 × 10 × 2 = 420 ms for each 3D stack .", "A 150 ms pause was added between each time point .", "Dithered-mode stacks were deconvolved in 3D using the Richardson–Lucy algorithm with experimentally measured point spread functions for each color .", "Granule cross-sectional area was calculated using ImageJ using maximum-projection images from two embryos .", "3D-SIM mode images were acquired as in Chen et al . ( 2014 ) , with 5 phase SIM .", "For the first image in Figure 5E , 71 z planes were acquired with 50 ms exposure per phase and a 0 . 15 μm z step between planes .", "For the other images in Figure 5E , 71 z planes were acquired with 20 ms exposure per phase and a 0 . 2 μm z step .", "SIM images were reconstructed as in Gao et al . ( 2012 ) .", "Note that the granules shown in Figure 5E span only 7–10 z planes , corresponding to 1–1 . 75 s acquisition time .", "We confirmed that during this time , the granules were stationary on the scale of the SIM excitation pattern , allaying any concerns of motion-induced artifacts in the SIM reconstructions .", "Photon counts were calculated according to the formula P ( photo ) = CF × ( C − off ) /Q ( lambda ) , where CF is the conversion factor ( electron/count ) , C is output count of the selected pixel , off is the background of the camera , and Q ( lambda ) is a curve based on the wavelength .", "Signal to noise ratio was calculated as sqrt ( P ) ." ] ]
[ "RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components .", "The molecules and reactions that drive these dynamics in vivo are not well understood .", "In this study , we present evidence that a group of intrinsically disordered , serine-rich proteins regulate the dynamics of P granules in C . elegans embryos .", "The MEG ( maternal-effect germline defective ) proteins are germ plasm components that are required redundantly for fertility .", "We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2APPTR−½ .", "Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly .", "Using lattice light sheet microscopy on live embryos , we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule .", "We conclude that , despite their liquid-like behavior , P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation ." ]
[ "For a gene to be expressed as a protein , its DNA is first used as a template to make a molecule of RNA , which is then translated to make the protein .", "In most cells , RNA molecules concentrate into aggregates called RNA granules .", "These granules contain both RNA and proteins that bind to RNA and are used to transport , store , and regulate the translation and breakdown of RNA molecules .", "Unlike many other structures within cells , RNA granules are not surrounded by a membrane; and the molecules that hold RNA granules together are not known .", "P granules are a type of RNA granule that is found in the germ cells ( the cells that go on to form eggs and sperm ) of a microscopic worm called C . elegans .", "When a C . elegans embryo is still a single cell , P granules move throughout the cell and the P granules at the front of the cell dissolve , while those at the back condense .", "As such , when the single-celled embryo divides , the front forms a cell without P granules ( that will go on to form the tissues of the worm's body ) and the back becomes a P granule-containing germ cell .", "Two proteins called MBK-2 and PPTR-1 have opposite effects on P granules: MBK-2 causes P granules to dissolve , while PPTR-1 makes them form .", "MBK-2 is an enzyme that adds phosphate groups onto other proteins , whereas PPTR-1 is part of an enzyme that removes such groups .", "Wang et al . have now searched for proteins that interact with MBK-2 and PPTR-1 in order to identify the molecules that regulate the assembly of P granules .", "They found that a group of proteins , known as MEG proteins , are acted upon by both of these proteins .", "Wang et al . found that MBK-2 adds phosphate groups to MEG proteins , which encourages granules to disassemble , while PPTR-1 removes these groups to promote granule assembly .", "Wang et al . generated mutant worms that lacked each of the MEG proteins .", "These mutant worms had fewer and smaller P granules than normal worms .", "Without MEG proteins , P granules failed to assemble or disassemble normally and the worms were infertile .", "Using high resolution microscopy , Wang et al . observed that the MEG proteins wrap around the P granules and that one of the MEG proteins—called MEG-3—follows an almost ribbon-like path that surrounds and enters each granule .", "These observations suggest that the MEG proteins stabilize RNA granules by forming a cage-like scaffold around each granule .", "How the MEG proteins—which are predicted to lack a fixed or ordered three-dimensional structure and show no similarity to proteins with known functions—assemble into a scaffold will be the focus of future studies ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology" ]
A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy
elife-09347-v1
[ [ "The cytoplasm of living cells is highly dynamic and yet exquisitely organized .", "Maintenance of this state requires a constant input of energy and a metabolism that is far from thermodynamic equilibrium .", "However , organisms typically live in unpredictable environments and frequently experience conditions that are not optimal for growth and reproduction .", "Under such conditions , cells must protect themselves by entering into a non-dividing state , generally referred to as dormancy ( Lennon and Jones , 2011 ) .", "Dormancy is defined as a state of reversible cell cycle arrest with reduced metabolic activity and changes in cellular organization ( Lennon and Jones , 2011 ) .", "It often involves execution of a developmental program , which culminates in the formation of specialized cell types such as spores , seeds , or cysts .", "These cell types can endure long periods of nutrient starvation , low temperatures , and even desiccation .", "Dormancy is also accompanied by extensive changes in cellular architecture , some of which are drastic .", "For instance , dormant cells have a very low water content , their cytoplasm is densely packed , and they show strongly diminished intracellular dynamics ( Ablett et al . , 1999; Cowan et al . , 2003; Dijksterhuis et al . , 2007; Parry et al . , 2014 ) .", "However , how cells enter into and recover from such a state is still unresolved .", "The current paradigm of cellular biochemistry is based on studies in dilute solutions , often performed with only a handful of proteins .", "Findings made in such dilute regimes have been extrapolated to the cellular interior .", "In recent years awareness has been increasing that the cellular environment is very different from such dilute regimes .", "One reason for this is that the cytoplasm is densely packed with macromolecules .", "The overall concentration of macromolecules in the cytoplasm is estimated to be around 200–350 mg/ml ( Ellis , 2001; Zimmerman and Trach , 1991 ) , which amounts to a volume fraction of up to 40% .", "This dense packing of macromolecules is referred to as macromolecular crowding .", "How macromolecules remain soluble at such high concentrations inside a cell is unknown , but it presumably involves a fine balance of attractive and repulsive interactions between the different cytoplasmic components .", "The highly crowded conditions inside a cell generate an environment with specific physical properties .", "These properties have traditionally been explored by following the diffusive behavior of tracer particles or organelles .", "Such particle-tracking approaches have led to the realization that intracellular diffusion is anomalous in cells ( Dix and Verkman , 2008; Hall and Hoshino , 2010; Luby-Phelps , 2000; Tolić-Nørrelykke et al . , 2004 ) .", "Based on these findings , proposals have been made about the physical nature of the cytoplasm , which has either been described as a hydrogel ( Fels et al . , 2009 ) or , more recently , as a liquid at the transition to a glass-like state ( Parry et al . , 2014 ) .", "Because many metabolic reactions and signaling processes take place in the cytoplasm , changes in its physicochemical properties should have far-reaching effects on cellular function and survival .", "Recent findings indicate that the organization of the cytoplasm can change considerably , in particular under stress conditions such as starvation .", "In energy-depleted yeast cells , many proteins and RNAs assemble into microscopically visible structures ( Laporte et al . , 2008; Narayanaswamy et al . , 2009; Noree et al . , 2010; O'Connell et al . , 2012; Sagot et al . , 2006 ) .", "These structures may constitute storage depots for proteins and RNAs ( Daignan-Fornier and Sagot , 2011; Laporte et al . , 2008; Sagot et al . , 2006 ) .", "Indeed , several metabolic enzymes assemble into filamentous structures in response to starvation , and the formation of these filaments leads to enzymatic inactivation ( Petrovska et al . , 2014 ) .", "Importantly , the enzymes contained in these filaments can be reused , when cells escape from dormancy .", "This suggests that cells may regulate the structure of the cytoplasm to enter into and exit from a metabolically inactive state .", "In this study , we demonstrate that , in acidic environments , entry into dormancy is triggered by an influx of protons that promotes a transition of the cytoplasm from a fluid- to a solid-like state through widespread assembly of proteins into higher-order structures .", "We show that this transition arrests the movement of organelles and foreign tracer particles .", "We provide further evidence that this state of reduced intracellular mobility is required for survival of energy depletion stress .", "Thus , we propose that organisms have global control mechanisms in place to fine-tune the material properties of the cytoplasm , allowing them to enter into a protective solid-like state , when challenged by extreme environmental conditions ." ], [ "To investigate how eukaryotic cells enter into a dormant state , we focused on budding yeast , a single-celled organism , which can enter into dormancy upon depletion of energy ( De Virgilio , 2012; Gray et al . , 2004; Neiman , 2011; Valcourt et al . , 2012 ) .", "Because a strong reduction in intracellular dynamics is a hallmark of dormant cells ( Cowan et al . , 2003; Dijksterhuis et al . , 2007; Mastro et al . , 1984; Parry et al . , 2014 ) , we first compared the mobility of different cellular organelles in dividing and dormant yeast cells ( Huh et al . , 2003 ) .", "Induction of a dormant state was achieved by treating yeast cells with 2-deoxyglucose ( 2-DG , an inhibitor of glycolysis ) and antimycin A ( an inhibitor of mitochondrial respiration ) , a treatment that decreases cellular ATP levels by more than 95% ( Serrano , 1977 ) .", "Using single particle tracking ( SPT ) and mean squared displacement ( MSD ) analysis we found a striking reduction in intracellular movements upon energy depletion ( Figure 1A and B ) . 10 . 7554/eLife . 09347 . 003Figure 1 . Reduced mobility of organelles and foreign tracer particles under energy depletion conditions .", "( A ) Representative trajectories of a range of different organelle markers tracked under control conditions ( ● , cells in log-phase , upper panel ) and upon energy depletion ( ▲ , lower panel ) .", "Organelle markers are GFP fusion proteins expressed under control of their endogenous promoters ( Huh et al . , 2003 ) .", "( B ) Corresponding time- and ensemble-averaged MSD plots for both control ( ● ) and energy depletion ( ▲ ) conditions .", "( C ) Representative trajectories ( upper panel ) and corresponding MSD plots ( lower panel ) of the foreign tracer particle GFP-µNS ( see Figure 1—figure supplement 1–3 for details ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 00310 . 7554/eLife . 09347 . 004Figure 1—figure supplement 1 . GFP-µNS particles form discrete particles in the yeast cytoplasm ( left ) .", "These particles explore the cytoplasm over time ( middle and right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 00410 . 7554/eLife . 09347 . 005Figure 1—figure supplement 2 . GFP-µNS particles show size-dependent MSD over short ( left ) and long ( right ) observation times . The fluorescence intensity of particles was used as a proxy for particle size . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 00510 . 7554/eLife . 09347 . 006Figure 1—figure supplement 3 . Same data as in Figure 1—figure supplement 2 , shown as log-log plots ( left ) .", "The generalized diffusion constant ( right ) was obtained from the time-averaged MSD of individual trajectories at the lag time of t=2 s divided by 4tα , where α=0 . 88 is the power-law exponent of the time and ensemble averaged MSD for all particles .", "The fluorescence intensity of particles ( AU ) was used as a proxy for particle size . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 006 Tracking of endogenous particles provides only limited information on the material properties of the cytoplasm , because they are often membrane-associated and/or move by active transport .", "Therefore , foreign tracer particles are better suited as probes of the subcellular environment .", "However , direct injection methods of foreign particles , as they have been developed for mammalian cells , are not feasible for yeast cells because of their small size .", "We therefore adopted a technique that relies on a genetically encoded viral capsid protein ( µNS ) , which has been used successfully in bacteria ( Parry et al . , 2014 ) .", "We could show that GFP-µNS self-assembles into distinct particles in the yeast cytoplasm ( Figure 1—figure supplement 1 , Video 1 ) .", "These particles have bead-like properties with a size-dependent MSD and generalized diffusion coefficient K ( Figure 1—figure supplement 2 and 3 ) , indicating that they are a valuable tool to study the properties of the yeast cytoplasm .", "Using GFP-µNS particles and SPT , we found that energy depletion caused a similar reduction in the mobility of these foreign particles ( Figure 1C ) .", "Thus , we conclude that upon energy depletion , the cytoplasm of budding yeast transitions into a state with strongly reduced dynamics . 10 . 7554/eLife . 09347 . 007Video 1 . Time-lapse microscopy of GFP-µNS particles moving in the S . cerevisiae cytoplasm . To illustrate how particles explore the yeast cytoplasm over time , the fluorescence channel and the reference bright field channel were merged . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 007 In higher eukaryotes , ATP-driven processes exert fluctuating forces on the cytoplasm , which lead to random movements of particles and thus cytoplasmic mixing ( Brangwynne et al . , 2008 , 2009; Guo et al . , 2014 ) .", "These effects are predominantly driven by motor proteins , which are linked to the cytoskeleton .", "However , in contrast to mammalian cells , yeast cells have a cell wall , and thus only a rudimentary cytoskeleton , which is primarily based on actin .", "Importantly , the actin cytoskeleton of yeast disassembles upon starvation ( Sagot et al . , 2006 ) , suggesting that this event may be responsible for the reduced particle mobility by removing tracks for motor-based mixing .", "To test this , we depolymerized the actin cytoskeleton by adding the drug latrunculin A ( LatA ) to dividing yeast cells .", "Indeed , GFP-µNS particle mobility was reduced , but the effect was much less pronounced than under conditions of energy depletion ( Figure 2A ) .", "Next , we treated yeast cells with the drug nocodazole to inhibit microtubule-based motor movements .", "Again , we only observed marginal effects on particle mobility ( Figure 2B ) .", "This indicates that a lack of active motor-driven movements can only partially explain the reduced particle mobility . 10 . 7554/eLife . 09347 . 008Figure 2 . Energy depletion causes a drop in cytosolic pH , which may explain reduced particle mobility .", "( A ) MSD of GFP-µNS particles in untreated cells ( control ) , cells treated with 100 μM latrunculin A ( LatA ) and energy-depleted cells .", "( B ) MSD of GFP-µNS particles in untreated cells ( control ) , cells treated with 15 μg/ml nocodazol and energy-depleted cells ( C ) The cytosolic pH of yeast cells was measured in response to energy depletion in growth media with two different pHs .", "( D ) MSD of GFP-μNS particles tracked under the conditions shown in C . Cells were energy-depleted in growth medium without glucose containing 20 mM 2-deoxyglucose and 10 µM antimycin A . In panel A and B particles were tracked over a longer time and with lower time resolution ( 5", "s ) than in panel D . All MSD plots represent time- and-ensemble averaged MSDs and particles of all sizes were considered . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 00810 . 7554/eLife . 09347 . 009Figure 2—figure supplement 1 . Yeast cells expressing the pH sensor pHluorin2 in the cytoplasm ( left panel ) were used to generate a pH calibration curve ( right panel ) , as described in materials and methods and reported previously ( Brett et al . , 2005 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 009 Yeast typically live in acidic environments .", "The standard laboratory growth media therefore have a pH of around 5 . 5 ( see materials and methods for details ) .", "However , the cytosolic pH is kept in the neutral range by proton-translocating ATPases such as Pma1 , which use a large amount of energy to continuously pump protons out of the cell , thus preventing cytosolic acidification ( Orij et al . , 2011 ) .", "In agreement with this , previous studies have reported that energy depletion leads to a drop in cytosolic pH ( pHc ) ( Dechant et al . , 2010; Orij et al . , 2012 ) .", "Indeed , using a ratiometric , pH-sensitive variant of GFP ( Mahon , 2011 ) ( Figure 2—figure supplement 1 ) , we observed a significant pHc decrease from around 7 . 3 to around 5 . 8 in yeast cells that were energy-depleted in normal growth medium of pH 5 . 5 ( Figure 2C ) .", "If this drop in pHc was responsible for the reduced particle mobility , it should be possible to prevent particle immobilization by keeping the pHc in the neutral range .", "Indeed , when yeast cells were energy-depleted in growth medium of neutral pH , cytosolic acidification could be prevented ( Figure 2C ) and the reduction in particle mobility was much less pronounced ( Figure 2D ) .", "Thus , we conclude that strong energy depletion leads to a rapid drop in cytosolic pH , which in turn causes reduced particle mobility .", "We next tested whether direct manipulation of the cytosolic pH in the presence of an energy source is sufficient to induce reduced particle mobility .", "The protonophore DNP rapidly carries protons across the cell membrane and effectively equilibrates the intracellular with the extracellular pH ( Dechant et al . , 2010; Petrovska et al . , 2014 ) .", "This allowed us to manipulate the intracellular pH by keeping cells in DNP-containing buffers of different pH ( Figure 3A , left panel ) .", "Cells exposed to DNP-containing buffers generally showed a reduced particle mobility , when compared to cells growing in medium ( see Figure 1C ) , most likely because of direct effects of DNP on metabolism .", "However , the particle mobility was much more strongly reduced at pHc 6 and 5 . 5 than at pH 7 . 0 ( Figure 3A , right panel ) .", "To exclude possible secondary effects of DNP , we also used a mild membrane-permeable acid ( sorbic acid ) to alter pHc ( Orij et al . , 2009 ) and found that it had a similar effect on cytosolic pH ( Figure 3B , left panel ) and on particle mobility ( Figure 3B , right panel ) .", "These experiments were performed in the presence of glucose as an energy source , suggesting that the pHc change acts downstream of ATP depletion . 10 . 7554/eLife . 09347 . 010Figure 3 . Acidification of the cytosol reduces particle mobility .", "( A ) The cytosolic pH of yeast cells exposed to phosphate buffers of different pH containing 2 mM 2 , 4-dinitrophenol ( DNP ) and 2% glucose was measured over time in a microfluidic flow chamber ( left ) .", "The MSD of GFP-µNS particles tracked under the same conditions is shown on the right .", "( B ) The cytosolic pH of yeast cells exposed to synthetic complete medium ( pH ~5 . 5 ) containing increasing concentrations of sorbic acid was measured in a microfluidic flow chamber ( left ) .", "The MSD of GFP-µNS particles tracked under the same conditions is shown on the right .", "( C ) MSD of GFP-µNS particles in S . pombe cells .", "Particles were tracked in untreated cells ( control ) , energy-depleted cells and cells treated with phosphate buffers of different pH containing 2 mM DNP .", "( D ) MSD of GFP-µNS particles in D . discoideum cells .", "Particles were tracked in untreated cells ( control ) , energy-depleted cells and cells treated with Lo-Flo buffer of different pH containing 0 . 2 mM DNP .", "Cells were energy-depleted in growth media or buffer , respectively , without glucose containing 2-deoxyglucose and antimycin A . All MSD plots represent time-and-ensemble averaged MSDs and particles of all sizes were considered . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 010 Our experimental setup allows for rapid changes of the intracellular proton concentration by almost two orders of magnitude ( from 7 . 4 to 5 . 5 ) .", "To test whether such pronounced pH fluctuations affect cell viability , we exposed yeast cells to repeated pHc changes in a microfluidic chamber .", "Remarkably , pH changes of this magnitude did not affect the viability of yeast ( Video 2 ) .", "Moreover , when yeast cells were acidified with DNP , reduced particle mobility manifested within minutes , and it was readily reversed on a similar time scale ( Video 3 ) .", "Thus , pH-induced changes are readily reversible and well tolerated by yeast . 10 . 7554/eLife . 09347 . 011Video 2 . Brightfield time-lapse microscopy of S . cerevisiae cells growing in a microfluidic flow chamber . Cells were exposed to buffers of different pH containing 2 mM DNP as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01110 . 7554/eLife . 09347 . 012Video 3 . Fluorescence time-lapse microscopy of GFP-µNS expressing S . cerevisiae cells growing in a microfluidic flow chamber . Cells were repeatedly exposed to buffers of different pH containing 2 mM DNP as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 012 Next , we tested whether other eukaryotic organisms undergo similar changes .", "We focused on another fungus , fission yeast , and a protist , the social amoeba Dictyostelium discoideum .", "As S . cerevisiae , both organisms can enter into a dormant state ( Jímenez et al . , 1988; Sajiki et al . , 2009 ) , form spores upon starvation ( Egel et al . , 1994; Xu et al . , 2004 ) and undergo cytosolic pH fluctuations in response to energy depletion ( Gross et al . , 1983; Karagiannis and Young , 2001 ) .", "Consistent with this , both organisms showed reduced GFP-µNS particle mobility in an energy- and pH-dependent manner ( Figure 3C and D ) .", "Thus , we conclude that the pH-induced reduction in particle mobility is not limited to budding yeast , but also extends to other , distantly related organisms .", "Our findings so far show that the mobility of particles is reduced upon energy depletion and acidification of the cytoplasm .", "In Figure 4A , we show the MSDs and their subdiffusive scaling for different experimental conditions .", "Particles of all sizes were included in the analysis .", "We see a dramatic decrease in particle mobility for energy depleted and acidified cells .", "To gain more insight into the rheological properties of the cytoplasm , which might explain this behavior , we performed a comprehensive analysis of the particle trajectories .", "From a rheological point of view , the cytoplasm can be considered as an active viscoelastic material ( Guo et al . , 2014; Mizuno et al . , 2007 ) .", "The motion of an inert tracer particle in the cytoplasm results from the balance between stochastic driving forces and opposing material forces .", "Due to a possible non-thermal origin of stochastic forces in living cells , in general , a combination of passive and active microrheology experiments is required to quantify the material properties of the cytoplasm ( Guo et al . , 2014; Mizuno et al . , 2007 ) .", "However , active microrheology experiments in yeast are challenging , because of its small size and stiff outer cell wall .", "Nonetheless , a detailed analysis of particle trajectories in the passive microrheology approach provides first pointers towards changes in the physical properties of the cytoplasm .", "One example is the power spectral density ( PSD ) of particle displacements , which is related to the power spectrum of stochastic forces and the material properties ( Guo et al . , 2014 ) .", "The PSD can be calculated as a Fourier transform of the MSD and can , for our data in all conditions , be well approximated by a power law dependence P ( ω ) ∝ω−γ ( see Figure 4—figure supplement 1 ) .", "We find that the exponent γ is smaller for acidified and energy-depleted cells .", "For a viscoelastic material driven by thermal fluctuations , a decrease in γ would correspond to a transition from a fluid to a more solid-like state ( Squires and Mason , 2010 ) .", "Although we demonstrate that active cytoskeleton-dependent forces do not strongly affect mobility of particles , and we expect no active motion to occur in energy-depleted cells , the thermal nature of driving forces remains an assumption and therefore limits our interpretation of the PSD data .", "To obtain further insight into particle diffusion , we turn to the analysis of the displacement data on the level of individual trajectories and suggest a statistical model for the observed particle motion . 10 . 7554/eLife . 09347 . 013Figure 4 . Characterization of the acidified and energy-depleted cytoplasm from a particle perspective .", "( A ) The time- and ensemble-averaged MSD are shown as a function of lag-time , together with the fitted power-law scalings as dashed lines .", "We obtained the following power-law exponents α ≈ 0 . 73 ( DNP treated cells with external pH 6 . 0 ) , α ≈ 0 . 84 ( DNP treated cells with external pH 7 . 4 ) , α ≈ 0 . 64 ( energy depleted cells ) and α ≈ 0 . 88 ( log phase cells ) .", "( B ) The master curve for the probability density function of particle displacements as a function of rescaled displacement ( full lines ) for log-phase cells ( left side ) and energy-depleted cells ( right side ) .", "Symbols and colors indicate the probability density function extracted from the data at different lag-times after rescaling the displacements .", "This plot is symmetric for each condition .", "Plots were split at the dotted line to allow comparison of both datasets .", "The dashed line corresponds to a Gaussian distribution .", "( C ) Correlation of subsequent displacements ( defined as c=δx'→⋅δx→|δx→| , where δx→ and δx'→are the displacement vectors at two consecutive time intervals ) were calculated from trajectories recorded in control cells and energy-depleted cells for two different lag-times ( time used to calculate the displacement ) of 200 ms and 2 s .", "Correlations are plotted as a function of the initial displacement length |δx→| .", "By assuming a linear dependence c=−b|δx⃑| , the slope b quantifies the negative correlations of subsequent particle displacements and its magnitude increases from b ≈ 0 . 15 to b ≈ 0 . 34 upon energy depletion .", "The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 5 . 5 , pH 6 . 0 and pH 7 . 4 , respectively , containing 2 mM DNP and 2% glucose .", "The cells were energy-depleted in SD-complete medium without glucose containing 20 mM 2-deoxyglucose and 10 µM antimycin A . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01310 . 7554/eLife . 09347 . 014Figure 4—figure supplement 1 . Top: The power spectral density ( PSD ) of particle displacements is shown as a function of frequency ω .", "The dashed lines correspond to a power law scaling of the PSD as ω−γ with γ=1+α where α is the exponent in the power-law scaling of the corresponding MSD ( see Figure 4A ) .", "Bottom: The PSD after randomly reshuffling the trajectories .", "The dashed line corresponds to the scaling of Brownian motion ( γ=2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01410 . 7554/eLife . 09347 . 015Figure 4—figure supplement 2 . The cumulative distribution functions ( CDFs ) of particle displacements for some representative trajectories in all considered conditions ( log-phase , energy-depleted , and pH-adjusted to pH 6 . 0 and pH 7 . 4 ) .", "The dashed lines are fits to CDFs of Gaussian distributions .", "The inset shows the values of the non-Gaussian parameter ( defined in the Materials and methods section ) for each individual trajectory in the corresponding experimental conditions .", "For all conditions its value is close to zero , in agreement with Gaussian statistics of particle displacements . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01510 . 7554/eLife . 09347 . 016Figure 4—figure supplement 3 . Left: The probability density functions ( PDFs ) of particle displacements for all conditions ( log-phase , energy-depleted , and pH-adjusted to pH 6 . 0 and pH 7 . 4 ) and at two different lag times after rescaling the displacements both by tαand the generalized diffusion constant Kα .", "The dashed line is the PDF of the Gaussian distribution with the unit variance .", "Right: The unscaled PDFs of particles’ displacements for log phase and energy depleted cells for two lag times of 0 . 2 and 2 s . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01610 . 7554/eLife . 09347 . 017Figure 4—figure supplement 4 . The directional correlation function , defined as C ( nτ ) =⟨Δx→", "( t ) |Δx→", "( t ) |⋅Δx→ ( t+nτ ) |Δx→ ( t+nτ ) |⟩ , where τ is the lag-time , n is an integer , Δx→", "( t ) is the change in particle position between time t and t+τ and |Δx→", "( t ) | denotes the length of the vector Δx→", "( t ) , for all conditions ( log-phase , energy depleted , and pH adjusted to pH 6 . 0 and pH 7 . 4 ) as a function of nτ for lag-time τ=200 ms . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01710 . 7554/eLife . 09347 . 018Figure 4—figure supplement 5 . Correlations of subsequent displacements ( defined as c=δx'→⋅δx→|δx→| , where δx→ and δx'→ are the displacement vectors at two consecutive time intervals ) were calculated from trajectories recorded in cells adjusted to pH 7 . 4 and pH 6 . 0 , respectively , and plotted as a function of the initial displacement length |δx→| .", "The lag-time ( time used to calculate the displacement ) is 200 ms . The linear slope b quantifies the negative correlations of subsequent particle displacements and its magnitude increases from b≈0 . 15 to b≈0 . 32 upon acidification .", "The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 6 . 0 and pH 7 . 4 , respectively , containing 2 mM DNP and 2% glucose . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 01810 . 7554/eLife . 09347 . 019Figure 4—figure supplement 6 . Time-averaged ( solid lines ) and ensemble-averaged ( symbols ) MSDs of particles in control and energy-depleted cells , respectively ( left panel ) Time-averaged ( solid lines ) and ensemble-averaged ( symbols ) MSDs of particles in cells adjusted to pH 6 . 0 and pH 7 . 4 with DNP , respectively ( right panel ) .", "Grey areas indicate +/- SEM of the time-averaged MSD . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 019 It has been suggested that the trajectories of tracer particles in cells ( Jeon et al . , 2011; Tejedor et al . , 2010 ) and hydrogels ( Stempfle et al . , 2014 ) can be well described by the model of fractional Brownian motion ( fBm ) .", "In contrast to ordinary Brownian motion , the displacements in fBm are correlated in time .", "Positively correlated displacements lead to superdiffusion , whereas negative correlations lead to subdiffusion .", "As for ordinary Brownian motion , the distribution of displacements of individual particles performing subdiffusive fBm is Gaussian , but the width of the distribution increases with the lag time sub-linearly σi2", "( t ) =2dKα , itα ( Hofling and Franosch , 2013 ) .", "Here , α<1 is the subdiffusion exponent , d is the dimension of space , and Kα , i is the generalized diffusion constant .", "The subscripts i indicate the diffusivities of individual particle trajectories .", "We show that our displacement data are consistent with the model of fBm: For sufficiently long individual trajectories , the cumulative distribution functions ( CDF ) of displacements are well described by CDFs of the corresponding Gaussian distributions ( Figure 4—figure supplement 2 ) .", "Also consistent with the model of fBm , the combined ( ensemble ) distributions of all particle displacements measured for different lag times collapse onto each other after rescaling the displacements as x/tα/2 ( Figure 4B ) .", "The value of α is read out from the scaling of the MSD for the corresponding experimental condition ( see Figure 4A ) .", "Remarkably , the shape of this distribution is not Gaussian .", "However , if we additionally rescale the displacements of each trajectory by its corresponding generalized diffusivity Kα , i , the combined distributions collapse onto a Gaussian distribution with unit variance ( Figure 4—figure supplement 3 ) .", "This collapse shows that the non-Gaussian shape of the combined distribution is a result of the variation in individual particle diffusivities ( see materials and methods for details ) .", "The variability of generalized diffusivities could be due to differences in particle sizes , but it could also reflect variations in the properties of the particles’ microenvironments .", "Indeed , we find that even for particles of similar sizes the diffusivities vary strongly for all size groups ( see Figure 1—figure supplement 3 ) , suggesting that the heterogeneity of the particle microenvironment has a strong impact on particle mobility .", "To further test how the microenvironment of the particles changes upon energy depletion and acidification , we analyzed the correlations in the displacements of particles .", "Negative displacement correlations are the origin of subdiffusive fBm .", "Indeed , we found that subsequent particle displacements were negatively correlated ( see Figure 4—figure supplement 4 and material and methods for the definition of the correlation function ) .", "Interestingly , such negative correlations are a hallmark of particle motion in an elastic environment; particles surrounded by elastic structures tend to be pushed back to their original position .", "The further the particle is initially displaced , the stronger are the forces pushing it back in the subsequent time interval , which results in stronger negative correlations .", "Indeed , we find on average that the restoring motion is opposite to the initial step and is linearly proportional to the initial displacement length ( Figure 4C ) .", "In general , the slope of this linear dependence changes from 0 for viscous fluid to -1/2 for an elastic material ( Weeks and Weitz , 2002 ) .", "In our data , the magnitude of the slope b increases from b≈0 . 15 to b≈0 . 34 upon energy depletion and acidification ( Figure 4C and Figure 4—figure supplement 5 ) .", "This result is consistent with the idea that energy depletion and acidification increase the stiffness of the particles’ microenvironments and that the cytoplasm transitions from a fluid-like to a more solid-like state under these conditions .", "We next tested whether the transition of the cytoplasm to a more solid-like state , as proposed by our particle analysis , also manifests in global changes in the mechanical properties of cells .", "To experimentally address this question , we mechanically phenotyped budding yeast cells .", "This required enzymatic removal of the rigid cell wall , which provides mechanical stability to yeast cells , by a process known as spheroplasting .", "Spheroplasted budding yeast cells were investigated using atomic force microscopy ( AFM , [Radmacher , 2007] ) , the standard in cell mechanical characterization , and real-time deformability cytometry ( RT-DC , [Otto et al . , 2015] ) , a novel microfluidic technique with 100000 times higher throughput .", "The AFM-based indentation experiments , performed with 10 µm-sized spherical probes to test whole cell mechanics , revealed that acidified cells were about 2 . 5 times as stiff as control cells ( Figure 5A and Figure 5—figure supplement 1 ) .", "Importantly , the apparent elastic modulus we measured for spheroplasted cells was three orders of magnitude lower than for yeast cells surrounded by a cell wall ( Figure 5—figure supplement 1 ) , thus clearly showing that the cell wall had been completely removed .", "However , cells are usually viscoelastic , and the apparent elastic modulus , as extracted using the Hertz model , reflects their combined elastic and viscous response .", "To test whether the observed 2 . 5-fold increase in the apparent elastic modulus of spheroplasts at low pH could also be caused by a strong increase in the viscous resistance to deformation , we extracted the viscosity of the cells from the AFM indentation-retraction curves similar to a recently published method ( Rebelo et al . , 2013; for details see Methods section ) .", "We found that the viscosity even decreased from pH 7 . 4 to pH 6 . 0 ( Figure 5—figure supplement 2 ) .", "Together , the analysis of apparent elastic modulus and viscosity unambiguously demonstrates that the cell body transitions from a compliant , more viscous material to a stiffer , more elastic material at low pH . 10 . 7554/eLife . 09347 . 020Figure 5 . Mechanical characterization of acidified and energy-depleted cells .", "( A ) The apparent elastic modulus of S . cerevisiae spheroplasts ( without rigid cell walls ) at pH 7 . 4 ( E = 636 ± 16 Pa ( mean ± SEM ) ; N = 249 ) and pH 6 . 0 ( E = 1459 ± 59 Pa; N = 257 ) was measured by AFM-based indentation .", "The cytosolic pH of spheroplasts was adjusted with phosphate buffers of pH 6 . 0 and pH 7 . 4 , respectively , containing 2 mM DNP , 1% glucose and 1 M sorbitol .", "( B ) The same cells as in ( A ) characterized with real-time deformability cytometry ( RT-DC ) .", "Each measured cell results in a dot in this deformation-cell diameter plot .", "Also shown are 90% ( solid ) and 50% ( dashed ) density lines , and the histograms of size and deformation including Gaussian fits .", "( C ) The cell wall of rod-shaped S . pombe cells was removed under control , energy depletion , and pH-adjusted conditions .", "The cytosolic pH of cells was adjusted during spheroplasting with phosphate buffers of pH 5 . 5 and pH 7 . 4 , respectively , containing 2 mM DNP , 2% glucose , 1 M sorbitol and cell wall-digesting enzymes .", "Cells were energy-depleted in growth medium without glucose containing 20 mM 2-deoxyglucose and 10 µM antimycin A for 2 hr prior to spheroplasting .", "Energy depletion was continued during spheroplasting by including 20 mM 2-deoxyglucose and 10 µM antimycin A in the spheroplasting buffer .", "( D ) The roundness of more than 160 cells per condition at the start of the experiment and after 3 hr of incubation in the presence of cell wall digesting enzymes ( end ) was quantified .", "∗∗p<0 . 01; ∗∗∗p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02010 . 7554/eLife . 09347 . 021Figure 5—figure supplement 1 . ( Left ) The apparent elastic moduli of S . cerevisiae spheroplasts , incubated in phosphate buffers of different pH in the presence and absence of DNP , were determined by AFM-based indentation . Independent measurements were repeated at least four times , with a total of N = 242–409 cells , for each condition .", "Cells were significantly stiffer at pH 6 . 0 ( E = 1459 ± 59 Pa; mean ± SEM ) than at pH 7 . 4 ( E = 636 ± 16 Pa ) and in the presence of DNP .", "Without DNP there was no statistically significant difference in stiffness between extracellular pH 6 . 0 and pH 7 . 4 ( 975 ± 39 Pa and 947 ± 27 Pa , respectively ) .", "Boxes show the 25th , 50th ( the median ) , and 75th percentiles , whiskers the spread of the data ( excluding outliers ) , and a square the mean score for a group .", "Mann-Whitney-tests were used to statistically compare two groups .", "Asterisks *** indicate significance level p<0 . 001 .", "( Middle )", "The apparent elastic modulus of S . cerevisiae cells with and without cell wall was determined by AFM-based indentation .", "It is E = 1 . 3 ± 0 . 2 MPa ( mean ± SEM; N = 69 ) for cells with cell wall and E = 975 ± 39 Pa ( mean ± SEM; N = 409 ) for cells without cell wall ( same data as for pH 7 . 4 w/o DNP in the left panel ) .", "( Right )", "Typical force-distance curves are shown for both pH values in the presence of DNP .", "The cytosolic pH of spheroplasts was adjusted with phosphate buffers of pH 6 . 0 and pH 7 . 4 , respectively , containing 2 mM DNP , 1% glucose and 1 M sorbitol . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02110 . 7554/eLife . 09347 . 022Figure 5—figure supplement 2 . The viscosity of the spheroplasted cells , extracted from the AFM-based indentation-retraction curves , decreased from η = 90 ± 16 Pa s ( mean ± SEM; N = 31 ) at pH 7 . 4 to η = 70 ± 14 Pa s ( N = 23 ) at pH 6 . 0 . Boxes show the 25th , 50th ( the median ) , and 75th percentiles , whiskers the spread of the data ( and a few outliers ) .", "Asterisk * indicates significance level p<0 . 05 ( Mann-Whitney-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02210 . 7554/eLife . 09347 . 023Figure 5—figure supplement 3 . The volume of pH-adjusted ( left panel ) and sorbitol-treated ( right panel ) yeast cells was measured with an imaging-based method ( see materials and methods ) .", "The cytosolic pH was adjusted by treating cells with phosphate buffers of indicated pH containing 2 mM DNP and 2% glucose .", "Sorbitol treatment was done in synthetic complete medium .", "The term 'relative cell volume' is used to indicate that the volume of control cells ( Ctrl , cells in synthetic complete medium ) was normalized to 100% .", "More than n=160 cells were measured for each condition .", "Values are given as mean +/- SEM .", "** Indicates a p-value < 0 . 01 , *** a p-value < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02310 . 7554/eLife . 09347 . 024Figure 5—figure supplement 4 . Both low pH and sorbitol treatment cause a reduction in particle mobility . MSD of GFP-µNS particles in cells exposed to increasing concentrations of sorbitol and in cells adjusted to low pH . The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 5 . 5 containing 2 mM DNP and 2% glucose .", "Sorbitol treatment was done in synthetic complete medium containing the indicated amount of sorbitol . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02410 . 7554/eLife . 09347 . 025Figure 5—figure supplement 5 . The diffusivity of a mCherry-GFP fusion protein was measured with fluorescence recovery after photobleaching ( FRAP ) in cells exposed to different concentrations of sorbitol ( left panel ) , cells adjusted to different cytosolic pH ( middle panel ) , and in energy-depleted cells ( right panel ) .", "Sorbitol treatment was done in synthetic complete medium containing the indicated amount of sorbitol .", "The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 5 . 5 , pH 6 . 0 and pH 7 . 4 , respectively , containing 2 mM DNP and 2% glucose .", "Cells were energy-depleted in synthetic complete medium ( standard pH of 5 . 5 ) without glucose containing 20 mM 2-DG and 10 μM antimycin A . Each curve represents the average of more than 5 measurements in different cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02510 . 7554/eLife . 09347 . 026Figure 5—figure supplement 6 . Energy-depleted S . pombe cells retain a rod-like shape in the absence of a cell wall . Left: Energy-depleted S . pombe cells before ( top ) and after ( bottom ) cell wall removal .", "BF: Bright field image .", "Calcofluor white staining confirms the presence of a cell wall before and absence of a cell wall after enzymatic removal .", "Right panel: Averaged line profiles through cells ( red lines indicated on the left show examples of line scans in representative cells ) .", "Measurements were carried out for 10 individual cells before and after cell wall removal , respectively .", "Note the different axis scales indicating strong differences in calcofluor staining intensity .", "Measurements were carried out for 10 individual cells before and after cell wall removal , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02610 . 7554/eLife . 09347 . 027Figure 5—figure supplement 7 . Cell wall digesting enzymes work equally well in buffers of different pH . Top: Still images of cells incubated with and without ( Ctrl ) cell wall removing enzyme mix in buffers of indicated pH . No sorbitol was added in order to allow for cell lysis upon cell wall removal .", "Bottom left: Quantification of the lysing activity ( absorbance at 595 nm ) as a function of time .", "Absorbance only decreases after addition of the enzyme mix ( arrow , time 0 ) .", "Bottom right: The activity of the enzyme mix at different pH was derived from the plot shown on the bottom left by linear regression .", "Enzyme activity is given as the change of absorbance per minute ( Abs . /min ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02710 . 7554/eLife . 09347 . 028Figure 5—figure supplement 8 . Energy-depleted S . pombe cells were imaged by time-lapse bright field microscopy after addition of the cell wall removing enzyme mix . Still images show multiple events ( yellow arrows ) in which the cell body separates from the cell wall sheath and maintains its rod-like shape .", "Also see corresponding Video 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 02810 . 7554/eLife . 09347 . 029Figure 5—figure supplement 9 . Low pH adjusted , rod-shaped S . pombe spheroplasts round up when exposed to glucose-containing medium . Prior to imaging the spheroplast was kept in phosphate buffer of pH 6 . 0 containing 1 M sorbitol and 2 mM DNP and did not round up for several hours .", "At time point 0 min the buffer was replaced with EMM5 medium containing glucose and 1 M sorbitol .", "Sorbitol was added to osmotically stabilize the cells .", "Also see corresponding Video 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 029 The increase in stiffness was independently confirmed by RT-DC measurements , which showed that the mechanical deformability of yeast cells was significantly reduced upon cytosolic acidification ( Figure 5B ) .", "In the RT-DC assay , we also noticed that acidification was associated with differences in cell size , with acidified yeast being smaller ( equivalent diameter: 3 . 503 +/- 0 . 012 microns; mean +/- SEM; N = 2938 ) than control cells ( equivalent diameter: 3 . 724 +/- 0 . 014 microns; mean +/- SEM; N = 2354 ) ( Figure 5B ) .", "Overall , these whole cell measurements show that acidification changes the mechanical properties of the cells in line with a transition of the cytoplasm to a more elastic , solid-like state , which is accompanied by a reduction in cell volume .", "Our findings so far can be explained in two ways: First , cytosolic acidification could lead to a regulatory cell volume decrease , including water loss and increased macromolecular crowding ( Mourão et al . , 2014 ) .", "Second , acidification could trigger the formation of macromolecular assemblies , which provide increased mechanical stability to the cytoplasm .", "In this case , the cell volume reduction could be a result of the exclusion of water from these assemblies ( Cameron et al . , 2006; Cameron and Fullerton , 2014; Fullerton et al . , 2006; Thirumalai et al . , 2012 ) .", "To investigate which scenario might apply , we performed a series of experiments with budding and fission yeast .", "First , we determined the volume of budding yeast cells using an image-based approach ( see materials and methods for details ) .", "We found that the cell volume was reduced in a pH-dependent manner .", "After 30 min at pH 5 . 5 , the cell volume was reduced by ~7% ( Figure 5—figure supplement 3 ) .", "Cell volume changes can also be induced by altering the osmotic strength of the growth medium with sorbitol ( Miermont et al . , 2013 ) .", "Thus , we exposed yeast to different sorbitol concentrations to determine the concentration at which the cell volume was similar to that of acidified yeast .", "We found that at a sorbitol concentration of 0 . 8 M the cell volume decrease was of the same magnitude ( Figure 5—figure supplement 3 ) .", "As a next step , we compared the particle mobility of osmotically compressed and acidified yeast showing a similar decrease in cell volume .", "We found that under both conditions particle motion was strongly reduced ( Figure 5—figure supplement 4 ) .", "However , in osmotically compressed cells particles of all sizes still performed small movements .", "These movements could also be detected when yeast cells were exposed to a sorbitol concentration of 1 M , which triggers an even more pronounced cell volume reduction of ~30% .", "In contrast , particle motion was abolished in acidified yeast ( Figure 5—figure supplement 4 ) .", "This suggests that a regulatory cell volume decrease cannot fully explain the reduced particle dynamics of acidified yeast .", "To further investigate this , we compared the diffusivity of a mCherry-GFP fusion protein ( 54 kDa ) in energy-depleted , acidified and sorbitol-treated cells .", "The diffusion of mCherry-GFP was not affected in acidified or energy-depleted yeast ( Figure 5—figure supplement 5 ) , but strongly decreased in cells subjected to high levels of osmotic compression .", "This indicates the presence of a fluid phase that allows unimpaired diffusion of small macromolecules such as mCherry-GFP in cells exposed to low pH or energy depletion conditions .", "Thus , cytosolic acidification and osmotic compression seem to induce qualitatively different states of the cytoplasm .", "We next analyzed the mechanical stability of fission yeast cells .", "Fission yeast has an elongated shape , which is supported by the cell wall .", "However , when the cell wall is removed , fission yeast cells rapidly round up into a spherical shape ( Kelly and Nurse , 2011; Sipiczki et al . , 1985 ) .", "This process requires the cytoplasm to be in a fluid-like state , and it is most likely driven by the osmotic pressure of the cytoplasm and by the passive tendency of the cell to minimize its surface to volume ratio .", "Remarkably , when we spheroplasted energy-depleted yeast cells , they did not relax into spheres , but maintained their initial rod-like shape ( Figure 5C and D , Video 4 ) .", "This effect was not due to incomplete removal of the cell wall ( Figure 5—figure supplement 6 , 7 and 8 , Video 6 ) .", "Importantly , for wall-free cells , maintenance of the rod-like shape was pH-dependent and could also be induced by reducing the cytosolic pH with DNP ( Figure 5C and D , Video 5 ) . 10 . 7554/eLife . 09347 . 030Video 4 . Brightfield time-lapse microscopy of S . pombe cells during cell wall removal . Cells were imaged in EMM5 medium containing glucose ( control , left panel ) or in EMM5 medium without glucose containing 20 mM 2-desoxyglucose ( 2DG ) and 10 µM antimycin A ( right panel ) .", "Cell wall removing enzyme mix was added immediately before the recording was started .", "Medium contained 1 M sorbitol to osmotically stabilize the cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 03010 . 7554/eLife . 09347 . 031Video 5 . Brightfield time-lapse microscopy of S . pombe cells during cell wall removal . Cells were imaged in phosphate buffer of pH 5 . 5 ( right panel ) and pH 7 . 4 ( left ) containing 2 mM DNP and 2% glucose .", "Cell wall removing enzyme mix was added after 30 min of imaging .", "Buffers contained 1 M sorbitol to osmotically stabilize the cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 03110 . 7554/eLife . 09347 . 032Video 6 . Brightfield time-lapse microscopy of energy-depleted S . pombe cells during enzymatic cell wall removal . Cells were energy-depleted in EMM5 medium ( pH 6 . 0 ) without glucose supplemented with 20 mM 2-deoxyglucose and 10 μM antimycin A for 2 hr before imaging .", "Imaging was then done in cell wall removal buffer ( phosphate buffer pH 6 . 0 containing 20 mM 2-deoxyglucose and 10 μM antimycin A as well as cell wall removal enzymes ) .", "Rod-like cells are slipping out of what seems to be a sheath of cell wall material without changing their shape . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 032 To investigate whether rod-shaped spheroplasts would eventually round up into spheres , we observed them for extended times by time-lapse microscopy .", "However , the spheroplasts maintained their elongated shape for several hours and did not show signs of rounding up ( Video 4 and 5 ) .", "Given this remarkable cellular phenotype , we tested whether the cells are still alive and get softer when energy is provided and the internal pH rebounds to neutral values .", "Indeed , when acidified yeast cells were re-exposed to medium , the cells quickly became spherical and started to enter the cell cycle ( Figure 5—figure supplement 9 , Video 7 ) .", "Importantly , this rounding up process occurred in the presence of 1 M sorbitol , which was used to osmotically stabilize the cells .", "Under these conditions , yeast cells experience a substantial reduction in cell volume ( Figure 5—figure supplement 3 ) , suggesting that an increase in molecular crowding alone does not generate enough mechanical stability to keep the cells in a rod-like shape .", "Rather , these findings support our idea that cellular stiffening may involve the formation of rigid cytoplasmic structures , which dissolve when energy-depleted yeast re-adjust their cytosolic pH to neutral values .", "Thus , we conclude that the cytoplasm of energy-depleted cells undergoes a pH-dependent transition from a fluid- to a solid-like state , which may be accompanied by the formation of structures that significantly increase the mechanical stability of cells . 10 . 7554/eLife . 09347 . 033Video 7 . Brightfield time-lapse microscopy of a S . pombe spheroplast . The spheroplast was generated prior to imaging in phosphate buffer of pH 6 . 0 containing 1 M sorbitol , 2 mM DNP and cell wall digesting enzyme mix and kept in this cell wall removal buffer for 4 . 5 hr .", "Directly before the start of the recording , the spheroplast was washed with EMM5 medium containing 1% glucose and 1 M sorbitol .", "Buffer and medium contained 1 M sorbitol to osmotically stabilize the spheroplast . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 033 Which cytoplasmic structures could be underlying this remarkable change in cellular rigidity ?", "A large number of in vitro studies have shown that the solubility of proteins drops precipitously , when the pH of the solution approaches their isoelectric points ( Tanford and De , 1961 ) .", "Under these conditions , proteins interact with each other to form higher-order assemblies , which macroscopically manifest as structures with solid-like properties ( Boye et al . , 1996; Matsudomi et al . , 1991; Parker et al . , 2005; Renard and Lefebvre , 1992 ) .", "Thus , we reasoned that the densely packed cytoplasm of yeast cells undergoes a similar transition on a global scale .", "To investigate this possibility , we first analyzed the distribution of the isoelectric points of all proteins in the yeast proteome .", "In agreement with previous work ( Weiller et al . , 2004 ) , we found that the isoelectric points of yeast proteins are largely excluded from the neutral pH range and cluster into two peaks , one in the acidic and one in the basic range ( Figure 6A ) .", "Importantly , the acidic peak overlaps with the pH that cells experience under starvation conditions .", "This suggests that many proteins have a reduced net charge in energy-depleted cells ( Chan et al . , 2006 ) and thus become less soluble .", "This is in agreement with previous results , where it was shown that starvation triggers the assembly of many proteins into higher-order structures ( Narayanaswamy et al . , 2009; Noree et al . , 2010; Petrovska et al . , 2014 ) .", "Importantly , protein complexes remain intact in energy-depleted cells , as shown by the fact that the different proteins in a hetero-complex colocalize in the same structures ( Figure 6—figure supplement 1 ) .", "This suggests that the proteins assemble into structures in a native-like state , ensuring that this step is readily reversible .", "To investigate whether protein assembly and reduced particle mobility are temporally linked , we exposed yeast cells to pH manipulations in a microfluidic chamber and followed assembly and particle movement by fluorescence microscopy .", "Indeed , we found that these two events coincided ( Video 8 ) , suggesting a causal relationship . 10 . 7554/eLife . 09347 . 034Figure 6 . Acidification of the cytosol causes widespread assembly of cytoplasmic proteins .", "( A ) The isoelectric points and the molecular weight of all yeast proteins were computed from their primary amino acid sequence and plotted as a virtual 2D gel .", "The green line indicates optimal growth pH , the red line indicates pH reported for dormant yeast cells .", "( B ) We systematically tested the response of 68 cytoplasmic proteins to a drop in cytosolic pH . Shown are representative images of proteins that responded with assembly formation to low pH . The same proteins also form assemblies in yeast spores .", "( C ) The percentage of cells showing protein assemblies at high versus low pH was quantified .", "The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 5 . 5 and pH 7 . 4 , respectively , containing 2 mM DNP and 2% glucose . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 03410 . 7554/eLife . 09347 . 035Figure 6—figure supplement 1 . Response of 68 cytoplasmic proteins to a drop in cytosolic pH and to treatment with 1 M sorbitol . Representative images of proteins that respond with assembly formation to low pH were also tested for their response to 1 M sorbitol ( left ) .", "The percentage of cells showing protein assemblies at high versus low pH as well as in sorbitol-treated cells was quantified .", "pH data is the same as in Figure 6 and is shown for comparison .", "The cytosolic pH was adjusted as described in the legend of Figure 6 .", "Sorbitol treatment was performed in synthetic complete medium containing 1 M sorbitol . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 03510 . 7554/eLife . 09347 . 036Figure 6—figure supplement 2 . Different subunits of the hetero-pentameric eIF2B complex colocalize in the same filamentous structures , suggesting that these protein retain their native-like structure when they form higher-order assemblies . Cells were energy-depleted in phosphate buffer of pH 6 . 0 for 3 hours prior to imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 03610 . 7554/eLife . 09347 . 037Video 8 . Fluorescence time-lapse microscopy of Gcn3-GFP ( left side ) and GFP-µNS ( right side ) expressing yeast cells growing in a microfluidic flow chamber . Cells were exposed to a phosphate buffer of pH 5 . 5 containing 2 mM DNP and 2% glucose as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 037 Given these observations , we reasoned that many proteins might be able to form structures in a pH-dependent manner .", "Widespread formation of macromolecular protein assemblies could lead to considerable changes in the cellular architecture , and could trigger the formation of a percolated filamentous-colloidal network that would obstruct the movement of particles and provide mechanical stability to the cell .", "To investigate this possibility , we tested a set of 70 proteins that had previously been shown to assemble into higher-order structures upon starvation ( Narayanaswamy et al . , 2009; Noree et al . , 2010 ) .", "We found that the majority of these proteins formed structures upon acidification , whereas such structures were less abundant or absent at neutral pH ( Figure 6B and C ) or in 1 M sorbitol ( Figure 6—figure supplement 2 ) .", "Similar structures were observed in dormant yeast spores ( Figure 6B ) , which reportedly have a pH in the acidic range ( Aon and Cortassa , 1997; Barton et al . , 1980 ) .", "Thus , we conclude that many proteins assemble into higher-order structures in energy-depleted and acidified cells and that this causes extensive changes in the organization and material properties of the cytoplasm .", "A hallmark of dormant cells is that they can survive extended periods of energy depletion .", "We therefore wondered whether pH-induced formation of a solid-like cytoplasm promotes cellular survival .", "Indeed , when energy-depleted budding yeast cells were kept in the presence of neutral medium to prevent cytoplasmic acidification , they rapidly lost viability ( Figure 7A ) .", "Moreover , when we fixed the pHc in the acidic or neutral range in energy-depleted cells , only acidified S . cerevisiae ( Figure 7B ) and S . pombe ( Figure 7C ) cells survived .", "Thus , we conclude that the change in the physical properties of the cytoplasm is protective and that yeast cells use a simple physicochemical signal—the pH of the cytosol—to signal a depletion of energy and to regulate entry into a dormant state . 10 . 7554/eLife . 09347 . 038Figure 7 . Acidification of the cytosol promotes survival under energy depletion conditions .", "( A ) Growth assay of S . cerevisiae cells that were energy-depleted with 20 mM 2-DG and 10 µM antimycin A in growth medium adjusted to pH 7 . 0 and 6 . 0 , respectively .", "( B ) Similar to A , but cells were incubated in buffers of pH 7 . 0 or pH 6 . 0 , respectively , containing 2 mM DNP .", "( C ) Similar to A , but the experiment was performed with fission yeast .", "( D ) A flow chart showing the hypothetical sequence of events promoting entry into a dormant state . DOI: http://dx . doi . org/10 . 7554/eLife . 09347 . 038" ], [ "Many biochemical reactions inside a cell take place in the cytoplasm .", "Thus , changes in the physical or chemical properties of the cytoplasm will have far-reaching consequences for cellular metabolism and survival .", "Here , we demonstrate that adaptive changes in the cytosolic pH alter the material properties of the cytoplasm and arrest the diffusion of cellular organelles and foreign tracer particles ( see schematic in Figure 7D ) .", "We further show that pH-controlled macromolecular assembly drives a transition of the cytoplasm to a solid-like state , which provides protection and increased mechanical stability .", "Many previous studies have demonstrated a role for energy in controlling intracellular dynamics .", "In eukaryotic cells , ATP-driven motor proteins carry organelles and other cargo along cytoskeletal tracks to specific subcellular locations , thus regulating the distribution of cytoplasmic components ( Hirokawa et al . , 2009; Roberts et al . , 2013 ) .", "Recent findings also indicate that the movements of motor proteins generate random fluctuating forces , which drive diffusive-like non-thermal motion ( Brangwynne et al . , 2008 , 2009; Guo et al . , 2014 ) .", "These non-thermal force fluctuations facilitate the mixing of the cytoplasm and thus are important for cellular function .", "As a consequence , energy depletion in mammalian cells causes a cessation of motor movements and thus a strong impairment of intracellular motion , with a significant impact on the distribution of macromolecules in the cytoplasm .", "The diffusion of a particle in the cytoplasm is the result of two opposing forces: fluctuating forces of thermal or non-thermal origin , which transfer energy onto the particle , thus propelling its motion , and opposing material forces that restrict the movement of a particle and result from interactions of the particle with cytoplasmic components .", "Studies so far have largely focused on the driving forces of particle motion , and most prominently those that are dependent on ATP ( Brangwynne et al . , 2008 , 2009; Guo et al . , 2014; Parry et al . , 2014 ) .", "However , we now show that changes in the material properties of the cytoplasm can also significantly affect particle motion .", "These changes occur in an energy-dependent manner , indicating that energy not only regulates motor-driven diffusive processes , but also has an impact on the organization of the cytoplasm .", "In the organisms we investigate , these changes are largely independent of the cytoskeleton , but reflect the collective effects of many cytosolic proteins that assemble into higher-order structures .", "The chemistry and physics of proteins has been under investigation for many decades .", "For example , it is a well-known fact that changes in the salt concentration can have a strong impact on protein solubility .", "One parameter with particularly strong effects on protein solubility is the pH . When the pH of a solution approaches a protein’s isoelectric point , the solubility of the protein drops to a minimum ( Tanford and De , 1961 ) .", "The reason is that under these conditions proteins attain a low net charge and thus are subject to weaker repulsive interactions .", "As a consequence , attractive interactions dominate , which—provided that the protein is present at a high enough concentration—can trigger the assembly of proteins into higher-order structures .", "The pH-driven assembly of proteins can result in the formation of large structures with solid-like properties ( Boye et al . , 1996; Matsudomi et al . , 1991; Parker et al . , 2005; Renard and Lefebvre , 1992 ) .", "Thus , on the macroscopic level , protein assembly can exhibit signs of a phase transition .", "Although this tendency of proteins to form higher-order structures is well known , this knowledge has not yet been applied to the understanding of living organisms .", "One reason may be the widespread but misleading assumption that the physicochemical properties of the cytoplasm are invariant .", "However , in recent years , an increasing number of reports have shown that key physical and chemical parameters of the cytoplasm can fluctuate , especially under conditions of stress .", "In the case of cytosolic proton concentrations , these fluctuations can span almost two orders of magnitude ( our study and Imai and Ohno , 1995; Orij et al . , 2009; Valli et al . , 2005 ) .", "Given that the cytoplasm is highly crowded with proteins , it is not surprising that pH changes of this scale have strong effects on the physical properties and the dynamics of the cytoplasm .", "Previous studies have analyzed the distribution of the isoelectric points in the proteome ( Chan et al . , 2006; VanBogelen et al . , 1999; Weiller et al . , 2004 ) .", "These studies found that only few proteins have isoelectric points in the neutral pH range and that most proteins only become electrically neutral when the pH shifts to the acidic or basic range .", "Importantly , proteins become more insoluble when their net charge decreases .", "Thus , to maintain maximum proteome solubility , cells have to keep the cytosolic pH in the neutral range .", "However , when the pH of the cytosol becomes more acidic , as during starvation , a large fraction of the proteome will become less soluble .", "Consistent with this , we could previously show that cytosolic acidification triggers the assembly of a group of metabolic enzymes into higher-order structures and that assembly inactivates their enzymatic activities ( Petrovska et al . , 2014 ) .", "Here , we provide evidence that many more proteins assemble into microscopically visible structures upon acidification ( Figure 6B and C ) , and we propose that assembly of these and probably many other proteins promotes a transition of the cytoplasm to a more solid-like state .", "Importantly , pH-dependent assembly of proteins does not seem to go along with protein denaturation , as the proteins in these assemblies retain their native structure ( Petrovska et al . , 2014 ) and oligomeric states ( Figure 6—figure supplement 2 ) .", "This ensures that assembly formation can readily be reversed and that the cytoplasm can rapidly recover from pH-induced alterations , thus allowing swift reentry into the cell cycle .", "Most importantly , protein assembly and the solid-like state of the cytoplasm protect cells from the adverse effects of energy depletion stress ( Figure 7 ) .", "We do not yet know why these processes are protective , but we favor a combination of different explanations , such as energy conservation ( Bernstein et al . , 2006 ) , regulation of metabolism ( Petrovska et al . , 2014 ) , and potentially protection of macromolecules from damage .", "One of the hallmarks of dormant cells is a loss of water .", "Although we do not determine the water content of energy-depleted and acidified cells , we find that acidification goes along with a significant reduction of the cellular volume , which is consistent with a loss of water .", "Previous studies have shown that protein assembly leads to the exclusion of water ( Cameron et al . , 2006; Cameron and Fullerton , 2014; Fullerton et al . , 2006; Thirumalai et al . , 2012 ) .", "The released water becomes osmotically available and can be lost to the surrounding environment , inducing a compaction of the cytoplasm and a change in cell volume .", "Thus , we propose that the observed cellular shrinkage is to a large degree caused by the formation of cytoplasmic structures and a subsequent release of water .", "Additional evidence for this scenario comes from a study that characterized the cytoplasm as a material with distinctive gel-like properties ( Fels et al . , 2009 ) .", "The authors of this study found that the cytoplasm of mammalian cells behaves like a hydrogel , which can swell and shrink depending on its water content .", "Importantly , changes in the cytosolic pH could modulate swelling and shrinking ( Fels et al . , 2009 ) .", "This suggests that the cytoplasm with its many macromolecular components is inherently pH sensitive , a property , which may have been exploited repeatedly during evolution as a strategy for adaptation or survival .", "In fact , the germination of spores goes along with a drastic increase in water content ( Cowan et al . , 2003; Dijksterhuis et al . , 2007 ) and spores consistently have a pH in the acidic range ( Aon and Cortassa , 1997; Barton et al . , 1980; Busa and Crowe , 1983; Setlow and Setlow , 1980; van Beilen and Brul , 2013 ) .", "However , what is still unclear is how water is released from forming spores and re-enters into spores upon germination .", "Given our findings , we propose that the dehydration/rehydration cycle of spores is at least partially driven by changes in the cytosolic pH . A regulatory cell volume decrease with increased macromolecular crowding may also contribute to the water loss in dormant cells ( Mourão et al . , 2014 ) .", "Dissection of this important problem will require the use of sophisticated biophysical , biochemical , and genetic approaches .", "We show that the cytoplasm of energy-depleted cells transitions from a fluid- to a solid-like state .", "This transition was evident for the cytoplasm ( as determined by particle tracking ) and on the level of the entire cell ( as determined by cellular deformability assays ) .", "This is , to our knowledge , the first viscous and elastic characterization of S . cerevisiae spheroplasts by AFM-indentation .", "The elastic modulus ( on the order of 1 kPa ) is several orders of magnitude lower than what has been reported for intact yeast cells surrounded by a rigid cell wall ( about 500 kPa; [Pillet et al . , 2014] ) .", "A similar difference in stiffness between spheroplasts and intact cells with a rigid cell wall has previously been found in E . coli ( Sullivan et al . , 2007 ) .", "The increased mechanical stiffness at low pH was independently confirmed by a new microfluidic technique ( RT-DC ) .", "The transition from a compliant , more viscous cytoplasm to a stiff , elastic cytoplasm in energy-depleted yeast cells is in agreement with a model in which many proteins assemble into a dense network , thus restricting the diffusion of large particles .", "This network could have the overall physical properties of a glass , as recently proposed for bacteria ( Parry et al . , 2014 ) .", "Future studies will have to determine the molecular mechanisms and physical causes promoting the formation of a solid-like cytoplasm .", "In contrast to mammalian cells , yeast cells are much smaller in size .", "This may explain why yeast rely more strongly on thermal diffusion for macromolecular dispersal .", "However , this also means that yeast cells have to alter the material properties of the cytoplasm to restrict diffusion during dormancy .", "We believe this is achieved by promoting a pH-controlled transition to a solid-like state , which significantly changes the fluidity of the cytoplasm .", "Acidification of the cellular interior of yeast seems to occur through an influx of protons from the outside , suggesting that this transition is dependent on an acidic environment , which may be generated through the normal metabolic activity of yeast .", "Thus , we predict that single-celled organisms make extensive use of the pH responsiveness of the cytoplasm in order to protect themselves and regulate their metabolism .", "However , even multicellular organisms such as marine brine shrimp can enter into a dormant state in a pH-dependent manner ( Busa and Crowe , 1983 ) .", "Importantly , in this organism dormancy is induced through protons that are released from intracellular stores ( Covi et al . , 2005 ) , indicating that dependence on outside pH could be a peculiarity of yeast .", "Moreover , cytosolic pH changes have also been observed when organisms such as yeast and Dictyostelium are challenged with other types of stresses , such as heat stress or osmotic stress ( Pintsch et al . , 2001; Weitzel et al . , 1985; 1987 ) .", "Thus , global control over the material properties of the cytoplasm through simple physicochemical signals such as the pH could be a frequently used means to regulate cellular function in fluctuating environments ." ], [ "S . cerevisiae was grown at 25°C or 30°C in yeast extract peptone dextrose ( YPD ) , synthetic complete ( S-complete ) or synthetic dropout ( SD ) medium .", "Standard pH of SD media is around pH 5 . 5 .", "S . pombe was grown in either YE5 or EMM5 ( standard pH is 6 . 0 ) medium at 30°C .", "D . discoideum was grown in AX medium ( ForMedium , standard pH 6 . 0-6 . 5 ) at 23°C under light .", "A list of all S . cerevisiae strains used in this study can be found in Supplementary file 1 . S . pombe wild type strain L972 was used for spheroplasting and spotting experiments .", "The same strain was transformed with plasmid pDUAL2HFG-µNS-sfGFP for particle tracking experiments .", "D . discoideum wild type strain AX2-214 ( DictyBase ) transformed with plasmid pDM353-µNS-GFP ( Veltman et al . , 2009 ) was used for particle tracking experiments .", "A list of all plasmids used in this study can be found in Supplementary file 2 . All cloning was done using the Gateway cloning system ( Invitrogen ) as described previously ( Alberti et al . , 2007 ) .", "The intracellular pH of S . cerevisiae and S . pombe cells was adjusted by incubation in phosphate buffers of different pH in the presence of 2 mM 2 , 4-dinitrophenol ( DNP ) as described previously ( Dechant et al . , 2010; Petrovska et al . , 2014 ) .", "DNP was added to the buffers from a 0 . 2 M ( 100x ) stock solution in methanol .", "Alternatively , cytosolic acidification was achieved by incubation in SD medium containing 1 , 2 , or 6 mM sorbic acid .", "D . discoideum cells were pH adjusted by treatment with either 4 mM sorbic acid or 0 . 2 mM DNP in LoFlo medium ( pH 5 . 5 ) .", "For generation of pH calibration curves , cells were treated with 75 µM monensin , 10 µM nigericin , 10 mM 2-deoxyglucose and 10 mM NaN3 in buffers of pH 5 . 0 , 5 . 5 , 6 . 0 .", "6 . 5 , 7 . 0 , 7 . 5 , and 8 . 0 containing 50 mM MES , 50 mM HEPES , 50 mM KCl , 50 mM NaCl , 0 . 2 M ammonium acetate as described previously ( Brett et al . , 2005 ) .", "S . cerevisiae and S . pombe cells were energy-depleted by incubation in SD medium or EMM medium , respectively , without glucose containing 20 mM 2-deoxyglucose ( 2-DG , inhibition of glycolysis ) and 10 µM antimycin A ( inhibition of mitochondrial ATP production ) .", "This treatment causes a more than 95% reduction in cellular ATP ( Serrano , 1977 ) .", "D . discoideum cells were energy-depleted with 40 mM 2-DG and 200 µM azide in Soerensen-phosphate buffer ( pH 6 . 0 ) .", "To test the influence of the actin cytoskeleton on particle mobility , cells were treated with 100 µM latrunculin A ( LatA ) in SD medium for 30 min prior to imaging .", "To test the role of the microtubule cytoskeleton , cells were treated with 15 µg/ml nocodazole in SD medium for 1 hr prior to imaging .", "S . cerevisiae spheroplasts were generated by incubating cells in PBS containing 1 M sorbitol ( Sigma ) , 1% glucose and 0 . 25 mg/mL Zymolyase 100T ( USBiological ) at 25°C for at least one hour under shaking .", "S . pombe spheroplasts were generated , with minor adaptations , as described previously ( Kelly and Nurse , 2011 ) .", "Shortly , cells were incubated in buffers of different pH ( depending on experiment ) in the presence of 1 . 2 M sorbitol ( Sigma ) , 0 . 5 mg/mL Zymolyase 100T ( USBiological ) and 2 . 5 mg/mL lysing enzymes from Trichoderma harzianum ( Sigma ) .", "Wild type S . pombe L972 cells were grown in liquid EMM medium containing 0 . 5% glucose at 30°C overnight shaking at 200 rpm , diluted and re-grown to mid-log phase the next day .", "Cells were harvested by centrifugation , washed twice with medium or buffer containing 1 . 2 M sorbitol and applied to a 4-chamber glass-bottom dish ( Greiner BIO-ONE ) coated with concanavalin-A .", "For energy depletion experiments , cells were energy-depleted as described above prior to loading to the dish .", "Unbound cells were washed off with EMM medium or phosphate buffers containing 1 . 2 M sorbitol .", "Bound cells were covered with 400 µl of phosphate buffer of different pH containing 1 . 2 M sorbitol and either 2 mM DNP ( pH experiment ) or 20 mM 2-DG and 10 µM antimycin A ( energy depletion experiment ) .", "Cells were imaged for five frames before addition of 40 µl cell wall digesting enzymes ( final concentrations: 0 . 5 mg/ml Zymolyase 100T , 2 . 5 mg/ml lysing enzymes from Trichoderma harzianum ) .", "Spheroplasting and rounding up of cells was followed by time-lapse bright-field microscopy with a DeltaVision ( Applied Precision ) microscope ( Olympus IX70 stand , Olympus UPlanSApo 20x objective , CoolSnap HQ2 camera ) .", "S . cerevisiae wild type strain W303 , or S . pombe wild type strain L972 were grown overnight , diluted to OD600 ~ 0 . 1 the next morning and regrown to OD600 ~0 . 5 .", "Cells were harvested and resuspended in either phosphate buffers of pH 6 . 0 or pH 7 . 0 , respectively , containing 2 mM DNP ( S . cerevisiae and S . pombe ) or in S-medium without glucose containing 20 mM 2-DG and 10 µM antimycin A ( S . cerevisiae ) .", "Cells were then incubated under shaking at 25°C .", "Samples were taken after 2 , 24 and 48 hr , cells were washed once with H2O and subsequently spotted on YPD as five-fold serial dilutions .", "For cytosolic pH measurements a codon-optimized version of the ratiometric fluorescent protein pHluorin2 ( Mahon , 2011 ) was integrated into the W303 ADE+ genome at the trp locus .", "The protein was expressed under control of a GPD promoter .", "A pH calibration curve was obtained as described previously ( Brett et al . , 2005 ) , except that we used a microscopy-based fluorescence readout .", "Briefly , cells were incubated in buffers of different pH-containing proton carriers ( 75 μM monensin , 10 μM nigericin ) and inhibitors ( 10 mM 2-deoxyglucose ) to rapidly deplete cells of energy and allow for complete equilibration of the intracellular and extracellular pH . Cells were immobilized in 4-chamber dishes ( Greiner BIO-ONE ) with concanavalin A and imaged using DAPI/FITC ( Excitation: DAPI; Emission: FITC ) and FITC/FITC ( Excitation and emission: FITC ) filter sets on a DeltaVision ( Applied Precision ) microscope ( Olympus IX70 stand , Osram Mercury short arc HBO light source , 100x Olympus UPlanSApo objective , CoolSnap HQ2 camera ) .", "Six different Z-stacks each with 6 planes ( Z-resolution 0 . 5 µm ) were recorded for each pH condition .", "Imaging settings were: 10% excitation intensity , 0 . 1 s exposure time , 512x512 pixels , 2x2 binning .", "After background subtraction , the mean DAPI/FITC to FITC/FITC ratio was calculated from the intensity readouts of both channels and plotted against pH to obtain a calibration curve .", "Subsequent pH measurements were calculated from a fourth degree polynomial fit to the calibration curve .", "Time series of pH measurements were obtained using identical imaging settings and a CellASIC ( Millipore ) microfluidics flow setup combined with CellASIC ONIX Y04C microfluidic plates .", "For particle tracking experiments , samples were prepared in 4-chamber glass-bottom dishes ( Greiner BIO-ONE ) .", "Dishes were coated with concanavalin A coating solution for at least 30 min .", "Subsequently the coating solution was removed and the glass surface washed with H2O twice before adding 1 ml of a log phase yeast culture ( OD600 = 0 . 5 ) .", "Cells were allowed to settle onto the glass surface for 10 min .", "The supernatant was then removed and cells sticking to the surface were washed with appropriate medium or buffer twice .", "This normally results in a single layer of yeast cells that stick tightly to the glass surface .", "For control experiments cells were then incubated in 500 µl of S-complete medium for 30 min before imaging .", "When treated with DNP or sorbic acid cells were incubated in 500 µl of appropriate buffer or medium for 30 min before imaging .", "For energy depletion experiments , cells were incubated in energy depletion medium for 2 hr before imaging .", "Imaging was done on different microscope setups depending on requirements for image acquisition rate and camera chip size .", "Data with a time resolution of 5 s were recorded on a Deltavision ( Applied Precision ) microscope ( Olympus IX70 stand , Osram Mercury short arc HBO light source , Olympus UPlanSApo 100x oil objective , CoolSnap HQ2 camera , resulting pixel size ( x , y ) = 65 nm ) .", "Z stacks with 10 focal planes were collected at each time point .", "Imaging settings were: 10% excitation intensity , 0 . 08 s exposure time , 1024x1024 pixels , total imaging time 10 min .", "All data with a time resolution of 1 s were recorded on an Andor spinning disk confocal microscope ( Olympus IX81 stand , Andor iXon+ EMCCD camera , resulting pixel size ( x , y ) = 81 nm ) .", "Z-stacks with 16 focal planes were collected at each time point .", "Imaging settings were: 40% laser intensity , minimum possible exposure time ( ~16 ms ) , 512x512 pixels , total imaging time 20", "s . Data with 10 millisecond time resolution was recorded on an Andor Spinning disk setup ( Olympus IX71 stand , Olympus UPlanSApo 60x silicon oil objective , resulting pixel size ( x , y ) = 108 nm ) .", "Imaging was done in a single focal plane of 764x1190 pixels , which allowed us to track a reasonable number of particles at this frame rate in a single experiment .", "Imaging settings were: 15% laser intensity , 10 milliseconds exposure time , total imaging time of 10", "s . If recorded , Z stacks were sum-projected using the Fiji image-processing package ( Schindelin et al . , 2012 ) .", "All particle tracking was done with the MosaicSuite particle tracker ( Sbalzarini and Koumoutsakos , 2005 ) a Fiji plugin freely available from http://mosaic . mpi-cbg . de .", "The following settings were used for tracking data with 5 s time resolution: particle radius: 7 , cutoff: 0 , percent: variable , link range: 3 , displacement: 20 .", "Data with 1 s time resolution was tracked with: particle radius: 8 , cutoff: 0 , percent: variable , link range: 1 , displacement: 20 .", "Data recorded with 10 ms time resolution was tracked with: particle radius: 8 , cutoff: 0 , percent: variable , link range: 1 , displacement: 5 .", "The MosaicSuite particle tracker also measures particle intensities ( m0 ) during tracking .", "The mean intensity was computed from m0 for each trajectory and used as a proxy for particle size .", "Particle trajectories were binned into three roughly equally populated size bins ( small , medium , large ) to illustrate the dependence of the MSD on particle intensity .", "Computations and plotting were either done in R , making use of the plyr , reshape and ggplot2 ( Wickham , 2009 ) packages or in MATLAB .", "We imaged a list of 73 strains ( see Supplementary file 1 ) from the yeast GFP collection ( Huh et al . , 2003 ) under conditions of high and low intracellular pH . Samples were prepared in 4-chamber glass bottom dishes as described for the single particle tracking experiments .", "Cells were incubated in phosphate buffers of pH 5 . 5 or pH 7 . 4 , respectively , containing 2 mM DNP and 2% glucose for exactly 30 min before imaging .", "Imaging was done on a DeltaVision ( Applied Precision ) microscope ( Olympus IX70 stand , Osram Mercury short arc HBO light source , Olympus UPlanSApo 100x oil objective , CoolSnap HQ2 camera ) .", "Z stacks with 14 focal planes were collected at 6 points for each sample .", "Imaging settings were: 50% excitation intensity , 0 . 1 s exposure time , 512x512 pixels , 2x2 binning .", "Imaging of protein assemblies in yeast spores was done with similar settings .", "GFP-expressing yeast cells were used to determine the volume of cells using an imaging-based approach .", "Samples were prepared in 4-chamber glass bottom dishes as described for the single particle tracking experiments .", "In control experiments cells were subsequently incubated in 500 µl of S-complete medium for 30 min before imaging .", "For pH adjustment cells were incubated in 500 µl of phosphate buffers of pH 5 . 5 , 6 . 5 or 7 . 4 , respectively , containing 2 mM DNP and 2% glucose for exactly 30 min before imaging .", "For volume adjustment cells were incubated in S-complete medium containing 0 . 6 , 0 . 8 , 1 or 2 M sorbitol for exactly 30 min before imaging .", "Cells were imaged on an Andor spinning disk confocal microscope ( Olympus IX81 stand , Olympus UPlanSApo 100x oil objective , Andor iXon+ EMCCD camera , resulting pixel size ( x , y ) = 81 nm ) .", "Z-stacks were obtained with z=210 nm resolution .", "Z stacks were projected to obtain 2D maximum intensity projections , which were then processed further for image segmentation and object detection .", "For image segmentation , objects smaller than 10 pixels were considered to be noise and removed , and a structural filter of ellipsoid shape was applied to detect the foreground of the cells .", "The background was identified by computing the distance transform matrix of the foreground .", "Using the watershed transform matrix , the background markers were turned into regional minima , and the foreground image was segmented to obtain individual cells .", "The mask for the individual cells was then used to select the corresponding Z stack , and the pixels above the stack threshold were considered as resulting from the GFP signal of the cell .", "Finally , the empty vacuolar regions were filled , and the resultant image was counted for total number of pixels above threshold to compute the total cellular volume .", "Image processing and analysis was done in MATLAB .", "To obtain an accurate measurement of the cell volume , budding and overlapping cells were not quantified .", "The mobility of a mCherry-GFP fusion protein was measured using fluorescence recovery after photobleaching ( FRAP ) .", "Prior to imaging , yeast cells were either pH adjusted in phosphate buffers of pH 5 . 5 , 6 . 0 or 7 . 4 , respectively , containing 2 mM DNP and 2% glucose , or treated with SD-medium containing 0 . 8 , 1 . 0 , 1 . 5 or 2 . 0 M sorbitol , respectively , or energy-depleted in SD-medium without glucose containing 20 mM 2-DG and 10 µM antimycin A . Cells were then immobilized on a cover slip with concanavalin A coating solution and imaged on an Andor spinning disc microscope ( Nicon eclipse Ti stand , Nikon Plan Apo TIRF 100x oil objective , Andor iXon+ camera , resulting pixel size 70 nm ) equipped with a FRAPPA unit ( Andor ) .", "A single pixel region of interest was bleached with a 405 nm laser pulse ( 1 repeat , 40% intensity , dwell time 60 ms ) .", "Recovery from photobleaching was then recorded in a single focal plane with a time resolution of 5 . 4 ms ( EM gain 200 , laser intensity of 5% ) .", "Image analysis was carried out in FIJI .", "AFM-based indentation experiments were performed using a Nanowizard I ( JPK Instruments , Berlin ) in combination with the CellHesion module .", "Tip-less silicone cantilevers ( Arrow-TL1 , Nanoworld , Switzerland ) were equipped with a polystyrene bead of 10 µm diameter ( microParticles GmbH , Germany ) and calibrated prior to measurements using the thermal noise method .", "Cell-Tak ( Corning , USA ) was used for immobilization of spheroplasts ( Gautier et al . , 2015 ) .", "To determine the stiffness of single spheroplasts ( S . cerevisiae ) , the cantilever was positioned above individual cells and lowered with a speed of 10 µm/s .", "Force-distance curves were recorded ( maximum force 2 nN ) and analyzed using the JPK data processing software ( JPK instruments ) : Force-distance data were corrected for the tip-sample separation ( Radmacher , 2007 ) and fitted with the Hertz model for a spherical indenter ( Sneddon , 1965 ) .", "An effective probe radius was used according to the Hertz model for two spheres .", "A Poisson ratio of 0 . 5 was assumed .", "Experiments were carried out in phosphate buffer ( containing 1 M sorbitol and 1% glucose ) adjusted to pH 6 . 0 or pH 7 . 4 at room temperature both with and without DNP ( see Figure 5 and Figure 5—figure supplements ) .", "Reporting an apparent elastic ( Young’s ) modulus acknowledges the fact that several assumptions of the Hertz model ( isotropic , homogeneous , semi-infinite space ) are not met; but this still serves well for quantitative comparison of cells in different pH conditions .", "The Hertz model also assumes an elastic material , but cells are viscoelastic and an observed increase in apparent elastic modulus could also be caused by an increase in viscous resistance to deformation .", "To directly determine the viscosity η of spheroplasted cells from the recorded force-distance data , we adapted a method proposed by ( Rebelo et al . , 2013 ) .", "Briefly , this method extracts the viscosity η by comparing the dissipated energy during the indentation process , Wdiss , to the viscous work , Wv , which is modeled taking into account the indenter shape and indenter velocity .", "Wdiss corresponds to the area between the approach and retract force-distance curves , Wdiss=∫0δmax ( F ( app ) −Fret ) dδ , where δ is the tip-sample-separation , or indentation , and the superscripts app and ret indicate the forces recorded during approach and retraction , respectively .", "The viscous work is the integral of the viscous force Fv , which is described by Fv=2πη ( R−δ ) dδ/dt for a spherical indenter of radius R . Force-distance curves were smoothened using a median filter and a multi-exponential fit to compute the time-derivative of the indentation .", "Finally , the viscosity was calculated as η=Wdiss2π∫0δmax ( R−δ ) ( δ' ( app ) −δ' ( ret ) ) dδ , where δ'=dδdt .", "All calculations were implemented in Python .", "Real-time deformability cytometry ( RT-DC ) has recently been introduced as a method for high-throughput cell mechanical characterization ( Otto et al . , 2015 ) .", "Briefly , the experimental setup consists of an inverted microscope ( Zeiss , Axiovert200M ) , a high-speed video camera ( MC1362 , Mikrotron ) and a syringe pump ( NemeSys , Cetoni ) , which are assembled around a microfluidic chip .", "The chip is made of polydimethylsiloxane using soft lithography and its geometry is defined by two reservoirs connected by a 300 μm long constriction with a 10 μm x 10 μm squared cross-section .", "When a cell suspension is driven through the narrow channel , cells experience a significant hydrodynamic stress and the resulting deformation is captured and analyzed in real-time using the high-speed camera .", "Deformation , D , is quantified by the circularity c: D=1−c=2πAl , where A is the projected surface area and l the perimeter of the cell inside the channel .", "The more a cell deviates from an ideal circular shape the larger is D . For simplicity , the size measure reported is the diameter of an equivalent circle with area A . A typical experiment requires a cell concentration of 106 cells/ml and a minimal absolute sample volume of 100 μl .", "Here , S . cerevisiae spheroplasts were resuspended in PBS-methylcellulose medium adjusted to different pH , containing 1 M sorbitol and 1% glucose .", "Cells were drawn into a 1 ml syringe and connected to polymer tubing , which had been cleaned using 70% ethanol and 200 nm sterile-filtered ( Sigma Aldrich ) deionized water .", "After connecting tubing to the syringe pump and microfluidic chip a flow was stabilized for 1 min .", "Here , measurements were carried out at a constant flow rate of 0 . 012 μl/s .", "For reference , data are also acquired inside the reservoirs to ensure no deformed cell shape in the absence of mechanical stress ( data not shown ) .", "The ensemble-averaged mean squared displacement ( MSD ) was calculated as MSD", "( t ) =1N∑i=1N ( xi", "( t ) −xi ( 0 ) ) 2+ ( yi", "( t ) −yi ( 0 ) ) 2 where N is the number of particles , and xi", "( t ) and yi", "( t ) are the coordinates of particle i at time", "t . The time-and-ensemble averaged MSD , MSDτ , was computed as MSDτ", "( t ) =1N∑i=1N1m−t∑j=0m−t−1 ( xi ( t+τj ) −xi ( τj ) ) 2+ ( yi ( t+τj ) −yi ( τj ) ) 2 where t is the frameshift , N is the number of particles and m is the length of the particle trajectory .", "The maximum frameshift was limited to 1/3 of the full trajectory length .", "The subdiffusion exponent α was estimated by fitting the time-and-ensemble averaged MSD to a power law between 0 . 2-2", "s . Before fitting , the MSD was noise corrected assuming the positional noise of 11nm estimated from the power spectra .", "In this way , we obtained α ≈ 0 . 73 ( DNP treated cells with external pH 6 ) , α ≈ 0 . 84 ( DNP treated cells with external pH 7 . 4 ) , α ≈ 0 . 64 ( energy depleted cells ) and α ≈ 0 . 88 ( log phase cells ) .", "The power spectrum of displacements for a one-dimension signal x", "( t ) is given by x ( ω ) 2 , where the angular brackets denote an average over the ensemble , and x ( ω ) =∫0∞x", "( t ) eiωtdt .", "For the two-dimensional tracking data , the total power spectrum is given by the sum of the power spectra of each component as x ( ω ) 2+y ( ω ) 2 .", "The integral is approximated using the built-in MATLAB function fft ( Fast Fourier Transform ) .", "The tracking accuracy is estimated by considering the plateau of the power spectrum .", "The experimental power spectrum is given by x^ ( ω ) 2+y^ ( ω ) 2=x ( ω ) 2+y ( ω ) 2+x02+y02 , where x02+y02 is the power spectrum of the positional noise .", "This positional noise will manifest itself as a plateau in the power spectrum at high frequencies .", "We find that the plateau can be reproduced by generating Gaussian noise with standard deviation of σ≈11 nm .", "The true power spectrum is obtained by subtracting the power spectrum of this estimated positional noise from the experimentally obtained power spectrum .", "Thereafter , the low-frequency regime ( from 0 . 1 Hz to 5 Hz ) was fitted to a power law in a least square sense .", "The low frequency regime was used for the fitting as this procedure minimizes potential additional errors from the tracking procedure .", "To check whether the deviation of the slope of the PSD from the expected slope from Brownian motion is an artifact of the finite length of the trajectories or a result of correlations in the data , signifying a viscoelastic material state , the displacements of the individual trajectories were randomly reshuffled .", "This was done by randomly permuting the order of the displacements using the MATLAB function randperm .", "This way , subsequent displacements in the reshuffled trajectories are uncorrelated and the corresponding PSD should therefore scale as for Brownian motion .", "We find that this is indeed the case under all conditions ( Figure 4—figure supplement 1 ) .", "We find that the time-averaged and ensemble-averaged MSD agrees well under all conditions , implying that the process is stationary ( does not age ) on the experimental time-scale ( Figure 4—figure supplement 6 ) .", "A statistical process that is consistent with our experimental observations ( stationarity , subdiffusive scaling of the MSD , and an anomalous power-law scaling of the positional power spectrum ( Weiss , 2013 ) is fractional Brownian motion ( fBm ) .", "For fBm , the probability density function ( PDF ) of displacements is Gaussian , but the displacements are correlated , x", "( t ) x", "( s ) =Kα[tα+sα−|t−s|α] , where Kα measures the strength of the fBm and may depend on particle size and the local microenvironment .", "As a consequence of the Gaussian property , the PDF is completely determined by its second moment , proportional to the MSD of the particle .", "The correlations cause the MSD to increase as a power law , MSDτ", "( t ) =2dKαtα where d is the dimension of space , and 0<α<1 is the subdiffusion exponent .", "From this expression we see that Kα can be referred to as a generalized diffusion constant .", "In the following discussion , we consider the motion along the two coordinate axes as two independent realizations of the same random process .", "The motion is consequently analyzed as a one-dimensional process , d=1 .", "For one-dimensional fBm the PDF of displacements of each individual particle is given by Pi ( x , t ) =1 ( 4πKα , itα ) 1/2exp ( −x24Kα , itα ) .", "The lengths of the individual trajectories are too short ( ~1000 time points ) to provide a reliable estimate of the PDF on a single-particle level .", "A statistical measure that can be estimated also for small datasets ( Weiss , 2013 ) is the cumulative distribution function ( CDF ) .", "The CDF Fi ( x , t ) =∫−∞xPi ( x' , t ) dx' of a single particle trajectory is a measure for the probability that the particle makes a displacement not larger than x ( note that x has both negative and positive values ) .", "To build a CDF Fi ( x , t ) at a given moment of time ( t=2", "s ) for an individual trajectory we just count the number of displacements which are smaller than x and divide this number by the total number of displacements in a given trajectory .", "The CDFs are fitted to a Gaussian CDF with zero mean and variance given by the variance of the displacements in the array ( see Figure 4—figure supplement 2 ) .", "In addition , for each individual trajectory we can calculate the so-called non-Gaussian parameter γi=△x4i3△x2i2−1 , which vanishes for displacements △x with the Gaussian distribution .", "Indeed we see that for all experimental conditions this parameter is close to zero , see inset in Figure 4—supplement figure 2 . By rescaling the displacements by the lag time as x˜=x/tα/2 , the PDF of displacements at different times can be collapsed to a single master curve Gi ( x˜ ) =1 ( 4πKα , i ) 1/2exp ( −x˜24Kα , i ) .", "For an ensemble of N particles performing fBm in a heterogeneous environment , the total master curve is given by G ( x˜ ) = ( 1N ) ∑i=1NGi ( x˜ ) .", "To obtain the master curve G ( x˜ ) for the fBm process underlying the particle motion , we need to estimate the generalized diffusion constants Kα , i of fBm for each trajectory .", "The strength of the fBm is obtained as Kα , i=MSDτ , i", "( t ) 2tα , for a lag time t=2", "s . As we see on Figure 4B the master curve perfectly fits the ensemble data for the PDF of particle displacements .", "Moreover , if we rescale displacements of each trajectory by the corresponding generalized diffusion constant , they should all collapse on a single Gaussian distribution with a unit variance , and this is what we show in Figure 4—figure supplement 3 ) .", "For reference we also provide the unscaled PDFs of displacements for log phase and energy depleted cells for two lag times t=0 . 2 and 2 . 0 s , see Figure 4—figure supplement 3 , right panel .", "The directional correlation function of the displacements was calculated as C ( nτ ) =⟨Δx→", "( t ) |Δx→", "( t ) |⋅Δx→ ( t+nτ ) |Δx→ ( t+nτ ) |⟩ , where τ is the lag-time , n is an integer , Δx→", "( t ) is the change in particle position between time t and t+τ and |Δx→", "( t ) | denotes the length of the vector Δx→", "( t ) .", "Averaging is performed over the ensemble of particles and time", "t . To quantify the strength of correlations we considered two displacements δx→and δx'→ during two consecutive time intervals of length τ ( Weeks and Weitz , 2002 ) .", "We then calculate the projection of the second displacement onto the direction of the preceding one , c=δx'→⋅δx→|δx→| .", "If this value is negative , it indicates that the second displacement tends to move oppositely to the first .", "For small displacements , we expect this quantity to be a linear function of the initial particle displacement , c=-b| ( δx ) |⃑ .", "For a viscous material the slope b vanishes , whereas for an elastic material the slope is b=1/2 and is independent of the lag time τ for which the displacements are calculated .", "To find out how c depends on the magnitude of the initial displacement , we first extract all pairs of subsequent displacements at a certain lag time .", "Thereafter the projection is calculated for each pair of displacements .", "In order to consider the relation between the correlation c and the initial displacement length |δx'→| , these lengths were binned in 38 equidistant bins .", "The correlation was averaged in each bin to obtain the correlation c as a function of displacement length |δx'→| , see Figure 4C and Figure 4—figure supplement 5 ) .", "The linear scaling of c with |δx'→| and its independence on the lag time also rule out the localization error as a possible ( dominating ) source of negative correlations in the displacement data ." ] ]
[ "Cells can enter into a dormant state when faced with unfavorable conditions .", "However , how cells enter into and recover from this state is still poorly understood .", "Here , we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles .", "We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm , which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability .", "We further demonstrate that this transition is required for cellular survival under conditions of starvation .", "Our findings have broad implications for understanding alternative physiological states , such as quiescence and dormancy , and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state ." ]
[ "Most organisms live in unpredictable environments , which can often lead to nutrient shortages and other conditions that limit their ability to grow .", "To survive in these harsh conditions , many organisms adopt a dormant state in which their metabolism slows down to conserve vital energy .", "When the environmental conditions improve , the organisms can return to their normal state and continue to grow .", "The interior of cells is known as the cytoplasm .", "It is very crowded and contains many molecules and compartments called organelles that carry out a variety of vital processes .", "The cytoplasm has long been considered to be fluid-like in nature , but recent evidence suggests that in bacterial cells it can solidify to resemble a soft glass-type material under certain conditions .", "When cells become dormant they stop dividing and reorganise their cytoplasm in several ways; for example , the water content drops and many essential proteins form storage compartments .", "However , it was not clear how cells regulate the structure of the cytoplasm to enter into or exit from dormancy .", "Now , Munder et al . analyse the changes that occur in the cytoplasm when baker’s yeast cells enter a dormant state .", "The experiments show that when yeast cells are deprived of energy – as happens during dormancy – the cytoplasm becomes more acidic than normal .", "This limits the ability of molecules and organelles to move around the cytoplasm .", "Similar results were also seen in other types of fungi and an amoeba .", "Munder et al . found that this increase in acidity during dormancy causes many proteins to interact with each other and form large clumps or filament structures that result in the cytoplasm becoming stiffer .", "A separate study by Joyner et al . found that when yeast cells are starved of sugar , two large molecules are less able to move around the cell interior .", "Together , the findings of the studies suggest that the interior of cells can undergo a transition from a fluid-like to a more solid-like state to protect the cells from damage when energy is in short supply .", "The next challenge is to understand the molecular mechanisms that cause the physical properties of the cytoplasm to change under different conditions ." ]
2016
[ "Introduction", "Results and Discussion", "Materials and methods" ]
[ "short report", "neuroscience" ]
Sexually divergent expression of active and passive conditioned fear responses in rats
elife-11352-v2
[ [ "In the laboratory , auditory or “cued” fear conditioning and extinction in rodents are the predominant tools for studying the neural mechanisms of learning and memory for aversive stimuli ( Blanchard and Blanchard , 1969; LeDoux , 2000; Maren , 2001 ) .", "In these assays , the strength of a tone-shock association is traditionally measured by the fraction of time during the conditioned stimulus ( CS ) that subjects exhibit freezing , defined as the cessation of all movement not required for respiration ( Fanselow , 1980 ) .", "Accordingly , low freezing is generally interpreted as reflecting a weak association and thus poor learning .", "Likewise , low freezing after extinction is taken to indicate successful suppression of the conditioned response , a new memory ( Quirk and Mueller , 2008 ) .", "However , by their construction , these traditional assays are insensitive to alternative expressions of fear , such as escape .", "Most studies of fear conditioning and extinction in rodents use exclusively male subjects ( Lebron-Milad and Milad , 2012 ) .", "The few studies that directly compare conditioned freezing responses in males and females produced mixed results ( Shansky , 2015 ) but most frequently reported lower freezing in females ( Gupta et al . , 2001; Maren et al . , 1994; Pryce et al . , 1999 ) .", "Whether this effect reflects genuine learning deficits in females , or is related to sex differences in fear response strategies is unknown .", "For example , females reliably exhibit heightened ambulation in a wide variety of common behavioral tests ( Archer , 1975; Fernandes et al . , 1999; File , 2001; Seney et al . , 2012 ) , which may influence their selection of responses to threatening stimuli .", "To identify possible alternative fear response strategies , we evaluated locomotor activity in gonadally intact adult male ( n=56 ) and female ( n=58 ) Sprague Dawley rats as they were trained and tested in auditory fear conditioning ( 5 habituation CS followed by 7 CS-US pairs ) , extinction ( 20 CS ) , and extinction retention ( 3 CS ) tests across 3 days ( Gruene et al . , 2015 ) ( Figure 1a ) .", "In many animals , we qualitatively observed a rapid ‘darting’ behavior during fear conditioning tone presentation–a rapid , forward movement across the chamber that resembled an escape-like response ( illustrated in Figure 1b , and Video 1 ) .", "We quantified these responses by identifying and counting them as discrete events in traces of each animal’s velocity for all sessions using Noldus Ethovision software and custom Matlab code ( see source code 1 , Materials and methods , and Figure 1c ) .", "We calculated darting rate ( darts/min ) during four non-overlapping trial epochs:", "1 ) 60 s pre-CS period ,", "2 ) 30 s CS presentation ,", "3 ) 5 s “shock response” period , and", "4 ) 30 s post-shock period ( Figure 2a ) .", "This approach allowed us first to determine if darting reflected an alternate conditioned response , and second , whether the expression of conditioned darting predicted distinct behavioral patterns across fear conditioning and extinction . 10 . 7554/eLife . 11352 . 003Figure 1 . Darting is an active learned response to the CS that occurs primarily in female rats .", "( a ) Experimental timeline .", "( b ) Darts were characterized by a brief , high velocity movement across the test chamber .", "( c ) Velocity traces from a representative animal , demonstrating increase in conditioned darting events across fear conditioning trials .", "Asterisks denote events that reached criterion for darting during the CS .", "Time 0 denotes CS onset .", "( d ) Temporal organization of darting in all female rats .", "On the left is a two dimensional histogram of dart timing relative to the CS averaged over all females for 5 habituation trials and 7 conditioning trials on day 1 .", "Trial time is on the x-axis and colored bars denote the trial epochs we defined as CS ( green ) , shock response ( orange ) , and post shock response ( blue ) .", "Each row represents a CS trial ( habituation 1–5 , and conditioning 6–12 ) , and depicts average dart rate by the color in each 4-second bin according to the color bar .", "On the right are histograms of the temporal organization of darts averaged over the five habituation trials ( top ) and the last three conditioning trials ( bottom ) .", "Darts were detected and counted as described in Materials and methods .", "( e ) Temporal organization of darting in all male rats .", "Panels are organized as in", "( d ) .", "During habituation trials , darts occurred at low rates throughout the trial in both sexes .", "In contrast , after conditioning only females exhibited increased darting triggered by tone onset ( ‘CS’ ) and sustained darting after shock delivery ( ‘Post-shock’ ) .", "Both sexes darted in response to the shock itself ( Shock response ) .", "In both sexes , the first bin after the shock exceeds the limits of the y-axis . DOI: http://dx . doi . org/10 . 7554/eLife . 11352 . 003" ], [ "Prior to the initiation of shocks , darts were not temporally structured with respect to the CS .", "However , we found that females , but not males , exhibited increased dart frequency in response to CS onset during late trials ( Figure 1c–e ) , suggesting that darting is a learned response .", "Figure 1d , e represent dart frequency amongst entire female and male cohorts , respectively . 10 . 7554/eLife . 11352 . 005Figure 2 . Sex differences in darting responses during fear conditioning and extinction .", "( a ) The 4 fear conditioning epochs in which velocity was recorded .", "Graphs in c-f and i-j are color coded to match , and represent mean +/- SEM .", "( b ) In graphs c-f and i-j , females are represented by filled circle , males by an open square .", "( c ) Pre-CS ( final 60 sec before 1st CS presentation ) and CS dart rate ( darts/min ) during conditioning .", "( d ) number of darts observed during 5s shock ( US ) response periods .", "( e ) maximum velocity reached during 5s shock ( US ) response periods .", "( f ) mean dart rate observed during 30s post-shock period .", "( g ) and", "( h ) Pearson’s correlations of mean shock response velocity and total session dart count [note that visible male outlier was removed from analysis for being 6 SDs above mean total dart count . When included , r=0 . 34 , p<0 . 05] .", "( i ) Pre-CS and CS dart rate ( darts/min ) during Extinction .", "( j ) Pre-CS and CS dart rate ( darts/min ) during Extinction testing .", "*p<0 . 05; **p<0 . 01; *** p<0 . 001; ****p<0 . 0001 males vs . females . DOI: http://dx . doi . org/10 . 7554/eLife . 11352 . 005 We next compared darting in males and females across all test sessions .", "Females exhibited higher CS dart rates than males on all 3 days ( Figure 2c , i , j; conditioning: ns p=0 . 07 , Mann Whitney test .", "p<0 . 001 2-way ANOVA main effect of sex , F1 , 112=12 . 1; sex x trial interaction F11 , 1232=2 . 12 , p=0 . 02 Extinction: p=0 . 01 , Mann Whitney test .", "p<0 . 05 , 2-way ANOVA main effect of sex , F1 , 112=4 . 05 .", "Extinction test: p=0 . 008 , Mann Whitney test ( Pre-CS ) ; p<0 . 001 , 2-way ANOVA main effect of sex , F1 , 112=14 . 58 ( CS ) ) .", "Notably , CS dart rate in females increased as CS-US presentations progressed ( Figure 2c ) and decreased during extinction ( Figure 2i ) , again suggesting that darting may reflect an alternate expression of associative learning .", "During fear conditioning , although both males and females reliably darted during the shock response period ( Figure 2d; p<0 . 01 2-way ANOVA main effect of sex , F1 , 112=8 . 5 ) , shock-evoked darts in females reached higher velocities than darts in males ( Figure 2e; p<0 . 0001 2-way ANOVA main effect of sex , F1 , 112=20 . 35 ) .", "Additionally , females were more likely to dart during the 30s post-shock period than males ( Figure 2f; p<0 . 0001 2-way ANOVA main effect of sex , F1 , 112=23 . 27 ) .", "To determine whether an animal’s shock response velocity was related to its overall propensity to dart , we correlated the mean velocity reached across all 7 US presentations with total detected darts during fear conditioning .", "These measures were significantly correlated in females ( Figure 2g ) but not males ( Figure 2h ) , suggesting that in females only , an animal’s immediate reaction to an aversive stimulus may influence its future response strategies .", "We did not observe darting in all females , however , and so to identify possible behavioral markers and outcomes of darting , we divided animals into ‘Darter’ and ‘Non-darter’ subgroups .", "An animal qualified as a “Darter” if it exhibited at least one dart during fear conditioning tones ( CS ) 8–12 .", "CS 8 is the 3rd CS-US pairing , and the point at which we usually observe a robust increase in freezing in males .", "Therefore , only darts that occurred during this same time period were considered to reflect conditioned darting .", "Over 40% of females qualified as Darters ( Figure 3a ) , whereas approximately 10% of males qualified ( Figure 3f; chi-square = 13 . 8 , p=0 . 0002 ) .", "There was no effect of the estrous cycle on darting ( Figure 3 - supplement ) .", "Compared to Non-darters , female Darters exhibited greater shock response velocities ( Figure 3b; p=0 . 001 2-way ANOVA main effect of group F1 , 56=11 . 49 ) , as well as higher dart rates in the post-shock period ( Figure 3c; p<0 . 0001 2-way ANOVA main effect of group , F1 , 56=25 . 42 . ) , suggesting that female Darters have a more robust and protracted response to the shock .", "Importantly , female Darters did not exhibit higher dart rates during pre-CS periods or during CS-only habituation trials ( Figure 3d; p=0 . 65 , Mann Whitney test ) , suggesting that Darters are not simply more active overall , and were not pre-disposed to dart in response to the CS .", "During the CS , Darters exhibited increased darting as CS-US pairs progressed ( p<0 . 0001 2-way ANOVA group x trial interaction , F11 , 616=8 . 8; main effect of group F1 , 56=26 . 35 , p<0 . 0001 ) .", "During the Extinction Pre-CS period , Darters did not dart more than Non-darters ( p=0 . 38 Mann Whitney ) , but Darters exhibited increased darting during the first Extinction CS , suggesting that darting is a conserved conditioned response ( 2-way ANOVA interaction , F19 , 1064=1 . 584 , p=0 . 05; *p<0 . 05 Sidak’s post-hoc test ) . 10 . 7554/eLife . 11352 . 006Figure 3 . Darting subpopulations are greater in females and exhibit distinct behavioral patterns .", "( a ) and", "( f ) proportion of females and males that qualified as Darters .", "( b ) max velocity reached during shock response period", "( c ) mean dart rate ( darts/min ) observed during 30s post-shock period .", "( d ) Pre-CS and CS dart rate ( darts/min ) during conditioning , extinction , and extinction test .", "( e ) CS freezing in female Darters vs . Non-darters .", "( g ) Shock response velocity did not differ between male Darters and Non-darters .", "( h ) mean dart rate ( darts/min ) observed during 30s post-shock period .", "( i ) Pre-CS and CS dart rate ( darts/min ) during conditioning , extinction , and extinction test .", "( j ) CS freezing in male Darters vs . Non-darters .", "*p<0 . 05; **p<0 . 01; *** p<0 . 001; ****p<0 . 0001 Darters vs . Non-dartersDOI: http://dx . doi . org/10 . 7554/eLife . 11352 . 00610 . 7554/eLife . 11352 . 007Figure 3—figure supplement 1 . Distribution of animals in each estrous cycle phase did not differ between Darters and Non-darters . Chi square = 2 . 785 , p=0 . 42 . DOI: http://dx . doi . org/10 . 7554/eLife . 11352 . 007 We next asked whether CS darting during fear conditioning related to CS freezing behavior ( Figure 3e ) .", "In females , Darters and Non-darters did not differ in pre-CS or CS-only ( habituation ) freezing during fear conditioning .", "However , as CS-US pairings progressed , Darters froze less than Non-darters , suggesting that increased darting may prevent or compete with freezing responses p=0 . 02 2-way ANOVA group x trial interaction F11 , 616=2 . 16 .", "main effect of darting F1 , 56=4 . 18 , p<0 . 05 ) .", "Darters and Non-darters did not significantly differ in freezing during Extinction .", "However , Darters also froze less during the extinction test ( day 3; p<0 . 02 , 2-way ANOVA main effect of group F1 , 56=5 . 76 ) despite not exhibiting increased darting at that time , suggesting that darting during fear conditioning does not simply compete with an animal’s freezing response , but may also reflect an adaptive response that predicts positive outcomes after extinction learning .", "In the small subpopulation of male Darters , CS dart rate ( Figure 3i; Conditioning p<0 . 0001 , 2-way ANOVA group x trial interaction , F11 , 594=3 . 76 . Extinction: p<0 . 0001 , 2-way ANOVA group x trial interaction , F19 , 1026=3 . 17 ) and freezing ( Figure 3j; Conditioning: p<0 . 01 2-way ANOVA group x trial interaction F11 , 583=2 . 68 . Extinction and extinction test: No significant interaction or effects ) patterns during fear conditioning shared some characteristics with those in females .", "However , there are several notable distinctions between male and female Darters .", "First , CS dart rate in darting males was characterized by a steady low rate of darting across trials ( Figure 3i ) , instead of the increase across trials observed in females ( Figure 3d ) , suggesting that darting in males may not reflect a learned fear response , but general hyperactivity that results in less freezing .", "Second , unlike our observations in females , male Darters did not exhibit heightened shock response velocities ( Figure 3g ) or robust post-shock dart rates ( Figure 3h; p=0 . 01 2-way ANOVA group x trial interaction , F6 , 324=2 . 8 , no main effects ) compared to Non-darters .", "Third , male Darters did not exhibit lower freezing during extinction testing , suggesting that the potential long-term behavioral implications of darting during fear conditioning are stronger in females than in males .", "Together with the large observed sex difference in darting prevalence ( Figure 2a , f ) , these discrepancies suggest that there may be qualitative differences in the potential causes and effects of darting in males versus females .", "Further work will be necessary to determine whether the neurobiological basis of darting is comparable in males and females .", "In summary , our data show that during auditory fear conditioning , a substantial subpopulation of predominantly female rats exhibit an active conditioned response associated with reduced conditioned freezing throughout fear conditioning and extinction tests .", "To our knowledge , this is the first formal characterization of conditioned escape-like responses during classical fear conditioning , in which the shock cannot be avoided .", "In contrast , learned escape behavior has been well studied in Active Avoidance ( AA ) paradigms ( Galatzer-Levy et al . , 2014; Martinez et al . , 2013 ) , and although research into potential sex differences in AA is rare , females are reported to learn AA faster than males ( Dalla and Shors , 2009 ) , which is consistent with females preferring active fear responses over freezing .", "One potentially provocative finding here is that female Darters exhibited comparable freezing to Non-darters at the start of extinction , but enhanced extinction retention the following day .", "Importantly , lower freezing during extinction retention could not be explained by increased darting during this phase .", "This suggests that darting during fear conditioning does not interfere with the formation or memory of the tone-shock association , but may confer a long-term protective or adaptive state that promotes increased cognitive flexibility and thus enhanced extinction maintenance ( Maren et al . , 2013 ) .", "This effect is reminiscent of reports from Maier and colleagues , who have convincingly demonstrated that perceived “escapability” in a shock stress paradigm leads to enhanced AA in subsequent testing ( reviewed in Maier , 2015 ) .", "In a similar vein , increases in “active coping” behavior ( digging , rearing , wall-sniffing ) during a cued fear memory test are positively correlated with AA success ( Metna-Laurent et al . , 2012 ) .", "Recruitment of these active coping fear responses instead of freezing has been shown to involve neural transmission in the central amygdala ( Gozzi et al . , 2010 ) and depend on cannabinoid signaling ( Metna-Laurent et al . , 2012 ) , but to date have not been studied in female rodents .", "Importantly , these responses have not been demonstrated during fear conditioning learning , the stage at which darting appears to be most critical .", "Clearly , a great deal of work remains to dissect the neurobiological mechanisms that mediate darting , and to determine its relevance to other indices of active coping , especially in female model organisms .", "The finding that conditioned darting occurs primarily in females holds major implications for the interpretation of fear conditioning and extinction studies that use both male and female rats , suggesting that freezing alone may not be a complete measure of learned fear in female subjects .", "Specifically , female rats that exhibit low freezing levels during fear conditioning could be erroneously described as expressing low fear and/or poor learning , when in fact they have engaged darting responses .", "This phenomenon may also be clinically relevant , pointing to a sex-specific threat response that predicts enhanced extinction maintenance .", "Because the learning processes that underlie extinction form the basis for exposure therapy ( a common treatment for Post-Traumatic Stress Disorder [PTSD] ) , a better understanding of the mechanisms that drive darting could lead to improved exposure therapy success .", "Women are at a twofold risk for PTSD compared to men , and thus identification of the neurobiological factors that determine darting in females may provide insight into sex differences in coping strategies , as well as in stress susceptibility and resilience ." ], [ "Young adult ( 8–10 weeks ) male ( n=56 ) and female ( n=58 ) Sprague Dawley rats were individually housed in the Nightingale Animal Facility at Northeastern University on a 12:12 light:dark cycle with access to food and water ad libitum .", "All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Northeastern University Institutional Animal Care And Use Committee .", "All experimenters were female .", "Females were vaginally swabbed daily for two weeks to ensure normal estrous cycling .", "Collected cells were smeared on a microscope slide , stained with Nissl , and examined with a light microscope for cytology .", "Video files from FreezeFrame were extracted as QuickTime File Format ( . mov ) and then converted to MPEG-2 files using AVS Video Converter 9 . 1 ( Online Media Technologies LTD . 2014 ) .", "The MPEG-2 files were then run through EthoVision software ( Noldus ) , with a center point tracking with a velocity sampling rate of 3 . 75 .", "Velocity data were computed by Noldus software at 3 . 75 Hz sampling rate and exported to Matlab ( Mathworks ) .", "Darts were detected in the exported trace using the findpeaks function with a minimum velocity of 23 . 5 cm/s and a minimum interpeak interval of 0 . 8 s .", "The 23 . 5 cm/s threshold for darts was determined by cross-referencing velocity data with experimenter scoring of darting behavior .", "23 . 5 cm/s was the velocity at which all movements at that rate or higher were consistently scored as darts .", "These discrete events were registered to each trial and analyzed with custom Matlab software ( available as a source code 1 file ) .", "Darting , velocity , and freezing values during each epoch were averaged for each group and analyzed for each session ( fear conditioning , extinction , extinction test ) using 2-way repeated measure ANOVAs with factors of group and trial .", "Mann-Whitney t-tests were used for all Pre-CS comparisons .", "One male animal was removed from analysis because its total dart count was 6 standard deviations outside the mean ( shown in Figure 2h ) ." ] ]
[ "Traditional rodent models of Pavlovian fear conditioning assess the strength of learning by quantifying freezing responses .", "However , sole reliance on this measure includes the de facto assumption that any locomotor activity reflects an absence of fear .", "Consequently , alternative expressions of associative learning are rarely considered .", "Here we identify a novel , active fear response ( ‘darting’ ) that occurs primarily in female rats .", "In females , darting exhibits the characteristics of a learned fear behavior , appearing during the CS period as conditioning proceeds and disappearing from the CS period during extinction .", "This finding motivates a reinterpretation of rodent fear conditioning studies , particularly in females , and it suggests that conditioned fear behavior is more diverse than previously appreciated .", "Moreover , rats that darted during initial fear conditioning exhibited lower freezing during the second day of extinction testing , suggesting that females employ distinct and adaptive fear response strategies that improve long-term outcomes ." ]
[ "Animals can respond to fear in a variety of ways , such as by standing still ( freezing ) , or rapidly escaping from an apparent threat .", "Freezing is the most common measure of fear that has been used in research studies .", "However , since the vast majority of these experiments have used male animals , it is not clear if freezing is a sufficient measure of fear in females .", "To address this question , Gruene et al . analyzed different types of fear responses in large groups of male and female rats .", "The experiments used a technique called cued fear conditioning , which pairs a sound with a mild electrical shock to a foot .", "When rats learn that the sound predicts the shock , the sound alone causes them to produce a fear response .", "However , if the sound is then played repeatedly without a footshock , the rats learn to become less fearful of the sound in another learning process called “extinction” .", "The experiments found that females were four times more likely than males to display fear in the form of rapid movements ( referred to as “darting” ) .", "Animals that displayed darting were also less likely to freeze in response to the sound cue , which suggests that darting may represent an alternative fear strategy that is more common in females .", "During a subsequent extinction test , females that darted also displayed quicker reductions in both types of fear responses , which suggests that darting might be an active coping response that promotes long term reductions in fear .", "Gruene et al . ’s findings suggest that there are differences in the ways that males and females respond in fear of a threatening stimulus , and highlight the importance of analyzing a variety of fear responses in experiments .", "The next steps will be to understand the biological basis of the darting response in female rats ." ]
2015
[ "Introduction", "Results and discussion", "Materials and methods" ]
[ "cell biology" ]
An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis
elife-00829-v1
[ [ "Clathrin-mediated endocytosis ( CME ) , the major route of internalisation for transmembrane proteins into mammalian cells , controls many cellular processes from signalling and growth , to cell motility and organelle identity ( McMahon and Boucrot , 2011 ) .", "It has been known for decades that CME , like many other cellular processes , is inhibited during the early stages of mitosis ( Fawcett , 1965; Berlin and Oliver , 1980; Warren , 1993; Fielding et al . , 2012; Fielding and Royle , 2013 ) .", "However , the mechanism that underlies this mitotic inhibition is unclear ( Fielding and Royle , 2013 ) .", "During CME , the cargo that is destined for internalisation is clustered into pits via interaction with adaptor proteins , which in turn , recruit clathrin triskelia .", "The forming clathrin-coated pit is then invaginated further and eventually pinched off in a process dependent on the GTPase dynamin .", "The actin cytoskeleton may assist in these latter stages of vesicle formation ( Robertson et al . , 2009 ) .", "CME is shut down shortly after prophase entry and resumes in late anaphase where it is required for membrane dynamics in cytokinesis ( Berlin and Oliver , 1980; Schweitzer et al . , 2005 ) .", "In early mitotic cells , shallow clathrin-coated pits are seen at the plasma membrane suggesting that invagination and subsequent steps of CME are inhibited ( Pypaert et al . , 1987 ) .", "Two main mechanisms have been proposed .", "First , direct mitotic phosphorylation of the CME machinery decreases its activity ( Pypaert et al . , 1991; Chen et al . , 1999; He et al . , 2003 ) .", "In support of this , numerous endocytic proteins are phosphorylated during mitosis ( Dephoure et al . , 2008 ) , but it is not clear what effect , if any , these modifications have on CME .", "Second , that the increased membrane tension of mitotic cells prevents invagination during CME ( Raucher and Sheetz , 1999 ) .", "There is good evidence that the membrane tension is increased in early mitosis ( Stewart et al . , 2011a ) in a process driven by osmotic changes ( Habela and Sontheimer , 2007; Stewart et al . , 2011b ) .", "The precise mechanism for shutdown is yet to be determined .", "Despite a clear role in yeast , the actin cytoskeleton does not have an obligatory role in CME in human cells ( Engqvist-Goldstein and Drubin , 2003; Robertson et al . , 2009; Traub , 2011; Anitei and Hoflack , 2012 ) .", "Light and electron microscopy studies clearly show that it is recruited to a subset of CME events in human cells ( Merrifield et al . , 2002 , 2005; Collins et al . , 2011; Taylor et al . , 2011 , 2012 ) and that it is important for internalisation of pathogens such as Listeria ( Bonazzi et al . , 2011 ) .", "Interestingly , the requirement for actin in CME may be linked to membrane tension .", "Recent work has shown that in cells where membrane tension is increased , there is an increased requirement for actin ( Aghamohammadzadeh and Ayscough , 2009; Boulant et al . , 2011; Stachowiak et al . , 2013 ) .", "During mitosis the plasma membrane tension is increased as a result of osmotic changes ( Raucher and Sheetz , 1999; Stewart et al . , 2011a ) , so why is the actin cytoskeleton not deployed to assist CME as occurs in interphase cells ?", "We set out to discover the mechanism for mitotic shutdown of CME .", "We found two methods to ‘restart’ CME in mitotic cells , which effectively rule out direct mitotic phosphorylation of endocytic proteins as a mechanism for the mitotic shutdown of CME .", "Instead we propose that the actin machinery , which is engaged in assembling a stiff cortex in mitotic cells , is unavailable to rescue the inhibition caused by the increased membrane tension .", "This unmet requirement for actin in CME explains why endocytosis is inhibited in early mitosis ." ], [ "Previous reports indicated that due to osmotic changes in mitosis , the plasma membrane tension is increased ( Raucher and Sheetz , 1999; Stewart et al . , 2011b ) .", "We began by verifying this observation in HeLa cells .", "To measure apparent membrane tension , a polystyrene bead coated with concanavalin A was held in an optical trap , attached to the plasma membrane , and then pulled away from the cell surface to form a membrane tether ( Dai and Sheetz , 1995; Figure 1A , B ) .", "The displacement of the bead from the trap centre is the tether force and this is proportional to the plasma membrane tension ( Figure 1A ) .", "We found that the tether force was increased from 19 . 2 ± 1 . 5 pN in interphase cells to 27 ± 2 . 8 pN in mitotic cells at metaphase ( mean ± SEM , p=0 . 027 , Figure 1C ) .", "These measurements are in good agreement with previous measurements in other cell types ( Raucher and Sheetz , 1999 ) .", "Tether force is related to the force required to invaginate a clathrin-coated vesicle .", "This means that , in mitotic cells , comparatively more energy is required from the CME machinery as it tries to overcome an increased load .", "The shortfall in this energy may explain mitotic shutdown of CME and the question is: what is the molecular basis of this shortfall ?", "10 . 7554/eLife . 00829 . 003Figure 1 . Membrane tension in mitotic cells is elevated .", "( A ) Schematic diagram to show the principle of using tether force to measure membrane tension .", "Displacement of the bead from the trap centre ( Δx ) can be converted to a tether force measurement ( pN ) as the stiffness of the trap is known .", "This tether force is proportional to the membrane tension .", "( B ) Still video image of a membrane tension measurement in a mitotic cell .", "Note the thin tether is formed by moving the cell away from the bead held in the trap .", "( C ) A typical trace showing bead displacement from the trap centre and the tether force .", "The cell was moved towards the bead ( down arrow ) to eliminate tension and give a zero value .", "The cell is then moved away ( up arrow ) and tether force is re-established .", "( D ) Summary plot of tether force measurements from HeLa cells ( dots ) in interphase ( orange ) or at metaphase ( purple ) .", "The mean is shown by a bar .", "( E ) Plot of tether force relative to the average interphase measurement from the same experiment .", "Ncell = 13 or 11 , Nexp = 5 .", "*p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 003 Clathrin-coated structures ( CCSs , pits , and vesicles ) are present in mitotic cells with the pits being arrested at the cell surface in a shallow state ( Pypaert et al . , 1987 ) .", "Fluorescent transferrin binding to abundant transferrin receptors on the plasma membrane can be seen in these pits but the uptake of transferrin is prevented ( Figure 2A; Fielding et al . , 2012 ) .", "Our hypothesis was that differences in the proteomes of CCSs purified from interphase or mitotic cells would explain why clathrin-coated pits are arrested at the surface .", "For example , components of the CME machinery that are regulated might be present in interphase CCSs but could be less abundant in mitotic CCSs .", "To identify such differences , we prepared fractions enriched in CCSs from interphase or mitotic HeLa cells , according to established methods ( Borner et al . , 2006 , 2012 ) .", "These fractions were analysed by mass spectrometry and label-free quantitation ( Figure 2B ) .", "Over four independent experiments , we compared the relative abundance of 1253 proteins , only a subset of which are confirmed CCS proteins ( Borner et al . , 2006 , 2012 ) .", "The list of all abundances was used to characterise the variance of the dataset and identify outliers ( ‘Materials and methods’ ) .", "We found that cortactin was the protein most consistently reduced in mitotic CCSs compared to interphase ( LFQ intensity ratio = 46 . 2 ) .", "Cortactin is an activator of Arp2/3-dependent actin polymerisation .", "A list of bona fide CCS proteins ( Borner et al . , 2012 ) was therefore supplemented with components of the actin cytoskeleton that are recruited to CCSs ( Taylor et al . , 2011 ) .", "The relative abundance of these proteins is shown in Figure 2C .", "These data show that most of the core CME machinery is not altered significantly between CCS-containing fractions from interphase and mitotic samples .", "Consistent with previous results ( Chetrit et al . , 2011; Kozik et al . , 2013 ) , Dab2 was less abundant and PICALM more abundant in mitotic fractions .", "HIP1 and HIP1R appeared to be differentially regulated .", "These two proteins link the clathrin machinery with the actin cytoskeleton and are regulated by binding clathrin light chain ( Le Clainche et al . , 2007; Wilbur et al . , 2008 ) .", "The accumulation of HIP1R and the absence of cortactin in mitotic fractions were interesting , given that these two proteins have been shown previously to be coupled functionally ( Le Clainche et al . , 2007 ) .", "Other interesting differences , to be explored in the future , included the accumulation of NSF ( N-Ethylmaleimide-Sensitive Factor ) in mitotic CCSs ( LFQ intensity ratio = 0 . 077 ) . 10 . 7554/eLife . 00829 . 004Figure 2 . Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells .", "( A ) Confocal micrographs of a HeLa cell expressing GFP-tagged clathrin light chain a ( clathrin , green ) .", "The same cell is shown before and after application of transferrin-Alexa568 ( Tf , red ) .", "A single XY section is shown together with a YZ view through the cell centre ( right ) .", "Far right , 3D projection of the whole confocal series .", "( B ) Schematic diagram of the purification and proteomic analysis of clathrin-coated structures purified from cells in interphase or metaphase .", "( C ) Bar chart to show the comparative interphase/mitotic abundance for CCV proteins from the search list .", "The interphase or mitotic LFQ value for each protein derived from four separate experiments were compared .", "Red bars show proteins related to the actin cytoskeleton .", "Red circles indicate proteins verified in D . Inset: histogram to show the frequency of abundances for all proteins in the analysis .", "A single Gaussian function was fitted to the data , the mean and variance of which is shown in the main bar chart .", "Dotted line shows the average comparative abundance and the shaded area represents ± 2 standard deviations .", "( D ) Representative fluorescence micrographs to show the colocalisation of GFP-tagged clathrin light chain a ( clathrin , green ) in interphase or mitosis , with HIP1R-tDimer-RFP , tdTomato-HIP1 or mCherry-cortactin ( red ) .", "Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 004 We next verified the comparative proteomics results by visualising the co-localisation of clathrin with HIP1R , HIP1 , and cortactin in interphase and mitotic cells .", "All three proteins were colocalised at least partially with clathrin in interphase CCSs .", "In mitotic cells , HIP1R was found tightly associated with clathrin-coated pits at the cell surface ( Figure 2D ) .", "By contrast , HIP1 and cortactin were diffusely localised in mitotic cells and were lost from CCSs ( Figure 2D ) .", "These results indicate that the actin cytoskeleton is important for the mitotic shutdown of CME .", "Taken together , this suggested the following mechanism: it is predicted that actin is required to assist CME in mitotic cells due to the increase in membrane tension ( Aghamohammadzadeh and Ayscough , 2009; Boulant et al . , 2011 ) .", "If the actin cytoskeleton is engaged in the formation of a stiff mitotic cortex ( Bray and White , 1988; Cramer and Mitchison , 1997; Stewart et al . , 2011a ) , perhaps it is unavailable to assist CME in mitotic cells ?", "To test this idea , we developed two strategies to ‘restart’ CME in mitotic cells .", "Both methods work by allowing the actin cytoskeleton to engage in CME in mitotic cells .", "Our first strategy was to deplete Ect2 , a guanine nucleotide exchange factor effector for RhoA .", "Mitotic cells depleted of Ect2 showed similar CME to control cells in interphase ( Figure 3A ) .", "Quantification of transferrin uptake demonstrated that with either of two independent siRNAs against Ect2 , the mitotic shutdown of CME was reversed ( Figure 3A–C ) .", "Furthermore , this reversal could itself be blocked by the expression of siRNA-resistant GFP-Ect2 constructs ( Figure 3—figure supplement 1 ) .", "These experiments show that restoration of CME following Ect2 RNAi is specifically due to loss of Ect2 protein and not due to an off-target effect . 10 . 7554/eLife . 00829 . 005Figure 3 . Restoring CME in mitotic cells by depletion of Ect2 . ( A ) Representative confocal micrographs to show transferrin uptake ( Tf , green ) , actin organisation ( phalloidin , red ) and DNA ( blue ) in control or Ect2-depleted cells .", "For each condition , a section though the middle or base of the mitotic cell with a neighbouring interphase cell is shown .", "Cells were transfected with control siRNA or one of two Ect2 siRNAs ( Ect2_5 or Ect2_6 ) .", "Scale bars , 10 µm .", "( B ) Representative western blot to show the amount of Ect2 remaining after RNAi .", "Blots were probed for Ect2 and β-tubulin as a loading control .", "( C ) Bar chart to show normalised transferrin uptake in interphase and mitotic ( prometaphase ) cells as quantified by confocal microscopy .", "Ncell = 5–8 .", "( D ) Representative confocal micrographs to show the distribution of F-actin ( Phalloidin , red ) , G-actin ( DNase I , green ) and DNA ( blue ) in mitotic HeLa cells .", "The cells were transfected with control ( GL2 ) or Ect2 ( Ect2_5 or Ect2_6 ) siRNA .", "Scale bar , 10 µm .", "( E and F )", "Bar charts to summarise the quantification of F-actin ratio at the cell cortex vs the cytoplasm ( E ) or the amount of G-actin in the cytoplasm ( F ) .", "Ncell = 40 , Nexp = 3 .", "( G ) Summary of membrane tension measurements on mitotic HeLa cells transfected with control ( GL2 ) or Ect2 ( Ect2_5 ) siRNA .", "Relative tether force is shown for individual cells ( dots ) and the bar indicates the mean .", "( H ) Bar chart to show normalised transferrin uptake in interphase and mitotic ( prometaphase ) cells .", "Note the inhibition by latrunculin B ( LatB , 1 µM 30 min ) of restored CME in mitotic cells depleted of Ect2 .", "Ncell = 7–14 .", "All bars show mean ± SEM , *p<0 . 05; **p<0 . 01 , ***p<0 . 001 .", "One-way ANOVA with Tukey’s post-hoc test , comparison to control RNAi , interphase . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 00510 . 7554/eLife . 00829 . 006Figure 3—figure supplement 1 . Ect2 RNAi rescue experiment to show that restoration of CME in Ect2-depleted cells is due to loss of Ect2 . Representative fluorescence micrographs of a transferrin uptake experiment in mitotic HeLa cells .", "The cells expressed GFP or siRNA-resistant forms of GFP-Ect2 and were treated with control ( GL2 ) or Ect2 ( Ect2_5 or Ect2_6 ) siRNAs as indicated .", "Transferrin uptake is shown in red , the GFP construct is shown in green and DNA in blue .", "Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 006 It was described previously that Ect2-depletion inhibits the formation of the stiff actin cortex in mitotic cells ( Matthews et al . , 2012 ) .", "We therefore assessed actin availability to see if this was altered by Ect2 depletion .", "In mitotic cells depleted of Ect2 , the ratio of F-actin fluorescence at the cell cortex vs the cytoplasm was reduced compared with control RNAi cells ( Figure 3D , E ) .", "Analysis of cytosolic G-actin , visualised by DNase I staining ( Cramer et al . , 2002 ) revealed small but significant increases in the level of G-actin in mitotic cells depleted of Ect2 compared with control RNAi cells ( Figure 3F ) .", "These observations suggest that Ect2 depletion increases the availability of actin because the cortical F-actin is not efficiently remodelled into a cortex .", "We next measured the membrane tension of mitotic cells following depletion of Ect2 .", "The relative tether force in mitotic cells depleted of Ect2 was not significantly different from control RNAi mitotic cells ( 1 . 02 ± 0 . 15 , mean ± SEM , p=0 . 921 , Student’s t test ) ( Figure 3G ) .", "This ruled out the possibility that actin cytoskeleton changes in Ect2-depletion indirectly decrease membrane tension and therefore restore CME in mitotic cells .", "The measurement shows that membrane tension remains high in mitotic Ect2-depleted cells .", "Is the restored CME in Ect2-depleted mitotic cells actin-dependent ?", "To test this possibility , we treated cells with latrunculin B ( 1 µM ) , a toxin that promotes actin disassembly .", "We found that restored CME in Ect2-depleted mitotic cells was sensitive to latrunculin B , whereas CME in interphase was not ( Figure 3H ) .", "Note that there was no evidence of restarting CME in control mitotic cells treated with latrunculin B ( Figure 3H ) .", "This argues against the possibility that the remodelled actin cortex in mitotic cells directly inhibits CME , as its destruction by the drug did not result in endocytosis .", "These experiments show that restored CME in mitotic cells is different to CME in interphase cells: it is strictly actin-dependent .", "Together our data demonstrate that the actin cytoskeleton is required for CME in the face of increased membrane tension during mitosis .", "Our second strategy to ‘restart’ CME in mitotic cells was to use a constitutively-active form of the small GTPase Rap1 , Rap1 ( Q63E ) ( Dao et al . , 2009 ) .", "Rap1 is an activator of β1- , β2- or β3-containing integrins that connect to the actin cytoskeleton ( Kim et al . , 2011 ) .", "At mitosis onset , Rap1 is normally inactivated , allowing the disassembly of focal adhesions and actin stress fibres , resulting in cell rounding ( Dao et al . , 2009 ) .", "Expression of Rap1 ( Q63E ) , but not the inactive mutant Rap1 ( S17A ) , causes mitotic cells to remain flat and unable to form a rounded actin cortex ( Figure 4A; Dao et al . , 2009 ) .", "Measurement of the ratio of F-actin fluorescence at the cell cortex vs the cytoplasm showed a decrease in Rap1 ( Q63E ) -expressing cells relative to controls ( Figure 4B ) .", "The inhibition of cortex formation resulted in ∼20% more G-actin to be available in mitotic cells expressing Rap1 ( Q63E ) compared with control mitotic cells ( Figure 4C ) .", "Moreover , membrane tension remained high in mitotic cells expressing Rap1 ( Q63E ) ( Figure 4D ) .", "The relative tether force was 1 . 15 ± 0 . 08 ( mean ± SEM , p=0 . 21 , Student’s t test ) .", "These results indicate that , as with Ect2 depletion , the membrane tension remains high but the prevention of F-actin remodelling in mitosis has resulted in more actin becoming available to assist CME . 10 . 7554/eLife . 00829 . 007Figure 4 . Restoring CME in mitotic cells by expression of Rap1 ( Q63E ) .", "( A ) Representative confocal micrographs to show the distribution of F-actin ( Phalloidin , red ) , G-actin ( DNase I , green ) , and DNA ( blue ) in mitotic HeLa cells .", "The cells either expressed no protein , the constitutively active Rap1 ( Q63E ) or the inactive Rap1 ( S17A ) .", "Scale bar , 10 µm .", "( B and C )", "Bar charts to summarise the quantification of F-actin ratio at the cell cortex vs the cytoplasm ( B ) or the amount of G-actin in the cytoplasm ( C ) .", "Ncell = 18 .", "( D ) Summary of membrane tension measurements on mitotic HeLa cells expressing Rap1 ( Q63E ) compared to control .", "Relative tether force is shown for individual cells ( dots ) and the bar indicates the mean .", "( E ) Representative confocal micrographs of interphase ( orange ) or mitotic ( purple ) HeLa cells to show transferrin uptake ( Tf , green ) , cortactin immunofluorescence ( CTTN , red ) and DNA ( blue ) .", "The cells were transfected as indicated to express Rap1 ( Q63E ) and with control or either one of two CTTN siRNAs ( CTTN_5 or CTTN_6 ) .", "Scale bar , 10 µm .", "( F and G )", "Bar charts to show normalised transferrin uptake ( F ) or CTTN immunofluorescence ( G ) for each condition as indicated .", "Ncell = 10–29 .", "All bar charts in the figure show mean ± SEM .", "All bars show mean ± SEM , *p<0 . 05; **p<0 . 01 , ***p<0 . 001 .", "One-way ANOVA with Tukey’s post-hoc test , comparison to control ( B and C ) or normal HeLa , interphase ( F and G ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 00710 . 7554/eLife . 00829 . 008Figure 4—figure supplement 1 . Cortactin RNAi prevents the restoration of CME by Rap1 ( Q63E ) expression , and this effect is due to loss of cortactin . Representative confocal images of a rescue experiment to test for specificity of the cortactin RNAi phenotype .", "Cells either expressed no additional protein , mCherry-cortactin , Rap1 ( Q63E ) or both proteins; and were either treated with control ( GL2 ) or cortactin ( CTTN_5 ) siRNA .", "The mCherry-cortactin construct was from mouse and was resistant to CTTN_5 , which targets the human transcript .", "Transferrin uptake ( Tf , green ) , mCherry-cortactin ( if present–CTTN , red ) , and DNA ( blue ) .", "Cells were transfected as indicated to express Rap1 ( Q63E ) and with control or CTTN siRNA .", "Micrographs of mitotic ( purple , A ) or interphase ( orange , B ) HeLa cells are shown .", "Note that there is no restoration of mitotic CME in control RNAi cells expressing mCherry-cortactin .", "This indicates that mitotic shutdown of CME is not simply due to loss of cortactin .", "Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 008 To test if this manipulation also ‘restarts’ CME in mitotic cells , we next measured uptake of fluorescent transferrin .", "We found that mitotic cells expressing Rap1 ( Q63E ) exhibited good transferrin uptake and this was comparable to the amount seen in interphase cells ( Figure 4E , F ) .", "We next investigated the involvement of the actin cytoskeleton in restored CME in Rap1 ( Q63E ) -expressing cells .", "To do this , we depleted cortactin using RNAi ( Figure 4E , F ) .", "Depletion of cortactin blocked the restored CME in Rap1 ( Q63E ) -expressing cells ( Figure 4E , F ) .", "This is in contrast to interphase cells where depletion of cortactin had no effect on CME as measured by transferrin uptake ( Figure 4E , F ) .", "The cortactin-dependence of restored CME in Rap1 ( Q63E ) -expressing cells was not due to an off-target effect of RNAi .", "First , it could be demonstrated with two different siRNAs ( Figure 4E , F ) .", "Second , the effect could be rescued by expression of an siRNA resistant form of cortactin in Rap1 ( Q63E ) -expressing cells ( Figure 4—figure supplement 1 ) .", "This demonstrates that ‘restarted’ CME in mitotic cells is different from CME in interphase: it is dependent on cortactin , a protein that was lost from mitotic CCSs in our proteomic analysis ( Figure 2 ) .", "This indicated that using Rap1 ( Q63E ) expression to restart CME in mitotic cells and then inhibiting the restored CME using cortactin depletion , is analogous to the approach above using Ect2 RNAi and latrunculin B . Inhibition of actin function by latrunculin B or cortactin RNAi demonstrate that CME in mitotic cells requires actin and that Ect2 RNAi or Rap1 ( Q63E ) -expression permit the utilisation of actin to restore CME .", "Both strategies to restart mitotic CME support a model where an unmet requirement for actin in CME due to increased membrane tension , causes shutdown .", "Does ‘restarted’ CME in mitotic cells represent global restoration of CME , or is it specific to CME of the transferrin receptor ?", "To explore this question , we tested for restoration of internalisation of the three main endocytic motifs YXXΦ , [DE]XXXL[LI] , and FXNPXY using the CD8 reporter system ( Kozik et al . , 2010; Fielding et al . , 2012 ) .", "Confocal imaging of the steady-state distribution of CD8 chimeras in control mitotic cells showed no intracellular puncta and all CD8 reporters localised at the plasma membrane ( Figure 5 ) .", "In mitotic cells expressing Rap1 ( Q63E ) , there were numerous intracellular puncta consistent with CME of CD8 chimeras possessing any of the three main endocytic motifs ( Figure 5A ) .", "Note that this assay of endocytosis does not involve temperature shifts , chemical synchronisation or serum starvation , which have been proposed to affect CME ( Tacheva-Grigorova et al . , 2013 ) .", "Moreover , endocytosis of CD8 chimeras as measured by antibody labelling on live cells using a fluorophore-conjugated anti-CD8 antibody confirmed that uptake of CD8 chimeras with endocytic motifs occurred in Rap1 ( Q63E ) expressing mitotic cells ( Figure 5B ) . 10 . 7554/eLife . 00829 . 009Figure 5 . Internalisation of diverse cargo is restarted in mitotic Rap1 ( Q63E ) -expressing cells .", "( A ) Representative confocal images of control and Rap1 ( Q63E ) -expressing HeLa cells in mitosis that express the indicated CD8 chimera .", "The cells were fixed and stained for total CD8 to show the steady-state distribution of CD8 chimeras ( green; DNA , blue ) .", "( B ) Representative fluorescence micrographs of anti-CD8 antibody uptake experiments .", "Control and Rap1 ( Q63E ) -expressing HeLa cells in mitosis are shown that express the indicated CD8 chimera .", "Uptake was performed as previously described ( Fielding et al . , 2012 ) .", "Internalised CD8 is shown in green , and surface CD8 is shown in red , and DNA in blue .", "Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 009 An alternative explanation for CME shutdown in mitosis is the phosphorylation of endocytic proteins by mitotic kinases ( Pypaert et al . , 1991; Chen et al . , 1999; Fielding and Royle , 2013 ) .", "Our observations , that mitotic cells are competent for CME if the actin cytoskeleton is made available , runs counter to this hypothesis .", "We first tested if mitotic phosphorylation of endocytic proteins was maintained in cells with restored CME .", "We analysed the phosphorylation status of Dab2 by western blotting .", "Dab2 is a clathrin adaptor for cargo proteins with an NPXY endocytic motif ( Mishra et al . , 2002 ) and it is known to be phosphorylated in mitosis ( He et al . , 2003; Chetrit et al . , 2011 ) .", "Figure 6A shows that Dab2 is phosphorylated in mitosis and that this phosphorylation can be reversed by brief treatment of mitotic cells with the Cdk1 inhibitor flavopiridol ( 5 µM , 10 min ) ( Losiewicz et al . , 1994 ) .", "An identical situation was seen in lysates from cells expressing GFP-Rap1 ( Q63E ) ( Figure 6A ) .", "This suggests that CME of proteins with NPXY motifs can occur ( Figure 5 ) despite maintained mitotic phosphorylation of Dab2 by Cdk1 .", "Secondly , we tested whether inhibition of Cdk1 was sufficient to restart CME .", "Such restarting would be expected if mitotic phosphorylation inhibited the CME machinery directly .", "To do this , CME was measured using transferrin uptake and flow cytometry .", "The brief incubation of mitotic cells with flavopiridol ( 5 or 10 µM ) did not restart CME .", "Together , these results indicate that the direct mitotic phosphorylation of endocytic proteins cannot account for mitotic shut down of CME .", "It remains possible that phosphorylation of endocytic proteins may have a more minor , modulatory role in mitotic shutdown of CME , but we found no evidence that phosphorylation of the CME machinery renders it inactive .", "Moreover , it should be noted that cyclin B1-Cdk1 activity orchestrates mitosis and therefore drives the changes that result in the inhibition of CME at a higher level . 10 . 7554/eLife . 00829 . 010Figure 6 . Mitotic phosphorylation does not explain mitotic shutdown of CME .", "( A ) Western blot to show Dab2 phosphorylation and inhibition by the Cdk1 inhibitor flavopiridol ( 5 µM , 10 min ) .", "Interphase or mitotic cell lysates prepared from cells expressing GFP or GFP-Rap ( Q63E ) to restart CME in mitotic cells .", "Blots were probed for Dab2 and β-tubulin as a loading control , or Cyclin-B1 and Cdk1 .", "( B ) Bar chart to show the average transferrin uptake in interphase ( orange ) or mitotic ( purple ) populations of cells by flow cytometry .", "The mean ± SEM of three separate experiments are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 010 We set out to describe the mechanism by which cells shut down CME during early mitosis .", "Membrane tension is elevated in mitotic cells and presents a challenge to the CME machinery: it now requires the assistance of actin to overcome this tension and internalise receptors .", "A proteomic survey revealed a comparative loss of cortactin from mitotic CCSs , implicating the actin cytoskeleton as being key to the inhibition .", "We showed that mitotic shutdown of CME can be cancelled either by the loss of Ect2 or by constitutive activation of Rap1 , two manipulations that increase actin availability by affecting the formation of a rounded actin cortex in mitotic cells .", "Restored CME under these conditions was uniquely sensitive to latrunculin B-treatment or cortactin-depletion , respectively .", "This agrees with the idea that the actin cytoskeleton is required for CME in mitotic cells , but it is normally unavailable due to the formation of the cortex .", "The unmet actin requirement for CME in mitosis is shown schematically in Figure 7 . 10 . 7554/eLife . 00829 . 011Figure 7 . Model to show ‘two layer’ inhibition of CME during early mitosis . In interphase , CME is continually active and can deploy actin to assist endocytosis if the membrane tension is increased .", "The forces of CME , actin and membrane tension are represented as green , red and blue arrows , respectively .", "In mitosis , the membrane tension is increased and actin is required to assist CME , but it is also unavailable due to the formation of the actin cortex .", "Increasing actin availability , either by Ect2-depletion or expression of Rap1 ( Q63E ) , can restore CME in mitotic cells .", "The membrane tension remains high , but actin is available to assist CME by providing additional force .", "If the actin cytoskeleton is considered to be a component of the endocytic machinery then its unavailability can be regarded as being due to a ‘moonlighting’ function in the formation of a rounded cortex ( Royle , 2013 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00829 . 011 The mechanism for mitotic shut down of CME can therefore be thought of as a ‘two layer’ inhibition .", "The first layer of inhibition comes from an increase in membrane tension , mainly due to osmotic changes in mitotic cells ( Raucher and Sheetz , 1999; Stewart et al . , 2011a ) .", "In interphase cells , CME can overcome such increases in membrane tension by recruiting the actin cytoskeleton to assist in force production ( Aghamohammadzadeh and Ayscough , 2009; Boulant et al . , 2011 ) .", "However , in mitotic cells , the cytoskeleton is engaged in the formation of a rounded actin cortex and is thus unavailable to assist the CME machinery .", "This lack of actin availability represents a second layer of inhibition .", "Local nucleation of actin could therefore provide an override mechanism for receptor internalisation in mitotic cells .", "Recently , F-actin-dependent internalisation of the EGF receptor has been described ( Vehlow et al . , 2013 ) and this may explain how the EGF receptor can be internalised in mitotic cells albeit with delayed kinetics ( Liu et al . , 2011 ) .", "An interesting question for the future is how the CME machinery senses increased membrane tension in order to engage the actin cytoskeleton ." ], [ "Plasmids for expression of mCherry-tagged mouse CTTN ( ID27676 ) and mouse HIP1R tagged with tDimer-RFP ( ID27700 ) were from Addgene .", "HIP1 tagged at the N-terminus with tdTomato was a kind gift from Uri Ashery ( Tel Aviv University ) .", "GFP-tagged clathrin light chain a ( GFP-LCa ) was available from previous work ( Royle et al . , 2005 ) .", "GFP-tagged Human Ect2 ( isoform 2 ) and Rap1 plasmids , pRK5-Rap1-Q63E and pRK5-Rap1-S17A were kind gifts from Buzz Baum ( MRC-LMCB , London , UK ) ( Dao et al . , 2009 ) .", "For Ect2 rescue experiments , siRNA resistant forms of GFP-Ect2 were made introducing silent mutations in the Ect2 siRNA target regions by site-directed mutagenesis .", "GFP-Rap1Q63E was constructed by digestion of pRK5-Rap1Q63E with EcoRI and ligation into pEGFP-C3 .", "Rap1 constructs were expressed for 42 hr .", "The following siRNAs were used: CTTN ( QIAGEN , UK Hs_CTTN_5 SI02661960 or Hs_CTTN_6 SI02662485 ) , Ect2 ( QIAGEN , Hs_ECT2_5 SI02643067 or Hs_ECT2_6 SI03049249 ) at 100 nM for ∼66 hr or 42 hr , respectively .", "HeLa cells were cultured in DMEM containing 10% fetal bovine serum and 100 U/ml penicillin-streptomycin at 37°C , 5% CO2 .", "DNA transfections were performed with GeneJuice ( MerckMillipore , UK ) and siRNA transfections with Lipofectamine 2000 ( Life Technologies , UK ) according to the manufacturer’s instructions .", "For transferrin uptake analysis , HeLa cells were serum-starved in DMEM for 30 min at 37°C before Alexa-conjugated transferrin ( 50 µg/ml ) was added in serum-free DMEM .", "The cells were incubated for 7 min at 37°C to allow uptake and then placed on ice to halt endocytosis .", "Two acid-washes ( 100 mM Glycine , 150 mM NaCl in PBS , pH 3 ) were performed on ice for 5 min to remove surface-bound transferrin .", "Additionally , the cells were washed with PBS before and after each acid-wash to reduce surface binding of transferrin .", "Latrunculin B ( 1 µM ) was added in serum-free media during the serum-starvation step of the transferrin uptake assay .", "For hypertonic sucrose treatment , sucrose ( 0 . 45 M ) was added after 15 min of serum starvation .", "HeLa cells were fixed with 3% paraforamaldehyde/4% sucrose in PBS ( wt/vol ) for 15 min , permeabilised with 0 . 5% Triton X-100 in PBS ( vol/vol ) for 10 min , and then blocked for 30 min with 3% ( wt/vol ) BSA , 5% ( vol/vol ) goat serum in PBS .", "Primary antibodies ( anti-CTTN , ab81208; Abcam , UK ) were incubated for 2 hr in blocking solution .", "After washing , secondary antibodies ( anti-rabbit Alexa Fluor568 , 1:500 ) were added for 1 hr .", "Finally , after washing with PBS , the cells were mounted in ProLong Gold Antifade or Mowiol containing 10 μg/ml 4′ , 6-diamidino-2-phenylindole ( DAPI ) .", "Optionally , TRITC-phalloidin ( Sigma-Aldrich , UK ) was added at a 1:2000 dilution for 1 hr after the permeabilisation step .", "CD8 live-labelling immunofluorescence experiments were carried out exactly as described previously ( Fielding et al . , 2012 ) .", "For total CD8 immunostaining fixed cells were stained with anti-human CD8 Alexa488-conjugated antibody ( 1:200; MCA1226A488; Serotec , UK ) with no secondary antibody .", "To assess the distribution of G-actin and F-actin , the DNase I/phalloidin method was used ( Cramer et al . , 2002 ) .", "Briefly , the cells were fixed in 4% formaldehyde ( EM grade ) with 0 . 32 M sucrose in cytoskeleton buffer ( in mM: 10 Mes , 138 KCl , 3 MgCl2 , 4 EDTA , pH 6 . 1 ) for 20 min at room temperature .", "The cells were permeabilised and then blocked for 20 min at room temperature .", "Cover slips were incubated with 0 . 3 µM DNaseI-Alexa488 and 1:2000 Phalloidin-TRITC , for 20 min at room temperature .", "The cover slips were washed three times in PBS with 0 . 1% Triton X-100 and then mounted as above .", "The purification of fractions containing clathrin-coated structures was as described previously ( Borner et al . , 2012 ) .", "HeLa cells were plated onto 245-mm square cell culture plates ( ∼2 million cells/plate ) .", "Mitotic cells were synchronised by 24 hr in thymidine ( 2 mM ) , released for 3 hr and then 16 hr in nocodazole ( 100 ng/ml ) .", "This arrested ∼80% of cells in mitosis and nine plates of mitotic cells were prepared vs seven plates for interphase .", "Mitotic cells were harvested 6 days after plating by shake-off collected by centrifugation , supernatant removed and cells were resuspended in Buffer A ( 0 . 1 M MES , pH 6 . 5 , 0 . 2 mM EGTA , 0 . 5 mM MgCl2 , 0 . 02% NaN3 , 0 . 2 mM PMSF ) .", "Interphase cells were grown without synchronisation and were rinsed in Buffer A and scraped into Buffer A using a rubber policeman .", "The cells were lysed by 20 strokes in glass homogeniser ( Kontes #22 ) and centrifuged at 152000 gav for 30 min at 4°C .", "Supernatant was removed to ice and 5 mg/ml RNAse A ( Sigma-Aldrich ) was added for 1 hr then centrifuged at 99 , 000 gav for 40 min at 4°C .", "Pellet was resuspended in 0 . 75 ml Buffer A and an equal volume of 12 . 5% Ficoll/12 . 5% sucrose was added and mixed in microfuge tubes and then centrifuged at 14 , 000 rpm for 30 min at 4°C in Eppendorf 5417R .", "Supernatant was removed , placed in thick-walled centrifuge tubes , mixed with 4 vol of Buffer A and clathrin-coated structures were pelleted by spinning at 121500 gav for 75 min at 4°C .", "Supernatant was removed and pellets resuspended in 20 µl Buffer A . The protein content was tested by BCA assay and equal amounts of interphase and mitotic samples ( 25–50 µg ) were run on 4–12% gradient NuPAGE gels and stained with SimplyBlue SafeStain ( Invitrogen ) .", "Each lane was cut into 32 slices and then digested with trypsin .", "The resulting tryptic peptide mixtures in 0 . 05% trifluoracetic acid were analyzed by nanoACQUITY UPLC system ( Waters , UK ) , coupled to an LTQ Orbitrap XL mass spectrometer with a Proxeon nano-electrospray source .", "A 5 μl sample of the digest was injected into a BEH-C18 symmetry trapping column ( Waters ) in 0 . 1% formic acid at 15 μl/min before being resolved on a 25 cm × 75 μm BEH-C18 column ( Waters ) , in a 1–62 . 5% acetonitrile gradient in 0 . 1% formic acid , with a flow rate of 400 nl/min .", "Full scan MS spectra ( m/z 300–2000 ) were generated at 30 , 000 resolution , and the top five most intense ions were fragmented and subjected to MS/MS in the linear quadrapole ion trap .", "Ions were fragmented using collision-induced dissociation ( collision energy 35% , 30 ms ) .", "All spectra were acquired using Xcalibur software ( version 2 . 0 . 7 ) .", "RAW files were analysed using MaxQuant version 1 . 2 . 7 . 4 and searched against the Human IPI database , v . 3 . 77 .", "The proteingroups . txt file from a combined MaxQuant analysis of all four experimental runs was used to calculate the interphase/mitotic ratio of LFQ values for each protein ( Waanders et al . , 2009 ) .", "A Log2 transformation of each ratio was taken and used to construct a frequency histogram to which a Gaussian function was fitted to find the mean and variance of the entire dataset , and identify outliers ( µ ± 2σ ) .", "MaxQuant detected the same outliers when each experiment was analysed individually .", "Excluded from the graph are proteins whose LFQ value for either interphase or mitosis was 0 .", "This could be due to total loss of the protein or poor detection .", "The following proteins had large LFQ scores ( >1 × 106 ) in one sample but 0 in the other and may therefore represent genuine CCS proteins that are only present in interphase ( EPN1 , HEXB , CTSC , NUMB ) or in mitosis ( CFL2 , CLTCL1 ) .", "HeLa cells transfected to express GFP or GFP-Rap1Q63E were synchronised with 100 ng/ml nocodazole for 16 hr at 37°C .", "Flavopiridol ( 5 µM ) was added for 10 min immediately prior to lysis in RIPA buffer supplemented with PMSF ( 0 . 2 mM ) , NaF ( 30 mM ) , and okadaic acid ( 100 nM ) .", "Lysates were analysed by SDS-PAGE and western blotting .", "Commercially available antibodies were used to Dab2 ( sc-13982; Santa Cruz/Insight Biotech , UK ) , cyclinB1 ( 554179; BD Biosciences , UK ) , cdk1 ( ab18; Abcam ) , β-tubulin ( ab6046; Abcam ) , Ect2 ( SC-1005; Santa Cruz ) .", "Confocal imaging was done using a Leica confocal microscope SP2 with a 63X ( 1 . 4 NA ) oil-immersion objective , as described previously ( Fielding et al . , 2012 ) ; or using a Perkin–Elmer UltraView spinning disk confocal microscope system using 405 , 488 and 561 nm lasers and two Orca-R2 cameras ( Hamamatsu ) under the control of Volocity software ( Perkin–Elmer ) .", "Epifluorescence images were taken using a Nikon-Ti Eclipse microscope with a 60X ( 1 . 4 NA ) oil-immersion objective , DS-Qi1Mc camera and standard filter sets for visualisation of DAPI , GFP and Alexa568 .", "HeLa cells ( 400 µl at 5 × 105 cells/ml ) were plated onto 22 × 50 mm glass cover slips ( thickness 1 . 5 ) .", "Each cover slip was suspended on blu tac pedestals inside a 10-cm petri dish alongside a 35-mm petri dish with no lid that contained 3 ml DMEM to humidify the larger dish .", "After 6 hr , 800 µl full DMEM was added to the cells and incubated overnight .", "This ensured good attachment of cells and a clean , dry surface on the underside of the glass .", "Polybead carboxylate 1 µm microspheres ( Polysciences/Park Scientific , UK ) were coated with concanavalin A using a Polylink protein coupling kit .", "This gave 20 µg of protein per 1 mg of microparticle .", "For trapping experiments , the bead solution was diluted ( 1:20 ) in full DMEM .", "A microchamber was created by sealing a small cover glass over the slide on which the cells were grown using high vacuum grease ( Apiezon ) .", "Bead suspension ( 10 µl ) was introduced by capillary action and the microchamber was mounted onto a custom-made optical trap microscope , described elsewhere ( Carter and Cross , 2005 ) .", "The stiffness of the optical trap was calibrated for each experiment , typically ∼0 . 3 pN/nm .", "A single bead was captured and held on the cell surface at a distance of 4–8 µm from the substrate , for several seconds .", "The bead was then pulled away , forming a membrane tether ( >10 µm length ) , by moving the microscope stage under computer control at a constant velocity .", "The displacement of the bead in the optical trap ( and thus tether force ) was measured using a quadrant detector at 5–20 kHz .", "Once the displacement was established , the cell was moved back towards the bead faster than the tether recovery rate to provide a zero force reference ( see Figure 1C ) .", "The cell was then moved back away and tether force was re-established .", "Tension values were taken from a 1 s averaged before stage manipulation .", "All measurements were taken less than 30 s after tether formation , because actin polymerisation into the tether introduced inconsistent measurements in older tethers .", "Measurements from multiple tethers pulled from the same cell indicated high reproducibility and showed that a single force measurement was representative of the membrane tension of that cell .", "Occasionally , double tethers visible in DIC were pulled by a single bead and these gave a twofold increase in tether force from the same cell .", "Data from double tethers were not included here .", "As previously described ( Fielding et al . , 2012 ) , mitotic cells were synchronised with 100 ng/ml nocodazole for 18 hr and harvested by shake-off .", "Interphase cells were growing asynchronously and harvested by trypsinisation .", "The cells were spun down at 1000 rpm for 5 min and resuspended in serum free DMEM , and this step was repeated .", "For the mitotic samples , 100 ng/ml nocodazole was added to prevent progression through mitosis .", "After 20 min of incubation at 37°C , flavopiridol was added for a further 10 min at 37°C .", "The cells were moved to ice for 2 min then Alexa488-conjugated transferrin ( 50 µg/ml dilution ) added on ice for 5 min .", "Uptake was allowed for 7 min at 37°C .", "Then , the cells were pelleted , resuspended in acid wash buffer and incubated on ice for 5 min .", "After a second acid wash , the cells were pelleted and resuspended in 1% ( wt/vol ) BSA/PBS .", "Finally , the samples were analysed by flow cytometry ( FACSCalibur; Becton Dickinson ) .", "The forward scatter channel ( FSC ) was gated at 200 in order to exclude all particles smaller than cells from analysis .", "The side scatter channel ( SSC ) was adjusted to 320 V and the fluorescent channel ( FL1 ) to 385 V . The geometric mean of FL1 was used for the graphs .", "Transferrin uptake quantification was performed in ImageJ using confocal images , as previously described ( Fielding et al . , 2012 ) .", "For quantification of G-actin and F-actin fluorescence , a 24 pixel wide ROI was created at the cell border using a confocal z-section taken at the cell equator .", "A second ROI was created by placing a 50 × 50 ROI in the cytoplasm .", "The background-subtracted values were used to calculate the mean G-actin signal in the cytoplasm and a ratio of cortex/cytoplasm ratio for F-actin .", "All analysis , plots and statistical testing ( see legends for details ) were done in IgorPro 6 . 32A ( WaveMetrics ) .", "Figures were assembled in Adobe Photoshop and Illustrator CS5 . 1 ." ] ]
[ "Clathrin-mediated endocytosis ( CME ) is the major internalisation route for many different receptor types in mammalian cells .", "CME is shut down during early mitosis , but the mechanism of this inhibition is unclear .", "In this study , we show that the mitotic shutdown is due to an unmet requirement for actin in CME .", "In mitotic cells , membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load .", "However , the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment .", "We demonstrate that CME can be ‘restarted’ in mitotic cells despite high membrane tension , by allowing actin to engage in endocytosis .", "Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME , indicating that direct phosphorylation of the CME machinery does not account for shutdown ." ]
[ "The plasma membrane that surrounds a cell acts as a protective barrier that regulates what can enter or exit the cell .", "However , large molecules and other ‘cargo’ can get into a cell in a variety of ways .", "One of these routes—known as clathrin-mediated endocytosis—involves a receptor on the outside of the membrane grabbing hold of the cargo while a protein called clathrin forms a ‘pit’ beneath the receptor .", "This pit becomes deeper and deeper until the cargo is completely surrounded by clathrin-lined membrane and is brought inside the cell .", "This process has been studied over the past 50 years , and it is known that clathrin-mediated endocytosis is turned off when a cell begins to divide to produce new cells , and then turned back on when cell division has come to an end .", "However , there are competing theories as to exactly why this process stops when cell division starts .", "Now , Kaur et al . have investigated these theories by looking at the role that another protein , called actin , plays in turning off clathrin-mediated endocytosis .", "Actin is a molecule that forms a sort of scaffolding within the cell ( called the cytoskeleton ) , and it also guides the movement of molecules and larger structures within the cell .", "Further , when the cell membrane is being stretched , the actin cytoskeleton can assist the clathrin-mediated endocytosis machinery to pull cargo into the cell .", "So why doesn’t actin help with endocytosis during cell division ?", "The answer , Kaur et al . suggest , is that all the actin in the cell is needed by the cytoskeleton during cell division , so there is no actin available to perform other tasks such as clathrin-mediated endocytosis .", "Further experiments demonstrated that this form of endocytosis can be ‘restarted’ in dividing cells by treating the cells in a way that frees up some additional actin .", "The work of Kaur et al . also ruled out the theory that chemical changes to the endocytosis machinery disabled it during cell division .", "These findings have implications for the delivery of drugs , via endocytosis , to the rapidly dividing cells that are involved in diseases such as cancer ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cancer biology" ]
Overcoming mutation-based resistance to antiandrogens with rational drug design
elife-00499-v1
[ [ "The recent FDA approval of enzalutamide ( formerly MDV3100 ) confirms the continued critical role AR signaling plays in castration-resistant prostate cancer ( Tran et al . , 2009; Scher et al . , 2012 ) .", "In spite of these promising results , patient responses to enzalutamide are varied and often short lived .", "Reactivation of AR signaling has been implicated in resistance to previous antiandrogen therapy ( Linja et al . , 2001; Chen et al . , 2004 ) , and one well-documented mechanism of reactivation is point mutation in the ligand-binding domain ( LBD ) of AR ( Bergerat and Ceraline , 2009 ) .", "Many of these mutations broaden ligand specificity , and some confer resistance by converting the AR antagonist into an agonist of the mutant receptor ( Veldscholte et al . , 1990; Haapala et al . , 2001; Hara et al . , 2003 ) .", "Because previous work with targeted therapies that inhibit oncogenic kinases has shown that unbiased mutagenesis screens in preclinical models can identify a priori clinically relevant mutations that alter drug activity ( Azam et al . , 2003; Burgess et al . , 2005 ) , we designed a novel screening method to prospectively identify AR mutations that confer resistance to enzalutamide .", "Mutagenesis screens to identify kinase inhibitor-resistant alleles of kinase targets such as BCR-ABL have relied upon cytokine-dependent test cells that become cytokine independent after introduction of the target kinase .", "Cells expressing drug-resistant kinase alleles selectively expand in the presence of drug , allowing rapid identification of mutations that confer drug resistance .", "There is no comparable strategy available for antiandrogens because introduction of AR does not confer a comparable growth advantage in AR-negative cells .", "We instead chose to identify and select cell populations with persistent AR transcriptional activity in the presence of enzalutamide .", "We reasoned that targeting the biological process of interest ( transcriptional activation of a target gene ) , rather than a distal symptom of resistance to drug ( i . e . , persistent viability , elevated proliferation ) might identify resistant clones more quickly .", "To this end , an AR-regulated EGFP reporter , with a probasin promoter and PSA enhancer elements driving EGFP expression ( Pb . PSE . EGFP ) ( Chapel-Fernandes et al . , 2006 ) , was used to screen for and enrich cell populations bearing biologically active mutations ( Figure 1A ) . 10 . 7554/eLife . 00499 . 003Figure 1 . Mutagenesis screen for enzalutamide resistance identifies novel AR mutation .", "( A ) A cartoon of the AR mutagenesis screen developed to identify enzalutamide resistance mutations .", "Briefly , cells were cotransduced with a randomly mutagenized AR cDNA library ( AR* ) and EGFP reporter of AR activity ( Pb . PSE . EGFP ) , treated with 1 μM enzalutamide , and EGFP-positive cells were sorted using FACS .", "AR was PCR amplified and sequenced to identify relevant mutations .", "( B ) Representative FACS histograms showing the progressive enrichment of an EGFP-positive subpopulation of LNCaP/AR*/Pb . PSE . EGFP cells post multiple rounds of enzalutamide treatment and cell sorting .", "( C ) A Sanger sequencing trace of exon 8 within AR on the exogenous AR allele from LNCaP/AR*/Pb . PSE . EGFP cells after the fifth FACS sort .", "The position of the mutation is highlighted with an arrow .", "The alignment was performed against AR WT .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 00310 . 7554/eLife . 00499 . 004Figure 1—figure supplement 1 . Enrichment of AR W741C mutant expressing LNCaP-Pb . PSE . EGFP cells after bicalutamide treatment and EGFP sorting . Genomic DNA was isolated from LNCaP-Pb . PSE . EGFP cells ectopically expressing either wild-type ( WT ) AR or mutant AR W741C , or different ratios of mutant-to-WT , and quantitative PCR was performed to test the sensitivity of the W741C-specific primers .", "With starting ratios of 1:100 and 1:1000 mutant-to-WT , we treated these cell mixtures with 1 μM bicalutamide for 4 days , and then FACS-sorted those that maintained/induced EGFP expression .", "Sorted cells were expanded and the brief bicalutamide treatment and FACS-sorting was repeated ( four rounds ) .", "Genomic DNA was isolated from the sorted cell populations , and quantitative PCR was performed to test for enrichment of the W741C mutant cells .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 00410 . 7554/eLife . 00499 . 005Figure 1—figure supplement 2 . Endogenous AR target gene , PSA is induced by enzalutamide in FACS-sorted cells . Parental LNCaP-Pb . PSE . EGFP cells , and those overexpressing AR WT , the random AR mutant library ( Mut Lib ) , and cells after each sort were treated with 1 μM enzalutamide for 24 hr in media containing full serum .", "RNA was then collected , reverse transcribed , and quantitative PCR performed for AR target gene KLK3 ( PSA ) .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 00510 . 7554/eLife . 00499 . 006Figure 1—figure supplement 3 . Expression of EGFP and endogenous AR target gene FKBP5 remain AR-dependent in FACS-sorted cells . LNCaP-Pb . PSE . EGFP cells overexpressing AR WT and cells from the fifth sort of our screen were transfected with 10 nM of either a non-targeting siRNA ( siNT ) or a siRNA against AR ( siAR ) .", "They were also treated with either vehicle ( V ) or 1 μM enzalutamide ( E ) .", "After 4 days of enzalutamide treatment and siRNA knockdown , cells were collected for both ( A ) flow cytometric analysis of EGFP expression and ( B ) western blot analysis of the AR target gene FKBP5 , and to ensure we achieved good AR knockdown .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 00610 . 7554/eLife . 00499 . 007Figure 1—figure supplement 4 . AR F876L mutation accounts for 50% of AR in cells after fourth sort , and further enriched after the fifth sort . We PCR amplified AR from our LNCaP/AR*/Pb . PSE . EGFP cells after four rounds of enzalutamide treatment and FACS-sorting , and Sanger sequenced the PCR product .", "AR F876L ( T → C ) accounts for approximately 50% of the AR in these cells .", "This mutation is further enriched after the fifth sort , and accounts for approximately 80% of AR in that population of cells .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 007" ], [ "Proof-of-concept studies were conducted with LNCaP cells cotransduced with Pb . PSE . EGFP and AR W741C , a well-characterized mutation that converts the AR antagonist bicalutamide into an agonist ( Haapala et al . , 2001; Hara et al . , 2003 ) .", "With starting ratios of 1:100 and 1:1000 cells overexpressing AR W741C to wild-type AR , respectively , we sorted cells with FACS that maintained EGFP expression after acute bicalutamide exposure .", "After four rounds of bicalutamide ‘selection’ and sorting , LNCaP cells expressing AR W741C dominated the final populations ( Figure 1—figure supplement 1 ) .", "We next conducted the enzalutamide resistance screen with the Pb . PSE . EGFP reporter and a randomly mutagenized AR library .", "After five iterations of enzalutamide exposure and FACS sorting , we identified a population of cells with durable EGFP expression ( Figure 1B ) .", "Moreover , enzalutamide promoted AR transcriptional activity in these cells , reflected by induction of EGFP expression compared to vehicle control ( Figure 1B ) .", "Analysis of endogenous AR target gene expression confirmed that enzalutamide behaved as an agonist in the enriched cell population ( Figure 1—figure supplement 2 ) , and siRNA knockdown of AR showed that these pharmacologically induced changes remained AR dependent ( Figure 1—figure supplement 3A , B ) .", "To identify AR mutations in these cells , we amplified the exogenously expressed AR cDNA , and Sanger sequenced the PCR product .", "In two of three replicates , a single dominant point mutation emerged , resulting in the amino acid substitution F876L ( Figure 1C ) .", "Importantly , this mutation clearly enriched throughout the selection process ( Figure 1—figure supplement 4 ) .", "To validate the results of the screen , an AR F876L vector was engineered and transduced into parental LNCaP cells expressing the Pb . PSE . EGFP reporter .", "Treatment of these cells with enzalutamide resulted in a dose-dependent induction of EGFP expression ( Figure 2A , Figure 2—figure supplement 1 ) .", "We also introduced AR F876L cDNA into AR-negative CV1 cells along with an AR-dependent luciferase construct , and upon enzalutamide treatment , luciferase activity was induced ∼50-fold ( Figure 2B ) .", "These results were comparable to those seen with the previously reported AR mutations T877A and W741C , which confer agonism to hydroxyflutamide and bicalutamide , respectively .", "Moreover , enzalutamide treatment potently induced nuclear localization of AR F876L ( Figure 2—figure supplement 2A ) , and chromatin immunoprecipitation studies showed that enzalutamide recruited AR F876L to the enhancers of AR target genes ( Figure 2—figure supplement 2B ) .", "Consistent with these results , endogenous AR target gene expression was either no longer repressed by enzalutamide ( Figure 2C , left ) or strongly induced by enzalutamide in cells expressing AR F876L ( Figure 2C , right ) .", "A competition assay with 16β[18F]fluoro-5α-DHT ( 18F-FDHT ) , to measure relative AR binding affinity ( Tran et al . , 2009 ) , showed that enzalutamide binds with higher affinity to AR F876L than wild-type AR ( Figure 2—figure supplement 3 ) , similar to what has been shown for hydroxyflutamide and the AR T877A mutant ( Ozers et al . , 2007 ) .", "Notably , F876L similarly impacted the pharmacology of ARN-509 ( Clegg et al . , 2012 ) , a structurally discrete antiandrogen sharing the bisaryl-thiohydantoin core motif ( Figure 2B , Figure 2—figure supplements 4 , 5 ) . 10 . 7554/eLife . 00499 . 008Figure 2 . AR F876L mutation converts enzalutamide into an agonist and rescues enzalutamide-induced growth inhibition .", "( A ) A representative FACS histogram shows the induction of AR-dependent EGFP expression by enzalutamide in LNCaP-Pb . PSE . EGFP cells ectopically expressing AR F876L .", "The magnitude of induction by enzalutamide ( 10 μM ) is comparable to that conferred by the endogenous androgen DHT ( 1 nM ) .", "Enzalutamide treatment of LNCaP-Pb . PSE . EGFP cells ectopically expressing AR WT effectively suppressed EGFP expression .", "Geometric-mean fluorescence intensity for WT treated cells: vehicle ( 348 ) , enzalutamide ( 66 . 4 ) , DHT ( 1554 ) ; for F876L cells: vehicle ( 345 ) , enzalutamide ( 1051 ) , DHT ( 1699 ) .", "( B ) Cotransduction of CV1 cells with an AR-regulated firefly luciferase construct , a constitutive Renilla luciferase construct , and one of the indicated AR constructs , recapitulates the pharmacology observed in the EGFP reporter system .", "These cells were treated with vehicle ( DMSO ) , antiandrogens ( 1 μM ) , or the synthetic androgen R1881 ( 1 nM ) .", "A dual luciferase assay was conducted on cell lysates , the firefly signal was normalized to the constitutive Renilla activity , and the data are reported as relative light units ( RLUs ) .", "Notably , the bisaryl-thiohydantoin antiandrogens ( enzalutamide and ARN-509 ) effectively induce AR F876L transcriptional activity , while structurally discrete antiandrogens ( hydroxyflutamide and bicalutamide ) do not impact AR F876L activity in this assay .", "As expected , the transcriptional activity of AR W741C or AR T877A was induced by bicalutamide or hydroxyflutamide , respectively .", "( C ) Quantitative reverse transcription–polymerase chain reaction analysis of LNCaP/AR F876L cells shows that enzalutamide ( 1 μM ) can induce the expression of canonical AR-regulated gene products ( i . e . , PSA , TMPRSS2 , SGK1 , and FKBP5 ) .", "Relative gene expression post therapy for LNCaP/AR WT cells is included as positive controls .", "FL = AR F876L , data are normalized to GAPDH and represented as mean ± SD , n = 3 .", "( D ) Cell proliferation data shows that overexpression of AR F876L in a human prostate cancer cell line sensitive to enzalutamide therapy can rescue cell growth .", "VCaP cells overexpressing either AR WT ( solid lines ) or AR F876L ( dashed lines ) were cultured in media containing full serum , treated with either vehicle ( DMSO ) or 10 μM enzalutamide , and the viable cell fraction was determined at the indicated time points ( data is represented as mean ± SD , n = 3 ) .", "( E ) Cellular proliferation data shows that enzalutamide also rescues the growth of VCaP cells expressing AR F876L in androgen-depleted media .", "VCaP cells overexpressing either AR WT ( solid lines ) or AR F876L ( dashed lines ) were treated with vehicle ( DMSO ) , 1 nM DHT , or 10 μM enzalutamide , and the viable cell fraction was determined at the indicated time points ( mean ± SD , n = 3 ) .", "( F ) A time to progression study for mice bearing subcutaneous LNCaP/AR-WT ( solid lines ) or LNCaP/AR-F876L ( dashed lines ) xenografts further highlights the genotype-dependent pharmacology of enzalutamide .", "Inoculated animals were treated once daily through oral gavage with either vehicle or enzalutamide ( 30 mg/kg ) , and tumor size was monitored weekly ( 11–16 tumors per treatment group ) .", "While enzalutamide potently suppressed the growth of LNCaP/AR-WT tumors , LNCaP/AR-F876L tumors exposed to enzalutamide grew with kinetics roughly equivalent to either vehicle treatment arm .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 00810 . 7554/eLife . 00499 . 009Figure 2—figure supplement 1 . Dose-dependent induction of EGFP expression by enzalutamide in LNCaP-Pb . PSE . EGFP cells expressing AR F876L . LNCaP-Pb . PSE . EGFP cells ectopically expressing AR F876L were treated with vehicle , 1 or 10 μM enzalutamide for 4 days .", "Cells were then collected for FACS-analysis of EGFP expression .", "Geometric-mean fluorescence intensity is indicated in the table .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 00910 . 7554/eLife . 00499 . 010Figure 2—figure supplement 2 . Enzalutamide induces AR F876L nuclear translocation and DNA binding to AR enhancer elements .", "( A ) LNCaP cells were transfected with EYFP-tagged wild-type AR or AR F876L in androgen depleted media containing vehicle , 1 μM enzalutamide , or 1 nM DHT .", "Representative confocal images are shown .", "Average nuclear-to-cytoplasmic ratios for EYFP are displayed ( ±SD , n = 3 ) .", "( B ) LNCaP cells stably overexpressing either AR WT or AR F876L were cultured in androgen-depleted media for 4 days , then treated with vehicle ( VEH ) , 10 μM enzalutamide ( ENZ ) , or 1 nM DHT for 4 hr .", "AR chromatin immuoprecipitation was performed , and real-time PCR quantification of PSA enhancer and FKBP5 enhancer is shown ( percent input mean ± SD , n = 3 ) .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01010 . 7554/eLife . 00499 . 011Figure 2—figure supplement 3 . Enzalutamide binds to AR F876L with higher affinity than to AR WT . Representative competition binding curves , showing displacement of 18F-FDHT in LNCaP/AR WT and LNCaP/AR F876L cells by increasing concentrations of cold DHT or enzalutamide ( ENZ ) .", "The median inhibitory concentration ( IC50 ) values from this experiment are displayed ( error bars represent the SD of triplicate measurements ) .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01110 . 7554/eLife . 00499 . 012Figure 2—figure supplement 4 . AR F876L mutation converts ARN-509 into an AR agonist . LNCaP-Pb . PSE . EGFP cells ectopically expressing either AR WT or AR F876L were treated with vehicle or 10 μM ARN-509 .", "After 4 days of treatment , cells were collected for analysis of EGFP expression ( FL1-H ) , geometric-mean fluorescence is shown in the table below .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01210 . 7554/eLife . 00499 . 013Figure 2—figure supplement 5 . Dose-dependent agonism in AR F876L expressing cells treated with enzalutamide or ARN-509 . LNCaP cells ectopically expressing AR F876L were cultured in androgen-depleted media ( 10% CSS ) for 48 hr , then treated with the indicated dose of antiandrogen for 24 hr , and qRT-PCR was performed to assess the expression of the indicated AR target gene .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01310 . 7554/eLife . 00499 . 014Figure 2—figure supplement 6 . Ectopic expression of AR F876L in CWR22Pc cells confers resistance to enzalutamide and rescues growth in androgen-depleted media .", "( A ) CWR22Pc cells stably expressing either AR WT or AR F876L were plated in full serum media containing vehicle , 1 μM enzalutamide , or 10 μM bicalutamide .", "CellTiterGLO assay was performed on days 1 , 4 , and 7 to measure cell viability ( mean relative light units [RLU] ± SD , n = 3 ) .", "( B ) CWR22Pc cells stably expressing either AR WT or AR F876L were plated in full serum media containing vehicle , 1 μM enzalutamide , or 0 . 1 nM DHT .", "CellTiterGLO assay was performed on days 1 , 4 , and 7 to measure cell viability ( mean relative light units [RLU] ± SD , n = 3 ) .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01410 . 7554/eLife . 00499 . 015Figure 2—figure supplement 7 . Other amino acid substitutions at Phe876 modify the pharmacology of second-generation antiandrogens . ARE ( 4X ) -luciferase assay for additional F876 substitutions .", "CV1 cells were cotransfected with an ARE ( 4X ) -firefly luciferase construct , SV40 Renilla luciferase construct , and one of the designated AR constructs .", "The cells were treated with 10 μM of the indicated antiandrogens , and a dual luciferase assay was performed on the lysates , and normalized to Renilla luciferase ( mean ± SD , n = 3 ) .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01510 . 7554/eLife . 00499 . 016Figure 2—figure supplement 8 . Bicalutamide is a modest inhibitor of AR F876L transcriptional activity . Parental LNCaP-Pb . PSE . EGFP cells and those transduced with AR WT or AR F876L were treated with vehicle , 1 or 10 μM bicalutamide ( BIC ) for 4 days , and then collected for flow cytometric analysis of EGFP expression ( FL1-H ) .", "Geometric-mean fluorescence intensity of EGFP is displayed in the table below each histogram plot . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 016 In vitro growth assays were conducted to examine the consequences of AR F876L expression on enzalutamide sensitivity in prostate cancer cell lines .", "Although enzalutamide treatment potently inhibits the growth of parental VCaP cells ( Tran et al . , 2009 ) , overexpression of AR F876L entirely reversed this phenotype ( Figure 2D ) .", "Enzalutamide also rescued the growth of VCaP/AR F876L cells in androgen-depleted media , similar to that seen with the endogenous androgen DHT ( Figure 2E ) .", "Finally , these results were recapitulated in CWR22Pc cells , another prostate cancer cell line that is sensitive to enzalutamide ( Figure 2—figure supplement 6A , B ) .", "In vivo , LNCaP/AR cells overexpressing either AR WT or F876L were grafted subcutaneously into castrate SCID mice and time to tumor emergence/progression was determined in the presence or absence of drug .", "While the growth of wild-type AR tumors was almost completely inhibited by enzalutamide treatment , tumors expressing AR F876L grew rapidly in the presence of enzalutamide , similar to vehicle-treated tumors of either genotype ( Figure 2F ) .", "We next asked whether the F876L mutation spontaneously arises in antiandrogen-sensitive human prostate cancer models after prolonged treatment with enzalutamide or ARN-509 .", "After culturing CWR22Pc cells in vitro with enzalutamide for several months , more than 50% of the cells expressed the F876L mutation ( Table 1 ) .", "Prolonged culture of these cells with ARN-509 also selected for a small population ( ∼1 . 3% ) expressing AR F876L .", "In vivo , long-term enzalutamide or ARN-509 therapy in mice bearing LNCaP/AR xenograft tumors also resulted in the outgrowth of tumor cell populations expressing AR F876L ( Table 1 ) .", "Sequencing revealed that AR F876L predominated in one tumor ( ∼71% ) , was present at low frequency in four other tumors ( ∼1 to 2% ) , and that a distinct amino acid substitution at this residue , F876I , was enriched in one enzalutamide-resistant tumor ( Table 2 ) .", "To further explore the function of Phe876 as the ‘gateway’ residue governing enzalutamide and ARN-509 pharmacology , we used site-directed mutagenesis to make additional amino acid substitutions at residue 876 .", "A conservative F876Y substitution did not alter the pharmacology of either drug , but aliphatic substitutions structurally similar to F876L , such as F876I ( also found in one xenograft with acquired resistance ) , conferred agonism to both enzalutamide and ARN-509 ( Figure 2—figure supplement 7 ) .", "Notably , we observed that bicalutamide did not induce AR F876L transcriptional activity in our luciferase reporter assay , either at low ( Figure 2B ) or at high ( Figure 2—figure supplement 7 ) concentrations , suggesting that it retains weak antagonist activity against this mutant .", "We conducted EGFP reporter assays to determine if AR F876L transcriptional activity is inhibited by bicalutamide , and found that while at low doses , it is an effective inhibitor of AR F876L , it loses potency at higher concentrations ( Figure 2—figure supplement 8 ) .", "We also observed only minimal growth inhibition in CWR22Pc cells expressing AR F876L with bicalutamide treatment ( Figure 2—figure supplement 6A ) .", "These data , along with the knowledge that AR overexpression is a common resistance mechanism in patients with CRPC ( Linja et al . , 2001 ) and confers partial agonism on bicalutamide ( Chen et al . , 2004 ) , suggest that bicalutamide is not a viable treatment option for patients who fail on enzalutamide due to AR F876L mutation .", "That F876L so dramatically impacted the pharmacology of enzalutamide and ARN-509 suggested that a clear structural change in the drug–receptor complex might be occurring .", "This consideration prompted us to investigate the structural basis of this antagonism-to-agonism conversion .", "Because a crystal structure depicting AR bound to an antagonist does not yet exist , we performed structural modeling using ligand docking and molecular dynamics ( MD ) simulations ( Karplus and McCammon , 2002; Jorgensen , 2004 ) .", "In designing the study , we noted that both enzalutamide and ARN-509 share identical A-rings with bicalutamide ( Figure 3A ) and its derivative S1 that were respectively cocrystallized with the LBD of AR W741L and AR WT in agonist conformations ( PDB ID 1Z95 and 2AXA ) ( Bohl et al . , 2005a , 2005b ) .", "2AXA was chosen as a structural template as it bears fewer amino acid substitutions compared to 1Z95 .", "After initial quantum-mechanical geometry optimization of the small molecules , each was independently docked into AR WT or AR F876L with the mutually shared A-ring overlaid with that of S1 , whereupon 10-ns explicit-solvent MD simulations were performed . 10 . 7554/eLife . 00499 . 019Figure 3 . Molecular dynamics simulations predict a novel binding mode for bisaryl-thiohydantoin antiandrogens and the basis for agonism toward AR F876L .", "( A ) Structures of the antiandrogens bicalutamide ( top ) , enzalutamide ( middle ) , and ARN-509 ( bottom ) oriented to highlight the common and discrete regions of the molecules .", "The A-D annotation of the rings is indicated .", "( B ) A magnified view of the cocrystal structure of AR W741L ( gray ) and bicalutamide ( gold ) shows the antiandrogen's spatial relationship to the H11 and H12 pockets and residue F876 ( blue ) .", "In this agonist conformation , the C-ring of bicalutamide does not interact with F876 .", "( C ) A magnified view of the initial-docked models of enzalutamide ( gold ) and ARN-509 ( cyan ) calculated using coordinates from 2AXA in which residue 741 is a tryptophan .", "The model suggests that the loss of torsional freedom imposed by the thiohydantoin B-ring imposes conformational restrictions on the antagonists that force the C-ring toward F876 and the ‘H11 pocket' . ( D ) The lowest-energy 10-ns MD models for enzalutamide with AR WT ( red ) and AR F876L ( cyan ) overlaid on 1Z95 ( gray—agonist reference structure ) . The F876L mutation allows for cooperative changes in neighboring residues which , when bound to enzalutamide , enable H12 to adopt a more agonist-like conformation . ( E ) An analogous view of the lowest-energy 10-ns MD models for ARN-509 with AR WT ( red ) and AR F876L ( cyan ) overlaid on 1Z95 ( gray ) shows a similar effect for F876L on the positioning of H11 and H12 . These simulations also point to the comparatively larger dislocation in H12 by ARN-509 in AR WT , presumably owing to favorable steric interactions between the spirocyclobutyl ring and H12 . AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 01910 . 7554/eLife . 00499 . 020Figure 3—figure supplement 1 . Overlay of predicted enzalutamide or ARN-509 simulations and solved agonist crystal structure . The coordinates for three 10-ns simulations with the indicated receptor ( cyan ) and drug compound ( gold ) were overlaid on the 1Z95 structure ( gray ) to highlight structural differences between the agonist conformation of the AR W741L/bicalutamide complex . Note the evidence of H12 dislocation in AR WT/antiandrogen complexes that is less evident for AR F876L . Bicalutamide was deleted for visual clarity . For the simulations conducted for the enzalutamide/AR F876L complex , one suspected outlier ( red ) was detected among the initial three MD simulations . Five additional simulations were conducted , and they recapitulated the majority tendency observed for the initial MD simulations . AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02010 . 7554/eLife . 00499 . 021Figure 3—figure supplement 2 . A zoomed in view of the H11 pocket from the enzalutamide and ARN-509 MD simulations . The lowest-energy 10-ns MD models for enzalutamide ( A ) and ARN-509 ( B ) with AR WT ( red ) and AR F876L ( cyan ) overlaid on 1Z95 ( gray—agonist reference structure ) , with an inset showing a zoomed in view of the region around residue 876 , that includes distances between close hydrophobic atoms on each receptor:ligand pair . AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 021 The docked enzalutamide and ARN-509 molecules demonstrated strikingly different interaction patterns with AR compared to bicalutamide ( Figure 3B , C ) . Notably , the thiohydantoin B-ring prevents the compound from accessing the ‘H12 pocket’ occupied by bicalutamide .", "Instead , the conformationally restricted thiohydantoin forces the C-ring to bind a region near the C terminus of helix 11 and the loop connecting helices 11 and 12 , that we termed the ‘H11 pocket’ .", "As seen in the MD simulations using the WT receptor with enzalutamide and ARN-509 ( Figure 3D , E [in red] , Figure 3—figure supplement 1 ) , accommodation of the C-ring in this region is coupled to significant conformational rearrangements of residues on H11 and the H11–H12 connecting loop that prevents H12 from adopting the agonist conformation required for efficient coactivator recruitment .", "To investigate how the F876L mutation might alleviate antagonism , we performed similar MD simulations using the F876L receptor ( Figure 3D , E [in cyan] , Figure 3—figure supplement 1 ) .", "For WT receptors in complex with enzalutamide and ARN-509 , the average RMSDs to the crystal agonist conformation for the helix 11 terminus ( residues 875–882 ) were measured at 2 . 24 and 1 . 94 Å , respectively , and those for the helix 12 terminus ( residues 893–900 ) were 1 . 81 and 2 . 08 Å , respectively .", "For the F876L mutants , in comparison , the average RMSDs for helix 11 were somewhat lower at 1 . 01 and 1 . 70 Å for enzalutamide and ARN-509 , respectively , and those for helix 12 went down to 1 . 37 Å for both ligands .", "The results demonstrate that despite inducing similar dislocations in the H11 pocket , the mutation allows the receptor to reposition H12 in a more agonist-like conformation that is compatible with coactivator recruitment ( Figure 3D , E , Figure 3—figure supplement 1 ) .", "A close look in the H11 pocket ( Figure 3—figure supplement 2A , B ) indicated the following: ( 1 ) F876 in AR WT likely interacts with the C-ring end of enzalutamide or ARN-509 through favorable pi stacking or van der Waals contacts , and ( 2 ) the loss of such favorable contacts upon F876L mutation concurrently affects the conformational choices of helices 11 and 12 , which interact through both bonded and nonbonded forces .", "Importantly , these structural modeling results are consistent with the differential resistance profiles for enzalutamide and bicalutamide involving residues 741 and 876 , respectively ( Figure 2B ) .", "Another notable insight from these simulations was that the substituent on position 4 of the B-ring ( i . e . , the geminal [gem]-dimethyl group on enzalutamide , the spirocyclobutyl ring on ARN-509; Figure 4A ) was predicted to lie in close proximity to residues on H12 of the mutant receptor ( Figure 3D , E ) .", "Moreover , the steric girth of the substituent appeared to impact the positioning of H12 , as the bulkier spirocyclobutyl moiety on ARN-509 elicited greater H12 displacements in AR WT than did enzalutamide's gem-dimethyl group ( Figure 3E ) . 10 . 7554/eLife . 00499 . 022Figure 4 . A focused chemical screen identifies novel antagonists of AR F876L .", "( A ) A tabular summary of the bioactivity of the novel antiandrogens in the LNCaP/AR/Pb . PSE . EGFP cell-based assay shows the importance of a carefully designed D-ring for competent inhibition of AR F876L .", "Antagonism is indicated with a ‘−’ symbol , and agonism is indicated with a ‘+’ sign .", "The asterisk is situated over the shared carbon atom in position 4 that joins the bisaryl-thiohydantoin scaffold to the respective ‘substituent’ .", "The source data is outlined in Figure 4—figure supplement 1 .", "( B ) A proliferation assay for VCaP prostate cancer cells overexpressing either wild-type AR ( left ) or AR F876L ( right ) shows that ( ± ) -DR103 effectively inhibits the growth of both models , while enzalutamide and the close structural analogue DR101 only inhibit the growth of VCaP/AR WT .", "Data are reported as mean ± SD , n = 3 .", "( C ) A view of the lowest-energy conformations of enzalutamide ( cyan ) , ARN-509 ( gold ) , and ( S ) -DR103 ( red ) in complex with AR F876L highlights the greater dislocation of H12 and the loop between H11 and H12 uniquely conferred by ( S ) -DR103 .", "The color scheme invoked for AR F876L matches the respective antiandrogen .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02210 . 7554/eLife . 00499 . 023Figure 4—figure supplement 1 . EGFP reporter assay for AR activity with DR series compounds . LNCaP-Pb . PSE . EGFP cells ectopically expressing either AR WT or AR F876L were treated with vehicle ( DMSO ) or 10 μM of the indicated DR-series compound .", "After 4 days of treatment , cells were collected and FACS analysis for EGFP expression was performed .", "Geometric-mean fluorescence intensity is indicated in the adjacent table . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02310 . 7554/eLife . 00499 . 024Figure 4—figure supplement 2 . ( ± ) -DR103 effectively competes with DHT for AR binding and induction of AR-regulated luciferase . CV1 cells were transfected with AR WT or AR F876L , 4XARE-luciferase , and SV40 renilla luciferase expression constructs .", "They were cultured in androgen-depleted media supplemented with the indicated androgen/antiandrogen for 36 hr , and a dual-luciferase assay was performed on cell lysates .", "Luciferase activity is represented by relative light units ( RLU ) ± SD , n = 3 .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02410 . 7554/eLife . 00499 . 025Figure 4—figure supplement 3 . ( ± ) -DR103 is a more potent antagonist for AR F876L than AR WT . CV1 cells were transfected with AR WT or AR F876L , 4XARE-luciferase , and SV40 renilla luciferase expression constructs .", "They were cultured in androgen-depleted media supplemented with the indicated androgen/antiandrogens for 36 hr and a dual-luciferase assay was performed on cell lysates .", "DHT concentration was 1 nM .", "Luciferase activity is represented by relative light units ( RLU ) ± SD , n = 3 .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02510 . 7554/eLife . 00499 . 026Figure 4—figure supplement 4 . Growth inhibition of CWR22Pc cells overexpressing AR WT or AR F876L with ( ± ) -DR103 treatment . CWR22Pc cells ectopically expressing wild-type AR or AR F876L , cultured in full-serum-containing media , were treated with vehicle ( DMSO ) or 10 µM of enzalutamide or DR103 .", "CellTiterGLO assay was performed on days 1 , 4 , and 7 to determine cell viability ( relative light units [RLU] ± SD , n = 3 ) .", "AR: androgen receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02610 . 7554/eLife . 00499 . 027Figure 4—figure supplement 5 . Novel antiandrogen , ( ± ) -DR103 , efficiently inhibits AR signaling and induces PARP cleavage in cells expressing both AR WT and AR F876L . VCaP cells ectopically expressing either AR WT or AR F876L were treated for 4 days with vehicle or 10 µM of the indicated antiandrogen ( VEH = vehicle , DMSO; ENZ = enzalutamide ) in media containing 10% FBS .", "Whole-cell lysates were analyzed by western blot .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02710 . 7554/eLife . 00499 . 028Figure 4—figure supplement 6 . DU145 cells treated with ( ± ) -DR103 and DR104 display no significant growth inhibition . DU145 cells were cultured in full-serum-containing media with 10 μM of the indicated antiandrogens .", "CellTiterGLO assay was performed on days 0 , 3 , and 6 to determine cell viability ( relative light units [RLU] ± SD , n = 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02810 . 7554/eLife . 00499 . 029Figure 4—figure supplement 7 . Overlay of predicted DR103 simulations and solved agonist crystal structure . An overlay of three 10-ns MD simulations for ( S ) -DR103 docked either in AR WT or AR F876L shows the dislocation of H12 in space compared to 1Z95 ( gray ) , consistent with the pharmacological model predicted by previous MD simulations for enzalutamide and ARN-509 in AR WT .", "The predicted conformations of the AR variants are highlighted in cyan , and the respective conformations of ( S ) - and ( R ) -DR103 are represented in gold .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 02910 . 7554/eLife . 00499 . 030Figure 4—figure supplement 8 . A zoomed in view of the H11 and H12 pockets of the MD simulations for AR F876L . A magnified view of the lowest energy conformations of enzalutamide ( cyan ) , ARN-509 ( gold ) , and ( S ) -DR103 ( red ) in complex with AR F876L , showing the structural framework and interactions in close proximity to the antagonist C and D-rings ( with distances measured ) .", "AR: androgen receptor; WT: wild-type . DOI: http://dx . doi . org/10 . 7554/eLife . 00499 . 030 To restore the positioning of H12 into an antagonist conformation for AR F876L , we designed and synthesized a series of analogues bearing saturated hydrocarbon spirocycles of incrementally greater size and complexity on the B-ring of the enzalutamide scaffold ( DR100-103 , Figure 4A ) .", "We defined these ring extensions on the B-ring as the D-ring ( Figure 3A ) .", "The merit of this approach was also supported by prior medicinal chemistry , which had shown that discrete bisaryl-thiohydantoin compounds bearing similar D-rings , were effective antagonists of AR WT ( Jung et al . , 2010 ) .", "In this regard , we were optimistic that , minimally , the larger inhibitors we designed should be tolerated within the ligand-binding pocket .", "Consistent with this precedent , the DR100-103 series inhibited the transcriptional activity of AR WT in the EGFP reporter assay ( Figure 4A , Figure 4—figure supplement 1A , B ) .", "Whereas DR100-102 behaved as strong agonists for AR F876L ( Figure 4—figure supplement 1A ) , ( ± ) -DR103 potently inhibited the mutant receptor and antagonized DHT induction ( Figure 4—figure supplements 1 and 2 ) .", "We also found that ( ± ) -DR103 was a more potent inhibitor of AR F876L than AR WT ( Figure 4—figure supplement 3 ) , a phenomenon we are currently working to understand .", "This striking structure–activity relationship prompted us to empirically investigate the significance of the position of the gem-dimethyl group on the D-ring of ( ± ) -DR103 .", "Remarkably , a compound with the gem-dimethyl group on position 4 ( rather than 3/5 ) of the D-ring ( DR104 ) was a modest agonist of AR F876L ( albeit an antagonist of AR WT ) .", "Moreover , a compound with gem-dimethyl groups at the 3 and 5 positions of the D-ring ( DR105 ) inhibited AR F876L ( and AR WT ) ( Figure 4—figure supplement 1C ) , further underscoring the biological importance of the steric interactions brought about by these moieties in the context of the mutant receptor .", "Encouragingly , transplanting the D-ring from ( ± ) -DR103 onto the ARN-509 scaffold ( [±]-DR106 ) also resulted in AR F876L inhibition ( Figure 4—figure supplement 1B ) .", "We interpret this result to be supportive of the model advanced by the previous MD simulations , as the F876L substitution appeared to impact the ability of enzalutamide and ARN-509 to induce H12 conformational choices in a roughly equivalent manner .", "Finally , to underscore the importance of the steric interactions conferred by the D-ring , we synthesized DR107 , a compound built on the enzalutamide scaffold bearing only hydrogen atoms at position 4 on the B-ring .", "This molecule was an agonist both for AR WT and AR F876L ( Figure 4—figure supplement 1C ) , pointing directly to the pharmacological significance of interactions between H12 and the substituent at the position 4 of the B-ring .", "In line with this pharmacology , ( ± ) -DR103 inhibited the growth of prostate cancer cell lines expressing both the WT and mutant receptor ( Figure 4B , Figure 4—figure supplement 4 ) .", "( ± ) -DR103 also inhibited endogenous AR signaling and induced PARP cleavage ( Figure 4—figure supplement 5 ) .", "DR101 , a close structural analogue that behaved as an agonist for AR F876L , did not inhibit cell growth at equivalent doses ( Figure 4B ) .", "Finally , the dose of ( ± ) -DR103 required to observe antiproliferative effects ( 10 μM ) did not impact the growth of DU145 ( an AR-null human prostate cancer cell line ) , supporting the specificity of the antiandrogen ( Figure 4—figure supplement 6 ) .", "Structural modeling studies for ( ± ) -DR103 reinforced our pharmacological model for AR antagonism by bisaryl-thiohydantoins .", "Unlike the results for enzalutamide and ARN-509 , MD simulations using ( S ) -DR103 suggested that an agonist-like conformation of H12 cannot be achieved for either WT or mutant AR ( Figure 4C , Figure 4—figure supplement 7 ) .", "The modeling study instead showed that the D-ring on ( S ) -DR103 was capable of directly displacing the N-terminal residues of H12 .", "A magnified view of the H12 pocket ( Figure 4—figure supplement 8 ) shows that ( S ) -DR103 occupies a region in the H12 pocket that neither enzalutamide nor ARN-509 can access , thus imposing antagonist-like dislocation of helix 12 .", "Whereas in the H11 pocket ( Figure 4—figure supplement 8 ) , ( S ) -DR103 did not show a significant difference in binding with residue L876 compared to either enzalutamide or ARN-509 , which suggests that the restored antagonism was not achieved by simply regaining interactions at the mutation site in AR .", "Similar MD simulations for the complex of AR F876L and ( R ) -DR103 showed a slightly less pronounced H12 dislocation ( Figure 4—figure supplement 7 ) , suggesting that the two enantiomers might cause different levels of antagonism ." ], [ "As is evident from previous work with ABL kinase inhibitors for chronic myeloid leukemia and antivirals for HIV and hepatitis ( Shah et al . , 2004; Glickman and Sawyers , 2012 ) , understanding mechanisms of drug resistance is a crucial first step in developing strategies to prevent or overcome it .", "With its recent approval , the case for defining mechanisms that overcome enzalutamide therapy is timely and compelling .", "Because AR mutations are a cause of clinical resistance to antiandrogens ( flutamide and bicalutamide ) ( Veldscholte et al . , 1990; Haapala et al . , 2001 ) , and previous work has shown that clinically relevant mutations can be discovered from screening platforms in preclinical models , we prospectively searched for such mutations in the context of enzalutamide using a novel saturation mutagenesis approach .", "This screen revealed that mutation of Phe 876 to Leu converts enzalutamide and ARN-509 into AR agonists and confers resistance to drug-induced growth inhibition in vitro and in vivo .", "Importantly , this mutation was also recovered ‘spontaneously’ from enzalutamide-sensitive cell line and xenograft models treated with prolonged enzalutamide therapy .", "That prostate cancer can spontaneously acquire gain-of-function mutations in AR ( rather than acquiring mutations that simply preclude inhibitor binding ) underscores the special challenge in pharmacologically overcoming this mechanism of resistance .", "By borrowing insight from studies of the progesterone receptor , showing that a single amino acid can determine sensitivity to RU486 , and structural analyses of the estrogen receptor ( Benhamou et al . , 1992; Shiau et al . , 1998 ) , our attention was immediately directed to establishing and testing a structural model of the AR/enzalutamide complex to explain enzalutamide's curious pharmacology in the context of AR F876L .", "Using MD simulations , a novel binding mode for the drug was identified , which provided a compelling explanation for how antagonism is retained against the bicalutamide-resistant Trp 741 mutation .", "More importantly , the MD simulations argued that an altered spatial orientation of enzalutamide within the AR LBD might explain the onset of agonism , as the F876L mutation appeared to reposition the drug to eliminate steric clashes that promoted H12 dislocation in AR WT .", "Reassuringly , several larger compounds that the MD simulations predicted could restore H12 dislocation ( the ‘D-ring’ series ) effectively antagonized AR F876L .", "Because the discovery of this mutation and its companion pharmacology provided the basis for our structural model , it is difficult to envision how the importance of the D-ring might have otherwise emerged from previous ( and ongoing ) chemical screening efforts .", "Among the ∼100 bisaryl-thiohydantoins published to date , several compounds bearing structurally similar moieties to our bioactive series were essentially indistinguishable from enzalutamide , ARN-509 , or other leading agents in conventional cell-based assays .", "Our own focused chemical screen further speaks to the unusually complicated pharmacobiology of AR F876L , as subtle changes in the position of geminal dimethyl moieties on DR103-5 radically impacted the respective bioactivity of the drugs .", "Our success predicting the pharmacology of candidate inhibitors with MD simulations argues for a novel workflow by which in silico screening could guide future antiandrogen drug discovery ( pending a cocrystal structure of an AR/antagonist complex ) .", "The data indicating that ∼50% of patients fail to respond to enzalutamide has somewhat overshadowed the importance of the discovery of the bisaryl-thiohydantoin chemotype for AR , and the ongoing enthusiasm for developing better drugs based on this motif is most visibly reflected by the clinical trial with ARN-509 .", "In this regard , our structural model provides a powerful tool to further refine the chemotype into drug candidates with improved properties .", "Collectively , these findings demonstrate the importance of coordinated mutagenesis , structural modeling , and medicinal chemistry studies in designing drugs against an important cancer target for which appropriate drug affinity and binding conformation are mutually indispensible for competent inhibition .", "We are optimistic that discovery of the AR F876L mutation will facilitate solution of the enzalutamide/AR complex by X-ray crystallography .", "As for the potential clinical impact of a priori discovery of drug–resistance mutations to novel cancer drugs , our previous experience with the ABL kinase inhibitor dasatinib in chronic myeloid leukemia serves as an example .", "Within 2 years of reporting dasatinib-resistant mutations in BCR-ABL in a preclinical model , analogous mutations were recovered from dasatinib-resistant chronic myeloid leukemia patients ( Burgess et al . , 2005; Shah et al . , 2007 ) .", "We hope that this report will guide a similar search for AR mutations in prostate cancer patients who develop clinical resistance to enzalutamide .", "Routine rebiopsy of tumor tissue in men with castration-resistant prostate cancer is challenging due to the high frequency of osteoblastic bone lesions consisting primarily of stromal tissue .", "Blood-based assays for AR mutation detection may be a compelling alternative , based on recent success in detection of tumor-specific mutations in circulating plasma DNA from patients with other cancers ( Diehl et al . , 2008; Leary et al . , 2012 ) ." ], [ "Fetal bovine serum ( FBS ) and charcoal-stripped dextran-treated fetal bovine serum ( CSS ) were purchased from Omega Scientific ( Tarzana , CA ) .", "Bicalutamide and hydroxyflutamide ( LKT Labs , St . Paul , MN ) , DHT ( Sigma , St . Louis , MO ) , and R1881 ( Perkin Elmer , Waltham , MA ) were commercially obtained; all other ligands were synthesized at MSKCC .", "Serial dilutions of all drugs were made using DMSO .", "Antibodies used for immunoblot assays were β-actin ( AC-15; Sigma ) PARP ( #9541; Cell Signaling Technology , Danvers , MA ) , FKBP5 ( IHC-00289; Bethyl , Montgomery , TX ) , β-tubulin ( D-10 ) , and androgen receptor ( N-20 ) ( both from Santa Cruz Biotechnology , Dallas , TX ) .", "Protein lysates were prepared in M-PER protein extraction reagent ( Pierce , Rockford , IL ) .", "The chromatin immunoprecipitation assay was conducted using a kit ( Upstate , Billerica , MA ) .", "Nontarget and human AR siRNA pools were from the ON-TARGETplus collection ( Dharmacon , Waltham , MA ) .", "LNCaP/AR cells were previously described ( Tran et al . , 2009 ) , and CWR22Pc cells ( Dagvadorj et al . , 2008 ) were provided by Marja T Nevalainen ( Thomas Jefferson University , Philadelphia , PA , USA ) .", "All other cell lines were obtained from ATCC ( Manassas , VA ) .", "All LNCaP and CWR22Pc derived cells were maintained in RPMI + 10% FBS .", "All CV1 and VCaP derived cell lines were maintained in DMEM + 10% FBS .", "All oligos were ordered from Operon Biotechnologies , Huntsville , AL .", "For the analogue syntheses , all chemicals were acquired from Sigma-Aldrich at highest purity available and were used without further purification .", "Chromatography was done using Merck grade silica gel 60 , and reactions were monitored by LC-MS ( Waters Autopure and Acquity systems in reverse phase and with mass , evaporative light scattering , and diode array detections ) .", "Proton NMR experiments were executed on Bruker Advance DRX running at 500 MHz , and fluorine NMR was run on the same machine but at 235 MHz .", "Chemical shifts are reported in parts per million relative to tetramethylsilane .", "The human AR cDNA plasmid , pWZL-AR , was provided by William Hahn ( Dana-Farber Cancer Institute , Boston , MA , USA ) .", "All mutant AR constructs were generated in pWZL-AR with the QuikChange II XL site-directed mutagenesis kit ( Agilent , Santa Clara , CA ) and primers designed using Agilent's online QuikChange Primer Design tool .", "Stable cell lines were generated by pantropic retroviral infection ( Clontech , Mountain View , CA ) and selected with blasticidin ( Invivogen , San Diego , CA ) .", "LNCaP cells were infected with the lentiviral AR-regulated EGFP reporter construct , Pb . PSE . EGFP ( Chapel-Fernandes et al . , 2006 ) , provided by Claude Bignon ( EFS Alpes Méditerranée , Marseilles , France ) .", "We then single-cell cloned the LNCaP-Pb . PSE . EGFP cells to reduce the heterogeneity in EGFP expression , and isolated a clone that had a high level of EGFP expression , which was modulated effectively by antiandrogens and AR agonists .", "This clone was used for all flow cytometry assays and for the FACS-based resistance screens .", "LNCaP-Pb . PSE . EGFP cells for flow cytometric analysis were treated with antiandrogens ( 1 or 10 µM ) for 4–6 days , changing media and drug every 2–3 days .", "Cells were collected using Accumax dissociation solution ( Innovative Cell Technologies , San Diego , CA ) , and dead cells were counterstained using TO-PRO3-Iodide ( Invitrogen , Grand Island , NY ) .", "EGFP expression was measured using the BD-FACSCalibur flow cytometer using the 488-nm laser and 530/30 bandpass filter to detect EGFP expression , and the 633-nm laser and 661/16 bandpass filter to detect TO-PRO3-Iodide labeled dead cells .", "For each sample , 2–5 × 104 cell events were collected and analysis was done using FlowJo software .", "FACS-sorting of LNCaP-Pb . PSE . EGFP cells was performed on a BD FACSVantage cell sorter .", "Dead cells were counterstained with DAPI ( Invitrogen ) .", "EGFP expression was detected using the 488-nm laser and 530/30 bandpass filter , and DAPI-labeled dead cells were detected using the 355-nm laser and 450/50 bandpass filter .", "We introduced four additional synonymous mutations into our pWZL-AR W741C construct to aid in distinguishing wild-type ( WT ) AR and AR W741C , using the QuikChange Multi Site-Directed Mutagenesis Kit ( Agilent ) .", "We then designed and optimized quantitative PCR primers across these mutation sites , so that they specifically amplified AR W741C .", "We overexpressed AR WT or AR W741C in our LNCaP-Pb . PSE . EGFP reporter cells , mixed different ratios of cells expressing either WT or W471C , treated these cells with 1 µM bicalutamide for 4 days , and FACS-sorted the cells that maintained/induced EGFP expression .", "Gates for EGFP positivity were set using WT or W741C expressing cells treated with bicalutamide .", "Sorted cells were expanded in culture ( without drug ) until they reached approximately 60 million cells , we then isolated gDNA and froze down a small fraction , and the brief bicalutamide treatment and sorting was repeated on the remainder .", "Our randomly mutagenized AR cDNA library was generated as follows: we transformed the DNA-repair-deficient Escherichia coli strain XL-1 Red ( Agilent ) with the pWZL-AR plasmid and plated them on ampicillin-agar bacterial plates .", "After a 36-hr incubation , colonies were collected by scraping , and plasmid DNA was purified using a plasmid MAXI kit ( Qiagen , Germantown , MD ) .", "This mutagenized AR plasmid stock was used to make pantropic retrovirus ( Clontech ) and infect LNCaP-Pb . PSE . EGFP cells at a MOI < 1 .", "Cells were selected for stable expression of our mutant pWZL-AR library using the blasticidin resistance cassette .", "Mutant library cells were cultured in 1 µM enzalutamide for 4–6 days , collected with Accumax and resuspended in Accumax containing 0 . 5% BSA and 10 mM HEPES .", "Cells that remained EGFP positive in the presence of enzalutamide were then FACS-sorted .", "Gates for EGFP positivity were set using LNCaP-Pb . PSE . EGFP cells transduced with the wild-type AR cDNA , treated with vehicle or 1 µM enzalutamide .", "Sorted cells were expanded in culture ( without drug ) until they reached approximately 60 million cells , we then isolated gDNA and froze down a small fraction , and the brief enzalutamide treatment and sorting was repeated on the remainder .", "We performed the screen in triplicate , with five rounds of FACS and expansion for each replicate .", "Exons 2 through 8 of the exogenously expressed AR cDNA were amplified from genomic DNA isolated from cells after each sort , by high-fidelity PCR ( Qiagen , Hotstar ) on a Mastercycler ( Eppendorf ) .", "The PCR product was subjected to bidirectional Sanger sequencing , using previously published primers ( Watson et al . , 2010 ) .", "Alignments were performed using SeqMan Pro ( DNASTAR ) , and Sanger traces were analyzed using 4Peaks software .", "Total RNA was isolated using the QiaShredder kit ( Qiagen ) for cell lysis and the RNeasy kit ( Qiagen ) for RNA purification .", "We used the High Capacity cDNA Reverse Transcription Kit ( Applied Biosystems , Grand Island , NY ) to synthesize cDNA according to the manufacturer's protocol .", "Quantitative PCR was done in the Realplex MasterCycler ( Eppendorf ) using the Power SYBR Green PCR Mastermix ( Applied Biosystems ) .", "Quantitative PCR for each sample was run in triplicate and each reaction contained 1 μl of cDNA in a total volume of 20 μl .", "PCR quantification was done using the 2-ΔΔCt method with normalization to GAPDH as described ( Applied Biosystems ) .", "All primers were used at a final concentration of 500 nM and are listed 5′ to 3′: GAPDH-Forward: GAAGGTGAAGGTCGGAGTC; GAPDH-Reverse: GAAGATGGTGATGGGATTTC; PSA-Forward: GGTGACCAAGTTCATGCTGTG; PSA-Reverse: GTGTCCTTGATCCACTTCCG; Tmprss2-Forward: CACTGTGCATCACCTTGACC; Tmprss2-Reverse: ACACGCCATCACACCAGTTA; Fkbp5-Forward: TCCCTCGAATGCAACTCTCT; Fkbp5-Reverse: GCCACATCTCTGCAGTCAAA; SGK1-Forward: GCAGAAGGACAGGACAAAGC; SGK1-Reverse: CAGGCTCTTCGGTAAACTCG .", "LNCaP cells ( 107 cells/condition ) were grown in phenol red free RPMI media supplemented with 10% CSS for 4 days , then treated with DMSO , 10 µM antiandrogens , or 1 nM DHT for 4 hr .", "The cells were cross-linked using 1% paraformaldehyde ( Electron Microscopy Sciences , Hatfield , PA ) for 15 min , glycine was then added , and samples centrifuged ( 4°C , 2500 rpm , 5 min ) to stop further cross-linking .", "ChIP was performed according to manufacturer's protocols using a ChIP assay kit ( Upstate ) with an antibody for AR ( PG-21; Upstate ) .", "Immunoprecipitated DNA was amplified by quantitative real-time PCR ( ABI Power SYBR Green PCR mix ) .", "All primers were used at 500 nM and are listed 5′ to 3′: PSA enhancer-Forward: ATGTTCACATTAGTACACCTTGCC; PSA enhancer-Reverse: TCTCAGATCCAGGCTTGCTTACTGTC; FKBP5 enhancer-Forward: CCCCCTATTTTAATCGGAGTAC; FKBP5 enhancer-Reverse: TTTTGAAGAGCACAGAACACCT .", "LNCaP cells ( 106 cells/well of six-well plate ) were transfected with 2 μg AR-EYFP plasmid ( from Jeremy Jones and Marc Diamond , UCSF ) or AR . F876L-EYFP plasmid ( QuikChange II XL site-directed mutagenesis kit ) using FUGENE HD ( Roche , Indianapolis , IN ) .", "6 hr after transfection , media was removed and replaced with phenol red-free RPMI media supplemented with 10% CSS .", "The next day cells were split and plated onto poly-lysine-coated Nunc Labtek chamber slides in RPMI + 10% CSS containing DMSO , 1 µM antiandrogens , or 1 nM DHT .", "24 hr later , the cells were counterstained with NucBlue Live Cell Stain Hoechst 33342 ( Molecular Probes , Grand Island , NY ) fixed with 4% paraformaldehyde , and mounted with a coverslip .", "Images were taken on a Leica TCS SP5-II Upright confocal microscope ( MSKCC Microscopy Core and were analyzed for EYFP [AR] nuclear/cytoplasmic localization using ImageJ ) .", "CV1 cells ( 2 × 106 cells/10 cm plate ) were cotransfected with 50 ng of SV40 Renilla Luciferase , 5 μg of ARE ( 4X ) -Luciferase , and 10 μg of one pWZL-AR expression construct using Lipofectamine 2000 ( Invitrogen ) .", "Transfection media was removed 4–6 hr later and replaced with phenol red-free DMEM containing 10% CSS .", "The following day each plate was split into 48-well plates , in 10% CSS media , containing the indicated drugs in triplicate .", "Luciferase activity was assayed 24–48 hr later using Dual-Luciferase Reporter Assay System ( Promega , Madison , WI ) .", "The binding affinity of enzalutamide to AR WT and AR F876L , relative to dihydro-testosterone ( DHT ) , was determined using a competition assay in which increasing concentrations of cold competitor are added to cells preincubated with 18F-FDHT .", "LNCaP/AR WT or LNCaP/AR F876L cells were cultured in phenol red-free RPMI + 10% CSS for 2 days prior to the binding assay .", "Cells were trypsinized , washed in PBS , and mixed with 20 , 000 cpm 18F-FDHT and increasing amounts of cold competitor ( 10 pM–10 µM ) , in triplicate .", "The solutions were shaken on an orbital shaker at ambient temperature for 1 hr , then isolated , and washed with ice-cold tris-buffered saline using a Brandel cell harvester ( Gaithersburg , MD , USA ) .", "Samples were counted using a scintillation counter , and the specific uptake of 18F-FDHT was determined .", "These data were plotted against the concentration of the cold competitor to give sigmoidal displacement curves , and IC50 values were determined using a one-site model and a least squares curve fitting routine ( Origin; OriginLab , Northampton , MA , USA ) with the R2 of the curve fit being >0 . 99 .", "In vivo xenograft experiments were done by subcutaneous injection of 2 × 106 LNCaP/AR cells ectopically expressing AR WT or AR F876L ( 100 μl in 50% Matrigel [BD Biosciences , San Jose , CA] and 50% growth media ) into the flanks of castrated male SCID mice .", "Daily gavage treatment ( using a formulation of 1% carboxymethyl cellulose , 0 . 1% Tween-80 , 5% DMSO ) was initiated on the day of injection .", "Once tumors were palpable , tumor size was measured weekly in three dimensions ( l × w × d ) with calipers .", "All animal experiments were performed in compliance with the guidelines of the Research Animal Resource Center of the Memorial Sloan-Kettering Cancer Center .", "Xenograft experiments in which AR F876 mutations emerged after long-term treatment with second-generation antiandrogens were performed as follows: 2 × 106 LNCaP/AR cells ( Tran et al . , 2009 ) were injected subcutaneously into the flanks of castrated SCID mice .", "Treatment with 30 mg/kg enzalutamide or ARN-509 was initiated once tumors reached ∼300 mm3 , resulting in rapid tumor regression .", "After several months of continual dosing , these tumors regain the ability to grow .", "Once these ‘resistant’ tumors reached their original volume , the mice were sacrificed , and tumors collected for analysis .", "CWR22Pc cells were cultured in RPMI + 10%FBS containing 0 . 1 nM DHT and either 10 µM enzalutamide or ARN-509 .", "Treatment media was replaced every 4–5 days , and cells were passaged upon reaching confluence .", "Cell strains were designated as antiandrogen resistant when the time between consecutive passages was reduced to 4–6 days , which is a period of time equivalent to that of untreated CWR22Pc .", "Genomic DNA ( gDNA ) was isolated ( PureGene Core Kit A; Qiagen ) from resistant CWR22Pc cell lines or LNCaP/AR xenograft tumors .", "With 20 ng of gDNA as template , exon 8 of AR was PCR amplified with Kapa HiFi Ready Mix ( Kapa Biosystems , Woburn , MA ) .", "RNA was extracted from LNCaP/AR xenograft tumors , reverse transcribed ( High Capacity cDNA Reverse Transcription Kit; Applied Biosystems ) , and exons 2 through 8 of AR was PCR amplified using 200 ng cDNA as template ( Qiagen , HotStar ) .", "PCR reactions were cleaned up with AMPure XP ( Beckman Coulter Genomics ) , and pooled reaction yields were quantified using the Qubit fluorometer ( Invitrogen ) .", "Library preparation was done using Nextera DNA Sample Preparation kit ( Illumina ) and run on the Illumina MiSeq sequencer using the 2 × 250 paired-end cycle protocol .", "Genomic DNA was aligned to the hg19 build of the human genome using BWA ( Li and Durbin , 2009 ) with duplicate removal using samtools ( Li et al . , 2009 ) as implemented by Illumina MiSeq Reporter .", "cDNA FASTQ files were processed with a windowed adaptive trimming tool sickle ( https://github . com/najoshi/sickle ) using a quality threshold of 32 .", "The reads were then mapped to the human genome build hg19 with TopHat 2 ( Trapnell et al . , 2009 ) using known AR transcripts NM_000044 and NM_001011645 .", "Duplicates were then removed with Picard ( http://picard . sourceforge . net ) .", "Variant detection was performed using VarScan 2 ( Koboldt et al . , 2012 ) with thresholds of a minimum of 10 supporting variant reads and variant allele frequencies of at least 1% .", "No structures have been solved experimentally for enzalutamide or ARN-509 in complex with AR ( agonist or antagonist conformation ) .", "Therefore , three-dimensional structures of antiandrogens were first built using the computer program Gaussview ( version 4 . 1 . 2; part of the computer program Gaussian 03 ) ( Frisch et al . , 2004 ) and then geometrically optimized in a quantum mechanical force field at the level of restricted Hartree-Fock ( RHF ) 6-31g* using the program Gaussian 03 .", "The partial atomic charges were derived from the optimized structures by Restrained ElectroStatic Potential ( Bayly et al . , 1993; Cornell et al . , 1993 ) ( RESP ) fitting to the RHF/6-31g* potentials .", "The other parameters modeling the antiandrogens were taken from the CHARMm22 ( Momany and Rone , 1992 ) force field after assigning CHARMm22 atom types to antiandrogens with an in-house program .", "The initial AR–antiandrogen complex structures were then modeled with the molecular modeling program CHARMM ( Brooks et al . , 1983 , 2009 ) .", "Starting with the atomic coordinates of AR WT and the A-ring of S1 in the template crystal structure ( PDB accession code , 2AXA ) , the side chain of residue F876 was replaced with CHARMm22-parameterized side chain of a leucine when needed and the CH group on the A-ring was replaced with a nitrogen in cases of ARN-509 .", "The rest of each antiandrogen was ‘grown’ from the A-ring using the ideal unbound structures solved by geometry optimization .", "Missing side chain atoms were built using standard CHARMm22 parameters and hydrogen atoms were added with the HBUILD ( Brunger and Karplus , 1998 ) module of CHARMM .", "All these newly introduced atoms without three-dimensional crystal coordinates treated flexible and the rest under harmonic constraints with the force constant of 100 kcal/mol/Å2 , each AR–antiandrogen complex structure was energetically minimized with one round of 100-step steepest decent followed by two rounds of 100-step Adopted-Basis Newton–Raphson ( ABNR ) energy minimization .", "Harmonic constraints were reset at the beginning of each round of minimization .", "No nonbonded cutoff was used .", "Solvent effects were implicitly modeled in this stage with a distance-dependent dielectric constant .", "The all-atom MD simulations were performed with explicit solvent atoms using the program CHARMM ( version 36a1 ) .", "Each initial AR–antiandrogen model was first centered and overlaid with a rhombic dodecahedron-shaped water box ( edge length being 88 Å ) of approximately 47 , 000 equilibrated water molecules .", "Any water molecule whose oxygen atom was within 2 . 8 Å away from any non-hydrogen atom of AR or antiandrogen was removed .", "Proper amount of sodium and chloride ions were automatically added to achieve overall charge neutrality and physiological level of ion concentration ( 0 . 145 M ) .", "Their positions were optimized with 10 independent trajectories of randomly replacing water molecules and performing 50 steps of steepest decent and 125 steps of ABNR energy minimization .", "The molecular system including AR , antiandrogen , water , and ions was heated to 300 K and equilibrated with two rounds of 0 . 1-ns MD simulations under successively weaker harmonic constraints on AR or antiandrogen atoms .", "After the MD equilibration , three sets of random velocities were assigned to initiate three independent 10-ns MD productions .", "The MD equilibration and production were performed using the crystal form of rhombic dodecahedron ( RHDO ) and the canonical ensemble ( NVT ) .", "A nonbonded cutoff of 14 Å , periodic boundary conditions in conjunction with Ewald summation method , the leapfrog Verlet integrator , and the Hoover thermostat for pressure and temperature were used .", "The timestep was set at 2 fs .", "Parallel jobs for MD simulations were run on a computer cluster of Intel Xeon X5650 series ( 2 . 66 GHz and 4 GB memory per CPU ) .", "Structural models were visualized in a molecular graphics program , UCSF Chimera ( Pettersen et al . , 2004 ) .", "The default option used when aligning structures ." ] ]
[ "The second-generation antiandrogen enzalutamide was recently approved for patients with castration-resistant prostate cancer .", "Despite its success , the duration of response is often limited .", "For previous antiandrogens , one mechanism of resistance is mutation of the androgen receptor ( AR ) .", "To prospectively identify AR mutations that might confer resistance to enzalutamide , we performed a reporter-based mutagenesis screen and identified a novel mutation , F876L , which converted enzalutamide into an AR agonist .", "Ectopic expression of AR F876L rescued the growth inhibition of enzalutamide treatment .", "Molecular dynamics simulations performed on antiandrogen–AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism through repositioning of the coactivator recruiting helix 12 .", "This model then provided the rationale for a focused chemical screen which , based on existing antiandrogen scaffolds , identified three novel compounds that effectively antagonized AR F876L ( and AR WT ) to suppress the growth of prostate cancer cells resistant to enzalutamide ." ]
[ "Prostate cancer is the most commonly diagnosed cancer in men , and the second most lethal .", "All stages of prostate cancer depend upon male sex hormones , also known as androgens , to grow because these hormones bind and activate androgen receptors .", "A class of drugs termed ‘antiandrogens’ can effectively treat prostate cancer because they bind to androgen receptors without activating them , thereby preventing androgens from binding .", "However , the efficacy of even highly potent antiandrogen drugs , such as enzalutamide is short-lived in many patients , and understanding the biological mechanisms that cause drug resistance is one of the major objectives in translational prostate cancer research .", "Resistance can arise through mutations of the androgen receptor that result in the receptor being activated , rather than inhibited , by antiandrogen drugs .", "However , no such mutations are known yet for enzalutamide , and researchers are keen to understand whether they exist and , if so , to generate new drugs for prostate cancer that overcome them .", "To identify mutations that may lead to resistance , Balbas et al . designed a new screening method in human prostate cancer cells and showed that androgen receptors with a specific mutation ( called F876L ) can be activated by enzalutamide .", "More comprehensive biological studies showed that prostate cancer cells harboring the mutation continued to grow when treated with the drug .", "Balbas et al . also showed that this mutation can arise spontaneously in human prostate cancer cells treated long term with enzalutamide .", "Balbas et al . reasoned that the mutation likely altered the way enzalutamide binds to the androgen receptor , and used computer-guided structural modeling of the complex formed by the receptor and the drug to investigate how this might occur .", "These studies indicated that the region of the androgen receptor containing the F876L mutation comes into direct contact with the drug , and provided a structural explanation for the loss of inhibition .", "Because these studies showed how enzalutamide might bind to the androgen receptor , they also suggested ways in which enzalutamide could be chemically modified to restore its inhibitory activity against the mutant receptor .", "Balbas et al . then designed and synthesized a set of novel compounds , which the modeling data suggested could act as inhibitors of the mutant receptor .", "Several of these compounds inhibited the activity of both mutant and wild-type forms of the androgen receptor , and suppressed the growth of both enzalutamide-resistant and nonresistant prostate cancer cells .", "The work of Balbas et al . outlines a general screening strategy for the discovery of clinically relevant mutations in cancer genes , and shows how in silico technologies can accelerate drug discovery in the absence of a crystal structure of a protein–drug complex .", "It also emphasizes how understanding the manner in which a drug binds its target can stimulate rational design of improved drug candidates ." ]
2013
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "chromosomes and gene expression", "computational and systems biology" ]
NF-κB oscillations translate into functionally related patterns of gene expression
elife-09100-v2
[ [ "Genetic circuits are instrumental for cells to provide adequate transcriptional responses to different external and internal stimuli , but we are still far from a complete understanding of how they work .", "The growing availability of single-cell measurements is bringing unprecedented insights into the dynamics of transcriptional responses: although it was traditionally thought that gene circuits should provide a temporally stable response to constant external stimuli , there is increasing evidence that the dynamics of gene circuits is more elaborate , and is often characterized by pulses of activity ( Levine et al . , 2013 ) .", "A special case is oscillatory dynamics , in which the activity of some element in the genetic circuit varies periodically , and so does the output of the circuit .", "Paradigmatic oscillatory dynamics are associated with circadian clocks , which are present in a wide variety of organisms that synchronize their activities to the periodic variation in external daylight ( Bell-Pedersen et al . , 2005 ) .", "However , oscillatory dynamics with periods far shorter than 24 hr have been reported in genetic circuits of bacteria ( Suel et al . , 2007 ) , yeast ( Cai et al . , 2008; Hao and O'Shea , 2012 ) and mammalian cells ( Geva-Zatorsky et al . , 2006; Hoffmann et al . , 2002; Larson et al . , 2013; Nelson et al . , 2004; Shankaran et al . , 2009 ) .", "The dynamics of transcription factors in gene circuits can play a key role in information transmission through biochemical networks ( Selimkhanov et al . , 2014 ) by selectively modulating the expression of different genes ( Purvis and Lahav , 2013 ) .", "A paradigmatic example is how different p53 dynamics can selectively activate transcriptional programs that commit the cell to different cell fate decisions ( Purvis et al . , 2012 ) .", "A property of free oscillating systems ( or free oscillators , as analogy to simple mechanical oscillators such as the pendulum ) is entrainment , by which an oscillator can gradually modulate its phase and frequency thanks to a small perturbation ( forcing ) that is itself periodic and oscillating with a period resonant with the intrinsic period of the oscillator ( Pikovsky et al . , 2003 ) .", "A common forcing can also lead to the synchronous dynamics of multiple oscillators and thus to collective dynamical states .", "Entrainment was successfully reproduced in synthetic genetic oscillators , in which single bacterial cells ( each expressing a fluorescent reporter protein in response to a synthetic genetic circuit ) were entrained collectively to an oscillating provision of arabinose ( Mondragon-Palomino et al . , 2011 ) .", "The forcing to one oscillator can also be provided by other oscillators , and this coupling can lead to the emergence of different collective dynamical states characterized by different synchronous dynamics ( Pikovsky et al . , 2003 ) .", "Inter-cellular coupling has indeed been exploited to genetically engineer synchronous quorum sensing genetic oscillators in bacteria ( Danino et al . , 2010 ) .", "On the other hand , intra-cellular coupling leads to locked oscillatory states for different cell oscillators , as recently shown for the circadian rhythm and the cell cycle ( Bieler et al . , 2014 ) .", "In mammalian cells , NF-κB is a typical transcription factor that displays intrinsic oscillatory behavior .", "NF-κB plays key roles in inflammation , immune responses , development and cancer ( Chaturvedi et al . , 2011; Ghosh and Hayden , 2008; Karin , 2006; Ledoux and Perkins , 2014; Naugler and Karin , 2008 ) .", "Strictly speaking , NF-κB is a family of dimers encoded by 5 different genes , but in what follows we will refer to p65/RELA independently of the dimer it forms .", "In resting cells , p65 exists mostly within a cytoplasmic complex bound to the IκB inhibitors ( Hoffmann et al . , 2002 ) .", "Inflammatory signals like tumor necrosis factor alpha ( TNF-α ) or lipopolysaccharide ( LPS ) induce phosphorylation of IκB proteins by IKKs–upstream kinases in the signaling pathway– , ubiquitination and degradation of IκBs , and the release of active NF-κB that translocates into the nucleus to activate the expression of several genes , including those encoding for the IκB inhibitors ( Hoffmann et al . , 2002 ) .", "Re-expression of IκBs contributes to relocate NF-κB in the cytoplasm , which is an inhibitory feedback loop for the system .", "A second feedback loop is centred on the protein A20 ( Ashall et al . , 2009 ) , which upon activation of the system inhibits the IKKs .", "Thus , these two layers provide a nonredundant regulation of NF-κB response to external stimuli ( Werner et al . , 2008 ) .", "Of note , the IκB inhibitors also show variable sensitivity to different stimuli and respond on different timescales to fine-tune the signaling pathway ( Paszek et al . , 2010; Shih et al . , 2009 ) .", "This system of negative feedback loops ( summarized in Figure 1A upper panel ) provides both a tight control of the response to external stimuli and flexibility .", "As a consequence of this complex wiring , upon constant stimulation the nuclear concentration of NF-κB in each cell oscillates with heterogeneous dynamics according to each cell’s susceptibility and to the inherent stochasticity of the system ( Nelson et al . , 2004; Tay et al . , 2010; Zambrano et al . , 2014a ) .", "Due to such asynchrony at the single cell level , the NF-κB response appears almost non-oscillating at the cell population level ( Hoffmann et al . , 2002 ) , and it is difficult to correlate NF-κB oscillatory profiles with gene expression outputs . 10 . 7554/eLife . 09100 . 003Figure 1 . Periodic forcing turns damped heterogeneous oscillation into synchronous sustained oscillations .", "( A ) The activity of NF-κB is regulated through different negative feedbacks provided by the inhibitors IκB and A20 .", "The scheme at the bottom represents a generic forcing with periodically alternating TNF-α doses D1 and D2 of duration T2+T1 = Tf; Tf is the period of the forcing .", "( B , C )", "Oscillations observed in three GFP-p65 cells obtained by computing the nuclear to the cytoplasmic GFP intensity ( NCI ) for constant flow of 10 ng/ml TNF-α ( B ) and upon alternating doses D1=10 ng/ml TNF-α , D2=0 ng/ml and T1=T2 =45 min ( C ) .", "Each colour corresponds to a single cell trace .", "Oscillatory patterns can be effectively visualised using the phase ϕ of the oscillation , which is 2π in the maxima of the oscillatory peaks ( yellow ) and π in the local minima ( green ) in the colour phase-plot of ϕ ( t ) below the panels .", "Scale-bar for ϕ is on the right .", "( D ) Time lapse images of cells under constant stimulation displaying the characteristic heterogeneous nuclear-to-cytoplasmic translocations .", "( E ) Phase plot drawn for 50 cells , of 105 analysed , showing the asynchrony of the oscillations except for the first peak .", "( F ) Distribution of the experimentally computed period of the oscillations Texp , measured as the time between two consecutive oscillatory peaks .", "The distribution has a maximum at T0 =90 min , which corresponds to the natural period .", "( G ) Quantification of the height for each peak .", "( H ) Time lapse images of the cells under periodic stimulation , showing synchronous NF-κB translocations between cytoplasm and nucleus .", "( I ) Phase plot for 50 cells , of 206 analysed , showing a clear synchrony of the oscillations .", "( J ) Distribution of the period of the oscillations Texp .", "Texp corresponds almost perfectly to the period of the forcing .", "( K ) Quantification of peaks height variation as described in G; values for n>1 are slightly higher than those observed under constant stimulation .", "Figure supplements from 1 to 10 are provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00310 . 7554/eLife . 09100 . 004Figure 1—figure supplement 1 . Experimental set-up and quantification .", "( A ) GFP-p65 MEFs are plated in a microfluidics plate chamber .", "The perfusion barrier is visible .", "( B ) TNF-α flows and diffuses inside the chamber through the perfusion barrier , minimizing cells’ shear stress .", "( C ) Example of the nuclear segmentation .", "A square of the cytoplasm around the nucleus is used to compute the nuclear to cytoplasmic ratio of the intensities , an internally normalized measure .", "( D ) The cells were subjected to two pulses of TNF-α of 30 min separated by a washout of 12 hr .", "The resulting time series shows that cells responded to both pulses with similar intensity , indicating a negligible degradation of TNF-α in our system . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00410 . 7554/eLife . 09100 . 005Figure 1—figure supplement 2 . Peaks and phase calculation .", "( A ) Distribution of the noisy peaks values for unstimulated ( blue ) and stimulated cells ( red ) .", "A threshold θ=0 . 15 is enough to discriminate significant peaks from noisy peaks .", "( B ) Peaks ( blue triangles ) detected in a typical time series and ( C ) Phase of the oscillation inferred from the same time series .", "( D ) Selected NCI profiles upon automatic segmentation display a variety of dynamical phenotypes , including the previously oscillating and non oscillating cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00510 . 7554/eLife . 09100 . 006Figure 1—figure supplement 3 . Damped oscillations for constant TNF-α .", "( A ) NCI ( nucleus to cytoplasm intensity ) time series ( green ) and average ( black ) for cells under a continuous flow of 10 ng/ml TNF-α .", "Same cells as in Figure 1 .", "( B ) Distribution of peak numbers in 12 hr timespan for the cells in A: the average number of peaks ( n=4 ) indicates that there are no sustained oscillations in the 12 hr . ( C ) NCI time series ( green ) and average ( black ) for cells stimulated with repeated pulses of 45 min of 10 ng/ml TNF-α followed by a 45 min wash-out .", "( D ) The average number of peaks of these cells in 12 hr ( close to 8 ) indicates that NF-κB shows sustained oscillations under pulsed stimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00610 . 7554/eLife . 09100 . 007Figure 1—figure supplement 4 . Scheme of the simple ODE mathematical model used for NF-κB dynamics , with the feedbacks provided by IκBα and A20 . Green arrows from NF-κB indicate the contribution of NF-κB to the gene activation while red lines from IκBα represent the contribution to transcriptional repression .", "The red line from A20 represents the inhibition of IKK activation .", "This model gives rise to different dynamics for constant stimuli: damped oscillations that converge to a fixed point and sustained oscillations around an unstable fixed point as shown in Figure 1—figure supplement 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00710 . 7554/eLife . 09100 . 008Figure 1—figure supplement 5 . Numerical exploration of the mathematical model suggests that damped oscillations are predominant .", "( A ) Real and imaginary parts of each eigenvalue of the fixed points of the NF-κB dynamical system .", "Red dots are for eigenvalues of unstable fixed points , corresponding to sustained oscillations .", "( B ) The distribution of the number of imaginary eigenvalues in fixed points obtained from our set of parameter combinations .", "Most fixed points have four or more negative eigenvalues , indicating the coexistence of two or more characteristic frequencies .", "The distribution of the number of positive real parts of eigenvalues in fixed points obtained from our set of parameter combinations .", "This distribution suggests that sustained oscillations might be infrequent ( right ) .", "( C ) Mean and standard deviation of the parameter values giving rise to oscillating ( red ) and nonoscillating ( blue ) trajectories .", "The intervals are very similar except for parameters n and dA involved in the A20 negative feedback .", "( D ) Examples of the variety of trajectories found in our numerical exploration , including oscillatory trajectories ( red ) with different peaks and a variety of damped oscillating and non-oscillating dynamics ( blue ) .", "These computed trajectories are similar to those observed experimentally . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00810 . 7554/eLife . 09100 . 009Figure 1—figure supplement 6 . UV-photodamage is not detectable in imaged cells .", "The cells were imaged for 15 hr in the presence of Hoechst , fixed and immunostained for thymine dimers ( right panel , anti TDM-2 antibody , 20x obj; shown is a representative experiment of three performed ) .", "The last image of the GFP-p65 cells acquired at the end of the timelapse acquisition is reported in the middle panel .", "The green square in the Hoechst panel indicates the area enlarged in panel B . ( B ) Higher magnification of the selected area in panel A ( 63x obj ) .", "( C ) Higher magnification of the selected area in panel B ( 63x obj , zoom 5 ) .", "( D ) For comparison , GFP-p65 cells were plated in the presence of Hoechst and exposed to increasing doses of UVC and immunostained together with the cells in A . Images were acquired with constant settings in the same microscopy session .", "Due to the very low intensity , the signal has been enhanced .", "Fluorescence is almost undetectable in both non-UVC-exposed ( 0 J/m2 ) and non-imaged cells ( D and B , respectively ) .", "( E ) Higher magnification of the selected area in panel D ( 63x obj , zoom 5 ) , green square in 0 J/m2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 00910 . 7554/eLife . 09100 . 010Figure 1—figure supplement 7 . Ongoing DNA repair is not detectable in cells imaged with Hoechst staining and UV irradiation . Immunostaining for gammaH2AX , a marker indicative of active DNA damage repair , was used to assess genetic damage in unstimulated cells exposed or not to Hoechst staining and UV and/or GFP imaging for 3 hr in microfluidic plates .", "All the images were acquired during the same microscope session keeping the acquisition parameters rigorously constant .", "( A ) GFP-p65 cells treated with sub-toxic doses of doxorubicin ( 10 nM ) for 2 hrs were used as positive control to set up imaging conditions .", "Obj: 63x .", "( B ) Left panel contains the negative control: cells cultured in the microfluidic plate were not Hoechst stained , nor UV and GFP imaged .", "Middle and right panels: immunostaining of cells in microfluidic chambers that were Hoechst-stained but not imaged or both Hoechst-stained and UV-imaged , respectively .", "Obj: 63x .", "( C ) To further confirm the previous results , and to exclude phototoxicity of the 488 nm laser in GFP imaging , we show fields from a chamber that did not received Hoechst staining and imaging ( position 7 ) or was exposed to both ( position 1 ) in the same experiment ( 20x Obj ) .", "The panel on the right shows an enlargement of a small portion of position 1 ( green rectangle ) to which the adjacent area outside the imaging field and not exposed to GFP imaging has been added to exclude indirect photodamage ( Martin et al . , 2005 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01010 . 7554/eLife . 09100 . 011Figure 1—figure supplement 8 . TNF-dependent activation of apoptosis in GFP-p65 cells stimulated with increasing doses of TNF-α .", "Shown is a montage of imaging fields ( GFP channel ) extracted from representative time-lapse experiments for the indicated time-points .", "Frame number 0 , 10 , 30 , 60 , 90 and 120 correspond to 0 , 1 , 3 , 6 , 9 and 12 hr , respectively .", "Red frames focus on fields with detectable apoptotic cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01110 . 7554/eLife . 09100 . 012Figure 1—figure supplement 9 . UV and 488 laser imaging does not activate NF-κB nor produce altered NF-κB dynamics .", "( A ) NCI profiles of untreated cells imaged for 12 hr . ( B ) Correlation between NCI values and nuclear NF-κB content .", "( C ) Comparison of NCI and background-corrected nuclear intensity profiles obtained by manual segmentation of cells stimulated with 10 ng/ml TNF-α without Hoechst/UV imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01210 . 7554/eLife . 09100 . 013Figure 1—figure supplement 10 . GFP-p65 levels do not change in the cell population upon TNF-α stimulation . Quantitative immunoblot analysis was performed on cells cultured in 0 . 1% FCS plus Hoechst and stimulated with TNF-α for the indicated times .", "Lysates from cells were normalized for their DNA content ( De Toma et al . , 2014 ) , and the equivalent of 1 . 5 µg of DNA was loaded per well on 8% SDS-PAGE gels .", "The 95 kDa band corresponding to the GFP fusion ( 65 +29 kDa ) was detected with anti-p65 antibody ( Santa Cruz , Mab , Cat . # sc-372 , upper panel ) and quantified using the ECL Plex fluorescent western blotting system ( GE Healthcare ) .", "Sixteen-bit images were acquired with FLA-9000 ( Fuji Film ) ; signals were within the linear part of the dynamic range ( Celona et al . , 2011 ) .", "Quantification of western blot signals was performed with ImageJ software ( Rasband , http://rsb . info . nih . gov/ij ) .", "GFP-p65 quantities were expressed as fold change upon setting to 1 the p65 levels in unstimulated cells ( bottom panel ) .", "Shown is a representative experiment of the three performed .", "Statistical error: SD of technical replicates ( behind symbols ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01310 . 7554/eLife . 09100 . 014Figure 1—figure supplement 11 . Cell cycle analysis of cells exposed to Hoechst and TNF-α .", "To determine whether Hoechst staining might alter the cell cycle , the imaging culture medium ( 50 ng/ml Hoechst , 0 . 1% FCS ) was added to GFP-p65 MEFs for 2 hr ( preincubation ) .", "After that time 10 ng/ml TNF-α was added and cells harvested at the indicated timepoints ( magenta ) .", "For comparison a set of cell samples was left untreated and harvested at the same timepoints ( blue ) .", "In green , cells cultured in 10% FCS .", "Propidium Iodide staining was performed with standard protocols after cell fixation and cells analysed with an Accuri FACS instrument ( Becton-Dickinson ) .", "The fractions of cells in each cell cycle phase are reported .", "Shown is one of three representative experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 014 Obtaining synchronous NF-κB dynamics in cell populations is therefore important to study gene expression , and can give insights into the dynamics at a collective level .", "White’s group showed that short trains of TNF-α pulses produce rounds of synchronous NF-κB translocation; the stimulation frequency affected both the translocation amplitude and gene expression levels ( Ashall et al . , 2009 ) .", "A study performed while the present one was in progress indicated that entrainment of NF-κB oscillations at population level arises when 3T3 cells stably transfected with GFP-p65 are perturbed with regular trains of sawtooth-like profiles of TNF-α ( Kellogg and Tay , 2015 ) .", "Here , we used GFP-p65 mouse embryonic fibroblasts ( MEFs ) derived from knock-in mice and thus expressing physiological levels of p65 ( De Lorenzi et al . , 2009 ) .", "Using a microfluidic device , we exposed cells to well-defined , periodic TNF-α stimuli , and eliminated continuously both catabolites produced by the cells and secreted proteins , which might generate a secondary autocrine/paracrine response .", "We find that GFP-p65 MEFs lock their NF-κB oscillations to the periodic external signal , and become synchronized .", "However , the 1:1 locking is maintained over a wide variety of frequencies of the driving TNF-α stimulus , does not improve over repeated stimulation and actually disappears fast when the external stimulus ceases , all of which suggest that entrainment is not achieved .", "The mathematical model we developed indeed suggests that NF-κB can behave as a damped oscillator , analogous to a mechanical damped harmonic oscillator; damped oscillators do not entrain but follow external forcing while it is present .", "Taking advantage of the fact that GFP-p65 MEFs under periodic stimulation behave as a synchronous population , we analyzed the transcriptional output at genome-wide level .", "We find that one single NF-κB dynamics translates into 3 different dynamics of transcriptional regulation , all of which can be reproduced by our mathematical model; the key discriminator is the parameter representing mRNA degradation .", "Furthermore , the three dynamical patterns correspond to specific functions , which suggests that group of genes were positively selected by evolution ." ], [ "To characterize the response of the NF-κB oscillatory system to different external stimuli , we made use of GFP-p65 knock-in MEFs ( De Lorenzi et al . , 2009; Sung et al . , 2009; Zambrano et al . , 2014a ) cultured in a microfluidic device to control precisely the concentration and timing of the TNF-α stimulation ( Materials and methods and Figure 1—figure supplement 1A and B ) .", "Notably , the flow rate in the microfluidic device is constant , so that the concentration of TNF-α cannot change due to the activity of the cells , nor can the medium accumulate catabolites or secreted proteins .", "Constant stimulation of GFP-p65 knock-in cells in the microfluidic plate with a constant flow of 10 ng/ml TNF-α for up to 15 hr induced nuclear-to-cytoplasmic p65 oscillations ( Figure 1B; Video 1 and Figure 1—figure supplement 2D ) that are qualitatively similar to the heterogeneous oscillations observed with static stimulation ( Sung et al . , 2009; Zambrano et al . , 2014a ) .", "Several controls were performed to exclude possible damaging effects related to imaging .", "Cells under constant flow of fresh medium without TNF-α divide and show almost no cell death ( Video 2 ) ; cell death observed under continuous flow depends on the dose of TNF-α ( Figure 1—figure supplement 8 and Video 1 ) independently of the imaging conditions .", "UV-induced DNA damage due to imaging ( Cadet et al . , 2005 ) is negligible as assessed by immunostaining for thymine dimers ( Komatsu et al . , 1997; Sinha and Hader , 2002 ) .", "DNA Damage Response ( DDR ) is also negligible as assessed by immunostaining of imaged cells for gammaH2AX ( Marti et al . , 2006; Oh et al . , 2011; Staszewski et al . , 2008 ) ( Figure 1—figure supplement 5 and 6 ) .", "Moreover , neither Hoechst exposure nor TNF-α affect dramatically the cell cycle ( Figure 1—figure supplement 11 ) .", "The controls to exclude possible effects of the nuclear dye ( Ge et al . , 2013; Martin et al . , 2005 ) or photo-damage ( Cole , 2014 ) are described in Materials and methods and in figure captions . 10 . 7554/eLife . 09100 . 015Video 1 . Dynamics for constant flow of TNF-α .", "Imaging of GFP-p65 knock-in cells in the microfluidic chamber stimulated with a constant flow of 10 ng/ml TNF-α for 12 hr .", "The stimulus induced nuclear-to-cytoplasmic p65 oscillations that are qualitatively similar to the heterogeneous oscillations observed with static stimulation .", "Along time , it is possible to appreciate the increase of TNF-induced cell death and apoptosis . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01510 . 7554/eLife . 09100 . 016Video 2 . Dynamics for Hoechst-stained cells in the absence of TNF-α .", "Several controls were performed to exclude possible damaging effects related to imaging .", "This video shows that cells under constant flow of fresh medium without TNF-α divide and very few events of cell death are detectable .", "Left part: GFP channel; right part HOE channel .", "Twelve-hour imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 016 To analyse the oscillatory dynamics , we refined our recently published pipeline ( Zambrano et al . , 2014a ) to compute the nuclear to cytoplasmic intensity ( NCI ) of GFP-p65 fluorescence for hundreds of cells per condition ( Materials and methods and Figure 1—figure supplement 1C ) .", "Although this measure implies a partial segmentation of the cytoplasm and thus is more elaborate than the background-corrected mean nuclear intensity used recently by different authors ( Kellogg and Tay , 2015; Lee et al . , 2014; Lee et al . , 2009; Sung et al . , 2014 ) it is a ratio of intensities and thus it self-corrects for changes in image intensity in our experimental settings ( see e . g . Video 1 or Video 2 ) .", "Furthermore , provided that the total amount of p65 is constant ( Figure 1—figure supplement 10 ) , NCI ( t ) is a monotonic function of nuclear NF-κB ( Figure 1—figure supplement 9B ) so that oscillations are observed in the former if and only if they are present in the latter ( further details in Materials and methods ) .", "Finally , a rigorous description of the observed dynamics was achieved through the automatic identification of statistically significant peaks ( Materials and methods and Figure 1—figure supplement 2A and B ) .", "The timing between two consecutive peaks is the experimental period Texp .", "Texp has a distribution with a maximum at 90 min ( Figure 1F ) , which is the approximate intrinsic period reported for NF-κB oscillations that we called T0 ( Nelson et al . , 2004; Tay et al . , 2010; Zambrano et al . , 2014a ) .", "As observed for static stimulation ( Zambrano et al . , 2014a ) , the observed oscillations are very heterogeneous ( Figure 1B ) and asynchronous ( Figure 1—figure supplement 3A and Figure 1—figure supplement 2D ) .", "However , the variety of behaviors observed , including non-oscillating cells , is compatible with the dynamics observed in the same cells for static culture conditions , see ( Sung et al . , 2009; Zambrano et al . , 2014a ) .", "Similar behaviors are found in the absence of nuclear staining ( see Figure 1—figure supplement 9C ) , which led us to conclude that imaging conditions do not interfere with NF-κB signaling .", "The tail in the period distribution ( Figure 1F ) and the observed average of four peaks in 12 hr of stimulation ( Figure 1—figure supplement 3B ) , instead of the expected eight , indicate that in our cells the heterogeneous oscillations are damped and tend to converge to an equilibrium state under stimulation ( Figure 1B ) .", "Indeed , although oscillatory peaks are observed for most of the cells , they are infrequent and irregular for times beyond 6 hr ( Figure 1B and Figure 1—figure supplement 2D ) as previously reported for the same cells in static conditions ( Sung et al . , 2009; Zambrano et al . , 2014a ) .", "Hence , we describe here these heterogeneous oscillations as damped in contrast with the sustained oscillations , which continue regularly and unabated for a very long time in continuously stimulated cells , see for example ( Kellogg and Tay , 2015 ) .", "Damped oscillations can easily emerge in the NF-κB genetic circuit .", "Indeed , a minimal deterministic model that takes into account the basic elements of the NF-κB genetic circuit ( Zambrano et al . , 2014b ) ( Figure 1A , Figure 1—figure supplement 4 , see Materials and methods for a complete description of the model ) shows that different combinations of the parameters can lead to different dynamics .", "The model parameters that we used for our explorations are provided in Supplementary file 2 .", "We denote as PS those specifying the external signal and as PNF-κB those used to model the double IκB and A20 negative feedback; in our explorations , we allow them to vary differently depending on the associated uncertainty about their values ( Materials and methods and Supplementary file 2 ) .", "We generated a library of randomized parameters and found that the system presents a fixed point whose stability changes depending on the parameters ( details on the stability analysis are found in the Materials and methods section ) .", "The vast majority of parameter combinations give rise to damped oscillations ( when all the eigenvalues have all negative real parts , see Figure 1—figure supplement 5A , B ) .", "A smaller fraction of parameter combinations give rise to trajectories that converge to a stable limit cycle around the unstable fixed points ( so certain eigenvalues have positive real parts , see Figure 1—figure supplement 5A , B ) .", "Interestingly , parameters for oscillating and non-oscillating cells are in similar intervals suggesting that it is the precise combination of the parameters , rather than a single one , what determines the resulting dynamics ( see Figure 1—figure supplement 5C ) .", "Our simulations might also explain why other researchers found continuous periodic oscillations with T0 = 90 min under a constant flow of TNF-α ( Kellogg and Tay , 2015 , and see Discussion ) and the variety of damped oscillatory dynamics upon LPS recently reported for fibroblasts ( Cheng et al . , 2015 ) and macrophages ( Sung et al . , 2014 ) .", "Our exploration shows further how variations of the parameters can give rise to a variety of dynamics that reflects what we find in an isogenic population ( Figure 1B and Figure 1—figure supplement 2 ) .", "Considering the heterogeneity of dynamics , to better visualize the collective oscillatory state of the population in each condition , following Mondragon-Palomino et al . , 2011 , we computed and represented the phase of the oscillation ϕ ( t ) for each cell by detecting peaks and setting ϕ=0 ( 2π ) at the maximum of each peak and ϕ ( t ) =π in the minimum between two peaks ( phase plots for the green time series are depicted in Figures 1B , C . See also Materials and methods and Figure 1—figure supplement 2B , C ) .", "Time series for single cells ( Figure 1B and C ) were converted to phase plots ( Figure 1E , I ) where each row represents one cell .", "Thus , oscillatory peaks can be easily observed .", "In the phase plot , the first response to constant TNF-α right after t=0 hr is synchronous in the population , but this synchrony is quickly lost , as previously reported ( Nelson et al . , 2004; Tay et al . , 2010; Zambrano et al . , 2014a ) .", "To investigate the response of the NF-κB oscillator to perturbations in the cell’s environment , we switched periodically the stimulus concentration in the culture chambers in the microfluidics apparatus .", "TNF-α switching between doses D1 and D2 occurs in less than 1 min and generates a tightly controlled square profile of stimulation .", "Stimuli were applied for intervals of time T1 and T2 .", "We refer to Tf = T1+T2 as the period of the forcing and to D1–D2 as the amplitude of the forcing ( Figure 1A , lower panel ) .", "We started our analysis by applying a periodic stimulation of 90 min , which is close to the intrinsic period of the NF-κB oscillatory system .", "Single-cell traces are provided in Figure 1C for cells stimulated with D1 =10 ng/ml TNF-α , D2 =0 ng/ml with Tf=90 min . ( T1=45 min and T2 =45 ) .", "Peaks are present in all the forcing cycles ( Figure 1C and Figure 1—figure supplement 3C ) and synchronous , in contrast to the asynchronous peaks observed in constantly stimulated cells ( Video 3 ) .", "Hence , we observe in this condition – a square forcing – a forcing-induced synchronous dynamics reminiscent of that obtained by applying short trains of pulses ( Ashall et al . , 2009 ) .", "Our synchronous oscillations are visually apparent both in time-lapse images ( Figure 1H ) and phase plots ( Figure 1I ) .", "Texp is sharply distributed around 90 min ( Figure 1J ) , corresponding to the expected eight peaks in 12 hr ( Figure 1—figure supplement 3D ) .", "The height of the first peak ( Figure 1K ) is similar to the height of the first peak under constant stimulation ( Figure 1G ) , indicating that the experimental conditions are highly comparable .", "The height of the subsequent peaks is slightly higher for periodically stimulated cells but still heterogeneous . 10 . 7554/eLife . 09100 . 017Video 3 . Dynamics for Tf=90 min . Imaging of cells stimulated with D1 =10 ng/ml TNF-α , D2 =0 ng/ml and Tf=90 min . ( T1=45 min and T2 =45 ) .", "Peaks are present in all the forcing cycles and synchronous .", "Twelve-hour imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 017 Taken together , these results indicate that a periodic external stimulus can lock in step a population of cells in a long series of synchronous NF-κB oscillations , in clear contrast with the remarkable oscillatory asynchrony and heterogeneity under constant stimulation .", "We then systematically analysed the response to variable forcing amplitudes , by applying different concentrations of TNF-α .", "The phase plots obtained for TNF-α ranging from 10 to 0 . 1 ng/ml with D2 = 0 ng/ml show that the coherence of the oscillations decreases with decreasing doses of TNF-α but still persists at low doses ( Figure 2A and Figure 2—figure supplement 1 ) .", "Texp is less sharply distributed around 90 min as the forcing amplitude decreases .", "Concomitantly , a second peak corresponding to multiples of Tf becomes progressively more conspicuous ( Figure 2B ) , suggesting that for lower doses of TNF-α an increasingly larger fraction of cells do not respond with a peak to some of the forcing cycles .", "In the plots of peak maxima ( Figure 2C ) the height of the first peak is not reproduced by the second and the third peaks , compatible with what reported for this forcing period ( Ashall et al . , 2009 ) .", "However the heights converge under repeated forcing to a constant value for all the forcing amplitudes .", "This is typical of nonlinear dissipative oscillators in the presence of periodic forcing ( Goldstein et al . , 2001 ) . 10 . 7554/eLife . 09100 . 018Figure 2 . Synchronous oscillations arise for different forcing amplitudes .", "( A ) Representative phase plots for 25 cells ( out of 216 , 80 , 188 , 263 , 225 cells analysed ) stimulated with D1=10 , 1 , 0 . 5 , 0 . 25 , 0 . 1 ng/ml TNF-α , D2=0 ng/ml , for Tf =90 min , T1=T2=45 min . ( B ) Distributions of the periods for the cells shown in panel ( A ) ; distributions become narrower as the dose D1 increases .", "The appearance of a second peak at Texp=3 hr at lower doses means that in some cycles a fraction of cells miss a peak and the interval to the next one is double .", "( C ) Quantification of height of the nth peak in the different conditions considered in ( A ) .", "By decreasing the stimulus amplitude , the ratio tends to stabilize to a constant value .", "( D ) Distribution of the phase difference Δϕ for the forcings considered: Δϕ becomes narrower as the forcing amplitude is increased .", "( E ) The synchrony intensity η , an entropy-based measure on how widely distributed the values of Δϕ are , increases with the amplitude of D1 for Tf =90 min . ( F ) The synchrony intensity ηn computed using only the peaks observed in each forcing cycle shows that the synchrony does not increase with the successive cycles of forcing .", "Figure supplements from 1 to 3 are provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01810 . 7554/eLife . 09100 . 019Figure 2—figure supplement 1 . NCI and average dynamics for different forcings .", "NCI of single cell time series ( green ) and average ( black ) for the indicated doses , ( A ) to ( C ) plots correspond to a forcing of 90 min .", "Cell numbers as in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 01910 . 7554/eLife . 09100 . 020Figure 2—figure supplement 2 . Distribution of the phase differences for cells stimulated with constant TNF-α .", "The distribution is almost flat and the synchronization intensity η is very low .", "Cell numbers as in Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02010 . 7554/eLife . 09100 . 021Figure 2—figure supplement 3 . Dynamics of alternating doses Tf = 90 min and D2 > 0 . Panels in each row ( left to right ) represent the single cell NCI dynamics ( green ) plus the average ( black ) and the detected peaks ( triangles ) ; the phase plot; the phase difference distribution ( with the synchrony intensity value η ) and the experimental period distribution .", "( A ) D1=7 . 5 , D2=2 . 5 , ( B ) D1=6 , D2=4 and ( C ) D1=5 . 2 , D2=4 . 8 ( in ng/ml TNF-α ) .", "Twentyfive cells are reported in the phase plots out of 111 , 78 , 66 analysed , respectively .", "T1 = T2 = 45 min . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 021 The degree of synchrony for each experimental setting of forcing can be evaluated considering the distribution of the phase difference Δϕ between the timing of each oscillatory peak and the beginning of the forcing ( see Materials and methods ) .", "For asynchronously oscillating cells under constant TNF-α stimulation , such distribution is flat ( Figure 2—figure supplement 2 ) .", "When cells are stimulated with different forcing amplitudes , the distribution of Δϕ is narrower for higher doses of TNF-α ( Figure 2D ) , indicating a higher degree of locking of the oscillations to the forcing for increasing amplitudes .", "We then compared the degree of synchrony of the cell populations using the synchrony intensity η , an entropy-based quantifier of the distribution of the phase difference Δϕ that is 0 for flat distributions and 1 for delta-like distributions ( Mondragon-Palomino et al . , 2011 , and Materials and methods ) .", "Synchrony intensity increases with the dose , but is nonzero even when D1=0 . 1 ng/ml TNF-α ( Figure 2E ) .", "To understand if D2=0 was a necessary condition for synchrony we applied D2>0 .", "Results show that the synchrony is maintained when D1 is threefold D2 , while for smaller differences synchrony is almost lost ( Figure 2—figure supplement 3 ) .", "We next investigated whether synchrony improved under repeated forcing .", "We computed ηn , the synchrony intensity at the n-th cycle of forcing ( i . e . Δϕ computed for peaks in the time intervals [ ( n–1 ) Tf , nTf ) for n≥1 ) .", "We find that ηn does not increase ( Figure 2F ) in successive cycles of external forcing , meaning that the oscillations do not become more synchronous as the system is perturbed repeatedly .", "Taken together , the above results indicate that NF-κB dynamics adapts to varying amplitudes of periodic inputs .", "However the system does not seem to learn from the previous periodic forcing cycles .", "We then tested the adaptability of the NF-κB system to different forcing periods Tf , ranging from 0 . 5T0 to 2T0 .", "We first considered periodic perturbation of Tf=2T0=180 min , with T1=30 min and T2 =150 min , and TNF-α doses D1 ranging from 10 ng/ml to 0 . 1 ng/ml , D2 =0 ng/ml .", "Oscillations are locked to the forcing even for D1 =0 . 1 ng/ml ( Figure 3A , see also Figure 3—figure supplement 1 , A–C and Video 4 ) .", "The use of T1=30 min assures the existence of sharp oscillatory and transcription peaks ( see below ) and also leads to synchrony of the oscillations for Tf=T0 ( see Figure 3—figure supplement 3 , bottom panels ) .", "For all doses we find a bimodal distribution of the oscillation periods , with an overall maximum at 180 min and a much smaller relative maximum at 90 min ( Figure 3C ) .", "This indicates that after a single pulse of forcing some cells oscillate a second time with a period similar to the intrinsic one .", "However , a closer analysis of peak maxima for time intervals of the form [ ( n–1 ) T0 , nT0 ) = [ ( n–1 ) Tf/2 , nTf/2 ) ( Figure 3E ) reveals that peaks arising right after each periodic stimulation ( even n ) are higher than the rare ones ( odd n ) arising when stimulation is absent . 10 . 7554/eLife . 09100 . 022Figure 3 . Cells adjust oscillations to different periods for a wide range of forcing amplitudes . Representative phase plots for 25 cells stimulated with D1=10 , 1 , 0 . 1 ng/ml TNF-α ( out of 151 , 77 , 123 cells analysed , respectively ) D2=0 ng/ml for ( A ) Tf =180 min with T1= 30 min and ( B ) Tf =45 min with T1=22 . 5 min ( analysed 101 , 112 , 119 cells ) .", "( C , D )", "Plots showing the distributions of the periods for the conditions given in panels ( A ) and ( B ) , respectively .", "( E , F )", "Average peaks height in the intervals [ ( n–1 ) T0 , T0 ) and [ ( n–1 ) T0/2 , nT0/2 ) , for A and B , respectively .", "In ( E ) , even n correspond to peaks right after stimulation , odd n correspond to the small peak arising between two consecutive stimulations .", "( G , H )", "Distribution of the phase difference Δϕ for the forcings in A and B: Δϕ has narrower distributions for higher doses .", "( I ) The synchrony intensity η grows with the doses for Tf=180 min ( blue ) and Tf=45 min ( red ) .", "( J , K )", "Synchrony intensity plots show that ηn does not increase as successive cycles of forcing are applied to the system , both for Tf=180 min and Tf=45 min , respectively .", "All the analyses included all the tracked cells with no preselection of the responding ones .", "Figure supplements 1 to 3 are provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02210 . 7554/eLife . 09100 . 023Figure 3—figure supplement 1 . NCI and average dynamics for different forcings .", "NCI of single cell time series ( green ) and average ( black ) for the indicated doses and timings .", "( A–C ) correspond to a forcing of 180 min , while ( D–F ) to a forcing of 45 min .", "D1 is indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02310 . 7554/eLife . 09100 . 024Figure 3—figure supplement 2 . Dynamics of alternating doses Tf=180 min and D2>0 . Panels in each row ( left to right ) represent the single cell NCI dynamics ( green ) plus the average ( black ) and the detected peaks ( triangles ) ; the phase plot; the phase difference distribution ( with the synchrony intensity value η ) and the experimental period distribution .", "( A ) D1=10 , D2=0; ( B ) D1=7 . 5 , D2=2 . 5; ( C ) D1=6 , D2=4 and ( D ) D1=5 . 2 , D2=4 . 8 ng/ml TNF-α .", "Number of cells analysed: 102 , 68 , 47 , 70 , respectively .", "T1=30 min , T2=150 min . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02410 . 7554/eLife . 09100 . 025Figure 3—figure supplement 3 . Dynamics for cells synchronised with T1=30 min and different TNF concentrations . NCI and average dynamics for 45 and 90 min forcing amplitudes , with TNF-α stimulation of 10 and 1 ng/ml , are reported here to integrate results presented in main Figures 2A and 3B .", "The aim was to assess synchronization for a constant T1 value of 30 min as opposed to 45 and 22 min .", "Each row shows ( A ) the phase plots ( approximately 150 cells analysed per condition ) , ( B ) the single-cell NCI dynamics ( green ) plus the average ( black ) and the detected peaks ( blue triangles ) for a specific condition of synchronization .", "( C ) The experimental period distribution ; ( D ) the phase difference distribution ( with the synchrony intensity value η .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02510 . 7554/eLife . 09100 . 026Video 4 . Dynamics for Tf=180 min . Imaging of cells stimulated with D1 =10 ng/ml TNF-α , D2 =0 ng/ml and Tf=2T0=180 min ( T1=30 min and T2 =150 ) .", "Oscillations are locked to the forcing .", "Twelve-hour imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 026 Oscillations in each condition show a good degree of synchrony , as described by the distributions of the phase differences Δϕ , which are remarkably narrow for all doses ( Figure 3G ) ; the synchrony intensity η is nonzero even for D1 = 0 . 1 ng/ml and increases with the dose ( Figure 3I , blue line ) .", "When D2>0 ng/ml we found synchronized oscillations for D1 at least three times D2 ( Figure 3—figure supplement 2 ) .", "We considered also the ability of the NF-κB system to respond to stimulations of periodicity below the intrinsic one .", "We selected Tf=T0/2=45 min , but T1=30 min led to a poor synchrony ( see Figure 3—figure supplement 3 , top panels ) .", "We therefore reduced T1 to 22 . 5 min , and thus T2=22 . 5 , which proved to be a sufficiently long resetting of the external signal; in these conditions we obtained a sharply defined dynamical response .", "Oscillations are locked in step with D1=10 and 1 ng/ml ( Figure 3B , Video 5 ) , whereas synchronization is weaker for D1=0 . 1 ng/ml ( this is also evident in the average NCI dynamics , see Figure 3—figure supplement 1 , D–F ) .", "The experimentally determined oscillatory period Texp ( Figure 3D ) is narrowly distributed around Tf=45 min for D1=10 ng/ml , but we also find a small peak around Texp=90 min , indicating that some cells skip the oscillation elicited by the forcing .", "The peak height for each forcing cycle tends to stabilize to a constant value and to be lower for lower forcing amplitude , although the differences are small ( Figure 3F ) .", "Also in this case , the synchrony correlates with the dose: in fact , the phase difference Δϕ tends to be more narrowly distributed for higher values of D1 ( Figure 3H ) , and the synchrony intensity η grows with the dose ( Figure 3I , red line ) . 10 . 7554/eLife . 09100 . 027Video 5 . Dynamics for Tf=45 min . Imaging of cells stimulated with D1 =10 ng/ml TNF-α , D2 =0 ng/ml and Tf=T0/2=45 min ( T1=22 . 5 min and T2 =22 . 5 min ) .", "Such short wash-out provides a sufficient resetting of the external signal; in these conditions we obtained a sharply defined dynamical response and oscillations are locked in step .", "Twelve-hour imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 027 It has been recently reported that NF-κB oscillations can be synchronized by entrainment ( Kellogg and Tay , 2015 ) .", "However , in our experiments the synchrony intensity for each cycle , ηn , does not increase when the system is forced repeatedly , either with periods of 90 , 180 or 45 min ( Figures 2F , 3J , K ) , in contrast to what is observed for entrained oscillators ( Mondragon-Palomino et al . , 2011 ) .", "Furthermore , when oscillators are entrained by external forcing , their oscillations also show m:n resonant patterns ( m oscillations for each n cycles of the forcing ) in so-called Arnold tongues ( Pikovsky et al . , 2003 ) .", "However , we only observed 1:1 synchronization patterns for all the periods of forcing ( Figure 3A , B ) , with no clear evidence of the 2:1 and 1:2 patterns expected for entrainment .", "We thus investigated further whether the synchronization mechanism we observed does correspond to entrainment .", "Once the forcing ceases , entrained oscillators dephase gradually , losing their sharply defined common entrained oscillatory period .", "We then investigated whether our cells kept a memory of the synchronous dynamics once the periodic forcing ceased .", "The phase plots for 75 cells forced with Tf=45 min D1 =10 ng/ml ( Figure 4A , washing out after 16 forcing cycles ) , and for Tf=90 min with D1 =1 ng/ml ( Figure 4B , washing out after eight forcing cycles ) , indicate that when the external stimulus stops , cells lose quickly their synchrony .", "This is also evident from single-cell NCI traces and from the peaks detected in the same conditions ( Figure 4C and D respectively ) .", "Indeed , only for Tf=45 min we observed small peaks after the last cycle of the forcing , but these peaks are on average 90 min away from the last forced peak , and correspond to the intrinsic oscillatory period of our oscillator .", "The quick loss of synchrony can also be observed in the evolution of the synchrony quantifier ηn for the two last cycles of the forcing ( Figure 4E , n=1 , 2 ) and the two subsequent ( virtual ) ones ( Figure 4E , n=3 , 4 ) .", "This is also the case when considering cells forced with Tf=90 min and a high stimulus amplitude D1 =10 ng/ml , after which we apply a constant flow of 10 ng/ml TNF-α: cells present a last well-defined translocation ( Figure 4F and 4G ) and rapidly dephase ( Figure 4H ) . 10 . 7554/eLife . 09100 . 028Figure 4 . Cells do not keep a memory of the synchronous oscillatory dynamics .", "( A , B )", "Phase plots of the last two oscillation cycles for Tf =45 min ( D1=10 ng/ml ) and Tf =90 min forcing ( D1= 1 ng/ml ) ( number of forcing cycles are indicated above ) followed by a period of 3 hr with no stimulation ( 75 cells are displayed out of 106 and 197 cells analysed , respectively ) .", "( C , D )", "NCI time series at single cell level ( green lines ) for the two conditions ( A ) and ( B ) .", "Blue triangles indicate the peaks considered in the computing .", "The thick black line is the average NCI , showing a small peak 90 min after the last forced peak for Tf =45 min ( C ) , compatible with the natural timescale of the free oscillations .", "( E ) The synchrony intensity ηn for the last two forced peaks ( n=1 , 2 ) and for the peaks detected in the absence of the stimulus ( n=3 , 4 ) illustrates the fast loss of synchrony .", "( F ) Phase plots of the last two oscillation cycles for Tf =90 min ( number of forcing cycles are indicated above ) ( D1=10 ng/ml ) followed by 4 . 5 hr flow of 10 ng/ml TNF-α ( 75 cells are displayed out of 149 cells analysed ) .", "( G ) NCI time series at single-cell level ( green lines ) .", "Blue triangles indicate the peaks considered in the computing .", "The thick black line is the average NCI , showing a small peak 90 min after the last forced peak , compatible with the natural timescale of the free oscillations .", "( H ) The synchrony intensity ηn for the last two forced peaks ( n=1 , 2 ) and for the peaks detected in the presence of the stimulus ( n=3 , 4 ) illustrates the fast loss of synchrony .", "Figure supplements from 1 to 4 are provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02810 . 7554/eLife . 09100 . 029Figure 4—figure supplement 1 . Dynamics of alternating doses Tf=60 min and D2>0 . Panels in each row ( left to right ) represent the single cell NCI dynamics ( green ) plus the average ( black ) and the detected peaks ( triangle ) ; the phase plot; the phase difference distribution ( with the synchrony intensity value η ) and the experimental period distribution .", "( A ) D1=10 , D2=0; ( B ) D1=7 . 5 , D2=2 . 5; ( C ) D1=6 , D2=4 and ( D ) D1=5 . 2 , D2=4 . 8 ( in ng/ml TNF-α ) .", "Analysed cells: 102 , 91 , 70 and 104 , respectively .", "T1=30 min , T2=30 min . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 02910 . 7554/eLife . 09100 . 030Figure 4—figure supplement 2 . The model predicts that NF-κB oscillations follow the forcing period . We computed the period to which the NF-κB system converges ( average timing between peaks in a 15-hr timespan ) for different periods of the forcing and various forcing amplitudes .", "A representative plot of the results obtained is shown: we keep PNF-κB constant while varying the forcing parameters PS .", "D1 varies between 0 and Dmax=2 and T1=T2=Tf/2 .", "The ratio Tnumerical/Tf between the computed NF-κB period and the forcing period is very close to one , as expected for damped oscillations when periodically forced .", "Only for high values of the period the ratio is close to 0 . 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03010 . 7554/eLife . 09100 . 031Figure 4—figure supplement 3 . Interpretation of the Tnumerical/Tf ratios close to 0 . 5 in Figure 4—figure supplement 2 .", "This ratio is due to the presence of smaller peaks between the bigger post-stimulation peaks in our simulations .", "An example of our simulations for NCI ( green line ) and the forcing ( red line ) is shown .", "Notice that the smaller peaks tend to decrease in time .", "This dynamics is reminiscent of the one observed for Tf=2T0=180 min in our experiments , see Figure 3A . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03110 . 7554/eLife . 09100 . 032Figure 4—figure supplement 4 . Sawtooth-like profiles lead to heterogeneous but synchronous dynamics in GFP-p65 cells .", "Each row shows", "( i ) the phase plot for 25 cells ( Approximately 100 cells were analysed per condition ) ,", "( ii ) the single cell NCI dynamics ( green ) plus the average ( black ) and the detected peaks ( blue triangles ) ,", "( iii ) the experimental period distribution and", "( iv ) the phase difference distribution ( with the synchrony intensity value η ) for a specific condition ofstimulation with a sawtooth profile , generated by a 15 min pulse followed by no washout .", "The TNF-α concentrations used are indicated for each row .", "For forcings of 60 and 90 min we observe synchronization similar to the one obtained for alternating doses this is more blurred for forcing of 180 min . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 032 Kellogg and Tay ( 2015 ) found no synchrony for Tf=60 min .", "However , we found that our cells can actually be synchronized under periodic stimulations with Tf=60 min ( T1 =T2 =30 min ) when D1 =10 ng/ml ( Video 6 ) and D2 =0 ng/ml ( Figure 4—figure supplement 1A ) .", "Furthermore , we found that synchrony is still visible for D2>0 ng/ml ( Figure 4—figure supplement 1B–D ) , as we had observed for other stimulation periods .", "The difference might arise from the fact that we use a forcing different from the sawtooth-like TNF-α profile used by Kellogg and Tay ( 2015 ) , which is obtained by periodically and quickly replacing the medium in contact with the cells with fresh TNF-α .", "The sawtooth concentration profile is assumed to arise due to a clearance process of TNF-α by degradation and internalization .", "We then applied sawtooth forcing to our cells ( Figure 4—figure supplement 4 ) .", "Cells lock their oscillations to the sawtooth forcing of periods Tf=60 min and Tf=90 min at the doses considered .", "Interestingly , the synchronization is lost for Tf=180 , suggesting that autocrine-paracrine signalling might introduce a distortion in the cell environment , which is reduced when the medium is changed more frequently .", "Alternatively , the different geometry of our microfluidic with respect to the one used in Kellogg and Tay ( 2015 ) might lead to a slower TNF-α degradation and produce profiles closer to static concentration than to oscillatory stimulation .", "The profiles observed are indeed similar to some of the dynamics observed for alternating doses of TNF-α with D2>0 ng/ml , such as those for Tf=60 min ( Figure 4—figure supplement 1B–D ) , Tf=90 min ( Figure 2—figure supplement 3 ) and Tf=180 min ( Figure 3—figure supplement 2 ) , with compatible values of η .", "However , it is clear that with sawtooth forcing of Tf=60 min the synchrony is strong , at variance with the results reported by Kellogg and Tay ( 2015 ) . 10 . 7554/eLife . 09100 . 033Video 6 . Dynamics for Tf=60 min . GFP-p65 cells can be synchronized under periodic stimulations with Tf=60 min ( T1 =T2 =30 min ) when D1 =10 ng/ml and D2 =0 ng/ml .", "Twelve-hour imaging . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 033 Overall , we conclude that the synchronization mechanism that we observe is not entrainment .", "Rather , it is similar to that of simple mechanical damped oscillators , such as the damped harmonic oscillator , which after a perturbation tend to relax to an equilibrium state .", "When these mechanical systems are challenged with an external periodic forcing , the period of the oscillations tends to match the period of the forcing ( Pikovsky et al . , 2003 ) .", "In fact , our minimal mathematical model of the NF-κB circuit ( Figure 1—figure supplement 4 ) reproduces damped oscillations ( e . g . Figure 1—figure supplement 5A , B , D ) .", "In these conditions , the period of the periodically forced system converges to the period of the forcing ( Figure 4—figure supplement 2 ) .", "Of note , the model reproduces the small peaks appearing between two forcing cycles ( Figure 4—figure supplement 3 ) , similar to the small peaks that we observed for the same forcing as in Figure 3A .", "When behaving as a damped oscillator , the NF-κB system is well suited to quickly synchronize its oscillatory period to input signals of a wide variety of timescales .", "This is probably a desired feature for a system that underlies responses to environmental challenges , which do not come with a particular periodicity .", "This is the reverse of what might be desirable for circadian clocks , whose design should privilege the entrainment to the 24-hr light/darkness period .", "To understand how the oscillatory pattern of NF-κB affects its biological output , we focused on the transcription dynamics of genes controlled by NF-κB .", "The heterogeneity of NF-κB dynamics in the cell population under constant stimulation makes it difficult to establish a direct correspondence between NF-κB activity and transcription .", "However , under conditions of synchronous oscillation the average dynamics corresponds to the dynamics of single cells .", "This is for example the case for cells stimulated with D1 =10 ng/ml D2=0 ng/ml TNF-α , T1=30 min and T2 =150 min ( Tf= 2T0 ) ( Figure 5A ) .", "Numerical simulations with a simple mathematical model of transcription under the control of the regulatory network using parameters from our previous work ( Figure 5—figure supplement 1 , Materials and methods and Zambrano et al . , 2014b ) suggested that for some genes there should be a clear coordination between the input pulsed signal , the oscillating dynamics of NF-κB and the transcriptional output ( Figure 5B ) . 10 . 7554/eLife . 09100 . 034Figure 5 . Synchronous NF-κB oscillations lead to population-level coordinated transcription .", "( A ) NCI plot of single cell oscillations ( green lines ) and population average ( black lines ) for cells stimulated with D1=10 ng/ml TNF-α , D2=0 ng/ml , T1=30 min and T2=150 min .", "The open circles represent the fitting obtained using our minimal mathematical model .", "( B ) The mathematical model predicts waves of transcription ( orange plot , right ) coordinated with the stimulus ( red plot , top ) and p65 oscillations ( green plot , left ) .", "( C , D ) q-PCR time course of nascent and mature mRNAs for the prototypical early and late genes IκBα and Ccl5 , respectively .", "( E , F )", "Transcription profiles for mature IκBα ( red ) and Ccl5 ( blue ) RNAs ( dots ) can be accurately fitted ( lines ) by our minimal mechanistic mathematical model .", "The fittings were performed by keeping common the parameters regulating the external signal ( PS ) and the dynamics ( PNF-κB ) in ( A ) , ( E ) and ( F ) but using different gene expression parameters PG for ( E ) and ( F ) .", "Figure supplements 1 to 2 are provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03410 . 7554/eLife . 09100 . 035Figure 5—figure supplement 1 . Mathematical model based on ODEs to fit single genes transcription; parameter names are reported . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03510 . 7554/eLife . 09100 . 036Figure 5—figure supplement 2 . Hoechst staining does not affect transcription . Quantitative PCR for nascent ( left ) and mature ( right ) IκBα ( upper panels ) and Ccl5 ( bottom panels ) in GFP-p65 MEFs cultured in 10% FCS or 0 . 1% FCS plus Hoechst .", "The cells were stimulated with TNF-α for the indicated times .", "The plots represent the average of two independent experiments .", "Bars: SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 036 To validate the model’s prediction , we tested RNA transcription in the synchronous oscillatory condition shown in Figure 5A .", "RNA samples were prepared at time 0 and 20 , 40 and 60 min after each TNF-α pulse .", "Quantitative RT-PCR was performed for both the nascent unspliced and the mature mRNA forms of the prototypical early and late genes IkBa and Ccl5/RANTES , respectively ( Figure 5—figure supplement 2 and Materials and methods ) .", "Nascent transcription of both genes starts synchronously with each TNF-α pulse and p65 nuclear localization ( Figure 5C , D ) , and the levels of nascent transcripts clearly oscillate .", "Mature transcripts of the early gene IκBα follow the oscillatory dynamics , show transcription-coordinated splicing and do not accumulate after stimulus wash-out ( Figure 5C ) .", "On the contrary , the mature form of the late gene Ccl5 accumulates slowly and progressively along 12 hr ( Figure 5D ) .", "This latter observation extends the notion that the splicing rate for inflammatory genes plays a key role in regulating the timing of gene expression , as previously reported ( Hao and Baltimore , 2013 ) .", "The induction levels for IκBα in the first hours hardly differ from those under chronic stimulation ( ( Hao and Baltimore , 2013 ) ; Figure 5 and Figure 5—figure supplement 2 ) .", "Interestingly , we also find oscillatory dynamics of IκBα and Ccl5 nascent transcripts when cells are stimulated with a forcing period of Tf= 90 min , although the oscillations are less prominent than for Tf=180 min .", "The expression of Ccl5 for Tf= 90 min grows monotonically ( Figure 7—figure supplement 1 ) .", "We then asked whether our minimal mathematical model ( Figure 5—figure supplement 1 and Figure 1—figure supplement 4 ) could simultaneously fit NCI dynamics ( Figure 5A ) and transcription profiles .", "Our model of transcription can be interpreted as a population-level NF-κB–driven telegraph model ( Suter et al . , 2011 ) that includes non-cooperative transcription activation by NF-κB ( Giorgetti et al . , 2010 ) and the role as transcriptional repressor reported for IκBα ( Arenzana-Seisdedos et al . , 1995; Kellogg and Tay , 2015; Lipniacki et al . , 2004; Lipniacki et al . , 2006a; Lipniacki et al . , 2006b; Tay et al . , 2010 ) ; we denote the parameters used to fit the transcription as PG ( details in the Materials and methods section ) .", "The fittings were performed by keeping constant the parameters of the external signal PS ( which is externally imposed ) and using the same parameters regulating NF-κB dynamics , PNF-κB in Figure 5A , E , F , while using different individual transcription parameters PG for each gene ( Figure 5E , F ) .", "Despite being much simpler than other existing models of NF-κB dynamics ( Ashall et al . , 2009; Tay et al . , 2010 ) , our model fits faithfully the observed transcription dynamics for both IκBα ( Figure 5E ) and Ccl5 ( Figure 5F ) .", "Overall , our results show that the expression of two different genes can follow different dynamical patterns even if the entire cell population is effectively locked to the dynamics of the transcription factor that controls both genes .", "Genome-wide profiling represents the logical further step to understand whether additional NF-κB regulated genes follow the diversified dynamics observed for Nfkbia/IκBα and Ccl5 .", "We performed microarray analysis on RNA purified from GFP-p65 knock-in cells under either constant or pulsed TNF-α stimulation ( Zambrano et al . , 2016 ) .", "Transcriptional outputs were subjected to unsupervised clustering of standardized profiles to group the transcription profiles by their shape , while minimizing differences in fold-changes ( Materials and methods ) .", "For constantly stimulated cells , gene expression profiles reproduce well the previously observed kinetics ( Rabani et al . , 2011; Sivriver et al . , 2011 ) , in which the maximum expression level is achieved with different timings for different genes , and no evidence of the oscillatory dynamics of NF-κB is discerned ( Figure 6—figure supplement 1 ) .", "We then quantified genome-wide expression profiles in the population shown in Figure 5 , which oscillates synchronously in the forcing regime of Tf=180 min ( Materials and methods ) .", "We found 970 genes whose expression responds to TNF-α stimulation , of which 499 increase and 471 decrease relative to unstimulated cells .", "Genes distributed in six highly homogeneous clusters; a higher number of clusters did not reduce significantly the inter-cluster distance ( Figure 6—figure supplement 2A ) .", "Three of the clusters contain genes with increasing expression and three contain genes with decreasing expression ( Figure 6A , Figure 6—figure supplement 2 ) .", "The clusters display profiles that range from the oscillatory dynamics of IκBα ( Cluster 1 ) to the slowly increasing dynamics of Ccl5 ( Cluster 3 ) .", "Notably , while genes contained in the three clusters of increasing expression are enriched with known NF-κB target genes ( 10-14 < p < 0 . 05 , Fisher’s exact test , Materials and methods and Figure 6—figure supplement 3 ) , clusters with decreasing expression are not significantly enriched .", "The same is observed in chronic stimulation ( Figure 6—figure supplement 4 ) . 10 . 7554/eLife . 09100 . 037Figure 6 . Synchronized NF-κB dynamics translates into functionally different dynamical patterns of gene expression , each corresponding to distinct pathways .", "( A ) Clusters 1–6 were obtained by an unsupervised k-means-like clustering from the genome-wide transcription profiling of samples harvested in the experiment shown in Figure 5A .", "Line colours are indicative of the membership value of each gene ( colour scale at the bottom ) .", "Three clusters contain genes with increasing expression ( 1–3 ) and three with decreasing expression ( 4–6 ) .", "On the y-axis , standardized expression profile in arbitrary units ( see Materials and methods ) .", "( B ) Plots show single-gene mRNA traces ( median: thick blue line; 85% and 15% intervals , thin blue lines ) .", "The time courses can also be fitted using our minimal mathematical model: shown is the median of the single-gene fits ( thick red line ) and the 85% and 15% intervals ( thin red lines ) .", "Fittings were performed using the same parameters for the external signal ( PS ) and the dynamics ( PNF-κB ) as in Figure 5 , but using different gene expression parameters PG for each gene .", "( C ) Top five pathways of hierarchical level 2 and 3 in the Reactome database significantly enriched in each dynamical cluster .", "Dot sizes are proportional to the percentage of genes in the cluster belonging to that pathway .", "Dot colours identify the corresponding p-values ( p-value < 0 . 05 is set as threshold ) .", "Scale bars on the right .", "( D , E )", "Heatmaps shows the degree of overlap at gene level ( D ) and pathway level ( E ) between each of the 6 clusters .", "Colour scale bar on the right .", "Figure supplements from 1 to 6 are provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03710 . 7554/eLife . 09100 . 038Figure 6—figure supplement 1 . Transcription in cells chronically stimulated with TNF-α .", "Four clusters are obtained from the standardized gene expression profiles for cells stimulated with a constant concentration of 10 ng/ml TNF-α .", "Each line represents a gene .", "Lines colors: blue and red indicate low and high membership values , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03810 . 7554/eLife . 09100 . 039Figure 6—figure supplement 2 . Mathematical validation of clustering .", "( A ) Minimal inter-centroid distance as a function of the number of clusters considered for Figure 6 .", "( B ) Principal Component Analysis based on two components segregates clusters containing genes with increased transcription ( 1 , 2 and 3 ) from those with decreased transcription ( 4 , 5 and 6 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 03910 . 7554/eLife . 09100 . 040Figure 6—figure supplement 3 . Cluster enrichment analysis for NF-κB targets in genes clustered and displayed in Figure 6 .", "Tf=180 with T1=30 min and T2=150 min .", "Two lists of NF-κB targets were considered .", "( A ) Gilmore’s web-site ( www . bu . edu/nf-kb/ ) , ( B ) from ( Li et al . , 2014 ) .", "Significance is shown as -Log10 ( p-value ) , a dashed line marks the threshold of significance at p=0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 04010 . 7554/eLife . 09100 . 041Figure 6—figure supplement 4 . Cluster enrichment analysis for NF-κB targets in genes clustered and displayed in Figure 7 ( constant stimulation ) .", "Two lists of NF-κB targets were considered: ( A ) Gilmore’s web-site ( www . bu . edu/nf-kb/ ) .", "( B ) from ( Li et al . , 2014 ) .", "Significance is shown as –Log10 ( p-value ) , a dashed line marks the threshold of significance at p=0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 04110 . 7554/eLife . 09100 . 042Figure 6—figure supplement 5 . Distribution of fitting distances . Distance distribution for single-gene fittings of genes with increased ( left ) or decreased ( right ) transcription in cells stimulated with a period of 180 min . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 04210 . 7554/eLife . 09100 . 043Figure 6—figure supplement 6 . Degradation rates values are the key parameter to reproduce different gene expression patterns .", "Upper panels: RNA degradation rates inferred from our model for the gene clusters , for a period of 180 min .", "Lower panels: example fittings of two genes for each cluster .", "Of note , the degradation rate is very high for oscillating genes in Cluster 1 .", "Clusters 2 and 3 containing genes with “rapidly-increasing and slowly-decreasing” transcription or “slowly-increasing” transcription are characterised by degradation rates two orders of magnitude lower .", "Genes with decreasing transcription and belonging to Clusters 4–6 have very low RNA degradation rates . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 04310 . 7554/eLife . 09100 . 044Figure 6—figure supplement 7 . Fitting of transcription data from the 180 min synchronization experiment with an alternative model of transcription . The data were also fitted using a model in which IκBα ( A ) does not act as a transcriptional repressor in the feedbacks nor ( B ) in the expression of each single gene , while the role of NF-κB as transcriptional activator ( green arrows in ( A ) ) is preserved .", "( C ) Gene expression data from the experiment in Figure 6 were fitted using the models of ( A ) and ( B ) analogously to our fittings of Figures 5 and 6 : with a common set of PNF-κB while each gene is fitted with its own PG .", "Again , the average relative error is lower for up-regulated genes .", "( D ) Plots show single-gene traces ( grey ) of all mRNA ( median: thick blue line; 85% and 15% intervals , thin blue lines ) .", "We show the median of the single-gene fits ( thick red line ) and the 85% and 15% intervals ( thin red lines ) .", "( E ) Histograms on the bottom part show the distribution of the degradation rates obtained from the fitting for each dynamical cluster .", "Here , again we have that the degradation rate is the key parameter to reproduce the gene expression patterns observed . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 044 We tested if our model would also reproduce the patterns of gene expression dynamics at a genome-wide level in these conditions , keeping the same parameters PS and PNF-κB as in Figure 5 but using different gene expression parameters PG for each gene .", "Indeed , the model fits well the dynamical patterns observed experimentally in clusters with increasing expression ( Figure 6B , grey lines ) .", "The median and the intervals of observed and simulated single gene expression levels match remarkably well ( Figure 6B , red and blue lines respectively , individual fittings are shown in Figure 6—figure supplement 6 , bottom-left panels ) ; the average relative error per timepoint is less than 10% ( Figure 6—figure supplement 5 , left , further details on the fittings and metrics used are given in Materials and methods ) .", "Genes in the clusters with decreasing expression cannot be fitted with the same accuracy ( the average relative error per timepoint is above 10% , see Figure 6—figure supplement 5 , right , individual gene fitting examples shown in Figure 6—figure supplement 6 , bottom-right panels ) , further suggesting that these genes are not controlled directly by NF-κB .", "Indeed , an analysis of the parameters show that gene expression is essentially fitted for those genes as a simple RNA degradation with nearly no contribution of the gene activation/inactivation processes ( low Kon , G and Koff , G , see Figure 5—figure supplement 1 and Materials and methods ) .", "Interestingly , our mathematical model indicates that for a given stimulus impinging on NF-κB , the degradation rate of the mRNA is the key parameter for producing the most distinct dynamical profiles .", "Of note the highest degradation rates are specific for oscillating genes , while those for slowly increasing genes are two orders of magnitude lower ( Figure 6—figure supplement 6 , top panels ) .", "This further underlines the importance of mature RNA processing and degradation in the definition of different patterns of gene expression .", "Although the role of IκBα as a repressor has been proposed in a number of theoretical analyses ( Kellogg and Tay , 2015; Lipniacki et al . , 2004; Lipniacki et al . , 2006a; Lipniacki et al . , 2006b; Tay et al . , 2010 ) only limited evidence of this role is experimentally available ( Arenzana-Seisdedos et al . , 1995 ) .", "Thus , to investigate the role of IκBα in patterning gene expression , we built a model where IκBα does not act as a direct repressor ( Figure 6—figure supplement 7A , B ) .", "This alternative model fits the gene expression dynamics of each cluster well ( Figure 6—figure supplement 7E ) , and again better for genes with increasing transcription than for those decreasing ( Figure 6—figure supplement 7C ) .", "The mRNA degradation rate is again the key parameter to discriminate between the different patterns of gene expression ( Figure 6—figure supplement 7D ) .", "While our fittings do not provide conclusive evidence on the role of IκBα as a transcriptional repressor , they suggest that this is not needed .", "Finally , to understand if the different dynamics observed with Tf=90 min were also present in other conditions , we used the same clustering approach for gene expression profiles of cells stimulated with Tf=90 min , D1 =10 ng/ml and D2 = 0 ng/ml ( as in Figure 1C ) .", "Oscillations in transcript levels were observed , although they were not as conspicuous as for Tf=180 min ( Figure 7A ) , probably due to the fact that 90 min is not long enough for most transcripts to degrade .", "Enrichment of target genes is similar to the two previous cases ( Figure 7C , D ) . 10 . 7554/eLife . 09100 . 045Figure 7 . Genome-wide clustering for Tf=90 min .", "( A ) Clusters obtained by unsupervised k-means clustering from the genome-wide transcription profiling of cells perturbed with a forcing of 90 min , D1=10 and D2=0 ng/ml of TNF-α .", "Lines colours: blue and red indicate low and high membership values , respectively .", "( B ) Top five pathways of hierarchical level 2 and 3 in the Reactome database significantly enriched in each dynamical cluster .", "Dot sizes are proportional to the percentage of genes in the cluster belonging to that pathway .", "Dot colours identify the corresponding p-values ( p-value<0 . 05 is set as threshold ) .", "Scale bars on the right .", "( C , D )", "Enrichment analysis for NF-κB targets from the clusters displayed in panel A . Two lists of NF-κB targets were considered: left: Gilmore’s web-site ( www . bu . edu/nf-kb/ ) ; right: data from Brasier and Kudlicki groups ( Li et al . , 2014 ) .", "Significance is shown as -Log ( p-value ) , a dashed line marks the threshold of significance at p=0 . 05 .", "( E , F )", "Heatmaps show the degree of overlap at gene level ( E ) and pathway level ( F ) between each of the 6 clusters .", "( G ) Overlap at a gene level between clusters obtained for 180 min and for 90 min .", "It is particularly high for Clusters 1 , those of oscillating genes .", "Colour scale bar on the right .", "Figure supplement 1 is provided . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 04510 . 7554/eLife . 09100 . 046Figure 7—figure supplement 1 . Synchronous NF-κB oscillations arising with 90 min forcing lead to population-level coordinated transcription .", "Q-PCR time-course of nascent and mature mRNAs for the prototypical late and early genes Ccl5 and IκBα are shown .", "PCR assays were performed on the same samples used for genome-wide transcription profiling shown in Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 046 We then performed a pathway analysis to determine if each of our dynamical clusters were enriched in functionally related pathways ( see Materials and methods ) .", "Indeed , this is the case ( Figure 6C ) : Cluster 1 is broadly related to chemokines and chemokine receptors and contains so-called early genes , Cluster 2 to the immune system and so-called intermediate genes , and Cluster 3 to extracellular matrix rearrangements and so-called late genes ( Rabani et al . , 2011; Tian et al . , 2005a; 2005b ) .", "Thus , the clustering also indicates a precedence order in the articulation of the cell's response .", "Genes with decreasing expression comprise pathways related to metabolism ( Cluster 4 ) , cell cycle ( Cluster 5 ) and chromosome maintenance ( Cluster 6 ) .", "The overlap between clusters is minimal both at gene and pathway level ( Figure 6D and Figure 6E , respectively , and Materials and methods ) .", "The correspondence between dynamics and gene function is also fulfilled in cells forced with Tf=90 min ( Figure 7B ) , with a low intercluster overlap at gene level ( Figure 7E ) , and slightly higher overlap at pathway level ( Figure 7F ) .", "Interestingly , there is a good correspondence between clusters identified upon both 180 and 90 min forcing .", "In particular Cluster 1 ( the oscillating cluster ) contains almost exactly the same genes in both conditions ( Figure 7G ) .", "Taken together , the above results indicate that different gene expression dynamics identify functionally related categories of genes , suggesting a strict relationship between dynamics and function in the cell's response to external stimuli ." ], [ "Our results show that single cells activate NF-κB signalling synchronously to external TNF-α stimulation , and that repeated forcing elicits synchronous NF-κB oscillations at population level .", "However , NF-κB does not behave as a free oscillator: no cell oscillates constantly in the presence of continuous stimulation ( Figure 8A and B left , green lines ) .", "Furthermore , synchronization among cells does not improve upon repeated forcing ( “training” ) , and single cells trained for a dozen forcing cycles stop oscillating and dephase ( “forget” ) as fast as cells stimulated only once .", "Finally , cells can be synchronized with a wide variety of forcing periods and forcing amplitudes , and synchronize to the external forcing ( Figure 8A and B right , green lines ) without showing any preference for periods resonating with the NF-κB intrinsic period of 90 min; synchronization is more pronounced with high than with low stimulation amplitude . 10 . 7554/eLife . 09100 . 047Figure 8 . NF-κB behaves as a damped oscillator that can synchronize to time-varying external stimuli to produce functionally related transcriptional outputs .", "( A ) The NF-κB system is able to provide different responses to different inputs , from constant ( left ) to time-varying ones ( right ) .", "( B ) Our cells show damped oscillations to a constant stimulus ( left , green lines ) , although for other cell types sustained oscillations might be possible ( magenta line ) .", "Damped oscillations can adapt to timevarying inputs ( right ) and give rise to synchronous oscillations .", "( C ) These synchronous oscillations produce different patterns of gene expression , from oscillating ( left , orange lines ) to slowly increasing ( right , blue lines ) and intermediate dynamics ( pink lines , centre ) .", "We find that each kind of dynamics is typical for genes involved in different cellular functions . DOI: http://dx . doi . org/10 . 7554/eLife . 09100 . 047 The ability of NF-κB to oscillate in tune with a forcing of 45 min illustrates well the plasticity of synchronization: 45 min is equivalent to one half of the intrinsic period , and entrained cells would skip one forcing period out of 2 ( following the 1:2 resonance ) , but our data do not support this interpretation , since cells only infrequently skip a forcing period .", "Simulations with our mathematical model can actually reproduce the ability of the system to synchronize to different forcing periods .", "The ability of our cells to synchronize to a 60 min periodic forcing points in the same direction .", "Thus , we find that our GFP-p65 knock-in cells are not entrained , as do not satisfy the essential precondition – sustained oscillations – and two critical tests for entrainment – increasing synchronization upon repeated forcing , and synchronization to the natural frequency of the internal oscillator ( Pikovsky et al . , 2003 ) .", "Our cells appear closer to the classical textbook example of a damped harmonic oscillator ( Goldstein et al . , 2001 ) , whose frequency corresponds to the frequency of the external stimulation , and whose synchronization to the external stimulus increase monotonically with stimulus amplitude .", "We interpret the damped oscillator behaviour of the NF-κB system as an intrinsic characteristic of the NF-κB system which allows it to adapt to a wide variety of inputs and to quickly reset .", "In fact , pathogens and inflammatory signals do not come in regular periodic patterns , and a carefully calibrated inflammatory response that would adapt itself to any irregular pattern of stimulation would be positively selected for .", "We explored our minimal mathematical model to understand why we observed damped oscillations whereas Kellogg and Tay observed sustained oscillations under constant stimulation , a behaviour that presumably is the root of the different synchronization mechanisms observed .", "Our simulations suggest that sustained oscillations would not be the norm , but rather the result of a specific set of parameters ( see Figure 1—figure supplement 5A , B ) .", "Indeed , the NF-κB network has an equilibrium that depending on the combinations of parameters can be stable , giving rise to damped oscillations that converge to it , or unstable , giving rise to a stable cycle around it ( see e . g . Figure 8B , left , magenta line ) ; transitions between these two states can be mediated by a Hopf bifurcation .", "The simulations performed with our simple model can thus reconcile our experimental observations with the detection of sustained oscillations for different cells ( Kellogg and Tay , 2015 ) , but also with the rich variety of damped oscillatory dynamics reported for other cell types in recent works ( Cheng et al . , 2015; Sung et al . , 2014 ) .", "Other theoretical analyses have identified the amount of NF-κB expressed by a specific cell as the determinant of the Hopf bifurcation and the stability of the oscillator ( Mothes et al . , 2015 ) .", "Notably , we used cells with physiological and uniform p65 levels .", "We also note that the interplay between different feedback loops , including the A20 feedback ( Werner et al . , 2008 ) can vary between cell types , leading to different degrees of dampening , as observed experimentally by selective knock-outs ( Hoffmann et al . , 2002 ) ; dissecting the contribution of each of these feedbacks will be the subject of future experimental and theoretical work .", "The oscillatory behaviour of NF-κB consists in periodic relocalization between the nucleus and the cytoplasm , and entails corresponding cycles of NF-κB binding to cognate binding sites in the genome .", "Thus , NF-κB driven transcription should be pulsatile as well , and in fact we detect cycles in the levels of elongating transcripts in the prototypical early and late NF-κB-controlled genes IkBα and Ccl5 .", "However , whereas mature mRNA oscillates for IκBα , it slowly accumulates for the late gene Ccl5 , and this held true for different frequencies of the periodic stimulation .", "Genome-wide analysis of dynamics , using unsupervised clustering of whole transcriptome profiles , identified six highly homogeneous sets ( “clusters” ) , three containing genes with increasing transcription and three containing genes with decreasing transcription .", "While the genes with increasing transcription are predicted NF-κB targets , those in the decreasing sets are not .", "The train of p65 nuclear translocations is thus decoded by the cell into different transcriptional dynamics: genes in Cluster 1 display a clear and sustained oscillation in transcript level; instead , transcripts for genes in Clusters 2 and 3 accumulate fast or increase slowly and steadily over many cycles of NF-κB oscillations , respectively ( see Figure 8C ) .", "The result is that each new wave of TNF-α elicits on Cluster 1 genes almost the same responses as the previous waves , and no memory is kept of the past; the expression of genes of Clusters 2 and 3 allows a long-lasting response that integrates over time the responses to previous waves .", "All three dynamics of genes with increased expression could be reproduced by our minimal mathematical model of transcription , which incorporates the features of the paradigmatic telegraph model ( Suter et al . , 2011 ) .", "Our model also suggests that mRNA degradation is the key parameter that allows a functional and temporal shaping of the NF-κB response , in particular for transcripts in Cluster 1 ( Figure 6—figure supplement 6 , top-left panel ) .", "The computational results fit well with the fast turnover of early transcripts , whose degradation depends on positive regulators like Zfp36 ( Rabani et al . , 2011 ) .", "Of note , Zfp36 is in Cluster 1 , and oscillates sharply and synchronously with the forcing; this might contribute further to the shaping of peaked responses .", "Overall , our most remarkable finding is that NF-κB oscillations drive the expression of distinct sets of genes whose expression dynamics and functions correlate: the transcripts that oscillate encode mostly cytokines and cytokine receptors , the ones that rise fast and decrease slowly encode proteins involved in immunity , and the ones that rise steadily encode proteins involved in the rearrangement of the extracellular matrix ( Figure 8C ) .", "While steady-rise and rise/decline programs of gene expression can be operated by non-oscillatory transcription factors , oscillating transcription programs can be best achieved with oscillating transcription factors .", "We note that the transcripts that oscillate are involved in the decision of cells of whether to move or not , which has to be refreshed repeatedly over time .", "The oscillatory dynamics of NF-κB allows time to be segmented in units of 90 min cycles , with the option to resume chemokine-directed migration after each single cycle .", "As seen from this perspective , the operation of the NF-κB system as a damped , fast-detuning oscillator implies that in the presence of constant stimulation each motile cell will decide for itself , without synchrony to nearby cells .", "In contrast , in a situation where stimuli change over time beyond a certain threshold – at least three-fold variation in amplitude – cells would be broadly coordinated .", "Uncoordinated movement may maximize the volume randomly patrolled by cells , while coordinated movement may be optimal to reach a specific target location .", "We thus speculate that NF-κB oscillations may have adaptive value in achieving oscillatory expression of genes involved in movement and direction , and the cells’ decision between uncoordinated or collective action .", "In conclusion , we suggest that the oscillatory dynamics of NF-κB ( and other transcription factors ) is a means of segmenting time to provide opportunity windows for decision ." ], [ "GFP-p65 knock-in fibroblasts were provided by M . Pasparakis ( details in De Lorenzi et al ( 2009 ) ; Sung et al ( 2009 ) ) and cultured in phenol-red free DMEM , with 10% FCS , 50 µM β-mercaptoethanol , 1x L-glutamine , 1x sodium pyruvate , 1x non-essential amino acids , and 1x pen/strep in standard tissue culture plastic .", "GFP-p65 fibroblast cultures were started from original aliquots frozen upon arrival from Pasparakis' lab and tested every week to exclude mycoplasma contamination using approved kits .", "Since there are no approved STR profiling protocols for mouse cells ( ( Yu et al . , 2015 ) and http://www . atcc . org/Global/FAQs/0/A/STR%20Testing%20Service%20-%20STR%20on%20mouse%20cell%20lines . aspx ) , we carefully tested our cells for the presence of non-green cells ( not expressing GFP-p65 ) that could represent a cross-contamination of the original culture .", "For imaging experiments , cells were plated one day before the experiment in CellASIC™ ONIX M04S-03 Microfluidic Plates at low density to avoid confluence on the day of the experiment ( e . g . Figure 1—figure supplement 1A ) .", "These plates consist of chambers for cell culture connected through microfluidic channels to a series of reservoirs containing media with selected concentrations of stimuli that can be flown through the chambers .", "Of note , to avoid cell stress or toxicity , the microfluidic plates are primed with 10%FCS in DMEM for 2–4 hr before cell plating .", "Before imaging , DMEM-0 . 1% FCS medium containing 50 ng/ml of the nuclear dye Hoechst33342 was replaced in the microfluidic chambers 3 hr prior to the experiment using the microfluidic platform , see details on the use of the CellASIC™ ONIX below .", "Mouse recombinant TNF-α ( R&D Systems ) was diluted in DMEM-0 . 1%FCS as specified in the text and added in the plate reservoirs .", "It is relevant to note that TNF-α activity is maintained after 12 hr incubation in the plate reservoirs at 37°C , indicating that TNF-α degradation is negligible when it is not in contact with the cells ( Figure 1—figure supplement 1D ) .", "The CellASIC® ONIX Microfluidic Platform allows to apply sharp stimuli by quickly replacing the medium in the cell chambers .", "The flexible proprietary software allows the delivery of medium containing different concentrations of TNF-α for specific time sequences for more than 10 hr while cell are imaged .", "The protocols are available upon request .", "The medium flows in the channels around the microfluidic chambers and diffuses through the perfusion barrier , minimizing the undesirable effect of shear stress ( Babini et al . , 2015 ) ( see Figure 1—figure supplement 1A , B ) .", "The small volume of medium in the chambers ( less than 1 µl ) is replaced fast even at low pressures , as confirmed by the sharp oscillatory profiles obtained even for high frequency stimuli .", "In spite of the low pressure applied ( 1 psi ) , the flow rates of 10 µl/hr ( manufacturer's website ) are in the same order of magnitude as the 200 nl/min used in Kellogg and Tay ( 2015 ) .", "We also generated “sawtooth-like” profiles as in Kellogg and Tay ( 2015 ) by periodically replacing the medium with a 15 min pulse and stopping the flow .", "However , the cell-to-medium ratio in our culture chambers might be different and relevant for TNF clearance due to cell-linked decay needed for the sawtooth profile .", "Live cell imaging of GFP-p65 knock-in MEFs was performed using a Leica TCS SP5 confocal microscope with an incubation system where cells were stably maintained at 37°C in 5% CO2 .", "Time-lapse images were acquired at 6 min intervals for more than 15 hr .", "We used a low magnification objective ( 20x , 0 . 5 NA ) and an open pinhole ( Airy 3 ) , ensuring that the image depth ( 10 . 7 µm ) contains the thickness of the whole cell so that images are a record of the total cell fluorescence .", "GFP-p65 is imaged with the 488 nm Argon laser ( GFP channel ) while Hoechst 33342 stained nuclei are imaged with the low energy 405 nm UV diode laser at 5% of its maximum intensity ( HOE channel ) .", "Images were acquired as 16 bit , 1024×1024 , TIFF files .", "Experiment replicates were acquired on different days starting from different batches of frozen cells ( samples provided as Videos 1–5 ) .", "Nuclei were stained with Hoechst 33342 for imaging , segmentation and tracking ( Zambrano et al . , 2014a ) without any interference with the natural signalling dynamics ( see below for a description of the controls performed ) .", "Stress from photo-damage during live-cell imaging can hamper cell biology studies ( Cole , 2014 ) , while nuclear dyes can interfere with the natural signaling dynamics ( Ge et al . , 2013; Martin et al . , 2005 ) .", "Hence different controls were performed to exclude distortions of the natural signalling in our imaging conditions .", "Staining was performed according to manufacturers’ instructions .", "For WB , we used an anti p65 Rab ( dil 1:1000 , #sc-372 C20 , Santa Cruz; Figure 1—figure supplement 10 ) .", "For immunostaining we used anti gammaH2AX Mab ( dil . 1:500; #05-636 , Millipore; Figure 1—figure supplement 7 ) and anti thymine dimers TDM-2 ( dil .", "1:2000 , Cosmo Bio , Japan ( Komatsu et al . , 1997 ) ; Figure 1—figure supplement 6 ) .", "UV-photodamage was induced with 254 nm UV irradiation using an UVC 500 Crosslinker , Amersham .", "Panels in Figure 1—figure supplements 6 and 7 are representative of 3 independent experiments .", "The following short description summarises the whole process in few sentences .", "Each passage will be then thoroughly described in the next paragraphs .", "The dynamics were quantified by computing the nuclear to cytoplasmic ratio of the intensities ( NCI ) of single cells , which is a measure robust against distortions and reflects faithfully the oscillations in the nuclear concentration of NF-κB oscillatory dynamics .", "This holds true provided that the total amount of p65 is constant , as we show for our cells under stimulation ( see Figure 1—figure supplement 9 ) , and is also assumed by mathematical models ( see Hoffmann et al ( 2002 ) and the more recent in Kellogg and Tay ( 2015 ) ) .", "Interestingly , p65 is not constant in macrophages stimulated with LPS ( Sung et al . , 2014 ) .", "The analysis of time series relies on the detection of significant peaks , performed following our previously discussed procedure ( Zambrano et al . , 2014a ) that allows to distinguish significant peaks from noisy peaks .", "Once peaks are detected we assign them a phase: 2π for the “maxima” and π for the “minima” between peaks .", "In order to assess the oscillations for a larger number of cells , we optimized our software ( Zambrano et al . , 2014a ) to calculate the nuclear to cytoplasmic ratio of the intensity ( NCI ) of NF-κB signal for hundreds of cells; NCI is a quantifier that has been used by other groups ( Ashall et al . , 2009; Nelson et al . , 2004 ) .", "As we argue below , thanks to the fact that the total amount of NF-κB is constant ( Figure 1—figure supplement 10 ) , NCI depends univocally and monotonically on the nuclear amount of NF-κB .", "More importantly , due to the fact that it is a ratio of intensities , it is robust and independent of slight changes in the focus and in the laser intensity , among other possible experimental distortions , which are observed in our setup .", "The same rationale brought us to use ratios of intensities in our previous works ( Sung et al . , 2009; Zambrano et al . , 2014b ) .", "Importantly , these distortions imply that the background-adjusted mean nuclear intensity used in other studies ( Kellogg and Tay , 2015; Lee et al . , 2014; Sung et al . , 2014 ) , although advantageous for other reasons ( it only requires the segmentation of the nuclei and estimation of the background ) would not be appropriate in our imaging experimental setup .", "We provide below a more detailed argumentation of these ideas .", "Following the notation of Zambrano et al ( 2014b ) we have that the intensity measured in pixel p of the image at time t in a time lapse experiment can be described as: ( Q1 ) I ( p , t ) =A ( p , t ) P ( p , t ) +Bp , t where P ( p , t ) is the amount of NF-κB in pixel p , A ( p , t ) is the amplification coefficient between the protein brightness and the amount of protein and B ( p , t ) corresponds to the background .", "In our experiments with time-varying intensities , it is clear that both A ( p , t ) and B ( p , t ) vary in time , and presumably also in space , due to laser variations and/or slight variations in the illumination uniformity .", "Our quantifier NCI , that we estimate using our software , is the ratio of the background corrected average intensities in the nucleus and the cytoplasm I ( t ) nuc and I ( t ) cyto respectively , and can be defined using this notation as: ( Q2 ) NCI ( t ) ≡I ( t ) nucI ( t ) cyto=ScytoSnucleus∑p∈nucleusI ( p , t ) -B ( p , t ) ∑q∈cytoplasmI ( q , t ) -B ( q , t ) =ScytoSnucleus∑p∈nucleusA ( p , t ) P ( p , t ) ∑q∈cytoplasmA ( q , t ) P ( q , t ) so we have that , if we consider that A ( p , t ) is approximately constant and equal to certain A ( t ) in the area occupied by the cell ( as it is in our images ) : ( Q3 ) NCI ( t ) ≅ScytoSnucleusA ( t ) ∑p∈nucleusP ( p , t ) A ( t ) ∑p∈cytoP ( q , t ) =ScytoSnucleus ( NF−κBnuc ( t ) ) ( NF−κBcyto ( t ) ) where Scyto and Snuc are the areas occupied by cytoplasm and the nucleus , respectively .", "Our quantifier is hence a good indicator of the ratio of the amount of NF-κB in the nucleus and in the cytoplasm .", "However the numerator and denominator of this ratio can fluctuate due to biological reasons and this might blur the existence of oscillations in the nuclear amount of NF-κB .", "But this is not the case , due to the fact that the total amount of NF-κB is constant for our stimulated fibroblasts .", "This has been assumed in different mathematical models present in the literature , from the seminal paper ( Hoffmann et al . , 2002 ) to more recent papers ( Kellogg and Tay , 2015 ) .", "Interestingly , though , this view has been challenged by the recent work of Sung et al . ( 2014 ) , which shows that in macrophages under LPS stimulation p65 is regulated by a positive feedback .", "Thus , to confirm that our assumption is reasonable , we quantified p65 by western blotting for our cells under 10 ng/ml TNF-α at several timepoints from 1 to 8 hr .", "The results are shown in ( Figure 1—figure supplement 10 ) , showing no significant change in the p65 levels upon stimulation .", "Hence , we consider our assumption valid .", "This implies that: ( Q4 ) NCI ( t ) =ScytoSnucleus ( NF−κBnuc ( t ) ) ( NF−κBTOT ) − ( NF−κBnuc ( t ) ) hence we can see that NCI ( t ) depends monotonously on ( NF−κBnuc ( t ) ) , see Figure 1—figure supplement 9B .", "For this reason , it is intuitively clear that each local maximum or minimum of ( NF−κBnuc ( t ) ) leads to a local maximum or minimum of NCI ( t ) .", "Hence , oscillations in the nuclear amount of NF-κB will be observed also using NCI ( t ) .", "We can put this mathematically by saying that oscillations in the nuclear concentration of NF-κB occur at times t for which the condition ( NF−κBnuc ( t ) ) '=0 .", "Similarly , oscillations in NCI will depend on the value of the derivative of NCI , that is: ( Q5 ) NCI' ( t ) ∝ ( ( NF−κBTOT ) − ( NF−κBnuc ( t ) ) +1 ) ( ( NF−κBTOT ) − ( NF−κBnuc ( t ) ) ) 2 ( NF−κBnuc ( t ) ) ' so from the above formula it is easy to see that: ( Q6 ) NCI' ( t ) =0↔ ( NF−κBnuc ( t ) ) '=0 Overall , then , imaging , mathematical and biochemical arguments confirm that computing NCI ( t ) is an adequate way to quantify oscillatory dynamics .", "Concerning the background-corrected average intensity , using the above notation it would be calculated as: ( Q7 ) I ( t ) nucleus=1Snucleus∑p∈nucleusI ( p , t ) −B ( p , t ) =1Snucleus∑p∈nucleusA ( p , t ) P ( p , t ) thus I ( t ) nucleus∝∑p∈nucleusP ( p , t ) =NF−κBnuc ( t ) for all t only if A ( p , t ) were constant in all time frames .", "As argued previously , this is not the case in our setting and that’s the reason why we preferred to use NCI .", "Finally , variations in the areas of the nucleus and the cytoplasm might introduce small distortions , although they typically vary seldom and slowly .", "Notice though that our software discards cells for which the areas change abruptly , which typically is an indicator of imminent mitosis or cell death .", "We cannot totally exclude variations of p65 at single cell level , provided that we measured it using a population assay; however , p65 turnover is known to be long , so its slow variation would contribute with a small term in the derivative and thus would just slightly distort the times for which peaks in NCI appear compared to those of the nuclear concentration .", "To conclude , we think that NCI would still capture the peaks and hence would be able to assess the cell's oscillatory state , which is our aim here .", "The data provided confirm the validity of NCI as a quantifier .", "First , we have that NCI ( t ) remains reasonably constant – except for some spontaneous oscillations , as previously reported ( Zambrano et al . , 2014b ) for cells that are not stimulated , as shown in Figure 1—figure supplement 9A .", "This is remarkable because the movie from which these series were obtained present considerable variation in the image intensity ( see the Video 2 ) .", "Along the same lines we do not find upwards or downwards average trends in our data of NCI time series for different conditions discussed in the main figures and in the figure supplements , which indicates further that the time series are properly normalized and reflect well the dynamics .", "As an additional confirmation , we have plotted together the average nuclear intensity and the NCI values obtained from manual segmentation in Figure 1—figure supplement 9C , that show the same kind of qualitative behavior as NCI but with different trends , as expected .", "A last indicator of the sensitivity of the quantifier comes from its ability to detect even small oscillatory peaks , see Figure 3 and Figure 3—figure supplement 1 as an example .", "Overall , then , NCI is a faithful measure of the oscillatory state of our cells .", "The major advantage of considering NCI with respect to the nuclear to total ratio used in Zambrano et al ( 2014a ) is that it does not require a perfect segmentation of the cytoplasm , which might be complicated when cells touch each other .", "We developed a software that uses this quantifier and is thus able to multiply by a factor of 2 the number of cells tracked , and by a factor of 1 . 5 the average tracking time with respect to the one described in Zambrano et al ( 2014a ) .", "The software used to calculate NCI is provided as source code and works as follows: for each time-lapse experiment , we have N frames images in the HOE channel and in the GFP channel , respectively .", "Nuclei were segmented and used for cell tracking following the procedure described in Zambrano et al ( 2014a ) .", "In order to estimate the average cytoplasmic intensity , the background was computed by taking a square area centred on the cell nucleus , dividing it in tiles and using the one with the smallest average intensity in the GFP channel to estimate the background intensity ( this procedure gives values compatible with the values obtained using a clustering-based algorithm ) .", "Points belonging to the cytoplasm are those around the nucleus in a size window equal to 1 . 5 times the size of the nucleus .", "The average cytoplasmic intensity is computed from the intensity in the GFP channel of the pixels from this “ring” .", "An example is shown in Figure 1—figure supplement 1D .", "Dividing or apoptotic cells were identified assessing their geometrical features ( abrupt changes in size of the nucleus and of the “cytoplasmic ring” ) and discarded automatically .", "To analyse the resulting NCI time series , we adapted our peak detection algorithm ( Zambrano et al . , 2014a ) .", "We consider peaks as a sequence of a local minimum , local maximum and local minimum .", "The value of the peak θ is defined by the height of the peak ( from the highest minimum to the maximum ) .", "A peak defined by only three consecutive time points is considered a noise peak .", "We plot the distribution of the noise peak values obtained from our unperturbed and chronic stimulation time series ( Figure 1—figure supplement 2A ) , for which we observe our previously reported spontaneous activations ( Zambrano et al . , 2014a ) .", "We find that these noisy peaks have a low value , and hence by considering as significant those with a value over θ>0 . 15 , we find a reasonably good compromise between the need to ignore noise peaks and the need to detect small peaks of valuable dynamical information .", "This was tested in a number of time series , and an example of time series with the significant peaks detected using this threshold is shown in Figure 1—figure supplement 2B .", "Calculations of magnitudes inferred from peaks take advantage of time series from cells that were tracked for at least 7 hr .", "Following ( Mondragon-Palomino et al . , 2011 ) we used the peaks to assign a phase value: peak maxima are assigned a phase 0 ( 2π ) and a phase π is assigned to peak minima .", "This was of particular interest to obtain an assessment of the oscillatory modes present in our system , provided that peaks can be very heterogeneous and plotting the peak height in colour-plots might make it difficult to appreciate the smaller ones and hence the possible resonant oscillatory patterns .", "An example of this transformation is shown in Figure 1—figure supplement 2C , which is the phase derived from the peaks obtained of the time series displayed in Figure 1—figure supplement 2B .", "As in Mondragon-Palomino et al ( 2011 ) we use the phase difference between the time series and the forcing to quantify the degree of synchrony .", "The phase difference Δφ was calculated from the timing ΔT between the beginning of each forcing cycle and the closest significant peak , as Δφ=2πΔT/Tf , where Tf is the forcing period .", "The entropy of the distribution of the phase is calculated as: S=−∑kpklogpk where pk is the probability of the kth bin ( we use eight bins for all the conditions considered ) .", "The synchrony intensity is then computed as: η=1−S/Smax where Smax is the value obtained for equal values of pk .", "Total RNA was isolated using the IllustraRNAspin Mini kit ( GE Healthcare ) , and complementary DNA ( cDNA ) was obtained by retro-transcription with Random Hexamers and SuperScript II Reverse transcriptase ( Invitrogen ) following the manufacturers’ instructions .", "Primers for the detection of mature transcripts were designed in adjacent exons and spanned the intervening intron; primers for nascent transcripts were located across an exon-intron boundary .", "Primer sequences are listed in Supplementary file", "1 . Quantitative real-time PCR was performed with SYBR Green I protocol using the LightCycler480 ( Roche ) .", "The results are shown as averages of three technical replicates .", "Analysis was performed with the –ΔCt method corrected for primer efficiencies ( Vandesompele et al . , 2002 ) and normalised with two reference genes ( Actb and Rplp1 ) ( Nordgard et al . , 2006 ) .", "Experiments were repeated twice .", "It is important to emphasize that we did not stain our cells nor did we illuminate them with our UV laser in our transcription experiments , neither in the RT-PCR assays nor in the Microarray experiments described below .", "The controls described previously show that the nuclear labelling and the imaging do not interfere with NF-κB signalling dynamics , so we expected the same for NF-κB-driven transcription .", "To further confirm this , we compared RT-PCR quantifications of transcription in cells stimulated with TNF and cultured in imaging medium ( 0 . 1% FCS+Hoechst ) or standard MEF medium ( 10% FCS ) .", "The results for nascent and mature IκBα and Ccl5 transcripts reported in Figure 5—figure supplement 2 show no difference in the transcriptional response in the two conditions .", "RNA samples were extracted using the IllustraRNAspin Mini kit ( GE Healthcare ) .", "Following extraction , RIN ( RNA Integrity Number ) was >9 ( BioAnalyser , Agilent RNA Nano Kit ) .", "RNA samples ( 500 ng ) were reverse transcribed with the IlluminaTotalPrep RNA Amplification Kit ( Ambion ) and copy RNA ( cRNA ) was generated with 14 hr in vitro transcription reactions and checked at the BioAnalyser .", "Washing , staining , and hybridization were performed according to standard Illumina protocols .", "cRNA samples were then hybridized to IlluminaBeadChip Array MouseRefSeq-8 v2 .", "BeadChips were scanned with BeadArray™ Reader in channel", "2 . The data have been uploaded on the Dryad Digital Repository ( Zambrano et al . , 2016 ) .", "Experiments were repeated twice .", "Genome Studio’s bead summary probe level data – not normalized and not background corrected – were analyzed using Bioconductor .", "We performed quality assessment by plotting the intensities of regular and control probes .", "Sample intensities were quantile normalized and filtered for expression and probe quality using beadarray R package: only probes with detection P-value <0 . 05 in at least one condition and whose categories is defined “Perfect” or “Good” were kept .", "Probes were labelled as deregulated if their absolute log2 fold change relative to the 0 time point were greater than", "1 . Clustering was performed applying a soft clustering of deregulated probes with the Mfuzz R package , which is suggested for microarray time course-data ( Kumar and Futschik , 2007 ) .", "The optimal number of clusters was assessed with the d . min function .", "To focus on the shape of the gene expression profiles , we standardised the gene expression profiles by taking the usual base-two logarithm and normalizing to obtain mean 0 and standard deviation", "1 . The algorithm then groups genes based on the Euclidean distance between profiles and the c-means objective function , which is a weighted square error function .", "Each gene is assigned a membership value between 0 and 1 for each cluster .", "Hence , genes can be assigned to different clusters in a gradual manner .", "The parameters m defines the degree of “fuzzification” .", "It is defined for real values greater than 1 and the bigger it is the more fuzzy the membership values of the cluster .", "We used an m value estimated by the “mestimate” function of 1 . 5 .", "Membership values indicate the similarity of vectors to each other defining a cluster cores .", "To extract list of genes belonging to the cluster cores , we used the “acore” function taking from each clusters all genes with a membership value of at least 0 . 5 .", "Pathway analysis of genes contained in each cluster was performed with the ClusterProfiling R packages using an adjusted p-value cut-off for enrichment <0 . 2 based on the hypergeometric distribution ( Yu , 2015 ) .", "We used for our analyses the Reactome database .", "In order to simplify the resulting output we plotted only the top five categories of the second and third level ( Yu , 2015 ) .", "Conversely , overlaps were calculated based on all categories of the second and third levels .", "The overlap coefficient ( or Szymkiewicz-Simpson coefficient ) between gene sets X and Y is given by: overlap ( X , Y ) =|X ∩ Y|min ( |X| , |Y| ) For statistical significance we performed a Fisher's Exact Test for evaluating if the resulting clusters where enriched for NF-κB's targets .", "The resulting p-value has been converted in a significance measure ( -Log10 ( p value ) ) .", "We used both a list taken from Li et al ( 2014 ) and from Thomas Gilmore’s website , Boston University ( http://www . bu . edu/nf-kb/gene-resources/target-genes/ ) .", "We propose here a mathematical model based on the one discussed in Zambrano et al ( 2014b ) that adds the layer of regulation by considering the negative feedback provided by the protein A20 that blocks IKK activation upon stimulation ( Figure 1—figure supplement 4 ) .", "For the sake of completeness , we describe briefly below the basic process that we considered , together with the biochemical rates involved ( values are given in Supplementary file 2 ) and a summary of our normalization procedure .", "Additional details on the motivations underlying certain selection of biochemical reactions and variables of interest can be found in that paper .", "Being a simple model , it provides important qualitative insights on the possible variety of single-cell dynamics , but we also use it to provide quantitative fittings of the average population dynamics and transcription .", "The biochemical reactions described here are essentially the basic ones given in the simple model given in Zambrano et al ( 2014b ) .", "The basic simplification of our model is to consider that free NF-κB is nuclear , while the complex with the inhibitor IκBα is cytoplasmic .", "The formation of the complex is given by ( NF-κB:IκBα ) NF−κB+IκBα→A ( NF−κB:IκBα ) that can also spontaneously dissociate ( NF−κB:IκBα ) →dNF−κB+IκBα An external signal can lead to the appeareance of IKKa that can free NF-κB by degrading of IκBα in the complex ( NF−κB:IκBα ) +IKKa→PNF−κB+IKKa and in its free form IκBα+IKKa→κPIKKa .", "The negative feedback loop is enabled by the fact that the gene encoding IκBα , Gα , can be activated by NF-κB Gα , OFF+NF−κB→Kon , IGα , ON+NF−κB while the inactivation is modulated by Gα , ON+IκBα→Koff , IGα , OFF+IκBα .", "Notice that we are assuming here that IκBα plays a role as transcriptional repressor , as suggested by experimental results ( Arenzana-Seisdedos et al . , 1995 ) and used in some mathematical models ( Kellogg and Tay , 2015; Lipniacki et al . , 2007; Tay et al . , 2010 ) , but not others ( Ashall et al . , 2009; Sung et al . , 2014 ) .", "We show later that this does not have a strong impact on the fittings , but to facilitate the correspondence with our previous work and in line with the mentioned modelling approach , we opt to keep this process for all the gene inactivations considered in most of our explorations .", "We only briefly explore the effect of setting the koff=0 in Figure 6—figure supplement 7 , results are detailed in the text .", "We also consider that the gene can be basally activated Gα , OFF→kon0 , IGα , ON and inactivated Gα , ON→koff0 , IGα , OFF Transcription ( mRNA production ) is given by Gα , ON→KRIGα , ON+IκBαRNA while the RNA degradation is given by IκBαRNA→dRI∅ .", "IκBα translation is given by IκBαRNA→KIIκBα while its spontaneous degradation is given by IκBα→dI∅ , a degradation that is also possible while it is forming the complex ( NF−κB:IκBα ) →γdINF−κB We have observed experimentally that even for constant stimulation different dynamics are possible , with trajectories showing different degrees of dampening and sometimes even irregular and non-oscillating profiles ( Figure 1—figure supplement 2D ) as pointed out in previous works ( Sung et al . , 2009; Zambrano et al . , 2014b ) .", "Our relatively simple model illustrates why this situation might arise and how a wider variety of dynamics might arise compared to the regular oscillations documented in the literature for more complicated models ( e . g . ( Ashall et al . , 2009; Tay et al . , 2010 ) ) .", "To illustrate this , we analyzed the dynamics for a constant external stimulus ( S ( t ) =2 h-1 ) and varying simultaneously each parameter of the parameter set PNF-κB according to their uncertainty degree D . We used them to generate trajectories and selected those giving rise to a response , i . e . those for which N ( t ) is bigger than 0 . 4 in the first 3 hr and whose average values in the last hours was smaller or equal than N=0 . 4 .", "To characterized in a systematic way their dynamics , we located the fixed point of Equations 1–8 and calculated the eigenvalues {λi} ( i=1 , . . . 8 ) of the Jacobian .", "In Figure 1—figure supplement 5A we plot the real and imaginary parts of each set of eigenvalues .", "We represent with a red dot the eigenvalues belonging to a set in which the real part of at least one of them is bigger than zero .", "This means that the fixed point for those parameters is unstable and hence trajectories converge to a stable limit cycle around it .", "In other words , for those parameter combinations sustained oscillations arise .", "The percentage of parameter sets giving sustained oscillations is below 10% ( Figure 1—figure supplement 5B , right panel ) , which make us conjecture that parameters giving rise to sustained oscillations are not the norm .", "We also plot in Figure 1—figure supplement 5C the parameter values giving rise to oscillating and non oscillating ( but responding ) trajectories .", "We note that the intervals are very similar except for parameters n and dA involved in the A20 negative feedback , which indicates that this feedback is critical in order to obtain a sustained or a damped oscillatory response .", "In previous works it was shown experimentally and through mathematical models that the interplay between the IκB and A20 regulatory modules regulate different phases of NF-κB response ( Werner et al . , 2008 ) .", "Our numerical results suggest that it also plays a key role in the type of dynamics observed ( oscillatory versus damped ) .", "Overall , this analysis further hints to the fact that it is the precise combination of parameters , rather than the precise single values , what determines the type of dynamics observed and might account for the different dynamics observed with respect to other groups ( Ashall et al . , 2009; Cheng et al . , 2015; Kellogg and Tay , 2015; Lee et al . , 2009; Sung et al . , 2009; Tay et al . , 2010 ) .", "Finally , we have to notice that being the system given by Equations 1–8 a dynamical system with real variables , the imaginary eigenvalues come in complex conjugate pairs .", "The distribution of parameter combinations with 2 , 4 and 6 imaginary eigenvalues are shown in Figure 1—figure supplement 5B ( left panel ) .", "Notice that an oscillatory frequency can be associated to each couple of complex conjugate pairs ( Goldstein et al . , 2001 ) , so having more than one of such couples means that the dynamics will combine different frequencies .", "To further illustrate all this , in Figure 1—figure supplement 5D we present examples of the variety of trajectories found .", "Some of them are oscillating ( red ) , showing both smooth and spiky oscillatory peaks .", "Many others are damped and in some cases the concurrence of two oscillating timescales , fast and slow , is evident .", "Some others present clearly non-oscillating profiles .", "Overall , our numerical exploration of our relatively simple models shows that different dynamics are possible in presence of constant stimulation , as observed in the experiments of this and previous works ( Sung et al . , 2009; Zambrano et al . , 2014a ) .", "The model simulation routine was implemented in C code language and compiled using gcc .", "Routines for parameter variation and time series analysis and stability analysis ( using the function fsolve of the Nonlinear Equations package ) where written using GNU Octave .", "Routines were written in GNU octave to fit the model to the data by combining Markov Chain Monte Carlo for initial exploration of the parameter space and a Levenberg–Marquardt algorithm .", "Parameters of Equations 1–8 were varied within the specified uncertainty degree .", "The goal is to minimize the distance between a given goal signal X={X ( tk ) } for k=1 , . . . , N and the theoretical values x={x ( tk ) } obtained from the model .", "We define the distance of a given fitting to the data as: dX , x=1N∑kX ( tk ) −xtkmax ( X ( tk ) , x ( tk ) ) When several data are fitted simultaneously , the total distance between model predictions and the data were obtained by summing each distance .", "Notice that the 1/N factor weighs for difference in the length of the data .", "For a given computations , this distance gives the average relative error per timepoint of the fitting .", "- To fit NCI , theoretical NCI profiles from the model were obtained as: NCI ( t ) =ScytoSnucleusN ( t ) 1−N ( t ) .", "where Scyto/Snuc is the ratio of the nuclear to total area of the cells in our image , which is estimated to be of around 1/3 .", "For parameter fitting of the gene expression obtained from Quantitative Real-Time and microarray experiments ( see below ) , the fold change levels predicted by the models where computed as R ( t ) /R ( 0 ) .", "For the simultaneous fitting of NCI , IκBα and Cccl5 mRNA shown in Figure 5 , common PNF-κB parameters where found while for IκBα and CCL5 mRNA we independently fitted the respective set of parameters PG .", "For the fitting of PNF-κB the parameters were varied within the limits given by their uncertainty degree .", "Fitting of the microarray data was performed by keeping constant the parameters PNF-κB obtained fitting the data of Figure 5 and varying PG ( those of ( 9–10 ) ) for each gene , as we did for IκBα and Cccl5 .", "Examples are shown in Figure 6—figure supplement 6 .", "Figure 6—figure supplement 5 shows that the distance between fitting and real data is better for genes with increased transcription with an average fitting error of 9% ." ] ]
[ "Several transcription factors ( TFs ) oscillate , periodically relocating between the cytoplasm and the nucleus .", "NF-κB , which plays key roles in inflammation and cancer , displays oscillations whose biological advantage remains unclear .", "Recent work indicated that NF-κB displays sustained oscillations that can be entrained , that is , reach a persistent synchronized state through small periodic perturbations .", "We show here that for our GFP-p65 knock-in cells NF-κB behaves as a damped oscillator able to synchronize to a variety of periodic external perturbations with no memory .", "We imposed synchronous dynamics to prove that transcription of NF-κB-controlled genes also oscillates , but mature transcript levels follow three distinct patterns .", "Two sets of transcripts accumulate fast or slowly , respectively .", "Another set , comprising chemokine and chemokine receptor mRNAs , oscillates and resets at each new stimulus , with no memory of the past .", "We propose that TF oscillatory dynamics is a means of segmenting time to provide renewing opportunity windows for decision ." ]
[ "The process of producing useful biological molecules from genes – known as gene expression – is not always simple .", "Many genes are part of complex circuits , some of which show regular patterns of activity in response to an environmental cue .", "For example , the expression of some genes is tied to the 24-hour daily cycle of light and dark .", "Transcription factors are proteins that control gene activation and expression , and some transcription factors periodically move in and out of the cell’s nucleus – the compartment of an animal cell that houses the vast majority of the genetic material .", "This behavior is known as oscillation .", "A transcription factor called NF-κB oscillates , changing between an inactive form outside of the nucleus and an active form inside .", "NF-κB plays important roles in inflammation and cancer , and is activated by cues from outside the cell .", "Some of the genes that the active form of NF-κB activates then produce molecules that inactivate NF-κB , thus helping to establish the oscillations .", "The benefits of the oscillations are not clear .", "However , recent studies suggest that environmental cues can cause small perturbations that gradually adjust the rate at which the oscillations occur , and in doing so , synchronize the oscillations amongst neighboring cells .", "By using embryo cells from genetically engineered mice , Zambrano et al . investigated how NF-κB oscillations get synchronized .", "The experiments showed that the activity of the NF-κB protein and the expression of the genes it controls synchronize across neighboring cells whenever the external environmental perturbations come in pulses .", "However , once the pulsed cues stop , this synchronization is quickly lost .", "In essence , the cells reset after each environmental cue with no memory of previous episodes of NF-κB activity .", "Further work revealed that the expression of the genes controlled by NF-κB also cycles and resets with each new environmental cue .", "However , the products of these genes accumulate in three different ways .", "Some accumulate quickly; some accumulate at a slow and steady pace; and some oscillate in amount , and this amount resets once the environmental cue has stopped .", "Each of these classes of gene products can be related to specific cell behaviors that activate sequentially on well-defined time schedules .", "Overall , Zambrano et al . suggest that the ability of NF-κB to reset its activity with each new environmental cue gives cells the opportunity to pause and adjust course .", "Zambrano et al . now plan to explore what happens to NF-κB synchronization in different cell types exposed to a collection of inflammatory stimuli .", "Along the same line , it will be worth exploring NF-κB behavior in cancer cells , where NF-κB activity is often out of control and drives unrestrained cell proliferation .", "These studies would contribute to a deeper understanding of cancer biology and to the identification of new treatments for the disease ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics", "microbiology and infectious disease" ]
Guanylate binding proteins directly attack Toxoplasma gondii via supramolecular complexes
elife-11479-v2
[ [ "IFNγ is an immunomodulatory cytokine that rapidly activates potent host cell effector mechanisms to confront a variety of intracellular pathogens ( Decker et al . , 2002 ) .", "Some of the most abundantly IFNγ induced proteins are the 65-kDa guanylate-binding proteins ( GBPs ) , which mediate cell-autonomous immunity ( MacMicking , 2012; Degrandi et al . , 2013; Pilla et al . , 2014; Meunier et al . , 2015 ) .", "GBPs are related to the dynamin super family of GTPases ( Praefcke and McMahon , 2004 ) and are highly conserved throughout the vertebrate lineage ( Vestal and Jeyaratnam , 2011 ) .", "The human genome harbors seven GBPs and at least one pseudogene , whereas the mouse genome contains 11 GBPs and two pseudogenes ( Kresse et al . , 2008; Olszewski et al . , 2006 ) .", "The gene loci of murine GBPs ( mGBPs ) are tandemly organized in clusters on chromosomes 3 and 5 ( Degrandi et al . , 2007; Kresse et al . , 2008 ) .", "GBPs contain a conserved GTPase-domain which binds guanine nucleotides with low affinities .", "This induces nucleotide dependent GBP multimerization and cooperative hydrolysis of GTP via GDP to GMP ( Praefcke et al . , 2004; Ghosh et al . , 2006; Kravets et al . , 2012; Prakash et al . , 2000b ) .", "Some GBPs are isoprenylated , endowing them with the ability to associate with intracellular membranous compartments ( Vestal et al . , 2000; Degrandi et al . , 2013 ) .", "Murine GBPs ( mGBPs ) exert a major impact on cell-autonomous restriction of Toxoplasma gondii ( Yamamoto et al . , 2012; Degrandi et al . , 2007; Selleck et al . , 2013; Degrandi et al . , 2013 ) .", "T . gondii is an apicomplexan protozoan parasite with a broad host range , is distributed worldwide and causes serious and often fatal infections in immunocompromised hosts ( Gazzinelli et al . , 2014 ) .", "T . gondii infection experiments in mice deficient for a cluster of mGBPs on chromosome 3 ( Yamamoto et al . , 2012 ) or solely for mGBP1 or mGBP2 ( Degrandi et al . , 2013; Selleck et al . , 2013 ) prove that mGBPs are essential immune effector molecules mediating antiparasitic resistance .", "In several cell types distinct mGBPs accumulate at the parasitophorous vacuole membrane ( PVM ) of T . gondii ( Degrandi et al . , 2007; Kravets et al . , 2012; Degrandi et al . , 2013 ) .", "In previous studies , introduction of point mutations into the key positions of the conserved motifs of the GTPase-domain ( R48A , K51A , E99A , D182N ) and the isoprenylation site of mGBP2 ( C586S ) , clearly showed that nucleotide binding , multimerization , GTP-hydrolysis and membrane anchoring , are essential for localization in vesicle-like structures ( VLS ) and for the recruitment of mGBP2 to the PVM of T . gondii ( Kravets et al . , 2012; Degrandi et al . , 2013 ) .", "However , the assembly of homo- and hetero-mGBP multimers , their composition in distinct subcellular compartments , localization-dependent multimerization as well as their requirement for replication control of T . gondii in living cells remained enigmatic .", "Therefore quantitative live-cell-imaging technologies were employed revealing seminal information on localization , interaction , concentration , structure and dynamics of biomolecules .", "To investigate the structure , composition and interaction of proteins , Förster resonance energy transfer ( FRET ) ( Giepmans et al . , 2006 ) is combined with Multiparameter fluorescence image spectroscopy ( MFIS ) ( Kudryavtsev et al . , 2007; Weidtkamp-Peters et al . , 2009 ) , which enables unique advances in FRET imaging .", "In MFIS , a variety of fluorescence parameters is monitored simultaneously with picosecond accuracy , allowing the determination of many fluorescence parameters in a pixel-wise analysis such as number of photons , anisotropies , fluorescence lifetimes , and signal ratios by statistically most efficient estimators ( Sisamakis et al . , 2010 ) and to plot distinct parameters in MFIS pixel frequency histograms .", "The combination of MFIS and FRET experiments ( MFIS-FRET ) enables a quantitative analysis of the biophysical properties of homomeric and heteromeric molecular complexes in living cells ( Stahl et al . , 2013 ) .", "This allows the identification and selection of pixel populations with unique properties for a detailed pixel-integrated analysis .", "Importantly , live cell measurements with MFIS can achieve the resolution and precision of traditional in vitro measurements of molecule ensembles with respect to the number of resolved species and rate constants .", "Here , by advanced biophysical MFIS-FRET technology , it is demonstrated that the GTPase activity and isoprenylation of mGBP2 are prerequisites for its multimerization .", "The multimerization is essential for control of T . gondii replication .", "Colocalization and MFIS analysis of mGBPs showed intermolecular interaction of mGBP2 with itself , with mGBP1 and mGBP3 , but not with mGBP6 in VLS in living cells .", "Interestingly , the interaction partnerships were recapitulated at the PVM of T . gondii .", "Moreover , characteristic interaction affinities of mGBP complexes were individually quantified .", "For the first time , we show that in the process of attacking T . gondii , mGBP2 directly targets the plasma membrane of the parasite after disruption and permeabilization of the PVM .", "These investigations enable a discrete understanding of the dynamics and intracellular interactions of mGBP effector molecules in T . gondii host defense ." ], [ "Site-directed mutagenesis of mGBP2 revealed that GTP-binding and hydrolysis as well as C-terminal isoprenylation affect the localization of mGBP2 in the cell ( Degrandi et al . , 2013; Kravets et al . , 2012 ) .", "However , the role of the GTPase activity and isoprenylation on the multimerization ability of mGBP2 in living cells is unknown .", "Therefore , MFIS-FRET measurements and fluorescence-anisotropy-based homo-FRET analysis were employed in living IFN-γ stimulated mGBP2-/- MEFs reconstituted either with GFP-fused mGBP2 WT protein ( hereafter referred to as G-mGBP2 MEFs ) or with one of the GTPase-domain mutants ( R48A , K51A , E99A , D182N ) or with the isoprenylation mutant ( C586S ) ( Figure 1a ) . 10 . 7554/eLife . 11479 . 003Figure 1 . Intracellular homo-multimerization of WT and mutant mGBP2 . All cells were pre-treated with IFNγ for 16 hr prior investigation", "( a ) Left panel .", "GFP fluorescence intensity ( SG , G ) images of GBP2-/- MEFs expressing G-mGBP2-WT ( G-mGBP2 MEFs ) , mutants ( R48A , K51A , E99A , D182N , C586S ) or GFP highlighted with selections of pixels within different cellular compartments .", "Right panel .", "MFIS 2D-histograms of GFP anisotropy ( rD ) on x axis vs . photon number per pixel on y axis , the frequency of pixels color coded from white ( lowest ) to black ( highest ) .", "This allows the identification and selection of pixel populations with unique fluorescence properties for a detailed subsequent pixel integrated analysis .", "The pixels with low photon numbers ( below 1000 ) are selected in red boxes ( defined as cytosol ) and those with more than 1000 photons in green boxes ( defined as VLS ) .", "Bars , 10 µm .", "( b ) Scheme of the principle of homo-FRET assays .", "Compared to G-mGBP2 monomers , rD in G-mGBP2 multimers decreases due to depolarization of GFP fluorescence while GFP SG , G increases .", "( c ) For specific compartments ( cytosol and VLS , respectively ) , the anisotropy values are averaged over all cells generally denoted as <rD>loc .", "<rD>loc and SG , G in cytosol and VLS were plotted for G-mGBP2-WT , and the K51A mutant and GFP in the cytosol .", "( d ) Mean anisotropy of averages over whole cells <rD>cell for G-mGBP2 WT and mutant proteins .", "GFP expressing cells served as controls ( ***p<0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 00310 . 7554/eLife . 11479 . 004Figure 1—figure supplement 1 . Biochemical properties and intracellular localization of the C586S mutant of mGBP2 .", "( a ) Nucleotide binding .", "A solution containing 0 . 5 µM mant-GTPγS , mant-GDP and mant-GMP was titrated with C586S mutant of mGBP2 .", "The fluorescence was excited at 355 nm and detected at 448 nm .", "The values were normalized to the fluorescence of the nucleotide alone .", "Dissociation constants are calculated from the fit of the binding curves as in ( Kravets et al . , 2012 ) .", "The results averaged over two to four experiments each are given in the Table 1 .", "( b ) GTP-hydrolysis .", "Concentration-dependent GTP-hydrolysis catalyzed by the C586S mutant was measured with a fixed concentration of GTP ( 1 mM ) at 37°C .", "The initial rates were measured ( < 30% GTP hydrolyzed ) from the linear parts of time-course experiments and normalized to the protein concentrations used ( specific activity ) .", "The specific activities were then plotted against protein concentrations .", "The data were fitted to a model describing the interaction of two molecules of mGBP2 , yielding KD ( µM ) and the maximal specific activity Kmax ( min-1 ) .", "The maximum specific GTPase activitiy , the dimer dissociation constant and the amount of GMP production are summarized in the Table 1 .", "( c ) Nucleotide-dependent multimerization .", "Size-exclusion chromatography of the C586S mutant of mGBP2 bound to GTPγS , GDP , GMP and in the nucleotide free state at 4°C .", "Elution of all proteins was followed using absorbance by 280 nm .", "The protein size was estimated by appropriate standard proteins and the absorbance values were normalized to the peaks of the curves .", "( d ) Intracellular localization of WT and C586S-mGBPs was analyzed by transduction of the GFP fusion constructs in mGBP2-/- MEFs .", "Cells were stimulated with IFNγ for 16 hr .", "Glass slides were analyzed by confocal microscopy .", "Bars , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 004 The mean steady-state anisotropy of GFP in the cytosol was experimentally determined as <rD>cytosol = 0 . 328 , which is in agreement with the value predicted by the Perrin equation ( Lakowicz , 2006 ) using the known mean global rotational diffusion time ρglobal ≈ 15 ns for freely diffusing GFP .", "When GFP is fused to mGBP2 , two opposing effects need to be considered ( Figure 1b ) .", "First , its rotational freedom is restricted and therefore rD increases; second , homo-FRET between G-mGBP2 complexes reduces rD by depolarization of the total GFP signal .", "Consequently , the average steady-state anisotropy of WT G-mGBP2 in the cytosol <rD>cytosol remained comparable to the value for free GFP ( Figure 1c ) .", "In contrast , the GFP signal intensity ( SG , G ) in VLS increased significantly , indicating an enrichment of mGBP2 molcules in these structures ( Figure 1c ) accompanied by a significant reduction of the average anisotropy <rD>VLS , suggesting an increased mGBP2 homo-multimerization ( Figure 1a , c ) .", "The nucleotide binding and hydrolysis impaired K51A mutant does not localize in VLS ( Kravets et al . , 2012 ) .", "This mutant showed a higher average anisotropy ( <rD>cytosol = 0 . 336 ) as compared to the cytosolic WT mGBP2 ( Figure 1a , c ) due to the absence of homo-FRET , proving its incapability to form multimers .", "Next , the mean anisotropies of averages over whole MEFs <rD>cell were determined ( Figure 1d ) .", "The hydrolytically impaired mGBP2 mutants R48A and E99A ( Kravets et al . , 2012 ) showed significantly increased <rD>cell values ( Figure 1a , d ) , further proving that the GTPase activity is essential for multimerization in living cells .", "The nucleotide binding deficient mGBP2 mutant D182N showed significantly increased <rD>cell value ( Figure 1a , d ) as compared to WT mGBP2 and mutants R48A and E99A reflects the low multimerization capability of this mutant .", "The recombinant isoprenylation mutant ( C586S ) did not show altered nucleotide binding , hydrolysis activity or multimerization of mGBP2 in cell-free analyses ( Figure 1—figure supplement 1 , Table 1 ) .", "Nevertheless , this mutant did not localize in VLS ( Degrandi et al . , 2013 ) and showed anisotropy values comparable to the dysfunctional K51A mutant ( Figure 1d ) . 10 . 7554/eLife . 11479 . 005Table 1 . Dissociation constants KD of mant-nucleotides for mGBP2 WT and C586S mutant determined by fluorescence titrations and GTPase activity parameters obtained by protein concentration-dependent hydrolysis . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 005Nucleotide bindingGTP-hydrolysismant-GTPγSmant-GDPmant-GMPKD ( µM ) KD ( µM ) KD ( µM ) Kmax ( min-1 ) Dimer KD ( µM ) GMP ( % ) WT0 . 450 . 5414 . 41020 . 02974C586S0 . 500 . 4515 . 51330 . 02672The % GMP indicates the relative amount of the two products , GDP and GMP Altogether , these data provide compelling evidence that nucleotide binding and membrane anchoring are prerequisites for multimerization of mGBP2 in living cells .", "The degree of multimerization of mGBP2 increases from cytosol to VLS .", "mGBPs were reported to be involved in rupture of T . gondii PVMs few hours after infection and are important for T . gondii control in vivo ( Degrandi et al . , 2013; Selleck et al . , 2013; Yamamoto et al . , 2012 ) .", "Previously , it could be determined that the GTPase activity as well as isoprenylation regulate the recruitment of mGBP2 to the PVM of T . gondii ( Degrandi et al . , 2013; Kravets et al . , 2012 ) .", "The next step therefore was to elucidate the impact of the GTPase activity and the isoprenylation of mGBP2 on the ability to multimerize at the PVM and to control intracellular T . gondii replication .", "Hence , G-mGBP2 MEFs as well as MEFs expressing GTPase and isoprenylation mutants were infected with T . gondii and analyzed by MFIS homo-FRET assays .", "Also , the ratio of replicative units , so called rosettes , versus single parasites was determined 32 hr after infection ( Figure 2 ) . 10 . 7554/eLife . 11479 . 006Figure 2 . Intracellular homo-multimerization of WT and mutant mGBP2 at the PVM of T . gondii and parasite inhibition . Cells were pre-treated with IFNγ for 16 hr prior infection with T . gondii ME49", "( a ) Left panel .", "GFP fluorescence intensity images of G-mGBP2-WT , mutants MEFs or GFP highlighted with selections of pixels with low and high numbers of photons .", "Blue boxes mark the PVM area .", "Bars , 10 µm .", "Right panel .", "MFIS 2D-histograms of GFP rD on x axis vs . photon number per pixel on y axis .", "The pixels with low photon numbers ( below 1000 ) are selected in red boxes and the pixels containing more than 1000 photons in green boxes .", "( b ) Mean values of <rD>loc and mean GFP SG , G were plotted for G-mGBP2-WT in the cytosol and at the PVM of T . gondii and for the K51A mutant and GFP in the cytosol .", "( c ) Mean anisotropy <rD>loc of WT and mutants in the cytosol and at the PVM ( blue boxes in", "( a ) ) .", "GFP expressing cells served as controls ( ns=not significant; *p<0 . 05; **p<0 . 01; ***p<0 . 0001 ) .", "( d ) Replication inhibitory capacity of G-mGBP2-WT and mutants .", "After fixation T . gondii were stained with the α-SAG1 antibody and the cell nuclei with DAPI .", "Slides were analyzed by confocal microscopy .", "Replication inhibition was calculated by the ratio of T . gondii single parasites versus replicative units ( rosettes ) in at least 100 infected cells ( ***p<0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 00610 . 7554/eLife . 11479 . 007Figure 2—figure supplement 1 . Spectroscopic characterization of G-mGBP2 WT in VLS in non-infected cells and at the PVM in T . gondii infected cells via homo-FRET assay . Average values of GFP fluoresecnce anisotropy ( rD ) and signal intensity ( SG , G ) over single-cell measurements are plotted , in which SG , G values are proportional to protein concentration .", "A much wider distribution of SG , G can be observed for G-mGBP2 localizing at the PVM ( blue circles ) comparing to the SG , G values for G-mGBP2 localizing in the VLS ( green squares ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 007 A marked decrease of fluorescence intensities of WT mGBP2 in the cytosol of infected cells ( Figure 2a , b ) compared to uninfected cells ( Figure 1c ) concurrent with a strong increase of the mGBP2 concentration at the PVM of T . gondii was observed along with a further decrease in anisotropy ( Figure 2a , b; Figure 2—figure supplement 1 ) .", "This raises the question on a distinct composition of the mGBP2 complexes at the PVM , which will be addressed below by pixel-integrated MFIS analysis .", "As shown previously , the enzymatically dysfunctional K51A and the isoprenylation C586S mutants showed nearly no recruitment to the PVM ( Kravets et al . , 2012; Degrandi et al . , 2013 ) .", "Interestingly , as shown here , the corresponding anisotropies ( Figure 2a–c ) did not significantly change in comparison to the uninfected situation ( Figure 1 ) .", "These mutants were incapacitated in controlling T . gondii replication ( Figure 2d ) .", "The R48A and E99A mutants , which have reduced capacity to recruit to the PVM ( Kravets et al . , 2012 ) , showed slightly increased anisotropy at the PVM as compared to WT mGBP2 ( Figure 2c ) and a reduced capability to restrict T . gondii growth ( Figure 2d ) .", "For the D182N mutant a higher anisotropy at the PVM in comparison to WT mGBP2 could be determined , suggesting a lower degree of multimerization .", "This correlated with insufficient control of T . gondii growth , comparable to the K51A and C586S mutants ( Figure 2d ) .", "In summary , it can be concluded that at the PVM the enrichment of mGBP2 is increased compared to VLS .", "Nucleotide binding , GTPase activity as well as membrane anchoring regulate the multimerization capability of mGBP2 at the PVM and are prerequisites for the control of T . gondii replication .", "Several members of the mGBP family localize in VLS in IFNγ stimulated cells ( Degrandi et al . , 2007 ) .", "However , it is unclear whether co-compartmentalization of mGBPs and molecular interactions between them in VLS occur .", "For this purpose , G-mGBP2 MEFs were cotransduced with mCherry fusion proteins of mGBP1 , mGBP2 , mGBP3 , mGBP5 , and mGBP6 ( hereafter referred to as G-mGBP2/mCh-mGBPx ) and confocal imaging studies were performed .", "( Figure 3 , Figure 3—figure supplement 1 ) .", "All of the analyzed mGBPs showed a vesicular distribution except for mGBP5 ( Figure 3 ) .", "A correlation of localization could be computed employing the Pearson´s coefficient , P . G-mGBP2/mCh-mGBP2 MEFs showed the most pronounced colocalization indicating that the fluorescence tags do not affect protein localization ( P = 0 . 758 ± 0 . 093 ) .", "Confocal images revealed a high correlation of G-mGBP2 positive VLS with mCh-mGBP1 ( P = 0 . 516 ± 0 . 132 ) and mCh-mGBP3 VLS ( P = 0 . 65 ± 0 . 121 ) .", "mCh-mGBP5 ( P = 0 . 108 ± 0 . 104 ) and mCh-mGBP6 ( P = 0 . 338 ± 0 . 126 ) scarcely overlapped with G-mGBP2 .", "Thus , the subcellular reservoir of mGBP1 , mGBP2 and mGBP3 differed from mGBP6 , whereas mGBP5 showed no compartmentalization . 10 . 7554/eLife . 11479 . 008Figure 3 . Intracellular colocalization of mGBP proteins . Subcellular localization of mGBPs was analyzed in G-mGBP2 coexpressing one of the mCh-mGBPs ( 1 , 2 , 3 , 5 or 6 ) .", "mCherry expressing cells served as controls .", "Cells were pre-treated with IFNγ for 16 hr .", "After fixation , nuclei were stained with DAPI .", "Glass slides were analyzed by confocal microscopy .", "Bars , 5 µm .", "Colocalization analysis was performed with Imaris ( Bitplane ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 00810 . 7554/eLife . 11479 . 009Figure 3—figure supplement 1 . Expression analysis of coexpressed mGBP proteins . Expression levels of mGBPs in postnuclear supernatants of mGBP2-/- MEFs reconstituted with G-mGBP2 and coexpressing one of the mCh-mGBPs ( mGBP1 , mGBP2 , mGBP3 , mGBP5 , mGBP6 ) were analyzed by Western Blotting .", "mCherry protein expressing cells served as controls .", "Cells were stimulated with IFNγ for 16 hr .", "Blots were stained with the α-mCherry antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 009 To elucidate whether the colocalization of mGBPs is due to specific protein interactions , MFIS-hetero-FRET measurements were performed using G-mGBP2 as donor and mCh-mGBPx as acceptors ( Figure 4 ) .", "In the FRET analysis GFP and mCherry fluorescence intensities ( FGand FR ) and the mean fluorescence-weighted donor lifetime <τD>f were determined for each pixel ( Figure 4a ) .", "By displaying the frequency of pixels in color scales for the two localizations ( red: cytosol , green: VLS ) , the VLS-population exhibits a correlated shift in the MFIS 2D-histogram of the FRET indicators FG/FR and <τD>f towards smaller values with respect to the population in the cytosol .", "This is a clear indicator for the presence of hetero-FRET , which proves the interaction between molecules .", "Furthermore , GFP rD was plotted versus <τD>f as well as the G-mGBP2 concentration ( CG-mGBP2 ) derived from FG ( see 'Determination of mGBP protein concentrations and binding curves' , Materials and methods section ) ( Figure 4b , c , Figure 4—figure supplement 1 ) .", "A <τD>f – rD diagram is essential to determine homo- and hetero-oligomerization between mGBPs sensed by hetero- and homo-FRET .", "Figure 4b illustrates the interpretation of a <τD>f – rD diagram based on the Perrin equation to visualize the effects on a donor-reference data set ( green circle ) by selective hetero- ( red sphere ) or homo-FRET ( yellow sphere ) or simultaneous homo- and hetero-FRET ( orange sphere ) .", "Comparing G-mGBP2 MEFs ( Figure 4c ) with G-mGBP2/mCh-mGBP2 MEFs , both homo- and hetero-FRET were visible for the latter cells indicated by a simultaneous reduction of <τD>f and an increase of rD .", "Moreover , analyzing the cells individually , the anisotropy dropped with increasing G-mGBP2 concentrations .", "The variation of mGBP2 concentrations between individual cells allowed the estimation of the spatially resolved apparent dissociation constant ( KD , app ) of the mGBP2 homomultimer of approx .", "9 μM in the VLS ( Figure 4c , upper right panel , black curve ) .", "Note that any interactions interfering with G-mGBP2 homomerization will result in a KD , app-curve shifted upwards ( purple curve ) . 10 . 7554/eLife . 11479 . 010Figure 4 . Intracellular homo- and hetero-multimerization of mGBPs . All cells were pre-treated with IFNγ for 16 hr prior investigation", "( a ) Left panels .", "GFP fluorescence intensity images of G-mGBP2 or G-mGBP2/mCh-mGBP ( 1 , 2 , 3 , 5 , 6 ) MEFs highlighted with selections of pixels with different intensities .", "Bars , 10 µm .", "Right panels .", "Two MFIS 2D-histograms of GFP fluorescence lifetimes ( <τD>f ) on y axes , GFP/mCherry fluorescence intensity ratios ( FG/FR ) or photon number per pixel ( N ) on x axes .", "The pixel populations locating in cytosol ( N < 1000: red island ) and VLS ( N > 1000: green island ) were separated according to photon numbers .", "( b ) Schematic 2D MFIS plot detailing the effects of hetero- and/or homo-FRET on a reference data set ( green circle ) .", "The average GFP <τD>f is plotted on the x axis from short to long , while the average steady-state rD is plotted on the y axis .", "For detailed explanation refer to results section .", "( c ) Upper panel .", "For individual G-mGBP2 , G-mGBP2/mCh-mGBP2or G-mGBP2/mCh-mGBP6 MEFs , mean values of rD in the cytosol ( empty squares ) and in the VLS ( solid squares ) were plotted against <τD>f and G-mGBP2 concentrations ( CG-mGBP2 ) .", "Lower panel .", "Mean anisotropy <rD>loc values ( average over all cells weighted by CG-mGBP2 ) were plotted against <τD>f or CG-mGBP2 .", "The two left panels contain an overlay calculated according to the Perrin equation: rD=r0/ ( 1+τDf/ρglobal ) with GFP fundamental anisotropy r0 = 0 . 38 and rotational correlation time ρglobal= 15 ns .", "The two right panels are overlaid with function curves plotting rD=rmax- ( rmax-rmin ) ·CG-mGBP2/ ( CG-mGBP2+KD , app ) which assumes a mGBP2 Langmuir binding model with an apparent dissociation constant KD , app .", "In all donor-only experiments the formation of mGBP2 homo-multimers could be described by KD , app = 9 μM , rmax = 0 . 32 and rmin = 0 . 22 ( black curve ) .", "If other interaction processes interfere with homo-FRET between G-mGBP2 proteins , this curve is shifted upwards ( violet curve ) while keeping KD , app invariant ( rmax = 0 . 345 and rmin = 0 . 245 ) .", "( d ,", "e ) εmix ( t ) and ε ( D , A ) ( t ) diagrams of a representative G-mGBP2/mCh-mGBP2 MEF", "( d ) and G-mGBP2/mCh-mGBP6 MEF", "( e ) .", "The drop in εmix ( t ) curves , as marked by the arrows , represents the species fractions of FRET-active complexes ( xFRET ) in the VLS ( green ) and in the cytosol ( red ) .", "In", "( d ) , the FRET rate constant ( kFRET ) in the cytosol is 0 . 09 ns-1 and in the VLS 0 . 20 ns-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 01010 . 7554/eLife . 11479 . 011Figure 4—figure supplement 1 . Intracellular homo- and hetero-multimerization of mGBPs in cells .", "( a ) For single IFNγ stimulated mGBP2-/- MEFs expressing G-mGBP2 alone or coexpressing G-mGBP2/mCh-mGBP5 , and G-mGBP2/mCh-mGBP6 , average values of rD in the cytosol ( empty ) and in the VLS ( solid ) were plotted against <τD>f or G-mGBP2 concentrations ( CG-mGBP2 ) .", "See the legend of Figure 4c for the description of the overlay curves in both panels .", "( b ) Corresponding plots as in", "( a ) for single cells expressing G-mGBP2 alone or coexpressing G-mGBP2/mCh-mGBP1 , G-mGBP2/mCh-mGBP2 and G-mGBP2/mCh-mGBP3 . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 01110 . 7554/eLife . 11479 . 012Figure 4—figure supplement 2 . Immunoprecipitation analysis of mGBP proteins .", "( a ) mGBP2-/- MEFs reconstituted with G-mGBP2 or GFP were stimulated with IFNγ for 16 hr , subsequently lysed and postnuclear supernatants were incubated o/n with G-sepharose beads and the α-GFP antibody at 4°C .", "IP probes were subjected to Western Blotting .", "Blots were stained with the α-mGBP2 , α-mGBP1 , α-mGBP3 , α-mGBP5 antibodies .", "( b ) Postnuclear supernantants of mGBP2-/- MEFs reconstituted with G-mGBP2 and coexpressing mCherry protein or one of the mCherry fused mGBPs ( mGBP1 , mGBP2 , mGBP3 , mGBP5 , mGBP6 ) were incubated o/n with GFP-Trap beads at 4°C .", "IP probes were subjected to Western Blotting .", "Blots were stained with the α-GFP and α-mCherry antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 012 To attain an overview of all experimental data , we computed the averaged values of <τD>f and fluorescence intensity weighted anisotropy <rD>loc for all cells of the specified FRET pair ( Figure 4c , lower panels ) .", "Both in cytosol and in VLS , the strongest fluorescence lifetime reduction compared to the donor-only sample could be measured for combinations of G-mGBP2 with mCh-mGBP2 and to a lesser extent for mCh-mGBP1 and mCh-mGBP3 ( Figure 4a , c ) , proving that mGBP1 , 2 , and 3 do not only colocalize but also directly interact .", "This could be confirmed by co-immunprecipitation ( co-IP ) experiments ( Figure 4—figure supplement 2 ) .", "Although no detectable lifetime reduction could be observed between G-mGBP2 and mCh-mGBP5 , data showed a higher anisotropy compared to the donor reference , indicating interference of mGBP5 with mGBP2 homomerization ( Figure 4c ) Also , co-IP of mGBP2 and mGBP5 was observed ( Figure 4—figure supplement 2 ) , suggesting a differing mode of interaction between mGBP2 and mGBP5 , which will be discussed below .", "No fluorescence lifetime reduction ( Figure 4c , left panel ) , interaction-induced anisotropy increase ( Figure 4c , right panel ) , or co-IP ( Figure 4—figure supplement 2 ) could be observed for mGBP2 and mGBP6 coexpressing cells .", "To elucidate the reason for the donor lifetime reduction in VLS by determining the fraction of FRET-active complexes ( xFRET ) together with their FRET properties given by the rate constants of FRET ( kFRET ) , pixel-integrated MFIS-FRET analysis was applied by computing the FRET-induced donor-quenching decay εmix ( t ) ( Equations 1–5 ) to graphically display the FRET effect ( Figure 4d ) .", "The larger drop of εmix ( t ) ( Figure 4d , upper panel ) directly shows the difference in xFRET which proves that more interacting mGBP2 complexes reside in the VLS than in the cytosol .", "The FRET-induced donor decay εmix ( t ) displays the interaction state of an ensemble of proteins , which includes both FRET-active and -inactive species .", "To separate the effects of both FRET-species on the decay , it is necessary to determine the characteristic kFRET of the populations in the cytoplasm and the VLS .", "The formally fitted decay curves ( Equations 1–5 ) of FRET-active complexes ε ( D , A ) ( t ) are separately plotted ( Figure 4d , lower panel ) , because this allows to remove the influence of the offset on the decay due to FRET-inactive species .", "The ε ( D , A ) ( t ) clearly differ for cytosol and VLS suggesting a higher degree of multimerization of mGBP2 in VLS .", "The εmix ( t ) -curve of a representative cell expressing G-mGBP2/mCh-mGBP6 ( Figure 4e ) had random fluctuations around 1 , which is consistent with the data in Figure 4a and c showing no FRET events and confirms the absence of heteromeric complexes .", "In summary , in the cytosol and VLS mGBP2 forms homo-multimers and hetero-multimers with mGBP1 and mGBP3 , but not with mGBP6 .", "Individual members of the mGBP family are able to recruit to the PVM ( Degrandi et al . , 2007 ) .", "To investigate the colocalization of several mGBPs at the PVM , G-mGBP2/mCh-mGBPx MEFs were infected with T . gondii .", "( Figure 5 ) .", "A colocalization of all investigated mGBPs with mGBP2 could be detected at distinct PVMs for each pairwise combination of proteins . 10 . 7554/eLife . 11479 . 013Figure 5 . Intracellular colocalization at the PVM of T . gondii and enrichment of mGBP proteins . Recruitment and colocalization of mGBPs was analyzed in G-mGBP2/mCh-mGBP ( 1 , 2 , 3 , 5 , 6 ) MEFs .", "mCherry expressing cells served as controls .", "Cells were stimulated with IFNγ for 16 hr and subsequently infected with T . gondii for 2 hr .", "After fixation , T . gondii were stained with an α-SAG1 antibody and cell and T . gondii nuclei with DAPI .", "Glass slides were analyzed by confocal microscopy .", "Bars , 5 µm .", "Colocalization analysis was performed with Imaris ( Bitplane ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 013 To investigate whether the colocalized mGBPs interact at the PVM , MFIS-FRET measurements were applied in G-mGBP2/mCh-mGBPx MEFs ( Figure 6 ) .", "A strong decrease of both FRET indicators , GFP fluorescence lifetimes <τD>f and intensity ratio FG/FR , could be detected in the cytosol and at the PVM of G-mGBP2/mCh-mGBP1 and G-mGBP2/mCh-mGBP2 MEFs and , to a lesser extent , in G-mGBP2/mCh-mGBP3 MEFs ( Figure 6a , b ) . 10 . 7554/eLife . 11479 . 014Figure 6 . Intracellular homo- and hetero-multimerization of mGBPs at the PVM of T . gondii . All cells were pre-treated with IFNγ for 16 hr prior investigation", "( a ) Left panels .", "GFP fluorescence intensity images of living G-mGBP2 or G-mGBP2/mCh-mGBP ( 1 , 2 , 3 , 5 , 6 ) MEFs infected with T . gondii highlighted with selections of pixels within different intracellular localizations .", "Right panels .", "Two MFIS 2D-histograms of GFP <τD>f on y axes , GFP/mCherry FG/FR and photon number per pixel ( N ) on x axes .", "The pixel populations locating in cytosol ( N < 1000: red island ) and at the PVM ( N > 1000: green island ) were separated according to photon numbers .", "( b ) Upper panel .", "For individual G-mGBP2 , G-mGBP2/mCh-mGBP2 or G-mGBP2/mCh-mGBP6 MEFs pixel averages of rD in the cytosol and at the PVM were plotted against <τD>f or CG-mGBP2 .", "Lower panel .", "Averages of <rD>loc were plotted against <τD>f and CG-mGBP2 .", "Please refer to Figure 4c for further information on the legend and overlaid curves .", "( c ,", "d ) εmix ( t ) and ε ( D , A ) ( t ) diagrams of a representative T . gondii infected G-mGBP2/mCh-mGBP2 MEF", "( c ) and G-mGBP2/mCh-mGBP6 MEF", "( d ) .", "The drop in εmix ( t ) curves , as marked by the arrows , represents xFRET at the PVM ( blue ) and in the cytosol ( red ) .", "The dashed curves representing the ε ( D , A ) ( t ) diagrams of G-mGBP2/mCh-mGBP2 interactions in the cytosol ( red ) and VLS ( green ) in uninfected cells are inserted for comparison from Figure 4d .", "In", "( c ) , kFRET at the PVM is 0 . 24 ns-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 01410 . 7554/eLife . 11479 . 015Figure 6—figure supplement 1 . Intracellular homo- and hetero-multimerization of mGBPs in T . gondii infected cells .", "( a ) For single IFNγ stimulated and T . gondii infected mGBP2-/- MEFs expressing G-mGBP2 alone or coexpressing G-mGBP2/mCh-mGBP5 , and G-mGBP2/mCh-mGBP6 , average values of rD in the cytosol ( empty ) and at the PVM ( solid ) were plotted against <τD>f or G-mGBP2 concentrations ( CG-mGBP2 ) .", "See the legend of Figure 4c for the description of the overlay curves in both panels .", "( b ) Corresponding plots as in", "( a ) for single cells expressing G-mGBP2 alone or coexpressing G-mGBP2/mCh-mGBP1 , G-mGBP2/mCh-mGBP2 and G-mGBP2/mCh-mGBP3 . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 01510 . 7554/eLife . 11479 . 016Figure 6—figure supplement 2 . Quantitative MFIS-FRET analysis of mGBP2 hetero-multimerization in living IFNγ stimulated cells .", "( a ) All the experiments on G-mGBP2/mCh-mGBP1 , G-mGBP2/mCh-mGBP2 and G-mGBP2/mCh-mGBP3 interactions were formally analyzed according to Equations 1–5 .", "Fit results of species fraction of FRET-active complex ( xFRET ) is plotted against G-mGBP2 and mCh-mGBPs concentrations determined in cytosol ( red ) , in VLS ( green ) and at PVM ( blue ) .", "The overlaid fuction curve plotting xFRET=S⋅CmCh−mGBP2/ ( CmCh−mGBP2+KD , app ) assumes a mGBP2 Langmuir binding model with apparent dissociation constant , KD , app = 9 μM , the same value as applied in Figure 4c and 6b .", "The scaling factor S = 0 . 64 was adjusted according to the saturation level of xFRET .", "( b ) For the same experiments as in", "( a ) , FRET rate constants ( kFRET ) are plotted versus G-mGBP2 and mCh-mGBPs concentrations .", "( c ) xFRET in", "( a ) is plotted versus total protein concentration .", "( d ) kFRET in", "( b ) is plotted versus total protein concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 016 For individual cells , MFIS diagrams plotting the rD values against donor lifetimes <τD>f and G-mGBP2 concentrations were generated ( Figure 6b upper panels , Figure 6—figure supplement 1 ) .", "The KD , app-curves describing the relationship between rD and CG-mGBP2 in uninfected cells ( Figure 4c ) fitted also very well to the infected situation ( Figure 6b ) .", "The averaged values of <τD>f , <rD>loc and CG-mGBP2 over individual cells are depicted in Figure 6b ( lower panels ) .", "An even stronger reduction in <τD>f was observed at the PVM for combinations of G-mGBP2 with mCh-mGBP2 and to a lesser extent with mCh-mGBP1 and mCh-mGBP3 as compared to the VLS in uninfected cells ( Figure 4c ) , proving that the observed colocalization at the PVM ( Figure 5 ) enables direct protein interactions .", "For G-mGBP2/mCh-mGBP5 MEFs the situation is more complex: in the cytosol the anisotropy was slightly increased but the donor lifetime was unchanged , whereas at the PVM an increase in anisotropy was absent ( Figure 6b , lower right panel ) .", "In G-mGBP2/mCh-mGBP6 MEFs no interactions were detected , neither in the cytosol nor at the PVM .", "The FRET-related donor quenching εmix ( t ) of one representative G-mGBP2/mCh-mGBP2 cell ( Figure 6c ) exhibited a larger drop , which indicates a higher xFRET , i . e . more interacting protein complexes were located at the PVM compared to VLS in uninfected cells ( Figure 4d ) .", "Nevertheless , their slopes ( kFRET ) of ε ( D , A ) ( t ) are comparable within the precision of the analysis ( Figure 6c , green dashed line ) , suggesting an unchanged local environment in the oligomer .", "Furthermore , the εmix ( t ) diagram for one representative G-mGBP2/mCh-mGBP6 cell revealed no interaction between these mGBPs .", "In conclusion , mGBP2 , besides its homo-interaction , directly interacts with mGBP1 and , to a lesser extent , with mGBP3 at the PVM .", "Although other mGBPs , such as mGBP5 and mGBP6 were recruited to the same PVMs , no direct interaction could be detected suggesting the formation of specific mGBP supramolecular complexes .", "In addition to the formal analysis by Equations 1–5 ( Figure 6—figure supplement 2 ) of the hetero-FRET data , an additional inspection of the time-resolved donor anisotropy ( rD ( t ) ) ( Figure 7a ) revealed that cells with a higher mGBP2 concentration ( CmGBP2 ) exhibited a larger drop in initial anisotropy , which is evidence for ultrafast depolarization processes due to the formation of densely packed mGBP2 homo-oligomers with multiple GFPs .", "These processes were too fast to be resolved by hetero-FRET analysis ( Figure 6c ) , but combining both homo- and hetero-FRET , global pattern based , pixel-integrated MFIS-FRET analysis could be performed to resolve the individual mGBP species ( Figure 7b and", "c ) and to characterize the composition of FRET-active homo- and hetero-complexes of mGBP2 ( Equation 6 , 7 ) for the distinct localizations .", "The information content in the experimental fluorescence decays is restricted by their noise ( Kollner and Wolfrum , 1992 ) .", "Given the limited amount of photons of the pixel-integrated fluorescence intensity histograms , the pattern fit uses structural information of molecular simulations ( Figure 7—figure supplement 1 ) to obtain population fractions of all species .", "The structural information is based on prior knowledge of the dimerization interface ( Vopel et al . , 2014 ) and on Monte Carlo simulations of the linkers connecting the fluorescent proteins to the GBPs ( see 'Monte Carlo sampling of the donor-acceptor conformational space of mGBP2 dimer' , Materials and methods section ) ( Evers et al . , 2006; Pham et al . , 2007 ) .", "The obtained species fractions of mGBP2 monomers , homo- or hetero-dimers and oligomers are displayed in Figure 7c .", "The homo- and hetero-dimer formation is very similar in G-mGBP2 MEFs and G-mGBP2/mCh-mGBP1 , 2 or 3 MEFs as expected for the highly conserved GTPase-domains of mGBPs .", "Dimeric complexes are primarily formed with a small fraction of monomers in the cytosol ( Figure 7c , middle panel , see methods , Equation 13 ) .", "The obtained KD , dim of ~24 nM is close to previous biochemical studies ( Kravets et al . , 2012 ) .", "In the VLS an equilibrium of mGBP dimers and oligomers existed which was shifted towards oligomers with increasing protein concentration so that , the fraction of oligomers at the PVM is even higher than in the VLS .", "However , the dissociation constants for oligomerization KD , oligo differ significantly between the mGBPs: 70 µM for G-mGBP2/mCh-mGBP1 , 8 µM for G-mGBP2/mCh-mGBP2 and 208 µM G-mGBP2/mCh-mGBP3 ( Figure 7c , lower panel ) . 10 . 7554/eLife . 11479 . 017Figure 7 . Species-resolved analysis of mGBP2 homo- and hetero-complexes .", "( a ) G-mGBP2 MEFs with higher concentration exhibited larger quasi instantaneous drop of rD ( t ) from its initial value of ~0 . 35 , which proves the appearance of a very fast depolarization process due to homo-FRET in mGBP2 oligomers .", "( b ) Distribution of FRET rate constants ( kFRET ) for mGBP2 dimer ( gray curve ) and oligomer species ( black symbols ) .", "Small ( black squares ) and large ( black dots ) oligomers , as formally differentiated in the pattern-based MFIS-FRET analysis , show generally higher kFRET than that of the mGBP2 dimer estimated by the MC simulation .", "( c ) Concentration dependence of the three mGBP species ( monomer , dimer and oligomer ) obtained by the global pattern fit ( Equations 6 and 7 ) of rmix ( t ) and εmix ( t ) for two localizations VLS and PVM .", "The line depicts the fit ( 'Pattern based pixel-integrated MFIS-FRET analysis' and 'Determination of dissociation constants' , Materials and methods section ) to the corresponding binding equilibrium with KD , dim , and KD , app-oligo ( values are given in the main text ) .", "( d ) Concentration dependence of FRET rate constants for mGBP2 oligomers which formally differentiated as small ( kOlig , s ) and large ( kOlig , l ) .", "( e ) kOlig , l versus the number of monomer units in mGBP2 multimers at the PVM determined by scanning FIDA ( see 'Scanning fluorescence intensity distribution analysis ( FIDA ) for determination of oligomer size' , Materials and methods section ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 01710 . 7554/eLife . 11479 . 018Figure 7—figure supplement 1 . Sample mGBP2 dimer conformations by MC molecular simulation .", "( a ) Conformational space sampled by the MC simulations of free mGBP2 is illustrated by the density of the GFP-chromophore , one conformation is shown using cartoon representation .", "( b ) Structural properties of a predicted mGBP2 dimer based on the crystal structure of the hGBP1 dimer ( PDB-ID 2BC9 ) .", "The characteristic FRET features of the dimer with flexibly linked fluorescent proteins can be predicted by calculating inter fluorophore distances from the space that is sterically accessible to the fluorescent proteins .", "The accessible space of attached fluorescent proteins ( green ( GFP ) and red ( mCherry ) is depicted as fuzzy cloud; ≥ 60% of all D-A configurations are FRET-inactive due to their large distances between the fluorophores , 'Monte Carlo sampling of the donor-acceptor conformational space of mGBP2 dimer' , Materials and methods section ) .", "( c ) Illustration of FRET parameter calculation on each sampled G-mGBP2/mCh-mGBP2 dimer conformation in the MC simulation .", "Vectors and coordinates in the figure are listed in supplementary file 1a .", "GFP: green , mCherry: red .", "( d ) Donor-acceptor orientation factor ( κ2 ) , spatial distance ( Rsim ) and FRET rate ( kdi ) were computed for each sampled mGBP2 dimer conformation , and their relation is plotted in the histogram .", "In the left panel , the overlay curve in black assumes that the Förster radius between GFP and mCherry is 52 Å , unquenched GFP fluorescence lifetime is 2 . 6 ns and <κ2> is 2/3 .", "The red line indicates the maximum resolvable FRET rate constant for our detection system ( 20 ns-1 ) .", "The area shade in grey indicates the irresolvable low FRET rate constant ( E < 1% , kdi < 0 . 004 ns-1 ) , in which the conformations constitute ~73% of the whole population .", "( e ) The donor-acceptor distance distribution ( RDA ) obtained from the Monte Carlo ( MC ) simulation of mGBP2 dimer ( blue ) and its optimized distance distribution according to the experimental data using maximum entropy method ( MEM-MC , in red ) , see the subsection 11 of Materials and methods for details .", "( f ) mGBP2 concentration determined by 2D FIDA analysis is plotted versus that directly derived from G-mGBP2 fluorescence intensity .", "( g ) A typical image showing the pixels at the PVM area which were analysed by scanning FIDA .", "The grey scale indicates acquired photon count per pixel .", "( h ) The corresponding 2D FIDA matrix analysing the fluorescence intensity in the green and red detection channel of", "( g ) ( the details of FIDA are given in 'Scanning fluorescence intensity distribution analysis ( FIDA ) for determination of oligomer size' , Materials and methods section ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 018 Global analysis of G-mGBP2 MEFs and G-mGBP2/mCh-mGBP2 MEFs , revealed the heterogeneity in size of the mGBP2 oligomers via the broad distribution of FRET rate constants for small and large oligomers , kOlig , s and kOlig , l , respectively ( Figure 7d ) .", "While kOlig , s did not change with increasing protein concentration , kOlig , l increased and reached a saturation level of ~15 ns-1 at ~50 µM ( Figure 7d , red line ) , which is expected for a maximal local packing of FRET acceptors around the donor ( see 'Maximum FRET rate constants' , Materials and methods section ) and proved the growth of oligomers .", "Notably FRET senses only the local environment in a distance range limited to ~10 nm , however the continuous increase in brightness suggests also the formation of larger oligomers .", "Therefore we introduced scanning fluorescence intensity distribution analysis ( FIDA ) ( ( Kask et al . , 2000 ) , 'Scanning fluorescence intensity distribution analysis ( FIDA ) for determination of oligomer size' , Materials and methods section ) to determine the mean number and brightness of the large oligomers for all pixels of the PVM in one infected MEF .", "The obtained oligomer brightness allowed us to derive the mean number of mGBP2 units in an oligomer using the specific brightness of one GFP under these measurement conditions .", "With increasing local mGBP2 concentration , scanning FIDA suggests also an increasing oligomer size ( Figure 7e ) .", "The mean number of mGBP2 monomer units in the oligomer ranges between 1000 and 6000 at the PVM .", "Remarkably the FRET rate constants in large oligomers kOlig , s saturated at approximated 2000 monomer units , which corresponds to a total local concentration of mGBP2 monomer units of ~ 30 µM ( Figure 7e ) .", "In summary , with increasing protein concentration the fraction of mGBP2 dimers decreases due to the formation of large oligomers of heterogeneous size .", "The formation of mGBP2 homo-oligomers is preferred over heteromers with mGBP1 and mGBP3 as KD , oligo dropped by a factor of 9 and 25 , respectively .", "The mean size of large mGBP2 oligomers can reach up to several thousand monomer units .", "mGBP2 was shown to rapidly accumulate at the PVM after active invasion of the parasite in IFNγ activated cells ( Degrandi et al . , 2013 ) .", "To further investigate the spatio-temporal behavior of mGBP2 , 3D live cell imaging was performed in mGBP2-/- MEFs stably expressing G-mGBP2 or mCh-mGBP2 ( Figure 8 and Videos 1–3 ) .", "mGBP2 localized in VLS of heterogeneous size , morphology , and velocity within the cytosol .", "In IFNγ stimulated uninfected cells the diameter of VLS reaches up to several microns .", "No obvious directional movement could be observed ( Video 1 ) .", "After T . gondii infection of IFNγ stimulated MEFs , mGBP2 accumulated rapidly at the PVM ( Figure 8a , b and Video 1 ) .", "Image analysis revealed that accumulation initiated simultaneously at different sites around the PVM ( Figure 8b ) .", "Quantification of the overall G-mGBP2 fluorescence in regions containing the PVM and the remaining cell revealed a constant reduction of the cytosolic and VLS G-mGBP2 concentrations after infection , paired with a reciprocal increase at the PVM ( Figure 8c ) .", "Thus , accumulation of mGBP2 at the PVM occurs by redistribution of the protein , leading to a depletion of mGBP2 reservoirs and a reduction of the number of VLS ( Figure 8d ) within the cytosol .", "However , no directional movement of VLS towards the parasite could be observed ( Video 1 ) . 10 . 7554/eLife . 11479 . 019Figure 8 . Live-cell imaging of mGBP2 in T . gondii infection .", "( a ) G-mGBP2 MEFs were treated o/n with IFNγ and infected with T . gondii ME49 .", "Living cells were observed by confocal microscopy at 37°C and a z-stack was recorded every 5–10 s .", "4D data were processed and rendered in normal shading mode ( upper panels ) and the DIC images are displayed ( lower panels ) for the indicated time points .", "One out of at least 3 similar experiments is shown .", "Bar = 5 µm .", "( b ) Magnification from Video 1 and Figure 8a of G-mGBP2 accumulation around two T . gondii parasites at time points indicated .", "Bar = 2 µm .", "( c ) Quantification of the total fluorescence intensity over the indicated voxels from Video 1 .", "Vertical lines indicate the time points of T . gondii infection of MEFs .", "One representative analysis out of at least 3 similar experiments is shown .", "( d ) Number of cytosolic VLS with at least approx .", "0 . 25 µm diameter from Video 1 over time .", "Fluorescence signals close to the T . gondii area were excluded from the analysis .", "Vertical lines indicate the time points of T . gondii infection of MEFs .", "One representative analysis out of at least 3 similar experiments is shown", "( e ) XY , XZ , and YZ projections of G-mGBP2 around one T . gondii PVM are shown for the indicated time points .", "Bar = 2 µm .", "( f ) Maximum intensity projections of mCh-mGBP2 around one T . gondii are shown for the indicated time points .", "Bar = 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 01910 . 7554/eLife . 11479 . 020Video 1 . mGBP2-/- MEFs transduced with G-mGBP2 were treated o/n with IFNγ and infected with T . gondii . The living cells were observed with a confocal microscope at 37°C and a z-stack was recorded every 5–10 s .", "4D data were processed and rendered in normal shading mode .", "Bar = 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 02010 . 7554/eLife . 11479 . 021Video 2 . mGBP2-/- MEFs transduced with G-mGBP2 were treated o/n with IFNγ and infected with T . gondii . The living cells were observed with a confocal microscope at 37°C and a z-stack was recorded every 5–10 s .", "4D data were processed and rendered as maximum intensity projection .", "Bar = 2 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 02110 . 7554/eLife . 11479 . 022Video 3 . mGBP2-/- MEFs transduced with mCh-mGBP2 were treated o/n with IFNγ and infected with T . gondii . The living cells were observed with a confocal microscope at 37°C and a z-stack was recorded every 5–10 s .", "4D data were processed and rendered as maximum intensity projection .", "Bar = 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 022 After accumulation of mGBP2 at the PVM of T . gondii , different fates of the parasite could be observed within the recording period by live cell imaging .", "mGBP2 remained at the PVM for more than 16 hr without any noticeable change in PVM or parasite morphology ( not shown ) , mGBP2 penetrated through the PVM into the vacuolar space and accumulated at the parasite membrane ( Figure 8e and Video 2 ) , or the mGBP2-associated PVM acquired a rounded shape immediately followed by disruption of the PVM and subsequent accumulation of mGBP2 at the parasite membrane ( Figure 8f and Video 3 ) .", "Importantly , the behavior of mGBP2 was independent of the mCherry or GFP fusion .", "Additionally , the events following mGBP2 recruitment to the PVM were documented and quantified .", "For this , IFNγ stimulated G-mGBP2 MEFs were infected with T . gondii for 6 hr , fixed and the plasma membrane of T . gondii was stained with anti-SAG1 .", "To determine the precise localization of mGBP2 at this time point , intensity profiles of G-mGBP2 and Alexa633-SAG1 were determined encompassing the PVM , the plasma membrane of the parasite and the cytosol of the parasite ( Figure 9 ) .", "A total of 110 intracellular mGBP2-positive T . gondii PVs out of two independent experiments were evaluated .", "About 1 . 8% of the parasites acquire mGBP2 on the plasma membrane without apparent loss of PV integrity ( Figure 9c ) .", "For 37 . 1% of counted parasites disruption of PVM and direct targeting of mGBP2 to the plasma membrane of the parasite was observed ( Figure 9b ) .", "The remaining 61 . 1% revealed mGBP2 targeting at the PVM without apparent disruption or permeabilization and targeting of the parasite plasma membrane ( Figure 9a ) .", "Occasionally , after 6 hr of infection , parasites with very aberrant SAG1 localization were observed , providing evidence that these parasites were already non-viable .", "In such cases G-mGBP2 fluorescence inside the cytosol of the parasite could be found , suggesting a loss of the membrane integrity of the parasite ( Figure 9d ) . 10 . 7554/eLife . 11479 . 023Figure 9 . Localization of mGBP2 at the PVM , the plasma membrane , or the cytosol of T . gondii . G-mGBP2 cells were stimulated with IFNγ for 16 hr and subsequently infected with T . gondii ME49 for 6 hr .", "After fixation , T . gondii were stained with an α-SAG1 antibody .", "Glass slides were analyzed by confocal microscopy .", "Bars , 2 µm .", "Profiles show individually normalized intensities of GFP ( mGBP2 , green ) or Alexa633 ( SAG1 , magenta ) fluorescence along the indicated white arrows .", "Black arrows indicate the localization of the T . gondii plasma membrane , as identified by the SAG1 staining .", "( a ) Example of mGBP2 accumulation at the PVM of T . gondii without disruption or permeabilization of the PVM .", "( b ) Example of mGBP2 accumulation at the plasma membrane of T . gondii with obvious disruption of the PVM .", "( c ) Example of mGBP2 accumulation at the plasma membrane of T . gondii without apparent PVM disruption .", "( d ) Example of T . gondii death and accumulation of mGBP2 in the cytosol of the parasite . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 023 As previously reported , a rapid colocalization of mGBP2 with the PV of T . gondii type II strain ME49 but not of T . gondii type I strain BK in IFN-γ–activated MEFs was observed ( Degrandi et al . , 2007 ) .", "After infection with T . gondii ME49 , selective permeabilization experiments revealed that immunofluorescence labeling of SAG1 at the T . gondii plasma membrane could be detected for mGBP2-positive PVMs in the absence of saponin .", "In contrast , after infection with the virulent BK T . gondii , almost no SAG1-labeled parasites could be detected ( Table 2 ) .", "Please note that after saponin permeabilization virtually all ME49 or BK parasites could be labeled with anti-SAG1 .", "This shows that targeting of mGBP2 to the PVM promotes permeabilization or disruption of the PVM , allowing influx of proteins into the PV space . 10 . 7554/eLife . 11479 . 024Table 2 . G-mGBP2 cells were stimulated with IFNγ for 16 hr and infected with T . gondii ME49 or BK strains for 2 hr .", "After fixation and permeabilization with the indicated amounts of saponin , T . gondii were stained with an α-SAG1 antibody and DAPI .", "T . gondii were counted and categorized according the indicated mGBP2 and SAG1 fluorescence .", "N . d = not detected . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 024ME49 T . gondiiBK T . gondiimGBP2+ SAG1-mGBP2+ SAG1+mGBP2- SAG1+mGBP2+ SAG1-mGBP2+ SAG1+mGBP2- SAG1+w/o Saponin50%38%12%n . d . n . d . 3%0 , 15% Saponinn . d . 57%43%n . d . 1%99% Additionally , we have monitored the influx of cytosolic mCherry protein into the PV space after PVM disruption of GFP-mGBP2 positive T . gondii PV ( Video 4 ) .", "This observation corroborates former experimental approaches , showing a disruption of the PVM after IRG recruitment ( Zhao et al . , 2009b ) . 10 . 7554/eLife . 11479 . 025Video 4 . mGBP2-/- MEFs transduced with G-mGBP2 and cytosolic mCherry were treated o/n with IFNγ and infected with T . gondii . The living cells were observed with a confocal microscope at 37°C . DOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 025 Taken together , these observations show direct evidence that mGBP2 promotes PVM permeabilization and disruption and provide novel evidence that mGBP2 translocates into the PV space targeting the plasma membrane of the parasite , presumably delivering a direct attack on the parasite ." ], [ "The localization , molecular dynamics , interactions , and the formation of mGBP supramolecular complexes in the context of defense against T . gondii could be directly visualized in living cells using MFIS and live cell imaging within this study .", "Our data demonstrate that GTP binding and hydrolysis as well as membrane anchoring enable the pre-assembly of multimeric complexes containing mGBP2 in VLS .", "mGBP2/mGBP2 , mGBP2/mGBP1 and mGBP2/mGBP3 complexes in the form of dimers and multimers with distinct composition are recruited at considerably high concentrations ( 10–200 µM ) to the PVM of T . gondii .", "Moreover , the GTPase activity and isoprenylation of mGBP2 are crucial for the control of T . gondii proliferation within the PV .", "Eventually , mGBP2 multimers target the plasma membrane of T . gondii , thus establishing the immune function of GBPs to directly attacking intracellular pathogens .", "To extract structural information from the MFIS-FRET data ( Kalinin et al . , 2012 ) , we performed Monte Carlo sampling of the donor-acceptor conformational space of the mGBP2 dimer to compute the expected FRET parameters ( 'Monte Carlo sampling of the donor-acceptor conformational space of mGBP2 dimer' , Materials and methods section , Figure 7—figure supplement 1b ) .", "The sterically accessible volume of flexibly attached fluorescent proteins ( green ( GFP ) and red ( mCherry ) ) are depicted as fuzzy clouds .", "The prediction that more than 60% of all D-A configurations are FRET-inactive due to their large distances between the fluorophores is confirmed by the formal MFIS-FRET analysis ( Figure 6—figure supplement 2d ) .", "Our data argue that GTP binding is a prerequisite to induce dimer-and multimerization of mGBP2 in living cells .", "Indeed , the simulated FRET parameters of the mGBP2 homodimer ( Figure 7—figure supplement 1b–d ) interacting via the GTPase domains are in good agreement with MFIS pixel integrated analysis ( Figure 4d , 6c , Figure 6—figure supplement 2 ) .", "Moreover , the K51A mutant , which is predicted to be predominantly nucleotide-free ( Kravets et al . , 2012 ) , shows higher anisotropy values compared to WT , is entirely delocalized in the cytosol , and is monomeric in living cells ( this study ) .", "However , GTPase-domain dimerization is not sufficient to determine the targeting of mGBP2 to the PVM .", "Interestingly , individual murine and human GBPs ( hGBPs ) harbor C-terminal CaaX motifs ( GBP1 , GBP2 , GBP5 ) , targeting them for isoprenylation , which provides anchorage to different membranous compartments distributed within the host cell ( Degrandi et al . , 2007; Britzen-Laurent et al . , 2010; Vestal et al . , 2000 ) .", "As described for hGBP1 , the dimerization of the GTPase-domains enables contact formation between the two C-terminal α13 helices resulting in a juxtaposition which is crucial for their membrane localization through the attached farnesyl groups ( Vopel et al . , 2014 ) .", "The purified CaaX mutant of mGBP2 ( C586S ) shows GTP binding and hydrolysis properties as well as nucleotide dependent dimerization like the WT protein ( Figure 1—figure supplement 1 ) .", "However , the C586S mGBP2 mutant renders the protein non-functional and it is found ubiquitously within the cytosol .", "Noteworthy , the isoprenylation mutant C586S shows similar localization and anisotropy values as the K51A mutant in living cells , also indicating a monomeric species .", "Altogether these studies suggest an assembly mechanism for mGBP2 complexes in living cells that connects the GTPase activity of mGBP2 with membrane association leading to the stabilization of mGBP2 multimers , which is essential for its biological function .", "Moreover , MFIS measurements with high-precision FRET and brightness analysis allowed us to characterize the dynamic equilibrium between mGBP2 multimers .", "Their size distribution is heterogeneous ranging from dimers to large multimers ( Figure 7c and d ) .", "The dependence of FRET rate constants on the mGBP2 concentration and their saturation level proves dense packing of the mGBP2 protomers in multimers ( 'Maximum FRET rate constants' , Materials and methods section ) as suggested for the related mechanochemical GTPase dynamin forming large helical oligomers ( Faelber et al . , 2011 ) .", "While FRET characterizes the molecular environment of GFPs , scanning FIDA shows that the average number of mGBP2 units in the oligomers can reach several thousands .", "Considering the predicted size of the mGBP2 monomer ( ~ 4 × 6 × 12 nm , according to PDB-ID 1F5N of hGBP1 ) , it is expected that the oligomers should reach a size of several hundred nanometers .", "Remarkably , confocal live cell imaging ( Figure 8e and Video 2 ) resolves the enrichment of mGBP2 at the PV membrane resulting in a rough surface with elongated very bright features , that are sufficiently large to be resolved by far field confocal microscopy .", "Figure 10 provides a scheme derived from the observed mGBP interactions in living cells with molecular resolution at various stages after T . gondii infection .", "Our hetero-FRET data of MFIS measurements clearly reveal interactions of mGBP2 in multimers with itself , mGBP1 , and , to a lesser extent , with mGBP3 but not with mGBP6 .", "However , the interplay between mGBP2 and mGBP5 is different .", "The two proteins can be coprecipitated ( Figure 4—figure supplement 2 ) , but the complex shows no FRET ( Figures 4 and 6 ) .", "Given the experimentally achieved concentrations in the cytosol and the corresponding enrichment in the VLS , the observation that fluorescence anisotropy of G-mGBP2 increased while its donor lifetime remained unchanged suggests either an interaction of mGBP2 and mGBP5 via adaptor molecules , so that they are not in close proximity and hence FRET inactive , or the rather unlikely case of an unfavorable static orientation of the fluorophores .", "It is noteworthy that , upon infection , oligomerization and accumulation of the mGBPs in VLS is reversible , so that the VLS serve as protein reservoir to accomplish a fast attack of the parasite after infection . 10 . 7554/eLife . 11479 . 026Figure 10 . Schematic view of mGBP dynamics and multimerization in T . gondii infected cells . For details see Results and DiscussionDOI: http://dx . doi . org/10 . 7554/eLife . 11479 . 026 Both mGBP1 and mGBP2 have been implicated in T . gondii defense in single gene deficient mice ( Degrandi et al . , 2013; Selleck et al . , 2013 ) .", "Since mGBP1 still recruits to T . gondii in mGBP2-/- cells ( Degrandi et al . , 2013 ) , the high level of colocalization and interaction between mGBP1 and mGBP2 and their important roles in T . gondii control strongly argue for a cooperative effect at the PVM of T . gondii .", "Interestingly , reconstitution of mGBP2 in mGBPchr3-deleted MEFs did not allow a sufficient control of T . gondii replication , while reconstitution of mGBP1 partially restored the WT phenotype ( Yamamoto et al . , 2012 ) .", "Although more studies on the hierarchy of mGBPs are needed to fully understand the individual roles of each GTPase , this might hint that mGBP2 acts in concert with mGBP1 and possibly other mGBPs to exert its molecular antiparasitic activity .", "The dissociation constant KD , oligo of mGBP2/mGBP3 heteromers is 25 times larger than that of mGBP2/mGBP2 homomers .", "Thus , it is not surprising that mGBP3 colocalized only partially in the same VLS ( Figure 3 , Figure 10 ) .", "Strikingly , mGBP6 , which also localizes in VLS and recruits to the PVM of T . gondii , is predominantly found in different VLS and shows no interaction with mGBP2 by FRET and co-IP .", "The different localizations of mGBP multimers argue for distinct individual functional roles in T . gondii immunity to be elucidated in the future .", "Recently , an essential function for the cassette of autophagy proteins , including Atg7 , Atg3 , and the Atg12-Atg5-Atg16L1 complex was demonstrated in cellular anti-T .", "gondii immunity by facilitating IRG and GBP recruitment to the PVM ( Ohshima et al . , 2014; Choi et al . , 2014; Haldar et al . , 2014 ) .", "This function appears to be independent of the classical autophagy degradation pathway ( Zhao et al . , 2008 ) , but rather to play a role in the delivery of effectors to pathogen containing vacuoles ( Selleck et al . , 2013 ) .", "Performing live cell imaging and MFIS analysis it could be shown that mGBP2 is loaded on the PVM of T . gondii within a few minutes post-infection , assembling a machinery of supramolecular complexes with mGBP1 and mGBP3 .", "It has been recently shown , that mGBP and IRG host proteins cooperate in destruction of PVs of T . gondii ( Haldar et al . , 2015; 2013; Yamamoto et al . , 2012 ) .", "Previous studies in astrocytes and macrophages infected with type II T . gondii strains have shown that IRGs participate in mediating vesiculation of the PVM , resulting in the exposure of disrupted parasites to the host cytosol ( Ling et al . , 2006; Martens et al . , 2005; Melzer et al . , 2008; Zhao et al . , 2009a; Zhao et al . , 2009b ) .", "However , no colocalization of IRG proteins with the parasite plasma membrane has been reported previously .", "Here , we unambiguously show that mGBP2 directly targets the membrane of the parasite after penetration or disruption of the PVM .", "Interestingly , GBP proteins , especially mGBP2 , were shown to stimulate caspase-11-dependent pyroptosis in macrophages infected with Gram-negative bacteria which reside in vacuoles .", "There , GBP dependent induction of lysis of the pathogen-containing vacuoles and release of cytoplasmic LPS leads to the activation of the noncanonical inflammasome ( Pilla et al . , 2014; Meunier et al . , 2014 ) .", "Strikingly , a novel study suggests a direct bacteriolytic function of mGBPs , releasing pathogen-associated molecular patterns into the cytosol , resulting in activation of the AIM2 inflammasome ( Man et al . , 2015; Meunier et al . , 2015 ) .", "Thus , based on our observations , it is likely that mGBPs promote not only disruption of the PVM , but also directly induce lysis of the plasma membrane of T . gondii .", "The hierarchy of events which might be involved in T . gondii targeting and elimination , such as autophagic degradation ( Choi et al . , 2014 ) and/or inflammasome activation ( Ewald et al . , 2014; Gorfu et al . , 2014; Meunier et al . , 2014; 2015 ) , have yet to be determined .", "These studies define mGBP2 as an important effector molecule of innate immunity in the host parasite interaction with apicomplexan parasites such as T . gondii , by providing seminal insight into its supramolecular assembly and cellular function .", "Further studies will be performed to address the question how this information can be exploited for anti-parasitic therapy ." ], [ "The WT ORF of mGBP2 ( NCBI accession numbers: mGBP-2 , NM_010260 . 1 ) was subjected to site directed mutagenesis ( QuikChange II Mutagenesis kit , Agilent , Germany ) for generation of GTPase mutants R48A , K51A , E99A and D182N ( Kravets et al . , 2012 ) and isoprenylation mutant C586S ( Degrandi et al . , 2013 ) in the pEGFP-C2 plasmid ( Clontech , France ) .", "The respective genes were then cloned into the pWPXL plasmid ( Trono lab , Switzerland ) as N-terminal GFP-fusion constructs .", "For the cloning of mCherry constructs , the pWPXL plasmid was modified by exchanging of the gene for GFP by the gene for mCherry .", "The ORFs of mGBP1 ( NM_010259 . 2 ) , mGBP2 , mGBP3 ( NM_001289492 . 1 ) , mGBP5 ( NM_153564 . 2 ) , mGBP6 ( NM_194336 . 2 ) were then cloned into the modified pWPXL plasmid as N-terminal mCherry-fusion constructs .", "The lentiviral envelope vector pLP/VSVG ( Invitrogen , Germany ) and the packaging vector psPAX2 ( Trono lab ) were used for the lentiviral genetic transfer .", "MEFs were cultured in Dulbecco's modified Eagle's medium ( DMEM , Invitrogen/Gibco , Germany ) supplemented with 10% ( v/v ) heat-inactivated low endotoxin fetal bovine serum ( FBS , Cambrex , Germany ) , 100 U/ml penicillin , 100 μg/ml streptomycin , 2 mM L-glutamine ( Biochrom , Germany ) and 0 . 05 mM β-mercaptoethanol ( Invitrogen/Gibco ) .", "Human foreskin fibroblasts ( HS27 , ATCC CRL-1634 ) were hold in culture in Iscove's Modified Dulbecco's Medium ( IMDM , Invitrogen/Gibco ) with the same supplementations .", "293FT cells were cultivated in DMEM supplemented with 10% FBS , 100 U/ml penicillin , and 100 μg/ml streptomycin .", "All recombinant lentiviruses were produced by transient transfection of 293FT cells according to standard protocols .", "Briefly , subconfluent 293FT cells were cotransfected with 20 µg of a plasmid vector , 10 µg of psPAX2 , and 5 µg of pLP/VSVG by calcium chloride precipitation in FBS free medium .", "After 6 hr medium was changed ( 10% FBS ) , and supernatants with recombinant lentivirus vectors were harvested 48 hr later .", "Alternatively , the trasfection was performed utilizing the jetPRIME trasfection reagent ( Polyplus , New York , NY ) in medium supplemented with 10% FBS .", "MEFs were seeded in 24 well plates ( Corning , Germany ) and transduced with 600 µl of lentivirus with 25 µg Polybrene ( Merck Millipore , Germany ) .", "After 4 hr of incubation medium was changed .", "The transduction efficacy was analyzed by flowcytometry .", "Subsequently , GFP or GFP/mCherry positive cells were sorted and cultivated .", "Tachyzoites from T . gondii strain ME49 were maintained by serial passage in confluent monolayers of HS27 cells .", "After infection of fibroblasts , parasites were harvested and passaged as described previously ( Degrandi et al . , 2007 ) .", "Cells were stimulated with 200 U/mL IFNγ ( R&D Systems , Minneapolis , MN ) 16 hr before infection .", "For immunofluorescence , MEFs were cultured in 24-well plates ( Falcon , BD Biosciences , Germany ) on cover slips ( ø 13 mm , VWR International , Germany ) and inoculated with freshly harvested T . gondii at a ratio of 50:1 .", "To remove extracellular parasites , cells were washed with PBS .", "Cells were fixed in 4% paraformaldehyde ( PFA , Sigma-Aldrich , Germany ) permeabilized with 0 . 02% saponin ( Calbiochem-Merck ) and blocked in 0 . 002% saponin with 2% goat serum ( Biozol , Germany ) .", "The outer membrane of T . gondii was visualized by anti-SAG1 ( Abcam , UK ) at a concentration of 1/700 .", "As secondary reagents , 1/200 concentrated Cy2-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG plus IgM ( Jackson ImmunoResearch Laboratories , UK ) were used .", "Nuclei were counterstained with 1/2500 4' , 6-diamidino-2-phenylindole ( DAPI , Invitrogen ) .", "The cover slips were fixed in fluorescence mounting medium ( Fluoromount-G , Southern Biotechnology Associates , Birmingham , AL ) .", "Fluorescence was visualized using a LSM780 confocal microscope ( Zeiss , Germany ) .", "Image analysis and processing was performed with the ZEN ( Zeiss ) and Imaris ( Bitplane , Switzerland ) software .", "Live cell imaging was performed using an LSM780 confocal microscope ( Zeiss ) at 37°C with 8% CO2 and humidity saturated air .", "Cells were cultured and imaged on imaging dishes CG ( MoBiTec , Germany ) with Phenol-free cell culture media .", "Image analysis was performed with the software ZEN ( Zeiss ) , Imaris ( Bitplane ) and AutoquantX3 ( MediaCy , Rockwell , MD/Bitplane ) .", "MFIS experiments ( Kudryavtsev et al . , 2007; Weidtkamp-Peters et al . , 2009 ) were performed with a confocal laser scanning microscope ( Olympus FV1000 , IX81 inverted microscope ) additionally equipped with a single photon counting device with picosecond time-resolution ( PicoQuant Hydra Harp 400 , PicoQuant , Germany ) .", "GFP was excited at 485 nm with a linearly polarized , pulsed ( 32 MHz ) diode laser ( LDH-D-C-485 ) at 0 . 4 μW at the objective ( 60x water immersion , Olympus UPlanSApo NA 1 . 2 , diffraction limited focus ) .", "mCherry was excited at 559 nm with a continuous-wave laser ( FV1000 ) at 0 . 54 µW at the objective .", "The emitted light was collected in the same objective and separated into its perpendicular and parallel polarization ( PBS 101 , Thorlabs , Germany ) .", "GFP fluorescence was then detected by SPADs ( PD5CTC , Micro Photon Devices , Italy ) in a narrow range of its emission spectrum ( bandpass filter: HC520/35 ( AHF , Germany ) ) .", "mCherry fluorescence was detected by HPDs ( HPMC-100-40 , Becker&Hickl , Germany ) , of which the detection wavelength range was set by the bandpass filters ( HC 607/70 , AHF ) .", "Images were taken with 20 μs pixel time and a resolution of 276 nm/pixel .", "With 485nm excitation , series of 40–100 frames were merged to one image and further analyzed with custom-designed software ( Widengren et al . , 2006 ) and at the web page ( http://www . mpc . hhu . de/software/software-package . html ) .", "From the recorded GFP ( SG ) and mCherry ( SR ) signal intensities , background intensities of the regions where no cells localize were subtracted to determine fluorescence intensities of GFP ( FG ) and mCherry ( FR ) respectively .", "To determine fluorescence anisotropy ( rD ) and fluorescence-weighted donor lifetimes ( <τD>f ) in each pixel , the histograms presenting the decay of fluorescence intensity after the excitation pulse were built with 256 bins and 128 ps per bin .", "The fitting procedures were described previously ( Stahl et al . , 2013; Kravets et al . , 2012 ) .", "In each obtained MFIS image , pixels in the VLS and in the cytosol in uninfected cells , and pixels at the PVM and in the cytosol in infected cells were separately selected according to fluorescence photon number ( Figures 1a , 2a , 4a and 6a ) .", "Photons from each pixel selection were integrated to an intensity decay histogram with 1024 bins and 32 ps per bin .", "The pixel-integrated histograms were formally fitted to quantitatively determine FRET parameters .", "In the model , fluorescence decay of FRET sample ( fmix ( t ) ) is the sum of FRET-quenched donor decay ( f ( D , A ) ( t ) ) weighted by its species fraction xFRET and unquenched donor decay ( f ( D , 0 ) ( t ) ) weighted by ( 1- xFRET ) : ( 1 ) f ( t ) = ( 1-xFRET ) ·f ( D , 0 ) ( t ) +xFRET·f ( D , A ) ( t ) Here , f ( D , 0 ) ( t ) could be pre-determined from donor-only measurements using a bi-exponential fit model: ( 2 ) f ( D , 0 ) ( t ) =∑mxD0 ( m ) ·exp ( -t·kD0 ( m ) ) in which m=2 because fluorescent proteins in living cells usually show a bi-exponential decay ( Suhling et al . , 2002 ) .", "Fit parameters in f ( D , 0 ) ( t ) include two normalized pre-exponential factors xD0 ( m ) ( ∑xD0 ( m ) =1 ) and two decay rate constants , kD0 ( m ) .", "These pre-determined parameters from donor-only measurements were then set as global restraints .", "The quenched donor decay f ( D , A ) ( t ) in Equation ( 1 ) is given by: ( 3 ) f ( D , A ) ( t ) =f ( D , 0 ) ( t ) ·exp ( -t·kFRET ) where kFRET is the FRET rate constant .", "The fitted parameters in the 1-kFRET model ( Equations 1–3 ) are xFRET and kFRET .", "This formal analysis revealed that mGBPs exhibit distinct FRET features in different cellular compartments ( Figure 6—figure supplement 2 ) .", "FRET-related donor quenching histogram ( εmix ( t ) ) was plotted to directly separate different molecular species and visualize FRET efficiency in the pixel-integrated data .", "εmix ( t ) is calculated as the ratio between normalized fluorescence decay of FRET sample , fmix ( t ) , and of donor-only sample , f ( D , 0 ) ( t ) : ( 4 ) εmix ( t ) =fmix ( t ) f ( D , 0 ) ( t ) =xFRET ε ( D , A ) ( t ) + ( 1-xFRET ) The drop on a εmix ( t ) diagram represents the species fraction of FRET-active complex , xFRET .", "In Equation ( 4 ) , ε ( D , A ) ( t ) is the ratio between f ( D , A ) ( t ) ( Equation", "3 ) and f ( D , 0 ) ( t ) ( Equation", "2 ) and describes the time-dependent occurrence of the FRET process .", "( 5 ) ε ( D , A ) ( t ) =f ( D , A ) ( t ) f ( D , 0 ) ( t ) =exp ( -t·kFRET ) To directly compare different experiments , ε ( D , A ) ( t ) diagrams were plotted in Figure 4d .", "A steeper slope in ε ( D , A ) ( t ) diagram indicates that the experiment showed higher kFRET .", "To resolve three characteristic protein species , namely mGBP monomer ( with specie fraction xmono ) , dimer ( xdi ) and oligomers ( xoligo ) by analyzing time-resolved anisotropy rmix ( t ) ( Equation", "6 ) and time-resolved FRET-induced donor decay εmix ( t ) ( Equation", "7 ) for homo- and hetero-FRET , respectively , both decays were fitted with a linear combination of three species-specific patterns .", "Homo-FRET .", "The rmix ( t ) of homo-FRET data was fitted with: ( 6 ) rmix ( t ) =r0· ( xmono+xdi ( ∫ p ( kdi ) e-2·kdi·tdkdi ) +xoligo ( xse-2·koli , s·t+xle-2·koli , l·t ) ) e-tρglobal Here p ( kdi ) is the FRET-rate distribution of mGBP2 dimer complex as determined by the conformational sampling of the sterically allowed space ( see Monte Carlo sampling of the donor-acceptor conformational space of mGBP2 dimer , Figure 7b and Figure 7—figure supplement 1d ) .", "kolig , s and kolig , l are formally assigned as the FRET rate constants of mGBPs oligomers of small and large sizes respectively ( Figure 7b ) , and xs and xl are their normalized fractions .", "It has to be considered that energy can be transferred in forward and backward direction which doubles the rate constants .", "The monomer is described by a constant offset , because there is no FRET .", "The fundamental anisotropy r0 for GFP molecules is known as 0 . 38 .", "The global rotational correlation time ρglobal was estimated as 15 ns given the molecular weight of G-mGBP2 fusion protein .", "Oligomer species which produced ultrafast decay components in rmix ( t ) resulted in a drop in the initial anisotropy ( Figure 6d ) .", "With the knowledge of r0 they can be determined together with other species in homo-FRET data .", "Hetero-FRET .", "An analogous analysis was applied to the hetero-FRET data .", "The εmix ( t ) ( Equation", "4 ) was fitted with: ( 7 ) εmix ( t ) =xmono+xdi∫ pkdie-kdi·tdkdi+xoligo , se-t·koli , s in which xoligo , s denotes the species fraction of small oligomers .", "In the case of hetero-FRET , donor molecules in large oligomers ( with species fraction xoligo , l ) could be strongly quenched by nearby acceptors up to nearly 100% and thus became irresolvable owing to the finite width of the instrument response function .", "Therefore the information of large oligomers in hetero-FRET data needed to be recovered according to the homo-FRET data .", "In the latter , the species fractions of small and large oligomers were found equal in various compartments .", "Based on the relation xoligo , s = xoligo , l the large oligomer fractions in hetero-FRET data were extrapolated .", "Moreover , such a coherent behavior between small and large oligomers indicated that they have a common origin; and the broad distribution of their rate constants showed that oligomers may consist of a variety number of units ( Figure 7b ) .", "Hence , it is more meaningful to combine both oligomer species and generally sort protein species as monomer , dimer and oligomer as displayed in Figure 6c .", "The fits were performed by custom software programmed in MATLAB .", "Based on the hGBP1 crystal structure ( Prakash et al . , 2000a ) homology models of the G-mGBP2 ( PDB-ID: 1F5N , 4EUL ) and mCherry-mGBP2 fusion protein ( PDB-ID: 1F5N , 2H5Q ) ( supplementary file 1a ) were constructed using MODELLER ( Fiser and Sali , 2003 ) .", "The homology models were protonated using PDB-ID 2PQR ( Dolinsky et al . , 2007 ) .", "Then the protonated full-length protein models were mapped to a reduced representation solely consisting of the C- , Cα- , N- , O- and the hydrogen atoms forming the NH-O bonds .", "The repulsion between the atom pairs ( O , N ) , ( C , O ) and ( C , N ) were modeled as repulsive quadratic potential ( Kalinin et al . , 2012 ) and the existing hydrogen bonds as simple scaled attractive potential ( 1/r ) preserving secondary structural elements .", "The sampling was performed on the ϕ and ψ torsion angles .", "In each iteration step the torsion angle of one amino acid was changed by random value taken from a Gaussian-distribution with a width of 0 . 025 rad .", "The sampling of the conformational space was restricted to the linkage region .", "Thus , only the internal coordinates of the connecting linker were altered while the internal coordinates of the beta-barrels as well as the internal coordinated of the mGBP2 model were kept constant .", "Given the sampled conformation of the mCh-mGBP2 and the G-mGBP2 constructs a putative head-to-head dimer structures was created by superimposing the LG-domains onto the LG-domains in the dimer structure of hGBP1 in presence of GppNHp ( PDB-ID: 2BC9 ) and discarding conformations with clashes ( Vopel et al . , 2014 ) .", "To calculate the donor-acceptor distance , Rsim , in every simulated structure , on each fluorophore , two Cα-atoms on the beta-barrel ( Asn122 and Asn147 on GFP , Tyr125 and Glu149 on mCherry ) were chosen ( Figure 7—figure supplement 1a , supplementary file 1a ) , so that the connecting vector of the two atoms is a good approximation of the transition dipole .", "The distance between the middle points of the connecting vectors of the donor and acceptor is taken as the distance between the chromophores ( RDA , sim ) .", "Supplementary file 1b lists out the detailed calculation steps to determine the ( RDA , sim ) and orientation factor ( κ2 ) .", "For each simulated mGBP2 dimer conformation , its kdi value was calculated according to: ( 8 ) kdi= ( 3/2 ) ·k2· ( 1/τD ( 0 ) ) · ( R0/RDA , sim ) 6 in which τD ( 0 ) = 1/k0 is 2 . 6 ns and the Förster radius ( R0 ) of GFP and mCherry is 52Å including κ2 = 2/3 .", "The donor-acceptor distance distribution obtained from the MC simulation of the mGBP2 dimer ( Figure 7—figure supplement 1e , blue curve ) was used as the prior input , and was further optimized according to experimental data measured in the cytosol using the maximum entropy method ( MEM ) ( Vinogradov and Wilson , 2000 ) .", "The optimized distance distribution ( MEM-MC ) is plotted in Figure 7—figure supplement 1e ( red curve ) .", "The difference between both distributions is primarily in the short distance range , because a small fraction of oligomers is present in the experimental data ( Figure 7c ) , but of course absent in the MC simulation of a dimer .", "The two distributions agree very well in the longer distance range , therefore the distribution from the MC dimer simulation ( kdi ) ( Figure 7b ) describes the experimental data in a valid manner .", "mGBP protein concentrations .", "The protein concentration is monitored via the fluorescence intensity of the fused fluorescent proteins .", "The detection volume of the MFIS microscope was calibrated by Fluorescence Correlation Microscopy ( FCS ) measurements of Rhodamine 110 ( Rh110 ) to determine its shape and size .", "The fitting model applied to the obtained FCS curve assumes a 3-dimensional Gaussian-shaped volume , and a single diffusing species including transitions to a triplet state as shown in ( Weidtkamp-Peters et al . , 2009 ) .", "From the Rh110 diffusion time of 32 μs and aspect ratio of 7 , the detection volume Vdet-GFP of GFP was determined as 0 . 5 fl .", "The detection volume of mCherry Vdet-mCherry is larger ( 0 . 8 fl ) because of the longer wavelength .", "The brightness of GFP or mCherry in living cells was individually characterized from FCS measurements of freely diffusing fluorescent proteins in the cytosol .", "By fitting the same model function as in Rh110 experiment , it was found that with 0 . 54 μW of 559 nm laser excitation at the objective , mCherry brightness is QmCherry = 0 . 1 kcpm ( kilo counts per molecule ) in the cytosol and that with 0 . 4 μW of 485 nm laser excitation , GFP brightness is QGFP = 0 . 56 kcpm in the cytosol .", "The average GFP fluorescence intensity of an image with GFP excitation was first corrected for detector dead time , and then the obtained intensity ( SG , Gm ) was further corrected for quenching effect due to FRET: ( 9 ) SG , Gu=SG , Gm ( 1-xFRET ) +xFRET· ( 1-E ) SG , Gu denotes unquenched GFP fluorescence intensity in the absence of hetero-FRET and was used to calculate the GFP concentration .", "The average mCherry fluorescence intensity of an image with mCherry excitation was first corrected for detector dead time ( Becker , 2005 ) , and then used to calculate the mCherry concentration with the determined detection volume and the mCherry brightness .", "The concentration of GFP ( CGFP ) was determined by ( 10 ) CGFP=SG , GuQGFP·Vdet-GFP=SG , Gu0 . 56kcpm·0 . 5fl The concentration of mCherry ( CmCherry ) was determined by ( 11 ) CmCherry=SR , RQmCherry·Vdet-mCherry=SR , R0 . 1kcpm·0 . 8fl We note that we do not measure intensities of single-molecule events as described by Wu et al . ( Wu et al . , 2009 ) but intensity averages of pixel populations so that it is sufficient to use an average brightness Q for the calculation of the fluorescent protein ( FP ) concentrations .", "In our pattern fits we usually find on average less than 10% of non-FRET species ( Figure 7c ) .", "From this we conclude that under our conditions with one photon excitation of donors with low irradiance ( as compared to the two photon excitation used by Wu et al . ( Wu et al . , 2009 ) and low FRET efficiency most of the mCherry molecules are active FRET-acceptors .", "The KD , dim of ~24 nM of mGBP2 dimerization determined in this way is very close to previous biochemical studies ( Kravets et al . , 2012 ) .", "In Equation 9 , the FRET-active species fraction ( xFRET ) is obtained from fitting of each measurement in pixel-integrated MFIS-FRET analysis using the 1-kFRET model ( Equations 1–3 ) .", "FRET efficiency , E , was calculated as: ( 12 ) E=1-∑mxD0 ( m ) ·kD0 ( m ) +kFRET-1∑mxD0 ( m ) ·kD0 ( m ) -1 Please refer to Formal pixel-integrated MFIS-FRET analysis for explanations on the symbols in Equations 9 and 12 .", "To quantify the dependence of the dimeric species fraction on the total protein concentration ( initial increase , stationary phase followed by a decrease ) the simplest possible model was used to approximate such a behavior .", "In this model the formation of a dimer and a subsequent formation of a tetramer formed by two dimers was assumed .", "The formation of a dimer and a tetramer can be described by two reactions with corresponding dissociation equilibrium constants: ( 13 ) A1+A1 ⇆ A2 KD , dim=c ( A1 ) c ( A1 ) c ( A2 ) , A2+A2 ⇆ A4 KD , oligo=c ( A2 ) c ( A2 ) c ( A4 ) For given of equilibrium constants and a total protein concentration cT=c ( A1 ) +2·c ( A2 ) +4·c ( A4 ) the species concentrations c ( A1 ) , c ( A2 ) , c ( A4 ) were determined numerically by solving the fourth polynomial equation cT ( A1 ) by a simple root-finding algorithm ( Ridders , 1979 ) and minimize the disagreement between the modeled species fractions and the fitted fractions by a Quasi-Newton method ( Shanno and Kettler , 1970 ) .", "This model of stepwise oligomer formation was extended by the stepwise binding of dimer in a non-cooperative fashion ( i . e . all equilibrium constants are equal to KD , oligo ) up to a dodecamer .", "If the total concentration of all oligomers ( 4–12 ) is used to display the binding isotherm , one obtains an only slightly broadened binding isotherm compared to the tetramer system .", "If this binding isotherm is fitted with the simpler tetramer model , a binding constant for dimer binding KD , app-oligo is obtained , which is slightly ( factor 1 . 6 ) larger than the simulated value .", "As no information on the cooperativity of binding and the spatially resolved GTP concentration was available , the formation of higher order oligomers was approximated by the minimal tetramer model for the following reasons: ( 1 ) FRET only senses its local environment ( i . e . a limited oligomer size ) thus the contribution of each monomer unit to the measured signal decreases with increasing oligomer size .", "( 2 ) This simple model reduces the number of fitting parameters to an adequate level given the spread of the data-points .", "To conclude , a simple model with a Langmuir binding isotherm ( i . e . non-cooperative binding ) describes all experiments very well .", "The simulation showed that the obtained apparent dissociation constant KDapp , oligo is a good approximation for the true KD , oligo .", "Note that the observed reduction in steady-state anisotropy ( rD ) for cells of high mGBP2 concentration as displayed in Figure 4c , was mainly due to the large drop in the initial anisotropy of their time-resolved anisotropy ( rD ( t ) ) as plotted in Figure 6d .", "Therefore the KD , app value ( 9 μM ) derived from rD in fact reports the mGBP2 oligomerization processes that could produce such ultrafast depolarizing effect , and is very close to the 8 μM obtained by fitting rD ( t ) with the species-resolved model .", "Hence , the two independent approaches interrogating the same oligomerization process delivered very consistent results , verifying the reliability of the analyses .", "Due to its inverse sixth-power distance dependence ( Equation 14 ) , FRET depends on molecular proximity and cannot occur between remotely located fluorescent proteins .", "Consequently , in large mGBP oligomers , the FRET-induced donor quenching will eventually saturate regardless of the presence of more acceptors simply because they are too distant .", "If assuming that the mGBP proteins are arranged homogeneously in mGBP oligomers , the maximum kFRET can be estimated following the ideas of T . Förster ( Förster , 1949 ) .", "Here , the case of a single donor is considered , the FRET rate constant kFRET from the donor to N surrounding acceptors is given by Equation 14 using the parameter in Equation 8 .", "( 14 ) kFRET , max=1τD ( 0 ) ∑k=1NR0RDA , k6 with RDA , k being the distance between the donor and the k-th acceptor kFRET .", "Assuming that the acceptors that attached on mGBPs are homogeneously distributed around the donor and closed packed with a minimum inter-fluorophore distance Rmin , which is ~26 Å given by the molecular dimensions of fluorescent proteins , a similar estimation of the maximum kFRET as in ( Förster , 1949 ) can be performed .", "To determine the maximum FRET-rate constant at which a donor molecule is quenched by multiple acceptors it was assumed that at saturation protein concentrations the space around the donor is fully filled by acceptors and the space that is occupied by the donor cannot be occupied by the acceptor .", "If a donor is homogenously surrounded by acceptors , given a constant molecular density ρ ( number of acceptors per volume ) , which are separated at least by a distance of Rmin from the donor , the FRET-rate constant is given by: ( 15 ) kFRET=1τ0∫Rmin∞ρ·4πR2R0R6dR=ρR06τ043π·1Rmin3=1τ0R06Rmin3·Rmol3 Rmol is the mean radius of the acceptor molecules and relates to molecular density ρ .", "Given the molecular structure of mCherry in mCh-mGBPs fusion proteins , Rmol is approximated by 31 Å .", "The minimum possible distance Rmin is given by the structure of the fluorescent proteins ( ~20–30 Å ) .", "Therefore , the maximum possible FRET-rate constant kFRET was approximated by ~15 ns-1 .", "To investigate the size ( composition ) of mGBP2 oligomer locating at the PVM which can exceed the detectable range of FRET technique ( > 10 nm ) , FIDA from ( Kask et al . , 2000 ) was adapted for imaging measurements and employed in infected G-mGBP2 expressing cells .", "Given the recorded photon trace in the image line of selected PVM area , 20 µs binned new sliding with 2 . 5 µs ( 1/8 × pixel time ) steps intensity traces were computed .", "Then a corresponding 2D matrix of green versus red photon counts from all the time windows is generated and analyzed by 2D FIDA .", "The average brightness , <Qoligo> , and average number , <Noligo> , of the mGBP2 oligomers could be determined .", "The average number of mGBP2 units ( Figure 7e ) per oligomer <NmGBP2> is calculated as the ratio of obtained <Qoligo> to single GFP brightness QGFP: ( 16 ) NmGBP2=QoligoQGFP Based on these two average numbers of oligomers and mGBP2 units per pixel and knowing the excitation volume of the setup , the average mGBP2 concentration <cmGBP2> is calculated as ( 17 ) cmGBP2FIDA=NoligoNmGBP2NA·Vdet where NA= 6 . 022 × 1023 mol−1 is the Avogadro‘s number and Vdet = 0 . 5 fl – excitation volume of the used laser .", "The mGBP2 concentration calculated from scanning FIDA was compared with that directly derived from the GFP intensity as a control .", "Figure 7—figure supplement 1f shows the good agreement between both methods ." ] ]
[ "GBPs are essential for immunity against intracellular pathogens , especially for Toxoplasma gondii control .", "Here , the molecular interactions of murine GBPs ( mGBP1/2/3/5/6 ) , homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational , Multiparameter Fluorescence Image Spectroscopy , and live cell microscopy methodologies .", "Control of T . gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus , enabling reversible oligomerization in vesicle-like structures .", "mGBP2 undergoes structural transitions between monomeric , dimeric and oligomeric states visualized by quantitative FRET analysis .", "mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane ( PVM ) as orchestrated , supramolecular complexes forming large , densely packed multimers comprising up to several thousand monomers .", "This dramatic mGBP enrichment results in the loss of PVM integrity , followed by a direct assault of mGBP2 upon the plasma membrane of the parasite .", "These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity ." ]
[ "A microscopic parasite called Toxoplasma gondii causes a serious disease known as toxoplasmosis in humans and other mammals .", "Once inside the body , the parasite can infect host cells , where it hides inside a cell structure called a vacuole .", "However , this triggers self-defense mechanisms in the infected cells that help to control the spread of the parasite in the body .", "Proteins called guanylate binding proteins – which are normally found as small units in healthy host cells – bind to each other and form larger “complexes” that promote immune responses in that particular cell .", "However , it was not known how the guanylate binding proteins congregate to form the complexes , or how this activates the cell’s defenses .", "Here , Kravets et al . use sophisticated fluorescence microscopy techniques with living cells to study the roles of guanylate binding proteins in immune responses during T . gondii infection .", "The experiments show that the proteins are stored as larger units in structures within healthy cells that allow them to relocate quickly to the vacuole when the parasite is detected .", "Once there , the guanylate binding proteins form large complexes that can contain thousands of protein units .", "The process requires energy that is released from the break down of a molecule called GTP , and specific chemical modifications to the guanylate binding proteins to allow them to bind to each other .", "Further experiments found that the guanylate binding proteins in the complexes assist in weakening the structure of the vacuoles , and that subsequently , one type of protein – called GBP2 – directly attacks the parasite itself .", "Kravets et al . ’s findings set the stage for the development of new therapies that help to fight T . gondii infections ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "genetics and genomics" ]
Large, three-generation human families reveal post-zygotic mosaicism and variability in germline mutation accumulation
elife-46922-v1
[ [ "In a 1996 lecture at the National Academy of Sciences , James Crow noted that ‘without mutation , evolution would be impossible’ ( Crow , 1997 ) .", "His remark highlights the importance of understanding the rate at which germline mutations occur , the mechanisms that generate them , and the effects of gamete-of-origin and parental age .", "Not surprisingly , continued investigation into the germline mutation rate has helped to illuminate the timing and complexity of human evolution and demography , as well as the key role of spontaneous mutation in human disease ( Scally and Durbin , 2012; Moorjani et al . , 2016; Deciphering Developmental Disorders Study , 2017; Yuen et al . , 2016; Acuna-Hidalgo et al . , 2016; Veltman and Brunner , 2012 ) .", "Some of the first careful investigations of human mutation rates can be attributed to J . B . S . Haldane and others , who cleverly leveraged an understanding of mutation-selection balance to estimate rates of mutation at individual disease-associated loci ( Haldane , 1935; Nachman , 2008 ) .", "Over half of a century later , phylogenetic analyses inferred mutation rates from the observed sequence divergence between humans and related primate species at a small number of loci ( Nachman and Crowell , 2000; Shendure and Akey , 2015 ) .", "In the last decade , whole genome sequencing of pedigrees has enabled direct estimates of the human germline mutation rate by identifying mutations present in offspring yet absent from their parents ( de novo mutations , DNMs ) ( Ségurel et al . , 2014; Scally and Durbin , 2012; Jónsson et al . , 2017; Goldmann et al . , 2016; Kong et al . , 2012; Roach et al . , 2010; Francioli et al . , 2015 ) .", "Numerous studies have employed this approach to analyze the mutation rate in cohorts of small , nuclear families , producing estimates nearly two-fold lower than those from phylogenetic comparison ( Roach et al . , 2010; Kong et al . , 2012; Jónsson et al . , 2017; Goldmann et al . , 2016; Scally and Durbin , 2012; Shendure and Akey , 2015; Turner et al . , 2017 ) .", "These studies have demonstrated that the number of DNMs increases with both maternal and paternal ages; such age effects can likely be attributed to a number of factors , including the increased mitotic divisions in sperm cells following puberty , an accumulation of damage-associated mutation , and substantial epigenetic reprogramming undergone by germ cells ( Jónsson et al . , 2017; Kong et al . , 2012; Goldmann et al . , 2016; Rahbari et al . , 2016; Crow , 2000; Gao et al . , 2019 ) .", "There is also evidence that the mutational spectra of de novo mutations differ in the male and female germlines ( Jónsson et al . , 2017; Goldmann et al . , 2016; Francioli et al . , 2015; Gao et al . , 2019; Agarwal and Przeworski , 2019 ) .", "Furthermore , a recent study of three two-generation pedigrees , each with 4 or five children , indicated that paternal age effects may differ across families ( Rahbari et al . , 2016 ) .", "However , two-generation families with few offspring provide limited power to quantify parental age effects on mutation rates and restrict the ability to assign a gamete-of-origin to ~20–30% of DNMs ( Rahbari et al . , 2016; Jónsson et al . , 2017; Goldmann et al . , 2016 ) .", "Here , we investigate germline mutation among families with large numbers of offspring spanning many years of parental age .", "We describe de novo mutation dynamics across multiple births using blood-derived DNA samples from large , three-generation families from Utah , which were collected as part of the Centre d'Etude du Polymorphisme Humain ( CEPH ) consortium ( Dausset et al . , 1990 ) .", "The CEPH/Utah families have played a central role in our understanding of human genetic variation ( Prescott et al . , 2008; 1000 Genomes Project Consortium et al . , 2015 ) by guiding the construction of reference linkage maps for the Human Genome Project ( Lander et al . , 2001 ) , defining haplotypes in the International HapMap Project ( International HapMap Consortium , 2003 ) , and characterizing genome-wide variation in the 1000 Genomes Project ( 1000 Genomes Project Consortium et al . , 2015 ) .", "The CEPH/Utah pedigrees are uniquely powerful for the study of germline mutation dynamics in that they have considerably more ( min = 4 , max = 16 , median = 8 ) offspring than those used in many prior studies of the human mutation rate ( Supplementary file 1 ) .", "Multiple offspring , whose birth dates span up to 27 years , motivated our investigation of parental age effects on DNM counts within families and allowed us to ask whether these effects differed across families .", "The structure of all CEPH/Utah pedigrees ( Supplementary file 1 ) also enables the use of haplotype sharing through three generations to determine the parental haplotype of origin for nearly all DNMs in the second generation .", "Using this large dataset of ‘phased’ DNMs , we can investigate the effects of gamete-of-origin on human germline mutation in greater detail .", "Finally , if a DNM occurs in the early cell divisions following zygote fertilization ( considered gonosomal ) , or during the proliferation of primordial germ cells , it may be mosaic in the germline of that individual .", "This mosaicism can then present as recurrent DNMs in two or more children of that parent .", "As DNMs are an important source of genetic disease ( Campbell et al . , 2014b; Campbell et al . , 2015; Biesecker and Spinner , 2013; Forsberg et al . , 2017; Acuna-Hidalgo et al . , 2016; Veltman and Brunner , 2012 ) , it is critical to understand the rates of mosaic DNM transmission in families .", "The structures of the CEPH/Utah pedigrees enable the identification of these recurrent DNMs and can allow one to distinguish mutations arising as post-zygotic gonosomal variants from those that are mosaic in the germline of the second generation ." ], [ "We sequenced the genomes of 603 individuals from 33 , three-generation CEPH/Utah pedigrees to a genome-wide median depth of ~30X ( Figure 1—figure supplement 1 , Supplementary file 1 ) , and removed 10 samples from further analysis following quality control using peddy ( Pedersen and Quinlan , 2017a ) .", "After standard quality filtering , we identified a total of 4 , 671 germline de novo mutations in 70 second-generation individuals , each of which was transmitted to at least one offspring in the third generation ( Figure 1a , Supplementary file 2 ) .", "Approximately 92% ( 4 , 298 of 4 , 671 ) of DNMs observed in the second generation were single nucleotide variants ( SNVs ) , and the remainder were small ( <=10 bp ) insertion/deletion variants .", "The eight parents of four second-generation samples were re-sequenced to a median depth of ~60X ( Figure 1—figure supplement 1d ) , allowing us to estimate a false positive rate of 4 . 5% for our de novo mutation detection strategy ( Materials and methods ) .", "Taking all second-generation samples together , we calculated median germline mutation rates of 1 . 10 x 10-8 and 9 . 29 x 10-10 per base pair per generation for SNVs and indels , respectively , which corroborate prior estimates based on family genome sequencing with roughly comparable parental ages ( Jónsson et al . , 2017; Kong et al . , 2012; Besenbacher et al . , 2016; Rahbari et al . , 2016 ) .", "Extrapolating to a diploid genome size of ~6 . 4 Gbp , we therefore estimate an average number of 70 . 1 de novo SNVs and 5 . 9 de novo indels per genome , at average paternal and maternal ages of 29 . 1 and 26 . 0 years , respectively ( Sasani , 2019 ) .", "We determined the parental gamete-of-origin for a median of 98 . 5% of de novo variants per second-generation individual ( range: 90 . 3–100% ) by leveraging haplotype sharing across all three generations in a family ( Kong et al . , 2012; Jónsson et al . , 2017 ) , as well as read tracing of DNMs to informative sites in the parents ( Figure 1b , Figure 1—figure supplement 2 ) .", "The ratio of paternal to maternal DNMs was 3 . 96:1 , and 79 . 8% of DNMs were paternal in origin .", "We then measured the relationship between the number of phased DNMs observed in each child and the ages of the child’s parents at birth ( Figure 2a ) .", "After fitting Poisson regressions , we observed a significant paternal age effect of 1 . 44 ( 95% CI: 1 . 12–1 . 77 , p<2e-16 ) additional DNMs per year , and a significant maternal age effect of 0 . 38 ( 95% CI: 0 . 21–0 . 55 , p=1 . 24e-5 ) DNMs per year ( Figure 2a ) .", "These confirm prior estimates of the paternal and maternal age effects on de novo mutation accumulation , and further suggest that both older mothers and fathers contribute to increased DNM counts in children ( Figure 2—figure supplement 1 ) ( Jónsson et al . , 2017; Goldmann et al . , 2016; Rahbari et al . , 2016; Wong et al . , 2016; Besenbacher et al . , 2015 ) .", "We next compared the paternal and maternal fractions of phased autosomal DNMs identified in the second generation across eight mutational classes ( Figure 2b ) .", "In maternal mutations , there was an enrichment of C > T transitions in a non-CpG context ( p=7 . 65e-6 , Chi-squared test of independence ) , and we observed an enrichment of T > G transversions in paternal mutations ( p=4 . 93e-3 , Chi-squared test of independence ) .", "Maternal and paternal enrichments of C > T and T > G , respectively , have been reported in recent studies of de novo mutation spectra , though the mechanisms underlying these observations are currently unclear ( Goldmann et al . , 2016; Jónsson et al . , 2017 ) .", "We additionally stratified second-generation individuals by the ages of their parents at birth and found no significant differences in the mutational spectra of children born to older or younger parents , though we may be underpowered to detect these differences in our dataset ( Figure 2—figure supplement 2 ) .", "A recent study of three two-generation pedigrees with multiple offspring suggested that the effect of paternal age on DNM counts in children may differ between families ( Rahbari et al . , 2016 ) .", "Given the large numbers of offspring in the CEPH/Utah pedigrees , we were motivated to perform an investigation of parental age effects on mutation counts within individual families .", "To measure these effects in the CEPH dataset , we first generated a high-quality set of de novo variants observed in the third generation , excluding recurrent ( mosaic ) DNMs shared by multiple third-generation siblings , likely post-zygotic DNMs ( Materials and methods ) , and ‘missed heterozygotes’ in the second generation ( 0 . 4% of heterozygous variants ) .", "The ‘missed heterozygotes’ represent apparent DNMs in the third generation that were , in fact , likely inherited from a second-generation parent who was incorrectly genotyped as being homozygous for the reference allele ( Materials and methods ) .", "In total , we detected 24 , 975 de novo SNVs and small indels in 350 individuals in the third generation ( Supplementary file 3 ) .", "Of these , we were able to confidently determine a parental gamete-of-origin for 5 , 336 ( median of 21% per third-generation individual; range of 8–38% ) using read tracing , and assign 4 , 201 ( 78 . 7% ) of these to fathers .", "Given the comparatively low phasing rate in the third generation , we focused our age effect analysis on the relationship between paternal age only and the total number of autosomal DNMs in each individual , regardless of parent-of-origin .", "Taking all third-generation individuals into account , we estimate the slope of the paternal age effect to be 1 . 72 DNMs per year ( 95% CI: 1 . 58–1 . 85 , p<2e-16 ) .", "Within a given family , maternal and paternal ages are perfectly correlated; therefore , the paternal effect approximates the combined age effects of both parents .", "When inspecting each family separately , we observed a wide range of paternal age effects among the CEPH/Utah families ( Figure 3 ) .", "To test whether these observed effects varied significantly between families , we fit a Poisson regression that incorporated the effects of paternal age , family membership , and an interaction between paternal age and family membership , across all third-generation individuals in CEPH/Utah pedigrees .", "As a small number of the CEPH/Utah pedigrees comprise multiple three-generation families ( Supplementary file 1 ) , we assigned each unique set of second-generation parents and their third-generation children a distinct ID , resulting in a total of 40 families ( Figure 3—figure supplement 1 ) .", "Overall , the effect of paternal age on offspring DNM counts varied widely across pedigrees , from only 0 . 19 ( 95% CI: −1 . 05–1 . 44 ) to nearly 3 . 24 ( 95% CI: 2 . 24–4 . 24 ) additional DNMs per year .", "A goodness-of-fit test supported the use of a ‘family-aware’ regression model when compared to a model that ignores family membership , even after accounting for variable sequencing coverage across third-generation samples ( median autosomal base pairs covered = 2 , 582 , 875 , 060; ANOVA: p=9 . 36e-10 ) .", "Moreover , we found that the interaction between paternal age and family membership improved the fit of the linear model ( p=0 . 043 , Appendix 1—table 1 ) , suggesting that inter-family variability involves differences in paternal age effects ( i . e . , the slopes of each regression ) .", "We note that the confidence intervals surrounding the slope point estimates for some CEPH/Utah families are quite wide , likely due to the small number of third-generation individuals ( with respect to count-based regression ) in each family , as well as some stochastic noise in the DNM counts attributed to each child ( Figure 3d ) .", "Nonetheless , family rankings based upon the effect of paternal age on DNM counts are stable and relatively insensitive to outliers ( Figure 3—figure supplement 2 ) .", "Finally , when compared to a multiple regression that includes the effects of both paternal and maternal age , a model that takes family membership into account remained a significantly better fit ( ANOVA: p=2 . 12e-5 ) .", "The high degree of correlation between paternal and maternal ages makes it difficult to tease out the individual contributions of each parent to the observed inter-family differences .", "Nonetheless , these results suggest the existence of substantial variability in parental age effects across CEPH/Utah families , which could involve both genetic and environmental factors that differ among families .", "Generally , studies of de novo mutation focus on variants that arise in a single parental gamete .", "However , if a de novo variant arises during or after primordial germ cell specification ( PGCS ) , that variant may be present in multiple resulting gametes and absent from somatic cells ( Rahbari et al . , 2016; Acuna-Hidalgo et al . , 2015; Campbell et al . , 2014b; Tang et al . , 2016; Jónsson et al . , 2018; Campbell et al . , 2015; Biesecker and Spinner , 2013 ) .", "These variants can therefore be present in more than one offspring as apparent de novo mutations .", "In each family , we searched for post-PGCS germline mosaic variants by identifying high-confidence DNMs that were shared by two or more third-generation individuals , and were absent from the blood DNA of any parents or grandparents within the family ( Figure 4a ) .", "Given the large number of third-generation siblings in each CEPH/Utah family , we had substantially higher power to detect germline mosaicism that occurred in in the second generation than in prior studies .", "In total , we identified 720 single-nucleotide germline mosaic mutations at a total of 303 unique sites , which were subsequently corroborated through visual inspection using the Integrative Genomics Viewer ( IGV ) ( Supplementary file 4 ) ( Thorvaldsdóttir et al . , 2013 ) .", "Of the phased shared germline mosaic mutations , 124/260 ( 47 . 7% ) were paternal in origin; thus , the mutations that occurred following PGCS likely occurred irrespective of any parental sex biases on mutation counts .", "Overall , approximately 3 . 1% ( 720/23 , 399 ) of all single-nucleotide DNMs observed in the third generation likely arose during or following PGCS in a parent’s germline , confirming that these variants comprise a non-negligible fraction of all de novo germline mutations .", "The mutation spectrum for non-shared germline de novo variants was significantly different than the spectrum for shared germline mosaic variants ( Figure 4b ) .", "Specifically , we found enrichments of CpG >TpG and C > A mutations , and a depletion of T > C mutations , in shared germline mosaic variants when compared to all unshared germline de novo variants observed in the third generation ( Figure 4b ) .", "An enrichment of CpG >TpG mutations in germline mosaic DNMs , which was also seen in a recent report on mutations shared between siblings ( Jónsson et al . , 2018 ) , is particularly intriguing , as many C > T transitions in a CG dinucleotide context are thought to occur due to spontaneous deamination of methylated cytosine ( Fryxell and Zuckerkandl , 2000 ) .", "Indeed , DNA methylation patterns are highly dynamic during gametogenesis; evidence in mouse demonstrates that the early primordial germ cells are highly methylated , but experience a global loss of methylation during expansion and migration to the genital ridge , followed by a re-establishment of epigenetic marks ( at different time points in males and females ) ( Seisenberger et al . , 2012; Reik et al . , 2001 ) .", "We also tabulated the number of each third-generation individual’s DNMs that was shared with one or more of their siblings .", "As reported in the recent analysis of germline mosaicism ( Jónsson et al . , 2018 ) , we observed that the number of shared germline mosaic DNMs does not increase with paternal age ( p=0 . 647 , Figure 4c , Materials and methods ) .", "Thus , a de novo mutation sampled from the child of a younger father is more likely to recur in a future child , as early-occurring , potentially mosaic mutations comprise a larger proportion of all DNMs present among the younger father’s sperm population ( Figure 4d ) .", "Conversely , a de novo mutation sampled from the child of an older father is less likely to recur , as the vast majority of DNMs in that father’s gametes will have arisen later in life in individual spermatogonial stem cells ( Figure 4d ) ( Campbell et al . , 2014a; Jónsson et al . , 2018 ) .", "Consistent with this expectation , we observed a significant age-related decrease in the proportion of shared germline mosaic DNMs ( p=1 . 61e-5 , Figure 4e ) .", "Although families with large numbers of siblings are expected to offer greater power to detect shared , germline mosaic DNMs , we verified that neither the mosaic fraction nor the number of mosaic DNMs observed in third-generation children are significantly associated with the number of siblings in a family ( Materials and methods ) .", "We further distinguished germline mosaicism from mutations that occurred before primordial germ cell specification , but likely following the fertilization of second-generation zygotes .", "De novo mutations that occur prior to PGCS can be present in both blood and germ cells; we therefore sought to characterize these ‘gonosomal’ variants that likely occurred early during the early post-zygotic development of second-generation individuals ( Besenbacher et al . , 2015; Campbell et al . , 2015; Campbell et al . , 2014a; Campbell et al . , 2014b; Rahbari et al . , 2016; Harland et al . , 2017; Jónsson et al . , 2018 ) .", "We assumed that these gonosomal mutations would be genotyped as heterozygous in a second-generation individual , but exhibit a distinct pattern of ‘incomplete linkage’ to informative heterozygous alleles nearby ( Materials and methods , Figure 5a ) ( Feusier et al . , 2018; Harland et al . , 2017; Jónsson et al . , 2018 ) .", "If these variants occurred early in development , and were present in both the blood and germ cells , we could also validate them by identifying third-generation individuals that inherited the variants with a balanced number of reads supporting the reference and alternate alleles ( Figure 5a ) .", "In total , we identified 475 putative autosomal gonosomal DNMs , which were also validated by visual inspection ( Supplementary file 5 ) .", "In contrast to single-gamete germline DNMs observed in the second-generation , gonosomal mutations appeared to be sex-balanced with respect to the parental haplotype on which they occurred; 52% ( 249/475 ) of all gonosomal DNMs occurred on a paternal haplotype , as compared to ~80% of germline DNMs observed in the second generation .", "Similarly , no significant enrichment of particular gonosomal mutation types was observed on either parental haplotype at a false discovery rate of 0 . 05 ( Figure 5b ) , though we found that T > A transversions are enriched in gonosomal DNMs when compared to single-gamete germline DNMs observed in the second generation ( p=2 . 32e-3 ) ( Figure 5c ) .", "Unlike single-gamete germline DNMs , there were no significant effects of parental age on gonosomal DNM counts ( maternal age , p=0 . 132; paternal age , p=0 . 225 ) ( Figure 5d ) .", "However , a recent study found tentative evidence for a maternal age effect on de novo mutations that arise in the early stages of zygote development ( Gao et al . , 2019 ) .", "As noted in this previous study , we are likely underpowered to detect a possible maternal age effect using the numbers of second-generation individuals in the CEPH/Utah dataset .", "Overall , our results demonstrate that over 9% ( 475/5 , 017 ) of all candidate autosomal germline mutations observed in the second generation were , in fact , post-zygotic in these second-generation individuals .", "Perhaps most importantly , approximately 6% of candidate de novo mutations detected in the second generation with an allele balance >= 0 . 2 ( 303/5 , 017 ) were determined to be post-zygotic , and present in both somatic and germ cells .", "This suggests that a fraction of many germline de novo mutation datasets are comprised of truly post-zygotic DNMs , rather than mutations that occurred in a single parental gamete .", "We note that our analysis pipeline may erroneously classify some gonosomal and shared germline mosaic DNMs .", "Namely , our count of gonosomal DNMs may be an underestimate , since our requirement that the second-generation individual be heterozygous precludes the detection of post-zygotic mosaic mutations at very low frequency in blood .", "Also , blood cells represent only a fraction of the total somatic cell population , and we cannot rule out the possibility that mosaicism apparently restricted to the germline may , in fact , be present in other somatic cells that were not sampled in this study ( Biesecker and Spinner , 2013 ) ." ], [ "Using a cohort of large , multi-generational CEPH/Utah families , we identified a high-confidence set of germline de novo mutations that were validated by transmission to the following generation .", "We determined the parental gamete-of-origin for nearly all of these DNMs observed in the second generation and produced estimates of the maternal and paternal age effects on the number of DNMs in offspring .", "Then , by comparing parental age effects among pedigrees with large third generations whose birth dates span as many as 27 years , we found that families significantly differed with respect to these age effects .", "Finally , we identified gonosomal and shared germline mosaic de novo variants which appear to differ from single-gamete germline DNMs with respect to mutational spectra and magnitude of the sex bias .", "Understanding family differences in both mutation rates and parental age effects could enable the identification of developmental , genetic , and environmental factors that impact this variability .", "The fact that there were detectable differences in parental age effects between families is striking in light of the fact that the CEPH/Utah pedigrees comprise mostly healthy individuals , and that at the time of collection they resided within a relatively narrow geographic area ( Malhotra et al . , 2005; Dausset et al . , 1990 ) .", "We therefore suspect that our results understate the true extent of variability in mutation rates and age effects among families with diverse inherited risk for mutation accumulation , and who experience a wide range of exposures , diets , and other environmental factors .", "Supporting this hypothesis , a recent report identified substantial differences in the mutation spectra of variants in populations of varied ancestries , suggesting that genetic modifiers of the mutation rate may exist in humans , as well as possible differences in environmental exposures ( Harris and Pritchard , 2017; Mathieson and Reich , 2017 ) .", "Another explanation ( that we are unable to explore ) for the range of de novo mutation counts in firstborn children across families is variability in the age at which parents enter puberty .", "For example , a father entering puberty at an older age could result in less elapsed time between the start of spermatogenesis and the fertilization of his first child’s embryo .", "Compared to another male parent of the same age , his sperm will have accumulated fewer mutations by the time of conception .", "Of course , this hypothesis assumes that for both fathers , three parameters are identical: the mutation rate at puberty , the yearly mutation rate increase following puberty , and age at fertilization of the first child’s embryo .", "Moreover , we note that replication errors are unlikely to be the sole source of de novo germline mutations ( Gao et al . , 2019 ) .", "Overall , the potential sources of inter-family variability in mutation rates remain mysterious , and we anticipate that future studies will be needed to uncover the biological underpinnings of this variability .", "Our observation of germline mosaicism , a result of de novo mutations that occur during or post-PGCS , has broad implications for the study of human disease and estimates of recurrence risks within families ( Jónsson et al . , 2018; Campbell et al . , 2014b; Biesecker and Spinner , 2013; Forsberg et al . , 2017; Krupp et al . , 2017 ) .", "If a de novo mutation is found to underlie a genetic disorder in a child , it is critical to understand the risk of mutation recurrence in future offspring .", "We estimate that ~3% of germline de novo mutations originated as a mosaic in the germ cells of a parent .", "This result corroborates recent reports ( Rahbari et al . , 2016; Jónsson et al . , 2018 ) and demonstrates that a substantial fraction of all germline DNMs may be recurrent within a family .", "We also find that the mutation spectrum of shared germline mosaic DNMs is significantly different than the spectrum for single-gamete germline DNMs , raising the intriguing possibility that different mechanisms contribute to de novo mutation accumulation throughout the proliferation of primordial germ cells and later stages of gametogenesis .", "For instance , the substantial epigenetic reprogramming that occurs following primordial germ cell specification may predispose cells at particular developmental time points to certain classes of de novo mutations , such as C > T transitions at CpG dinucleotide sites ( Gao et al . , 2019 ) .", "Recurrent DNMs across siblings can also manifest as a consequence of gonosomal mosaicism in parents ( Biesecker and Spinner , 2013; Jónsson et al . , 2018 ) .", "Although it can be difficult to distinguish gonosomal mosaicism from both single-gamete germline de novo mutation and germline mosaicism , we have identified a set of putative gonosomal mosaic mutations that are sex-balanced with respect to the parental haplotype on which they occurred , and do not exhibit any detectable dependence on parental age at birth .", "Both of these observations are expected if gonosomal mutations arise after zygote fertilization , rather than during the process of gametogenesis .", "We do , however , find that T > A transversions are enriched in gonosomal DNMs , as compared to DNMs that occurred exclusively in the germline of a parent .", "Overall , we estimate that approximately 10% of candidate germline de novo mutations in our study were , in fact , gonosomal mutations that occurred during the early cell divisions of the offspring , rather than in a single parental gamete .", "Prior work in cattle has estimated the fraction of mosaic DNMs that occur during early cell divisions to be even higher , suggesting that these mosaic mutations make up a large fraction of DNMs that are reported to have occurred in a single parental gamete ( Harland et al . , 2017 ) .", "These results underscore the power of large , multi-generational pedigrees for the study of de novo human mutation and yield new insight into the mutation dynamics that exist due to factors such as parental age and sex , as well as family of origin .", "Given that we studied only 33 large pedigrees , the mutation rate variability we observe is very likely an underestimate of the full range of variability worldwide .", "We therefore anticipate future studies of multi-generational pedigrees that will help to dissect the relative contributions of genetic background , developmental timing , and myriad environmental factors ." ], [ "Whole-genome DNA sequencing libraries were constructed with 500 ng of genomic DNA isolated from blood , utilizing the KAPA HTP Library Prep Kit ( KAPA Biosystems , Boston , MA ) on the SciClone NGS instrument ( Perkin Elmer , Waltham , MA ) targeting 350 bp inserts .", "Post-fragmentation ( Covaris , Woburn , MA ) , the genomic DNA was size selected with AMPure XP beads using a 0 . 6x/0 . 8x ratio .", "The libraries were PCR amplified with KAPA HiFi for 4–6 cycles ( KAPA Biosystems , Boston , MA ) .", "The final libraries were purified with two 0 . 7x AMPureXP bead cleanups .", "The concentration of each library was accurately determined through qPCR ( KAPA Biosystems , Boston , MA ) .", "Twenty four libraries were pooled and loaded across four lanes of a HiSeqX flow cell to ensure that the libraries within the pool were equally balanced .", "The final pool of balanced libraries was loaded over an additional 16 lanes of the Illumina HiSeqX ( Illumina , San Diego , CA ) .", "2 × 150 paired-end sequence data was generated .", "This efficient pooling scheme targeted ~30X coverage for each sample .", "Sequence reads were aligned to the GRCh37 reference genome ( including decoy sequences from the GATK resource bundle ) using BWA-MEM v0 . 7 . 15 ( Li , 2013 ) .", "The aligned BAM files produced by BWA-MEM were de-duplicated with samblaster ( Faust and Hall , 2014 ) .", "Realignment for regions containing potential short insertions and deletions and base quality score recalibration was performed using GATK v3 . 5 . 0 ( DePristo et al . , 2011 ) .", "Alignment quality metrics were calculated by running samtools ‘stats’ and ‘flagstats’ ( Li et al . , 2009 ) on aligned and polished BAM files .", "Single-nucleotide and short insertion/deletion variant calling was performed with GATK v3 . 5 . 0 ( DePristo et al . , 2011 ) to produce gVCF files for each sample .", "Sample gVCF files were then jointly genotyped to produce a multi-sample project level VCF file .", "We used peddy ( Pedersen and Quinlan , 2017a ) to perform relatedness and sample sequencing quality checks on all CEPH/Utah samples .", "We discovered a total of 10 samples with excess levels of heterozygosity ( proportion of heterozygous calls > 0 . 2 ) .", "Many of these samples were also listed as being duplicates of other samples in the cohort , indicating possible sample contamination prior to sequencing .", "We therefore removed all 10 samples with a heterozygous genotype proportion exceeding 0 . 2 from further analysis .", "In total , we were left with 593 first- , second- , and third-generation samples with high-quality sequencing data .", "We identified high-confidence de novo mutations from the joint-called VCF in the second and third generations as follows , using cyvcf2 ( Pedersen and Quinlan , 2017b ) .", "For each variant , we required that the child possessed a unique genotyped allele absent from both parents; when identifying de novo variants on the X chromosome , we required male offspring genotypes to be homozygous .", "We required the aligned sequencing depth in the child and both parents to be >= 12 reads , Phred-scaled genotype quality ( GQ ) to be >= 20 in the child and both parents , and no reads supporting the de novo allele in either parent .", "We removed de novo variants within low-complexity regions ( Li , 2014; Turner et al . , 2017 ) , and any variants that were not listed as ‘PASS’ variants by GATK HaplotypeCaller .", "Finally , we removed DNMs with likely DNM carriers in the cohort; we define carriers as samples that possess the DNM allele , other than the sample with the putative DNM and his/her immediate family ( i . e . , siblings , parents , or grandparents ) .", "We adapted a previously published strategy ( Jónsson et al . , 2017 ) to discriminate between ‘possible carriers’ of the DNM allele ( samples genotyped as possessing the de novo allele ) , and ‘likely carriers’ ( a subset of ‘possible carriers’ with depth >= 12 , allele balance >= 0 . 2 , and Phred-scaled genotype quality >= 20 ) .", "We removed putative DNMs for which there were any ‘likely carriers’ of the allele in the cohort .", "We then separated the candidate variants observed in the second-generation into true and false positives based on transmission to the third generation .", "For each candidate second-generation variant , we assessed whether the DNM was inherited by at least one member of the third generation; to limit our identification of false positive transmission events , we required third-generation individuals with inherited DNMs to have a depth >= 12 reads at the site and Phred-scaled genotype quality >= 20 .", "We defined ‘transmitted’ second-generation variants as variants for which the median allele balance across transmissions was >= 0 . 3 .", "One CEPH/Utah family ( family ID 26 ) contains only four sequenced grandchildren ( Supplementary file 1 ) ; therefore , we did not include the two second-generation individuals from this family in our analysis of DNMs observed in the second-generation , as we lacked power to detect high-quality transmission events .", "Because we were unable to validate DNMs observed in the third generation by transmission , we applied a more stringent set of quality filters to all third-generation DNMs .", "We required the same filters as applied to all second-generation DNMs , but additionally required that the allele balance in each DNM was >= 0 . 3 .", "We further required that there were no possible carriers of the de novo allele in the rest of the cohort .", "For each DNM in the third generation , we assessed if any of the third-generation individuals’ grandparents were genotyped as possessing the DNM allele; if so , we removed that DNM from further analysis ( see section entitled ‘Estimating a missed heterozygote rate’ ) .", "Finally , we removed a total of 319 candidate germline DNMs in the third generation after finding evidence that these were , in fact , post-zygotic mutations ( see section entitled ‘Identifying gonosomal mutations’ ) .", "To determine the parent of origin for each de novo variant in the second generation , we phased mutation alleles by transmission to a third generation , a technique which has been described previously ( Jónsson et al . , 2017; Kong et al . , 2012; Goldmann et al . , 2016; Rahbari et al . , 2016 ) ( Figure 1—figure supplement 2a ) .", "We searched 200 kbp upstream and downstream of each DNM for informative variants , defined as alleles present as a heterozygote in the second-generation individual , observed in only one of the two parents , and observed in each of the third-generation individuals that inherited the DNM .", "For each of these informative variants , we confirmed that the informative variant was always transmitted with the DNM; if so , we could infer that the heterozygous variant was present on the same haplotype as the DNM ( assuming recombination did not occur between the DNM and the flanking informative variants ) , and assign the first-generation parent with the informative variant as the parent of origin ( Figure 1—figure supplement 2a ) .", "For each second-generation DNM , we identified all transmission patterns ( i . e . , combinations of a first-generation parent , second-generation child , and set of third-generation grandchildren that inherited both the informative variant and the DNM ) .", "We only assigned a confident parent-of-origin at sites where the most frequent transmission pattern occurred at >= 75% of all informative sites .", "We additionally phased de novo variants in the second generation , as well as all DNMs in the third generation , using ‘read tracing’ ( also known as ‘read-backed phasing’ ) ( Jónsson et al . , 2017; Goldmann et al . , 2016 ) .", "Briefly , for each de novo variant , we first searched for nearby ( within one read fragment length , 500 bp ) variants present in the proband and one of the two parents .", "Thus , if the de novo variant was present on the same read as the inherited variant , we could infer haplotype sharing , and determine that the de novo event occurred on that parent’s chromosome ( Figure 1—figure supplement 2b ) .", "Similarly , if the de novo variant was not present on the same read as the inherited variant , we could infer that the de novo event occurred on the other parent’s chromosome .", "We were also able to determine the parent-of-origin for many of the shared germline mosaic variants by leveraging haplotype sharing across three generations ( Jónsson et al . , 2018 ) .", "If all third-generation individuals with a post-PGCS DNM shared a haplotype with a particular first-generation grandparent , we assigned that first-generation grandparent’s child ( i . e . , one of the two second-generation parents ) as the parent of origin .", "In the second generation , the read tracing and haplotyping sharing phasing strategies were highly concordant , and the parent-of-origin predictions agreed at 98 . 8% ( 969/980 ) of all DNMs for which both strategies could be applied .", "Given the filters we employed to identify high-confidence de novo mutations , we needed to calculate the fraction of the genome that was considered in our analysis .", "To this end , we used mosdepth ( Pedersen and Quinlan , 2018 ) to calculate per-base genome coverage in all CEPH/Utah samples , excluding low-complexity regions ( Li , 2014 ) and reads with mapping quality <20 ( the minimum mapping quality threshold used by GATK HaplotypeCaller in this analysis ) .", "For each second- and third-generation child , we then calculated the number of all genomic positions that had at least 12 aligned sequence reads in the child’s , mother's , and father's genome ( excluding the X chromosome ) .", "In the second generation , the median number of callable autosomal base pairs per sample was 2 , 582 , 336 , 232 .", "For each individual , we then divided their count of autosomal de novo mutations by the resulting number of base pairs , and divided the result by two to obtain a diploid human mutation rate per base pair per generation .", "The median second-generation germline SNV mutation rate was calculated to be 1 . 143 × 10−8 per base pair per generation .", "We then adjusted this mutation rate based on our estimated false positive rate ( FPR ) and our estimated ‘missed heterozygote rate’ ( MHR; see section entitled ‘Estimating a missed heterozygote rate’ ) as follows:adj_mu = mu * ( 1 - FPR/1 - MHR ) adj_mu = 1 . 143e-8 * ( 1–0 . 045/1–0 . 004 ) Using the full call set of de novo variants in the third generation ( excluding the recurrent , post-PGCS DNMs and likely post-zygotic DNMs ) we first fit a simple Poisson regression model that calculated the effect of paternal age on total autosomal DNM counts in the R statistical language ( v3 . 5 . 1 ) as follows:glm ( autosomal_dnms ~ dad_age , family = poisson ( link='identity’ ) ) This model returned a highly significant effect of paternal age on total DNM counts ( 1 . 72 DNMs per year of paternal age , p<2e-16 ) , but was agnostic to the family from which each third-generation individual was ‘sampled . ’ Importantly , a number of third-generation individuals in the CEPH/Utah cohort share grandparents , and may therefore be considered members of the same family , despite having unique second-generation parents ( Figure 3—figure supplement 1 ) .", "For all subsequent analysis , we defined a ‘family’ as the unique group of two second-generation parents and their third-generation offspring ( Figure 3—figure supplement 1 ) .", "In the CEPH/Utah cohort , there are a total of 40 ‘families’ meeting this definition .", "To test for significant variability in paternal age effects between families , we fit the following model:glm ( autosomal_dnms ~ dad_age * family_id , family = poisson ( link='identity' ) ) Which can also be written in an expanded form as:glm ( autosomal_dnms ~ dad_age + family_id + dad_age:family_id , family = poisson ( link='identity' ) ) To assess the significance of each term in the fitted model , we performed an analysis of variance ( ANOVA ) as follows:m = glm ( autosomal_dnms ~ dad_age + family_id + dad_age:family_id , family = poisson ( link='identity' ) ) anova ( m , test='Chisq' ) The results of this ANOVA are shown in Appendix 1—Table 1 .", "In summary , this model contained the fixed effect of paternal age , as well as different regression intercepts within each ‘grouping factor’ ( i . e . , family ID ) .", "Additionally , this model includes an interaction between paternal age and family ID , allowing for the effect of paternal age ( i . e . , the slope of the regression ) to vary within each grouping factor .", "To account for variable sequencing coverage across CEPH/Utah samples , we additionally calculated the callable autosomal fraction for all third-generation individuals by summing the total number of nucleotides covered by >= 12 reads in the third-generation individual and both of their second-generation parents , excluding low-complexity regions and reads with mapping quality <20 ( see section entitled ‘Calculating the rate of germline mutation’ ) .", "Since we only consider the effect of paternal age on the mutation rate , we can model the mutation rate ( mu ) as:mu = Bp * Ap +B0 Where Bp is the paternal age effect , Ap is the paternal age , and B0 is an intercept term .", "Therefore , the number of DNMs in a sample is assumed to follow a Poisson distribution , with the expected mean of the distribution defined as:E ( # DNMs ) = mu * callable_fractionE ( # DNMs ) = ( Bp * Ap + B0 ) * callable_fractionE ( # DNMs ) = ( Bp * Ap * callable_fraction ) + ( callable_fraction * B0 ) As our analysis only considers the effect of paternal age on total DNM counts , we can thus scale Ap ( paternal age at birth ) by the callable_fraction , generating a term called dad_age_scaled , and fit the following model , which takes each sample’s callable fraction into account:glm ( autosomal_dnms ~ dad_age_scaled + autosomal_callable_fraction +0 , family = poisson ( link='identity' ) ) Then , we can determine whether inter-family differences remain significant by comparing the above null model to a model that takes family into account:glm ( autosomal_dnms ~ dad_age_scaled * family_id + autosomal_callable_fraction + 0 , family = poisson ( link='identity' ) ) After running an ANOVA to compare the two models , we find that the model incorporating family ID is a significantly better fit ( ANOVA: p=9 . 359e-10 ) .", "We previously identified significant effects of both maternal and paternal age on DNM counts ( Figure 2a ) .", "Therefore , to account for the non-negligible effect of maternal age on DNM counts , we fit a final model that incorporated the effects of both maternal and paternal age , as well as family ID , on total DNM counts as follows:glm ( autosomal_dnms ~ dad_age +mom_age +family_id , family = poisson ( link='identity' ) ) We then performed an ANOVA on the model , and found that a model incorporating a family term is a significantly better fit than a model that includes the effects of paternal and maternal age alone ( p=2 . 12e-5 ) .", "To identify post-PGCS mosaic variants , we searched the previously generated callset of single-nucleotide DNMs in the third generation ( ‘Identifying DNM candidates’ ) for de novo single-nucleotide mutations that appeared in two or more third-generation siblings .", "As a result , all filters applied to the germline third-generation DNM callset were also applied to the post-PGCS mosaic variants .", "We validated all putative post-PGCS mosaic variants by visual inspection using the Integrative Genomics Viewer ( IGV ) ( Thorvaldsdóttir et al . , 2013 ) .", "In a small number of cases ( 32 ) , we found evidence for the post-PGCS mosaic variant in one of the two second-generation parents .", "Reads supporting the post-PGCS mosaic variant were likely filtered from the joint-called CEPH/Utah VCF output following local re-assembly with GATK , though they are clearly present in the raw BAM alignment files .", "We removed these 32 variants , at which an second-generation parent possessed two or more reads of support for the mosaic DNM allele in the aligned sequencing reads .", "To identify a paternal age effect on the number of post-PGCS DNMs transmitted to third-generation children , we tabulated the number of each third-generation individual’s DNMs that was shared with at least one of their siblings .", "We then fit a Poisson regression as follows , regressing the number of mosaic DNMs in each third-generation individual against their father’s age at birth:glm ( mosaic_number ~dad_age , family = poisson ( link='identity' ) ) We did not find a significant effect of paternal age ( p=0 . 647 ) .", "Using the predicted paternal age effects on germline DNM counts and post-PGCS DNM counts , we determined that the fraction of post-PGCS DNMs should decrease non-linearly with paternal age ( Figure 4e ) .", "Therefore , to assess the effect of paternal age on the fraction of each third-generation individual’s DNMs that occurred post-PGCS in a parent , we fit the following model:lm ( log ( mosaic_fraction ) ~dad_age ) We found a significant effect of paternal age on the post-PGCS mosaic fraction ( p=1 . 61e-5 ) .", "As we may be more likely to identify shared , post-PGCS DNMs in families with larger numbers of third-generation siblings , we additionally tested whether the fraction of post-PGCS DNMs in each child was dependent on the number of their siblings in the family by performing a correlation test as follows:cor . test ( mosaic_fraction , n_siblings ) We did not observe a significant correlation between a third-generation individual’s number of siblings and the fraction of their DNMs that was shared with a sibling ( p=0 . 882 ) .", "We also did not observe a significant correlation between a third-generation individual’s number of siblings and the total number of their DNMs shared with a sibling ( p=0 . 426 ) .", "To identify variants that occurred early in post-zygotic development , we identified de novo single-nucleotide variants in the second generation using the same genotype quality and population-based filters as described previously ( ‘Identifying DNM candidates’ ) .", "Then , to distinguish single-gamete germline de novo mutations from post-zygotic DNMs ( de novo mutations that occurred in the cell divisions following fertilization of the second-generation individual’s embryo ) , we employed a previously described method ( Harland et al . , 2017; Feusier et al . , 2018; Jónsson et al . , 2018 ) that relies on linkage between DNMs and informative heterozygous alleles nearby .", "In this approach , which is similar in principle to the strategy used for phasing germline second-generation DNMs , we first search ±200 kbp up- and down-stream of the de novo allele in the second-generation individual for ‘informative’ alleles; that is , alleles that are present in only one first-generation parent , and inherited by the second-generation child ( Figure 5a ) .", "Then , we identify all of the third-generation grandchildren that inherited the informative alleles .", "If all of the third-generation individuals that inherited the informative alleles also inherited the DNM , we infer that the DNM occurred in the germline of the first-generation parent with the informative allele .", "However , if one or more third-generation individuals inherited the informative alleles but did not inherit the DNM , we can infer that the DNM occurred sometime following the fertilization of the second-generation sample’s embryo .", "This is because the DNM is not always present on the background haplotype that the second-generation individual inherited from their informative first-generation parent .", "Using this approach , we do not apply any allele balance filters to putative gonosomal DNMs in the second generation , instead relying on linkage to distinguish them from germline DNMs .", "As with germline de novo mutations observed in the second-generation , to limit our identification of false positive events , we required third-generation individuals with inherited DNMs to have a depth >= 12 reads at the site , Phred-scaled genotype quality ( GQ ) >= 20 , and for the median allele balance across transmissions to be >= 0 . 3 .", "Additionally , we can use an orthogonal method to distinguish single-gamete germline DNMs from post-zygotic DNMs .", "In this second approach , we identify all heterozygous sites ± 500 base pairs ( approximately one read length ) from a DNM in a child .", "Then , by assessing the linkage of the DNM and heterozygous alleles , we look for evidence of three distinct haplotypes in the child ( Figure 5—figure supplement 1 ) .", "If we observe at least two reads supporting a third haplotype ( i . e . , reads that indicate incomplete linkage between the DNM and the informative heterozygous allele ) , we inferred that the DNM occurred post-zygotically in the child .", "We applied this method to all putative germline DNMs identified in the third generation , and discovered that 319 of apparent germline DNMs showed evidence of being post-zygotic mutations that occurred following the fertilization of the third-generation embryo .", "We removed these DNMs from all analyses of third-generation germline DNMs .", "We validated all putative gonosomal variants in the second generation by visual inspection using the Integrative Genomics Viewer ( IGV ) ( Thorvaldsdóttir et al . , 2013 ) .", "Infrequently , variant calling methods such as GATK may incorrectly assign genotypes to samples at particular sites in the genome .", "When identifying de novo variants , we require that children possess genotyped alleles that are absent from either parent; thus , genotyping errors in parents could lead us to assign variants as being de novo , when in fact one or both parents possessed the variant and transmitted the allele .", "Given the multi-generational structure of our study cohort , we were able to estimate the rate at which our variant calling and filtering pipeline mis-genotyped a second-generation parent as being homozygous for a reference allele .", "To estimate this ‘missed heterozygote’ rate in our dataset , we looked for any cases in which one or more third-generation individuals possessed a putative de novo variant ( i . e . possessed an allele absent from both second-generation parents ) .", "Then , we looked at the sample’s grandparental ( first-generation ) genotypes for evidence of the same variant .", "If one or more grandparents was genotyped as having high-quality evidence for the de novo allele ( depth >= 12 and Phred-scaled genotype quality >= 20 ) , we inferred that the variant could have been ‘missed’ in the second generation , despite being truly inherited .", "We estimate the missed heterozygote rate ( MHR ) to be 0 . 4% , by dividing the total number of third-generation DNMs with grandparental support by the total number of third-generation DNMs ( 100/25 , 075 ) .", "In a small number of CEPH/Utah pedigrees , some members of the first-generation ( grandparental ) generation were not sequenced ( 6 grandparents in five families , Supplementary file 1 ) .", "As a result these families are underpowered to detect evidence of third-generation DNM alleles in the first generation , and our MHR is likely a slight underestimate .", "In a separate set of sequencing runs , a total of 8 first-generation grandparents were re-sequenced to a greater genome-wide median depth of 60X ( Figure 1—figure supplement 1d ) .", "However , when variant calling and joint genotyping was performed on all 603 CEPH/Utah samples , the 30X data for these grandparents was used .", "Therefore , we sought to estimate the false positive rate for our de novo mutation detection strategy using the de novo mutation calls in the children of these eight first-generation individuals .", "For each of the children ( second-generation ) of these high-coverage first-generation individuals , we looked for evidence of the second-generation DNMs in the 60X alignments from their parents .", "Specifically , for each second-generation DNM , we counted the number of reads supporting the DNM allele in each of the first-generation parents , excluding reads with mapping quality <20 ( the minimum mapping quality imposed by GATK HaplotypeCaller in our analysis ) , and excluding bases with base qualities < 20 ( the minimum base quality imposed by GATK HaplotypeCaller in our analysis ) .", "If we observed two or more reads supporting the second-generation DNM in a first-generation parent’s 60X alignments , we considered the second-generation DNM to be a false positive .", "Of the 202 de novo mutations called in the four second-generation children of the high-coverage first-generation parents , we find nine mutations with at least two reads of supporting evidence in the 60X first-generation alignments .", "Thus , we estimate our false positive rate for de novo mutation detection to be approximately 4 . 5% ( 9/202 ) .", "Code used for statistical analysis and figure generation has been deposited on GitHub as a collection of annotated Jupyter Notebooks: https://github . com/quinlan-lab/ceph-dnm-manuscript ( Sasani , 2019; copy archived at https://github . com/elifesciences-publications/ceph-dnm-manuscript/blob/master/README . md ) .", "Data files containing high-confidence de novo mutations , as well as the gonosomal and post-primordial germ cell specification ( PGCS ) mosaic mutations , are included with these Notebooks .", "To mitigate compatibility issues , we have also made all notebooks available in a Binder environment , accessible at the above GitHub repository ( Sasani , 2019 ) ." ] ]
[ "The number of de novo mutations ( DNMs ) found in an offspring's genome increases with both paternal and maternal age .", "But does the rate of mutation accumulation in human gametes differ across families ?", "Using sequencing data from 33 large , three-generation CEPH families , we observed significant variability in parental age effects on DNM counts across families , ranging from 0 . 19 to 3 . 24 DNMs per year .", "Additionally , we found that ~3% of DNMs originated following primordial germ cell specification in a parent , and differed from non-mosaic germline DNMs in their mutational spectra .", "We also discovered that nearly 10% of candidate DNMs in the second generation were post-zygotic , and present in both somatic and germ cells; these gonosomal mutations occurred at equivalent frequencies on both parental haplotypes .", "Our results demonstrate that rates of germline mutation accumulation vary among families with similar ancestry , and confirm that post-zygotic mosaicism is a substantial source of human DNM ." ]
[ "Humans receive half of their DNA from each of their parents .", "However , this inherited DNA is not identical to the corresponding half of the parents’ genetic material .", "Instead , both the egg and the sperm that combine to generate an embryo carry so-called ‘germline de novo’ mutations that are not present in the rest of the parents’ cells .", "Although these de novo mutations are an important source of genetic diversity , they can also cause disease .", "Geneticists have a longstanding interest in how , when and at what rate germline de novo mutations arise .", "These questions are commonly addressed by analyzing the DNA of large cohorts of two-generation families .", "Now , Sasani et al . have used the genetic data of 33 families in Utah , United States , which all span three generations , to determine the rate at which de novo mutations appear .", "The analysis revealed that , on average , each person has around 70 de novo mutations that were not present in their parent’s genetic code .", "Sasani et al . also found that sperm and egg cells from older parents typically contain more de novo mutations .", "However , this effect varied substantially across the Utah families .", "In some families , an increase of one year in the parents’ age resulted in over three extra de novo mutations in their children .", "In others , the number of new mutations barely increased at all .", "In addition , Sasani et al . found that almost 10% of de novo mutations do not occur in the parents’ sperm or eggs , but happen in the embryo very soon after fertilization .", "These mutations can lead to ‘mosaicism’ , resulting in a person having a mutation in some , but not all of their organs and tissues .", "In some cases , this could cause an unknown number of sperm and egg cells to carry a mutation that others do not .", "This makes it hard to predict how likely two or more siblings are to inherit the mutation .", "This analysis reveals that parental age affects the number of de novo mutations in children , but this effect changes from family to family .", "This finding could point to genetic or environmental factors that alter the human mutation rate ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "chromosomes and gene expression", "structural biology and molecular biophysics" ]
Cryo-EM structure of the SAGA and NuA4 coactivator subunit Tra1 at 3.7 angstrom resolution
elife-28384-v2
[ [ "Cells execute precise programmes of transcription in response to environmental or developmental signals .", "These programmes are regulated by activator proteins which bind to specific DNA sequences and recruit coactivators that activate transcription by stimulating assembly of the basal transcriptional machinery and/or catalysing chromatin modifications at target genes ( Hahn and Young , 2011; Weake and Workman , 2010 ) .", "Coactivators often interact with multiple activators and are also targeted by signaling pathways , making them integrative hubs that interpret multiple inputs to modulate transcription ( Malik and Roeder , 2010; Rosenfeld et al . , 2006 ) .", "The yeast SAGA ( Spt-Ada-Gcn5-Acetyltransferase ) ( Grant et al . , 1997 ) and NuA4 ( Nucleosome-Acetyltransferase-of-histone-H4 ) ( Allard et al . , 1999 ) coactivators are conserved in all eukaryotes but are evolutionarily and mechanistically unrelated to each other; SAGA is a 19–20 subunit , 1 . 8 MDa complex that stimulates preinitiation complex formation by interaction with TBP ( Dudley et al . , 1999 ) , and contains H3 histone acetyltransferase ( HAT ) and H2B deubiquitinase enzymatic activities ( Daniel et al . , 2004; Henry et al . , 2003 ) , whereas NuA4 is a 13-subunit , 1 . 3 MDa complex that acetylates H4 and H2A ( Allard et al . , 1999; Grant et al . , 1997 ) .", "SAGA and NuA4 also have functions outside of transcription , with diverse but important roles in mRNA export ( Rodríguez-Navarro et al . , 2004 ) , DNA repair ( Bird et al . , 2002; Downs et al . , 2004 ) and telomere maintenance ( Atanassov et al . , 2009 ) .", "Despite their differences , both complexes have integrated the large Tra1 ( Transcription-Associated protein 1 ) subunit ( Allard et al . , 1999; Grant et al . , 1998; McMahon et al . , 1998; Saleh et al . , 1998; Vassilev et al . , 1998 ) , an essential and highly conserved 433 KDa protein ( Figure 1—figure supplement 1 ) that belongs to the Phosphoinositide 3-Kinase-related kinase ( PIKK ) family of cellular regulators , which includes mTOR , DNA-PKcs , ATM/Tel1 , ATR/Mec1 and SMG-1 ( Baretić and Williams , 2014; Lempiäinen et al . , 2009 ) .", "PIKKs are protein kinases that have diverse regulatory functions in transcriptional regulation , DNA repair , cell growth , metabolic control and mRNA surveillance but Tra1 is the only member that is catalytically inactive , due to loss of the DFG motif within the kinase active site ( Saleh et al . , 1998 ) ( Figure 1—figure supplement 2 ) .", "Although lacking catalytic activity , Tra1 is critical for coactivator function as it is a direct target for multiple activators ( Brown et al . , 2001 ) and enables the activities of SAGA and NuA4 to be directed at specific genes in order to stimulate their expression .", "Activators contain transactivation domains ( TADs ) which directly target coactivators ( Näär et al . , 2001; Ptashne , 1988 ) .", "Understanding the molecular mechanisms of TAD-coactivator interactions is a major challenge as TADs are poorly conserved , are often promiscuous and exhibit a strong compositional bias toward acidic , proline , glutamine or serine residues ( Mitchell and Tjian , 1989 ) , resulting in an intrinsically disordered protein region unless bound to a coactivator target ( Dyson and Wright , 2005 ) .", "In S . cerevisiae , activators such as VP16 , Gal4 , Gcn4 and Hap4 directly target Tra1 in vitro ( Brown et al . , 2001; Fishburn et al . , 2005; Herbig et al . , 2010; Knutson and Hahn , 2011; Reeves and Hahn , 2005 ) and in vivo ( Bhaumik and Green , 2001; Bhaumik et al . , 2004; Larschan and Winston , 2001; Lin et al . , 2012 ) , and the human homolog TRRAP interacts with the transcription factors c-Myc , E2F and E1A and is required for their stimulation of oncogenesis ( Ard et al . , 2002; Bouchard et al . , 2001; Deleu et al . , 2001; Kulesza et al . , 2002; McMahon et al . , 1998; 2000 ) , making Tra1/TRRAP a conserved activator target in all eukaryotes .", "Mutations of Tra1 have been described that affect HAT activity without affecting coactivator complex integrity , indicating roles beyond activator targeting ( Knutson and Hahn , 2011; Mutiu et al . , 2007 ) .", "Tra1 is also present in other chromatin-related complexes , including the SAGA-related complex SILK ( Pray-Grant et al . , 2002 ) and the ASTRA complex ( Shevchenko et al . , 2008 ) from yeast .", "Similarly , TRRAP is present in four different human coactivator complexes STAGA , TFTC , PCAF and Tip60 ( Murr et al . , 2007 ) .", "Interestingly , Schizosaccharomyces pombe contains two Tra1 paralogs which separately associate with SAGA and NuA4 ( Helmlinger et al . , 2011 ) .", "Both Tra1 and TRRAP are essential proteins; Tra1 is the only essential subunit of the SAGA complex ( Saleh et al . , 1998 ) and TRRAP disruption leads to early embryonic lethality in mice ( Herceg et al . , 2001 ) .", "The high level of conservation of Tra1 sequence and function from yeast to human , its requirement for cellular viability and its presence in multiple coactivator complexes highlights its pivotal role in regulating gene expression .", "However , the molecular mechanisms behind Tra1 function are poorly understood , and the reason for its common integration within multiple complexes is unclear .", "To elucidate these aspects of its function , we determined an atomic structure of the Tra1 protein ." ], [ "We over-expressed and purified S . cerevisiae Tra1 from its native host and determined its structure by single-particle cryo-EM to a resolution of 3 . 7 Å ( Figure 1—figure supplement 3 ) .", "Sidechains were visible throughout the reconstruction ( Figure 1—figure supplement 4A ) and an atomic model was built entirely de novo with 3474 residues ( 93% ) assigned with excellent stereochemistry ( Table 1 ) , representing the first atomic structure for this member of the PIKK family .", "270 residues were not resolved in the reconstruction , distributed across 15 chain breaks that are predicted to contain either loops or disordered protein .", "Tra1 has the domain structure characteristic of PIKK family proteins , consisting of HEAT , FAT , FRB , Kinase and FATC domains arranged sequentially from N- to C- terminus ( Figure 1A ) ( Baretić and Williams , 2014; Lempiäinen et al . , 2009 ) .", "Alpha-helical solenoid repeats account for 86% of its mass which are contributed by the HEAT and FAT domains , and contain 49 HEAT repeats ( labelled H1-H49 ) and 15 TPR repeats ( labelled T1-T15 ) respectively ( Figure 1B , D and Video 1 ) .", "These are followed by FRB , kinase and FATC domains at the C-terminus .", "Tra1 resembles a diamond ring , where the HEAT domain forms the ring , the FAT and FRB domains combine to form the setting , and the kinase and FATC domains represent the centre stone ( Figure 1C and Video 1 ) .", "Using that analogy , Tra1 can be broadly separated into four topological regions which we have termed Finger , Ring , Clasp and Head ( Figure 1A , C ) .", "The Finger , Ring and Clasp regions lie within the HEAT domain , whereas the Head region contains the FAT , FRB , Kinase and FATC domains ( Figure 1A ) .", "The Tra1 Head is therefore analogous to the Head or FATKIN ( FAT plus KINase ) regions defined for structures of ATM , DNA-PKcs , Mec1 , mTOR/Tor , Tel1 , and SMG-1 ( Aylett et al . , 2016; Baretić et al . , 2016; 2017; Lau et al . , 2016; Melero et al . , 2014; Rivera-Calzada et al . , 2005; Sawicka et al . , 2016 ) in that they all encompass the FAT , FRB , Kinase and FATC domains and represent the most structurally conserved feature amongst PIKK family members ( Figure 5—figure supplement 1 ) .", "The HEAT domain begins with the Finger , which consists of an alpha solenoid formed of N-terminal HEAT repeats H1-H16 ( Figure 1B and C ) , and is equivalent to the ‘Spiral’ region of mTOR/Tor and ATM , or the Arm/Bridge region of DNA-PKcs ( Sharif et al . , 2017 ) ( Figure 5—figure supplement 1 ) .", "Finger Repeats H1-H6 form a flap over the midpoint of the Ring and appear flexible , as suggested by local resolution analysis ( Figure 1—figure supplement 4B ) , and continues through H7-H16 which runs across the Ring toward the Head .", "H9 is an unusually large HEAT repeat and contains a 99-residue insertion ( residues 482 to 580 ) between its helices ( Knutson and Hahn , 2011 ) .", "Two-thirds of this insertion was resolved in the reconstruction and is an unusual feature , as the N-terminal helix of H9 extends across the Ring to contact the opposite side at H42-H43 ( Figure 1B ) .", "This interaction is corroborated by BS3-crosslinking experiments on the complete SAGA complex ( Han et al . , 2014 ) , but the function of the H9 insertion is unclear , given that it is poorly conserved ( Figure 1—figure supplement 1 ) and is not essential for viability ( Knutson and Hahn , 2011 ) .", "After H16 , the Finger solenoid is terminated by a 38-residue loop ( residues 960–996 ) containing a two-stranded beta sheet ( Figure 1—figure supplement 4A ) and a second solenoid is formed by repeats H17-H49 .", "This is the largest continuous solenoid in Tra1 and dominates the appearance of Tra1 , forming a large closed ring approximately 125 Å in diameter .", "This solenoid starts with the N-clasp ( H17-H18 ) , continues with the Ring region ( H19-H46 ) and ends with the C-clasp ( H47-H49 ) which abuts the N-Clasp to close the ring ( Figure 1C ) .", "The Clasp contains a significant proportion of insertions between its repeats , as predicted by sequence analysis ( Knutson and Hahn , 2011 ) , which form a set of interlocking loops that fix the Ring closed ( Figure 2A ) .", "The Ring has a cradle-like conformation ( Figure 1B ) and its juxtaposition with the Finger creates large solvent-accessible channels between them , creating a highly open conformation and a large surface area ( Figure 1C ) .", "As well as closing the Ring , the Clasp is also partly continuous with the FAT domain solenoid , in effect creating a ‘figure-of-eight’ conformation ( Figure 2—figure supplement 1 ) .", "Collectively , the N-Clasp , Ring and C-Clasp regions are topologically equivalent to the ‘Bridge’ , ‘Railing’ and ‘Cap’ regions defined for the HEAT domain of Tor ( Baretić et al . , 2016 ) and ATM ( Baretić et al . , 2017 ) , in that they separate the N-terminal Finger/Spiral from the C-terminal FAT domain , although their relative positioning is different .", "The FAT , FRB , Kinase and FATC domains combine to form the Head ( Figure 1A and C ) .", "The FAT domain contains 15 TPR repeats ( T1-T15 ) and together with the FRB domain , surround the Kinase and FATC domains .", "The kinase domain has a fold typical for PIKK and PI3K catalytic domains , superposing onto the mTOR ( Yang et al . , 2013 ) and DNA-PKcs ( Sibanda et al . , 2017 ) kinase domains with RMSDs ( Cα ) of 2 . 7 Å and 2 . 9 Å respectively .", "However , although the relative positions of the catalytic , activation and phosphate-binding loops of mTOR/DNA-PKcs are preserved in Tra1 ( Figure 2B ) , the critical residues required for ATP/Mg binding and catalysis are not conserved , and the Tra1 activation loop contains an 18-residue insertion compared to its counterparts in the catalytic PIKKs ( Figure 1—figure supplement 2 ) .", "The relative juxtaposition of FRB and kinase domains also differ as the DNA-PKcs and mTOR FRB domains are positioned away from the active site cleft , whereas the Tra1 FRB domain occludes it , contacting the LBE ( mLST8-Binding-Element ) on the opposite site of the cleft ( Figure 2B ) .", "These conformational differences between DNA-PKcs/mTOR and Tra1 likely reflect that Tra1 is a pseudokinase , allowing the divergence of its catalytic features .", "The FATC domain is integral to the kinase domain and is sandwiched between the LBE and the Kinase C-terminal lobe , forming a plug over a large hydrophobic cavity within the kinase domain ( Figure 2C ) .", "Disruption of this plug is likely to destabilise the kinase domain significantly and/or induce conformational changes in adjacent domains , explaining why mutations within FATC often result in loss of viability or decreased stability of Tra1 ( Hoke et al . , 2010 ) .", "Although the FATC domain is hypothesized to be critical for regulating catalytic activity of PIKKs ( Yang et al . , 2013 ) , its sensitivity to mutagenesis within the kinase-inactive Tra1 and its protection of the hydrophobic cavity from solvent suggests it also has a key role in maintaining structural integrity .", "Given the common presence of Tra1 in SAGA and NuA4 , a key question is how Tra1 is incorporated into each coactivator , and whether complex integration results in functional or mechanistic differences .", "To examine its interactions with the SAGA complex , Tra1 was fitted into a recent 30 Å negative stain EM reconstruction of wild-type S . cerevisiae SAGA ( EMD-2693 ) which exhibits a bilobal structure ( Durand et al . , 2014 ) .", "A unambiguous fit was found within ‘Lobe A’ ( Figure 3 and Video 1 ) and indicate that no gross conformational changes are required to fit Tra1 into this SAGA reconstruction .", "The remaining SAGA density is contained within the crescent shaped ‘Lobe B’ which accounts for the remaining SAGA subunits , hence the contact between lobes A and B represents a major interface between Tra1 and SAGA .", "However , the interface is small ( Figure 3 and Video 1 ) , demonstrating that Tra1 occupies a peripheral position within SAGA ( Setiaputra et al . , 2015; Han et al . , 2014; Wu et al . , 2004 ) and is not required for its structural integrity as a scaffolding platform ( Helmlinger et al . , 2011; Wu and Winston , 2002 ) .", "The interface is localized to one side of of Tra1 , primarily around the FAT domain at TPR repeats T1-T7 ( residues 2572–2830 ) but also at the C-terminal half of the Ring at repeats H41-H44 ( residues 2150–2350 ) ( Figures 1 and 3 ) , which clearly represent sites of intermolecular contact between Tra1 and the remaining SAGA subunits .", "This is supported by BS3-crosslinking experiments ( Setiaputra et al . , 2015; Han et al . , 2014 ) , which detected five Tra1 residues ( K2351 , K2713 , K2781 , K2808 and K2815 ) that lie adjacent to this interface , making intermolecular crosslinks to subunits Taf12 , Spt20 , Ada1 and Sgf73 ( Figure 3 ) .", "Given that Taf12 is the most frequently identified crosslinking partner of Tra1 ( accounting for 6/13 intermolecular crosslinks ) , we suggest that it lies within or close to the observed interface , which is consistent with an earlier proposed model of the SAGA complex determined by negative stain EM , albeit at lower resolution ( Wu et al . , 2004 ) .", "Similarly , as Ada1 can form a heterodimer with Taf12 ( Selleck et al . , 2001 ) , and its deletion causes the release of Tra1 from SAGA ( Wu and Winston , 2002 ) , it is also likely to lie close to this interface .", "The BS3-crosslinking experiments also detected three additional residues that make intermolecular crosslinks to Spt3 , Sgf73 and Taf12 , but are located distal from the observed interface , being located on the Finger ( K476 ) or on the opposite side of the FAT domain ( K3161 and K3175 ) ( Figure 3 ) .", "Although the identification of crosslinked amino acids can suffer from false positives , the position of these residues away from the main interface are not necessarily inconsistent with forming intermolecular contacts , as elements of SAGA that are less globular in structure and are poorly resolved by negative stain EM may project away from Lobe B to make contacts with Tra1 , such as extended loops or helices .", "To reveal potential activator binding sites , mutations that disrupt targeting by activators VP16 , Gcn4 , Rap1 , and Gal4 ( Brown et al . , 2001; Knutson and Hahn , 2011; Lin et al . , 2012 ) were mapped to the structure ( Figure 4A ) .", "Two Tra1 mutants defective for interaction with Gal4 contain five amino acid substitutions ( H400Y and D397N/S404F/D469N/V1115I ) ( Lin et al . , 2012 ) , four of which cluster at repeats H7-H8 within the N-terminal half of the Finger and are solvent exposed , indicating a binding site for the Gal4 activator ( Figure 4A ) .", "Importantly , these mutations are highly specific for Gal4 , and do not appear to affect interaction with other activators .", "Similarly , two deletion mutants of Tra1 ( ∆88–165 and ∆319–399 , located at H3 and H6-H7 respectively ) disrupt coactivator recruitment by activators Gcn4 and Rap1 ( Figure 4 ) but do not disrupt recruitment by Gal4 ( Knutson and Hahn , 2011 ) .", "These mutations are all located in the N-terminal half of the Finger but are specific for their affected activators , suggesting that the Finger contains multiple but distinct binding sites for different activators .", "Interestingly , the N-terminal half of the Finger contacts the Ring at repeats H31-H38 ( Figure 1B ) , which was found to mediate interactions between human TRRAP and c-Myc , specifically within repeats H36-H38 ( Park et al . , 2001 ) , and suggest that this pole of Tra1 ( i . e . opposite to the Head ) is generally targeted by activators .", "The juxtaposition between Finger and Ring in this region also forms a channel lined by positively charged residues contributed by both Finger and Ring ( Figure 4B ) , which may assist binding of the acidic transactivation domains frequently found in activators that target Tra1 .", "However , activators may also be targeted to other regions of Tra1 , as Gcn4 and Rap1 are disrupted by deletions in the Ring at H25-H26 ( ∆1424–1508 ) ( Knutson and Hahn , 2011 ) , mutations that disrupt VP16 are clustered around the Head ( Figure 4A ) , and in vitro experiments show VP16 interacts with the C-terminal regions of Tra1 ( Brown et al . , 2001 ) .", "Comparison of Tra1 to other PIKK structures show that mTOR ( Aylett et al . , 2016; Yang et al . , 2013 ) ( Figure 5—figure supplement 1 ) and ATM ( Wang et al . , 2016 ) have a similar arrangement of FAT , Kinase and FATC domains but the conformation of their HEAT domains differ significantly .", "This is unsurprising given that PIKKs have highly divergent functions , and typically form complexes with a diverse range of regulatory factors which often target the HEAT domain ( Aylett et al . , 2016; Baretić and Williams , 2014; Spagnolo et al . , 2012 ) .", "However , the entire Tra1 structure is strikingly similar to human DNA-PKcs ( Sibanda et al . , 2017 ) ( Figure 5 ) an essential DNA double strand break ( DSB ) repair factor .", "Despite only having having 18% sequence identity , both Tra1 and DNA-PKcs have similar ‘diamond ring’ topologies , and regions analogous to Finger , Clasp , Ring and Head can be defined for DNA-PKcs , resulting in the same relative positioning as Tra1 ( Figure 5 ) .", "The largest difference in conformation is between the Tra1 Finger , which is equivalent to the ‘N-terminal Unit’ ( Sibanda et al . , 2017 ) or ‘Arm/Bridge’ region ( Sharif et al . , 2017 ) defined for DNA-PKcs .", "Specifically , the N-terminal repeats of the Finger region in Tra1 form a flap over the Ring , whereas the equivalent zone DNA-PKcs forms an arch whose concave surface was hypothesized to be a DNA-binding site required for synapsis of a DSB within a DNA-PKcs dimer ( Sibanda et al . , 2017 ) .", "Although the equivalent region of Tra1 does not form an arch and cannot sterically accommodate duplex DNA , these repeats contain a highly positively charged surface ( Figure 4B ) and local resolution analysis suggests they are flexible ( Figure 1—figure supplement 4B ) , potentially indicating the presence of a nucleic acid binding site ." ], [ "Coactivators are far less numerous than activators , and represent hubs within transcriptional regulation .", "Understanding the molecular mechanisms behind coactivator function is essential for elucidating how complex programmes of gene expression are established .", "Although SAGA and NuA4 are well characterized in terms of their enzymatic activities and genome localization , their interactions with activators are poorly understood .", "Tra1 has been identified as a key activator target , but the molecular details of its interaction with activators and its parent complexes are yet to be determined .", "To provide insight into this aspect of coactivator function , we determined the structure of Tra1 by cryo-EM , and built an atomic model of the complete protein , allowing activator-disrupting mutations to be mapped to the structure and highlighting potential TAD binding sites .", "The model also fitted unambiguously into an existing reconstruction of SAGA to reveal its binding interface and integration within the complex .", "The integration of Tra1 within SAGA leaves Tra1 relatively unimpeded for activator binding , as its interaction occludes very little of its solvent accessible surface .", "Similarly , as its conformation within SAGA appears unchanged from apo-Tra1 ( Video 1 ) , this suggests that activators cannot discriminate SAGA from NuA4 via Tra1 targeting alone , and other subunits specific to SAGA may act as additional activator targets and provide specificity e . g . numerous activators can directly target the SAGA subunits Gcn5 , Ada1 , Taf6 and Taf12 ( Klein et al . , 2003; Reeves and Hahn , 2005; Zhang et al . , 2014 ) .", "It is possible that the presentation of Tra1 by NuA4 could restrict or alter its binding to activators in comparison to SAGA , but this remains to be determined; visual comparisons of our Tra1 2D class averages with those from a previously determined cryo-EM reconstruction of NuA4 ( Chittuluru et al . , 2011 ) ( Figure 3—figure supplement 1 ) shows that the entirety of NuA4 closely matches Tra1 in appearance , indicating that the remaining NuA4 subunits are highly dynamic and/or have dissociated in the reconstruction .", "As endogenous purifications of NuA4 do not co-purify subunits of SAGA ( and vice versa ) , Tra1 is unlikely to connect the two coactivators into a single complex and its presence in separate coactivators may result from overlapping contact sites with both SAGA and NuA4 , precluding their assembly around the same molecule of Tra1 .", "Hence the SAGA contact sites identified in Tra1 may also be exploited for NuA4 interactions which is supported by deletion mutations within Tra1 that simultaneously cause defects in SAGA and NuA4 assembly ( Knutson and Hahn , 2011 ) .", "Mutations that disrupt the interaction of Tra1 with Gal4 , Gcn4 , Rap1 and VP16 are spread across Tra1 , located within the Finger , Ring and Head regions , and are predominantly distal from the interface with SAGA .", "However , the precise mechanism of disruption remains unknown; as well as specifically abrogating a Tra1-activator interface , these mutations may also cause an allosteric change , affect protein stability or some combination thereof .", "Nevertheless , 4/5 of the amino acid substitutions that disrupt interaction with Gal4 are located in the Finger at solvent exposed sites , and three are spatially proximal to each other ( D397N/H400Y/S404F ) , ( Figure 4 ) , strongly suggesting the presence of a direct Gal4 interface .", "Additionally , of the three deletion mutants that affect Gcn4 and Rap1 , two are also located in the Finger adjacent to the Gal4 interface on its N-terminal side ( H3 and H6-H7 ) , hence we suggest that the Finger may function as a platform for interacting with multiple activators .", "The Finger is unlikely to be the only activator interaction site , given the location of the third deletion mutant in the Ring ( proximal to the Head at H25-H26 ) , the clustering of mutations that affect the interaction with VP16 within the Head , and an in vitro interaction between a C-terminal fragment of Tra1 and VP16 ( Brown et al . , 2001 ) .", "A critical feature of these mutations is the ability to disrupt specific activators whilst leaving others unaffected i . e . mutations that disrupt the interaction of Gal4 with Tra1 do not affect Gcn4 ( Lin et al . , 2012 ) and vice versa ( Knutson and Hahn , 2011 ) .", "Conversely , the mutations that affect Gcn4 also effect Rap1 , indicating that these activators target Tra1 in a similar manner .", "Although allosteric effects cannot be excluded , the simplest mechanism is that Tra1 harbors multiple interfaces for activators that can be specific for single activators ( Gal4 ) or bind multiple activators ( Gcn4 and Rap1 ) .", "The presence of multiple interfaces would therefore provide a mechanism for Tra1 to integrate signals from activators , allowing multiple activators to co-operate in stimulating transcription .", "Individual binding sites are also likely to vary in their affinity and kinetics of interaction , further tuning the strength of transcriptional activation .", "More intricate mechanisms can also be hypothesized , such as competition between different activators for the same binding site , or by allosteric changes upon activator binding that alter its interaction with other activators and/or coactivator components .", "Although Tra1 required no conformational changes to fit into SAGA ( Figure 3 ) , HEAT repeat proteins are highly flexible ( Kappel et al . , 2010 ) and Tra1 may undergo conformational changes upon interacting with other factors such as activators .", "In that regard , the N-terminal part of the Finger is the most structurally dynamic part of Tra1 ( Figure 1—figure supplement 4B ) and makes extensive contacts with the Ring , so activator binding in this location may induce conformational changes and stimulate allosteric changes within Tra1 that may exert effects on its parent histone-modification complex .", "The remarkable and unexpected structural homology to the DNA repair factor DNA-PKcs suggests that Tra1 has roles beyond transcriptional activation .", "Yeast lack a homolog for DNA-PKcs and the Tra1 parent complex NuA4 is required for double strand break ( DSB ) repair ( Bird et al . , 2002 ) , so it is tempting to speculate that Tra1 may have functional similarities with DNA-PKcs and a direct role in DNA repair .", "DNA-PKcs mediates ligation of double strand breaks ( DSBs ) by forming a synaptic complex with Ku70-Ku80 and aligning the broken DNA ends , whereupon its kinase becomes active and coordinates further assembly of the repair machinery ( Dobbs et al . , 2010 ) .", "Although Tra1 lacks the kinase activity that is crucial for DNA-PKcs function ( Chen et al . , 2005; Cui et al . , 2005 ) , the structural homology between Tra1 and DNA-PKcs suggests that Tra1 might retain the non-catalytic features of DNA-PKcs in binding nucleic acids and/or recruiting additional repair factors .", "Recruitment of active kinases may then substitute for lack of Tra1 catalytic activity , such as ATM which interacts with Tip60 ( the human homolog of NuA4 ) in human cells ( Sun et al . , 2005 ) .", "Although a direct role in DNA repair remains speculative , connections between Tra1 and DNA damage are already well established , as depletion of TRRAP compromises DSB repair ( Murr et al . , 2006; Robert et al . , 2006 ) and its parent complex Tip60 is recruited to DSBs in a TRRAP-dependent manner ( Murr et al . , 2006 ) , resulting in H4 acetylation that facilitates repair .", "TRRAP also forms a complex with the MRE11 , RAD50 , and NBS1 ( MRN ) complex ( Robert et al . , 2006 ) , a key sensor of DSBs that also recruits PIKK family member ATM to sites of DSBs ( Falck et al . , 2005 ) .", "In this manner , MRN could function analogously to Ku70-Ku80 , which recruits DNA-PKcs to sites of DSBs .", "Additionally , NuA4 can recognize DSBs directly ( Bird et al . , 2002 ) and SAGA and NuA4 preferentially acetylate the ends of a linear chromatin template ( Vignali et al . , 2000 ) , hence Tra1 may provide this recognition capability , given its homology to DNA-PKcs which has affinity for DNA ends ( Gottlieb and Jackson , 1993 ) .", "As a direct target of multiple activators , and as common component of SAGA and NuA4 , Tra1 is central to transcriptional regulation .", "However , its size and presence in additional chromatin-related complexes , and its homology to DNA-PKcs points to a role beyond activator targeting .", "The structure presented here is an important step toward discovering those roles , and further structural and biochemical studies of Tra1 bound to activators and/or its parent complexes will elucidate new mechanisms of its functions ." ], [ "The S . cerevisiae Tra1 coding sequence ( YHR099W ) from genomic DNA was PCR amplified and cloned into a galactose-inducible pRS424-based expression vector ( courtesy of K . Nagai , MRC LMB , Cambridge ) with a N-terminal 3xFLAG tag .", "The plasmid was transformed into S . cerevisiae strain BCY123 ( MATα pep4::HIS3 prb1::LEU2 bar1::HIS6 lys2::GAL1/10GAL4 can1 ade2 trp1 ura3 his3 leu23 , 112 ) and transformants selected on SC plates lacking tryptophan ( Yeast Nitrogen Base , Trp dropout mix ( Formedium Ltd . , UK ) , 2% glucose , 50 mg/ml adenine ) at 30°C for 2 days before making a glycerol stock for storage at −80°C .", "The following liquid shaker cultures were all made with the same media omitting agar , and incubated at 30°/185 rpm; a one litre pre-culture was prepared from the glycerol stock , and incubated overnight .", "The pre-culture was centrifuged and washed with sterile dH20 and used to inoculate a 24 litre expression culture with glucose replaced by 2% Raffinose to a starting OD of 0 . 1–0 . 2 and incubated until OD ~0 . 8 .", "Tra1 expression was then induced with 2% Galactose for 6 hr before harvesting by centrifugation .", "Cells were frozen in liquid N2 for storage at −80°C .", "Cells were thawed and resuspended in an equal volume of Buffer A ( 125 mM HEPES 8 . 0 , 250 mM NaCl , 1 . 5 mM MgCl2 , 10% glycerol , 0 . 1% IGEPAL CA-630 , 0 . 5 mM DTT ) supplemented with protease inhibitors ( 1 . 25x Roche cOmplete Ultra plus AEBSF ( 210 μM ) , Aprotinin ( 0 . 3 μM ) , Benzamidine ( 6 . 5 mM ) , Leupeptin ( 105 μM ) , E-64 ( 2 . 8 μM ) , PMSF ( 1 . 15 mM ) and Pepstatin ( 200 μM ) ) for pipetting into liquid N2 and subsequent lysis by cryo-milling ( SPEX 6870 freezer mill ) , followed by storage at −80°C .", "All following purification steps were completed at 4°C .", "Lysate powder was thawed , supplemented with Benzonase ( 1 . 5 μl/10 ml lysate , Sigma E8263 ) for 20 min , and sonicated before centrifugation ( Ti45 rotor , 35000 rpm , 120 min ) .", "Clarified lysate was filtered and adjusted to pH 8 . 0 with 1M HEPES ( pH 8 . 0 ) and incubated with 2 ml Anti-FLAG agarose ( Sigma A2220 ) for 3 hr before washing with modified buffer A ( as Buffer A but 50 mM HEPES pH 8 . 0 , 0 . 3 mM DTT , protease inhibitors cocktail ( Sigma S8830 ) ) and elution with 0 . 5 mg/ml 3x FLAG peptide ( Generon ) in the same buffer .", "FLAG eluates were pooled and diluted to match conductivity of buffer B ( 50 mM HEPES 8 . 0 , 100 mM NaCl , 1 . 5 mM MgCl2 , 0 . 5 mM DTT ) and applied to a MonoS 5/50 GL column ( GE Healthcare ) equilibrated in buffer B . The column was washed with buffer B before gradient elution to 1M NaCl in buffer B over 25 CV .", "Tra1 containing fractions were assayed by SDS-PAGE , and appeared in both flowthrough and elution fractions .", "These were pooled , diluted to match conductivity of buffer B and applied to a MonoQ 5/50 GL column ( GE Healthcare ) in the same manner .", "Tra1-containing elution fractions were pooled and spin concentrated ( Amicon Ultra ) for injection onto a Superose 6 10/300 GL column ( GE Healthcare ) equilibrated in buffer B containing 150 mM NaCl .", "Fractions containing monomeric Tra1 eluted at 14 ml ( Figure 1—figure supplement 3 ) and were pooled and spin concentrated to 0 . 1 mg/ml ( Amicon Ultra ) .", "Aliquots of the Tra1 preparation were placed on negatively glow discharged lacey grids with ultrathin carbon over holes ( Agar Scientific , UK ) and vitrified in liquid ethane using a Vitrobot Mark IV ( FEI , USA ) .", "Blotting was carried out at 4°C and 94% humidity .", "Due to the low protein concentration two subsequent applications of Tra1 were required to achieve the desired protein density on grids .", "Each application was followed by 20 s waiting time , with a short 0 . 5 s blotting after first application and 5 s blotting after the second .", "Data were acquired using a Titan Krios microscope ( FEI ) operated at 300 keV and equipped an energy filter ( Gatan GIF Quantum , USA ) .", "The images were collected with a post-GIF K2 Summit direct electron detector ( Gatan ) operating in counting mode at a nominal magnification of 130 , 000x , corresponding to 1 . 06 Å per physical pixel .", "An energy slit with a width of 20 eV was used during data collection .", "The dose rate on the specimen was set to 5 . 5 electrons per Å2 per s and a total dose of ∼44 e/Å2 was fractionated over 32 frames .", "Data were collected using EPU software ( FEI ) with a nominal defocus range set from −1 . 5 μm to −3 . 5 μm .", "Unless otherwise stated , RELION 2 . 0 ( Scheres , 2012 ) was used for all subsequent processing steps .", "MotionCor2 ( Zheng et al . , 2017 ) was used for patch-based motion correction of movie frames followed by CTFFIND4 ( Rohou and Grigorieff , 2015 ) to estimate the contrast transfer function ( CTF ) parameters of the corrected micrographs .", "An initial subset of the data was processed with Gautomatch ( Urnavicius et al . , 2015 ) , using an automatically generated Gaussian reference .", "After initial particle extraction and reference-free 2D classification , selected 2D classes were used as a template for further iterations of particle picking with Gautomatch , yielding 418 , 339 particles from 1733 micrographs .", "These were subjected to reference-free 2D classification , and particles contributing to the best 2D classes ( Figure 1—figure supplement 3D ) were selected for 3D refinement .", "A previously published 13 Å resolution cryo-EM density map of DNA-PKcs ( EMD-1102 ) was low-pass filtered to 40 Å and used as initial reference for 3D refinement , and the resulting consensus model was used as a reference map for 3D classification .", "The best 3D class containing 182 , 285 particles ( 44% of total ) was used to perform a 3D refinement run , resulting in a 3 . 9 Å map .", "Substitution of the particles contributing to this map by particles from dose-weighted images calculated by MotionCor2 provided a final reconstruction at 3 . 7 Å resolution after a last run of 3D refinement .", "Reported resolutions are based on gold-standard Fourier Shell Correlation ( FSC ) curves between independently refined half-maps , using the 0 . 143 criterion .", "The resulting maps from refinement were post-processed by RELION and sharpened by a negative B-factor using an automated procedure .", "The final map was highly detailed with clear density for strands , helices and loops ( Figure 1—figure supplement 4A ) .", "Sidechains were resolved throughout the reconstruction , allowing de novo building of 3474/3744 residues .", "Model building was performed with Coot ( Emsley et al . , 2010 ) and assisted by secondary structure predictions from PSIPRED ( Jones , 1999 ) , JPRED3 ( Cole et al . , 2008 ) , and also reported within ( Knutson and Hahn , 2011 ) .", "The abundance of helices amongst the solenoid repeats greatly assisted building and assignment of sequence register .", "Maps were B-factor sharpened with phenix . auto_sharpen ( 1 . 11 . 1–2575 ) ( Adams et al . , 2010 ) or filtered to 5 Å to provide extra guidance during model building .", "Local resolution variations were estimated within RELION .", "The model was refined with phenix . real_space_refine using secondary structure restraints .", "For cross-validation of the model , atomic positions were randomly perturbed by up to 0 . 5 Å to remove model bias from prior refinement against all the data , and subsequently refined against a single ( unmasked ) half-map using secondary structure restraints .", "The refined model was used for FSC calculations against the same half-map ( FSCwork ) , the withheld half-map ( FSCfree ) , and the combined map ( FSCtotal ) to monitor for overfitting ( Figure 1—figure supplement 3 ) .", "Refinement half-maps correspond to the same half-maps used during gold-standard FSC resolution estimation .", "Refinement/validation statistics are shown in table S1 .", "The fit of the Tra1 structure into a SAGA reconstruction ( EMD-2693 ) was performed with fitting tools implemented in Chimera ( Pettersen et al . , 2004 ) , and assessed by correlation score and visual appearance .", "Figures were generated with Chimera and PyMOL ( 1 . 8 , Schrödinger , LLC . ) ." ] ]
[ "Coactivator complexes SAGA and NuA4 stimulate transcription by post-translationally modifying chromatin .", "Both complexes contain the Tra1 subunit , a highly conserved 3744-residue protein from the Phosphoinositide 3-Kinase-related kinase ( PIKK ) family and a direct target for multiple sequence-specific activators .", "We present the Cryo-EM structure of Saccharomyces cerevsisae Tra1 to 3 . 7 Å resolution , revealing an extensive network of alpha-helical solenoids organized into a diamond ring conformation and is strikingly reminiscent of DNA-PKcs , suggesting a direct role for Tra1 in DNA repair .", "The structure was fitted into an existing SAGA EM reconstruction and reveals limited contact surfaces to Tra1 , hence it does not act as a molecular scaffold within SAGA .", "Mutations that affect activator targeting are distributed across the Tra1 structure , but also cluster within the N-terminal Finger region , indicating the presence of an activator interaction site .", "The structure of Tra1 is a key milestone in deciphering the mechanism of multiple coactivator complexes ." ]
[ "Inside our cells , histone proteins package and condense DNA so that it can fit into the cell nucleus .", "However , this also switches off the genes , since the machines that read and interpret them can no longer access the underlying DNA .", "Turning genes on requires specific enzymes that chemically modify the histone proteins to regain access to the DNA .", "This must be carefully controlled , otherwise the ‘wrong’ genes can be activated , causing undesired effects and endangering the cell .", "Histone modifying enzymes often reside in large protein complexes .", "Two well-known examples are the SAGA and NuA4 complexes .", "Both have different roles during gene activation , but share a protein called Tra1 .", "This protein enables SAGA and NuA4 to act on specific genes by binding to ‘activator proteins’ that are found on the DNA .", "Tra1 is one of the biggest proteins in the cell , but its size makes it difficult to study and until now , its structure was unknown .", "To determine the structure of Tra1 , Díaz-Santín et al . extracted the protein from baker’s yeast , and examined it using electron microscopy .", "The structure of Tra1 resembled a diamond ring with multiple protein domains that correspond to a band , setting and a centre stone .", "The structure was detailed enough so that Díaz-Santín et al . could locate various mutations that affect the role of Tra1 .", "These locations are likely to be direct interfaces to the ‘activator proteins’ .", "Moreover , the study showed that Tra1 was similar to another protein that repairs damaged DNA .", "These results suggest that Tra1 not only works as an activator target , but may also have a role in repairing damaged DNA , and might even connect these two processes .", "Yeast Tra1 is also very similar to its human counterpart , which has been shown to stimulate cells to become cancerous .", "Further studies based on these results may help us understand how cancer begins ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics" ]
Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation
elife-12792-v4
[ [ "The estrogen receptor α ( ERα ) is a ligand-activated nuclear hormone receptor and a major regulator of cell growth , survival , and metastasis in a large fraction of breast cancers .", "Inhibiting the action of ERα with selective estrogen receptor modulators ( SERMs ) or selective estrogen receptor degraders ( SERDs ) , or reducing endogenous estrogen levels with aromatase inhibitors ( AI ) , are effective treatments for many of these breast cancers ( Strasser-Weippl and Goss , 2005 ) .", "Due to their efficacy and broad therapeutic indices , antiestrogens can be administered sequentially for progressive disease over the course of several years ( Toy et al . , 2013 ) .", "Unfortunately , despite continued expression of ERα , the majority of metastatic breast cancers that initially respond to endocrine therapies become refractory .", "Recently , somatic mutations in the ERα gene ( ESR1 ) were linked to acquired resistance to endocrine therapies of breast cancer ( Toy et al . , 2013; Merenbakh-Lamin et al . , 2013; Robinson et al . , 2013; Li et al . , 2013; Jeselsohn et al . , 2014 ) .", "Approximately 25% of patients who previously received SERM/SERD/AI treatments for an average of five years presented with conserved somatic mutations that were not identified in primary ( untreated ) tumors .", "The most prevalent ERα point mutations were Y537S and D538G , while several others were identified at significantly reduced frequencies .", "Importantly , breast cancer cell-based studies revealed that the Y537S and D538G mutations conferred hormone-independent activation of ERα and reduced the inhibitory potency and efficacy of clinically prescribed SERMs and SERDs ( Toy et al . , 2013; Merenbakh-Lamin et al . , 2013; Robinson et al . , 2013; Li et al . , 2013; Jeselsohn et al . , 2014; Carlson et al . , 1997 ) .", "Notably , the constitutive activity and antagonist resistance of the Y537S and E380Q mutations were first described in cell models in 1996 ( Weis et al . , 1996 ) , and shortly thereafter , the Y537N mutation was found in a clinical sample of metastatic breast cancer ( Zhang et al . , 1997 ) .", "However , no clinical follow-up studies were reported until 2013 .", "The Y537S and D538G mutations are especially interesting because they occur at the N-terminus of Helix 12 ( H12 ) in the ERα ligand-binding domain ( LBD ) .", "Structurally , ERα LBD is an α-helical bundle , with the C-terminal helix , H12 , functioning as a key structural component of the activating function-2 ( AF-2 ) cleft that governs the agonist or antagonist state of the receptor .", "In the agonist conformation ( e . g . estradiol ( E2 ) -bound ) , H12 covers the ligand-binding pocket , docking between Helices 3 ( H3 ) and 11 ( H11 ) , thereby facilitating coactivator recruitment to the AF-2 cleft via canonical LXXLL coactivator sequence motifs .", "In contrast , in the antagonist state ( e . g . SERM-bound ) , H12 occupies the AF-2 cleft using its own LXXML sequence , thereby blocking coactivator recruitment and ERα action .", "In this study , biophysical assays reveal the impact of the Y537S and D538G mutations on ERα LBD ligand and co-regulator binding affinity .", "Additionally , x-ray crystal structures and atomistic molecular dynamics ( MD ) simulations uncover altered conformations adopted by the mutant receptors in the absence and presence of agonists and antagonists .", "Together , these findings present a molecular explanation for how the Y537S and D538G mutations elevate the basal or constitutive activity of ERα and confer resistance to the beneficial effects of the SERM , SERD , and AI therapies .", "A comprehensive understanding of how these and other gain-of-function mutations alter the structure and function of ERα is crucial to development of more efficacious and potent inhibitors to target these mutant receptors in the clinic ." ], [ "An established time-resolved Förster Resonance Energy Transfer ( tr-FRET ) assay that determines the affinity of the steroid receptor coactivator 3 nuclear receptor domain ( SRC3 NRD ) for the ERs was used to investigate differences among the WT , Y537S , and D538G ( Tamrazi et al . , 2005 , Jeyakumar et al . , 2011 ) .", "SRC3 was chosen because of its abundance in breast cancer cells and high affinity for ERα ( Liao et al . , 2002 ) .", "Table 1 summarizes all SRC3 coactivator binding affinities .", "SRC3 NRD bound to the E2-saturated WT ERα LBD with high affinity ( Kd = 2 . 67 ± 0 . 5 nM ) while no binding was detected in the absence of E2 or in the presence of the SERM 4-hydroxytamoxifen ( TOT; the active metabolite of tamoxifen ) ( Figure 1 ) .", "In contrast , the SRC3 NRD bound to Y537S and D538G ERα in the absence of E2 , with affinities of 13 . 6 ± 2 . 0 nM and 151 ± 20 nM , respectively , and the binding curves reached approximately 60% of the maximum ( Figure 1 ) .", "When Y537S and D538G were pre-saturated with E2 , the SRC3 binding curves reached the same maximum as WT with E2 , with the coactivator binding affinity for the mutants being comparable or slightly higher than WT ( WT EC50 = 2 . 67 ± 0 . 5 nM; Y537S = 0 . 59 ± 0 . 1 nM; D538G = 3 . 65 ± 0 . 4 nM ) ( Figure 1 ) .", "Neither the WT nor the mutants bound coactivator when pre-incubated with saturating concentrations TOT ( Figure 1 ) .", "To determine the potency of ligands to affect coactivator binding to the ER , ligand was titrated into a constant amount of SRC3 and ER and measured by tr-FRET .", "Addition of E2 resulted in increased coactivator affinity to the Y537S ( EC50 = 1 . 6 ± 1 . 2 nM ) and D538G ( EC50 = 2 . 2 ± 0 . 1 nM ) ERα LBD .", "Interestingly , the EC50 value was somewhat reduced for WT ( EC50 = 13 . 8 ± 0 . 9 nM ) ( Figure 1—figure supplement 1 ) .", "TOT abolished basal Y537S and D538G SRC3 binding in the absence of agonist .", "To mimic this reversal in WT , which does not bind SRC3 NRD without ligand , a low concentration of E2 was added to WT-ER to recruit SRC3 NRD to about 50% of maximal ( data not shown ) .", "As expected , titration of TOT reversed the binding of SRC3 NRD by the mutant ER and E2-primed WT .", "The EC50 values for suppressing SRC3 binding of the mutant ( done in the absence of agonist ) were comparable to the Ki values for WT .", "The Ki of TOT was 1 . 82 ± 0 . 30 nM for WT , 6 . 7 ± 0 . 40 nM for Y537S , and 0 . 79 ± 0 . 04 nM for D538G .", "Our earlier work demonstrated that SERMs were less potent in inhibiting the transcriptional activity of the ERα Y537S and D538G mutants compared to WT in breast cancer cells ( Toy et al . , 2013 ) .", "The binding affinities of E2 with the WT and mutant ERα LBDs were measured using radioligand bindingligand-binding assays ( Carlson et al . , 1997 ) .", "The affinity of E2 for WT-ER ( Kd = 0 . 26 ± 0 . 13 nM ) is approximately five-fold greater than for the mutants , Y537S ( Kd = 1 . 43 ± 0 . 55 nM ) and D538G ( Kd = 1 . 30 ± 0 . 63 nM ) ( Figure 2 ) .", "Table 2 summarizes all ligand-binding affinities for the WT and mutant ERα LBDs .", "A competitive radioligand-binding assay with 3H-E2 as tracer was used to measure the relative competitive binding affinities ( RBAs ) of TOT for WT and the mutant-ERs ( Katzenellenbogen et al . , 1973; Carlson et al . , 1997 ) .", "The Ki of TOT binding to WT was 0 . 337 ± 0 . 018 nM , whereas it was 2 . 61 ± 0 . 60 nM and 3 . 42 ± 0 . 5 nM for the Y537S and D538G mutants , respectively .", "Comparing the Ki values , it is notable that the affinity of TOT for the Y537S and D538G mutants is impaired approximately 8- and 10-fold relative to WT ( Table 2 ) .", "This reduced binding affinity is consistent with the published lower inhibitory potency of TOT on the mutants in breast cancer cells ( Toy et al . , 2013 ) .", "Figure 3 shows representative radiometric competitive binding measurements ." ], [ "Acquired resistance to endocrine therapies represents a substantial barrier toward obtaining prolonged remission of ER-dependent metastatic breast cancers for a significant population of patients .", "While somatic mutations in the androgen receptor are a known mechanism of acquired hormone therapy resistance in prostate cancer , somatic mutations in ESR1 have only recently been identified as an important mechanism of acquired endocrine therapy resistance in breast cancer .", "Subsequent studies have established Y537S and D538G as the two most common point mutations conferring hormone-independent activation and reduced SERM/SERD/AI inhibitory potency and likely efficacy ( Robinson et al . , 2013; Toy et al . , 2013; Jeselsohn et al . , 2014 ) .", "The clinical importance of these ESR1 mutations highlights the importance of understanding the mechanisms by which they influence ERα structure and function .", "Here , biochemical and biophysical techniques combined with x--ray crystal structures , and MD simulations provide a molecular explanation for how the Y537S and D538G point mutations in the ERα LBD alter the structure and function of the receptor .", "Coactivator binding assays show that these mutant LBDs recruit the SRC3 coactivator in the absence of hormone , while the unliganded WT LBD does not .", "Importantly , apo Y537S binds SRC3 NRD with a significantly increased affinity compared to D538G .", "This differential coactivator binding affinity likely accounts for the significantly increased constitutive transcriptional activity of Y537S versus D538G in breast cancer cell line reporter gene assays ( Toy et al . , 2013 ) .", "Figure 8 shows a model for aberrant ERα activity as a result of Y537S and D538G mutations in the recurrent anti-estrogen-resistant breast cancer cell .", "Ligand-binding assays demonstrate that both mutants possess a slightly reduced affinity for E2 and a significantly reduced affinity for TOT .", "Collectively , these data suggest that the combination of a recruitment of coactivator in the absence of hormone and a reduced TOT-binding affinity underlie the hormone therapy resistance conferred by these H12 ERα mutations .", "Comprehensive biophysical and structural investigations by proteolytic susceptibility assays , HDX-MS , x-ray crystallography , and MD simulations reveal how the Y537S and D538G mutations affect ERα in the apo , agonist , and antagonist-bound states , thereby providing a detailed structural explanation for the hormone-resistance conferred to the ERα .", "The Y537S and D538G mutations are located at or near H12 , a key molecular switch governing the ligand-regulated actions of ERα via AF-2 .", "Previously published apo and agonist-bound Y537S structures showed that S537 promotes the agonist conformation in the absence of ligand by forming a hydrogen bond to D351 ( Nettles et al . , 2008 ) , in the process facilitating a tighter packing of the H11-12 loop against the LBD .", "Similarly , our analysis of the agonist-bound and apo D538G structures show that this mutation relaxes the helical character at the start of H12 , thereby also relaxing the H11-12 loop and improving the packing of its hydrophobic side chains .", "Importantly , our data also show that binding of coregulator ( SRC3 ) further stabilizes H12 in the agonist conformation .", "While the Y537S and D538G mutants may work through different mechanisms , both stabilize the agonist state in the absence of hormone .", "The D538G mutation , however , appears to be less stabilizing , as reflected by the lower constitutive activity of D538G ERα in both biochemical and cell-based assays ( Toy et al . , 2013 ) .", "Examination of the molecular basis for reduced SERM potency and efficacy for mutant ERα LBDs reveals that this likely evolves from structural changes to the H11-12 loop , resulting in a decreased binding affinity of antagonist ligands and an altered , stabilized , antagonist conformation of H12 in the AF-2 cleft .", "Our biophysical studies indicate that the H11-12 loop and H12 are both altered when TOT is bound in the Y537S and D538G mutants compared to the WT .", "Further , when compared to the WT-TOT structure , the D538G-TOT structure shows an altered conformation of the H11-12 loop and H12 occupancy of the AF-2 cleft , and multiple conformations of the TOT ligand ( indicative of reduced influence on the H11-12 loop ) .", "Additionally , MD simulation of the Y537S-TOT complex shows that S537 might form a hydrogen bond with E380 that alters the antagonist conformation .", "Therefore , the reduced inhibitory potency of TOT stems from its reduced affinity for the Y537S and D538G mutants along with conformational changes to the antagonist state once it occupies the ligand-binding site .", "Taken together , these results suggest that the constitutive activity conferred by the Y537S and D538G mutations stems from the intrinsic ability of the mutant receptors to adopt a stable agonist conformation in the absence of hormone , thereby leading to enhanced recruitment of SRC3 coactivators and increased ERα transcriptional activity .", "This pre-organized agonist state contributes to a decreased affinity for hormone and especially for SERMs because the stabilized H12 agonist conformation restricts ligand access to the hormone-binding pocket .", "In addition to reduced ligand affinity , SERM action is further reduced by an altered antagonist state of H12 .", "Thus , recruitment of coactivators in the breast cancer cell is not inhibited as efficiently for the Y537S and D538G mutants as for WT ERα .", "One caveat to the approach described in this study is that ERα is a multi-domain protein and only the LBD was used for structural studies .", "To gain deeper insight into how these mutations affect full length ERα , further studies on intact multi-domain protein will be necessary .", "In addition , the effect of these mutations on the other aspects of ERα action including other hormone/SERM/SERD binding affinities , homodimer formation , DNA-binding , and stability ( in vitro and in vivo ) and whether these mutant receptors display a differential preference for a spectrum of coactivators must be investigated .", "Our findings suggest that SERMs and SERDs that are designed to specifically increase the dynamics of H12 might lead to drugs with increased potency .", "In this regard , our data show that the H11-12 loop plays an important and previously unrecognized role in regulating the behavior of H12 , an essential molecular switch that is allosterically controlled by ligand , which determines the differential ability of the ERα AF-2 to recruit coactivators and corepressors .", "Therefore , antagonists with improved inhibitory potency will increase the dynamic character of mutant H12 , an already appreciated aspect of SERD action ( Pike et al . , 2001 ) .", "Additionally , our work provides a biophysical hypothesis for why fulvestrant ( a SERD , known to disorder H12 ) was the only molecule which could completely ablate the transcriptional activity of the Y537S and D538G mutants in breast cancer cells while TOT ( a SERM ) could not ( Toy et al . , 2013 ) .", "Therefore , newly developed mixed SERM/SERDs and SERDs with improved pharmacokinetics and oral bioavailability over fulvestrant , such as AZ9496 , bazedoxifene , GDC910 , and RAD1901 , should be particularly effective against cancers expressing the Y537S and D538G ESR1 mutants ( De Savi et al . , 2015; Garner et al . , 2015; Lai et al . , 2015; Wardell et al . , 2013 ) .", "These compounds may prove invaluable for treating endocrine therapy-resistant ER+ breast cancers and also preventing or delaying the appearance of these somatic mutations in early-stage patients ." ], [ "Relative binding affinities ( RBA ) were determined by a competitive radiometric binding assay with 2 nM [3H]-E2 as tracer , as a modification of methods previously described ( Katzenellenbogen et al . , 1973: Carlson et al . , 1997 ) .", "Incubations were at 0°C for 18–24 hr .", "Hydroxyapatite was used to absorb the receptor-ligand complex , and unbound ligand was washed away .", "The determination of RBA values is reproducible in separate experiments with a CV of 0 . 3 .", "The IC50 values for inhibition of [3H]-E2 were converted to Ki values using the Cheng-Prusoff equation ( Ki = IC50/ ( 1 + conc . tracer/Kd tracer ) ) ( Cheng and Prusoff , 1973 ) ; this was necessary because the affinity of the [3H]-E2 tracer is different for WT and mutant ERs .", "The Kd of [3H]-E2 for the ERs was determined in a saturation-binding assay , as 0 . 26 ± 0 . 13 nM for the WT , 1 . 43 ± 0 . 55 nM for Y537S , and 1 . 30 ± 0 . 63 nM for D538G ( Figure 2 ) .", "For the saturation ligand binding ( Scatchard analysis ) , protein was diluted to 0 . 8 nM , in Tris-glycerol buffer ( 50 mM Tris pH 8 . 0 , 10% glycerol , with 0 . 01 M 2-mercaptoethanol and 0 . 3 mg/mL ovalbumin added ) and incubated with various concentrations of [3H]-E2 ( Perkin-Elmer , Waltham , MA ) in the absence or presence of a 100-fold excess of unlabeled ligand for 3–4 hr , at 0°C .", "Aliquots of the incubation solution were used to determine the total [3H]-E2 in the sample .", "The incubation solutions were then assayed by adsorption onto HAP ( hydroxyapatite , BioRad , Hercules , CA ) and the free estradiol was washed away .", "Data were processed by GraphPad Prism 4 according to the method of Scatchard ( Scatchard , 1949; Hurth et al . , 2004 ) .", "Protein was prepared and labeled as described above for the trFRET assays .", "It was incubated in t/g buffer with or without 1 μM of ligand , at room temperature for 1 hr .", "Then , 1 μg trypsin per unit of protein was added for 10 , 30 , 60 , and 300 min at room temperature according to previously established methods ( Tamrazi et al . , 2005 ) .", "FRET signal was measured using a Victor X5 plate reader as outlined above .", "The data , representing 2–3 replicate experiments , were analyzed using GraphPad Prism 4 , and are expressed as half-lives ( t1/2 ) .", "A parameter set was constructed for TOT .", "Its structure was optimized quantum mechanically at the level of restricted Hartree-Fock ( RHF ) 6-31g* using the computer program Gaussian 03 ( Gaussian 03 , Revision C . 02 , Frisch et al . , 2004 ) .", "The partial atomic charges of TOT were then derived with Restrained ElectroStatic Potential ( RESP ) ( Bayly et al . , 1993; Cornell et al . , 1993 ) fitting to the quantum mechanical RHF/6-31g* potential .", "The ideal geometry was defined as the optimized .", "The other molecular mechanical parameters were derived by assigning CHARMm22 atom types for TOT ( Momany and Rone , 1992 ) .", "The dimer with the least missing residues of the H11-H12 loop was selected from the D538G-TOT crystal structure and served as the template structure to model the Y537S-TOT dimer structure .", "The side-chain atoms at positions 537 and 538 were removed , and then desired side-chain atoms were placed with the other missing atoms using the default geometry parameters in CHARMm22 .", "Hydrogen atoms were placed with the hbuild module of the computer program CHARMM ( Brünger and Karplus , 1988; Vanommeslaeghe and MacKerell , 2012 ) .", "Missing residues ( loops ) in the starting crystal structure were optimized in three rounds ( 100 steps of the steepest descent method followed by two rounds of 100 steps of the adopted New-Raphson method ) with updated harmonic constraints on the other atoms .", "Then , all newly added atoms' positions were optimized in the same fashion .", "The resulting minimized structure was solvated with water molecules of 15 Å padding thickness from the molecular boundary and ionized to reach charge neutrality and the concentration of 0 . 145 M , both of which were done with VMD ( Humphrey et al . , 1996 ) .", "The system was minimized for 5000 steps before a 100-ns MD simulation using NAMD2 ( Phillips et al . , 2005 ) was performed ." ] ]
[ "Somatic mutations in the estrogen receptor alpha ( ERα ) gene ( ESR1 ) , especially Y537S and D538G , have been linked to acquired resistance to endocrine therapies .", "Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance .", "Here , we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα .", "We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist ( estradiol ) and antagonist ( 4-hydroxytamoxifen ) are reduced .", "Further , they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα , which leads to a stabilized agonist state and an altered antagonist state that resists inhibition ." ]
[ "Around one in every eight women will be diagnosed with breast cancer in their lifetime .", "Hormone-based therapies – also referred to antiestrogen drugs – target a protein called estrogen receptor alpha and are effective treatments for the majority of these cancers .", "Unfortunately , about half of patients will develop recurrent breast cancers even though the cancer continues to produce the target of the drugs .", "The estrogen receptor alpha drives breast cancer in a number of ways , many of which require the receptor to be activated by binding to the hormone estrogen .", "When estrogen binds it causes the receptor to change shape to expose a surface where other proteins called coactivators can bind .", "Once a coactivator is bound , the estrogen receptor is active and signals the cancer cell to grow , divide , invade local tissues , and spread to new sites in the body .", "Antiestrogen drugs competitively block the binding of estrogen to the receptor and cause the receptor to take on a different shape that inhibits the binding of the coactivator .", "However , recent studies identified mutations at specific sites in the gene that encodes estrogen receptor alpha in a large subset of patients with breast cancers that have spread .", "These mutations make the receptor resistant to antiestrogen drugs , and two mutations ( called Y537S and D538G ) account for approximately 70% of cases .", "However , it was not clear how these mutations altered the activity of estrogen receptor alpha at the molecular level .", "Fanning , Mayne , Dharmarajan et al . now show these two most common mutations allow estrogen receptor alpha to bind to the coactivator in the absence of hormone .", "This unfortunately also reduces the effectiveness of one of the mostly widely administered antiestrogen therapies – a drug called tamoxifen .", "However , Fanning , Mayne , Dharmarajan et al . also show that the newer and more potent antiestrogens that are currently under examination in clinical trials should be highly effective at treating the cancers with the mutated versions of estrogen receptor alpha .", "Applying the knowledge gained from these new findings toward the development of new antiestrogens could help reverse the impact of these common mutations .", "If successful , these new drugs will provide life-saving treatments for many breast cancer patients ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience", "genetics and genomics" ]
A genomic lifespan program that reorganises the young adult brain is targeted in schizophrenia
elife-17915-v1
[ [ "Identifying the genetic mechanisms that underpin brain ageing across the lifespan may provide explanations for the maturation of behaviours and age of onset of diseases .", "Longitudinal studies show cognition , emotion and personality emerge progressively during childhood and adolescence , with executive functions peaking in early adulthood ( Craik and Bialystok , 2006; De Luca et al . , 2003 ) .", "This coincides with the onset of some of the most devastating psychiatric disorders , most of which arise during later stages of brain development in the teenage years and early twenties ( Kessler et al . , 2007 ) .", "For example , impulse-control disorders arise in late childhood and early teen years , substance abuse peaks in the early twenties , and schizophrenia in the mid-twenties ( with a delay of around two years in females ) ( Häfner et al . , 1993 ) .", "Some monogenic neurological disorders also have early adult onset , such as Inclusion Body Myopathy associated with Paget disease Frontotemporal Dementia ( Watts et al . , 2004 ) and rapid-onset dystonia Parkinsonism ( Brashear et al . , 2012 ) .", "Why some brain disorders with a strong genetic component have a late developmental onset is unknown .", "The prevailing hypothesis for schizophrenia proposes that an early ( fetal ) insult or mutation renders the brain vulnerable to a secondary environmental insult that occurs in young adults , which then triggers the onset of psychosis ( Bayer et al . , 1999 ) .", "However , the finding that the age of onset for schizophrenia has a heritability estimated at 33% ( Hare et al . , 2010 ) , suggests that the timing may have a genetic basis .", "In recent years , there has been major progress in understanding the genetic basis of schizophrenia with the identification of many mutations and variants contributing to disease susceptibility .", "It is widely accepted that many mutations directly impact on synapse proteins , particularly those involved with postsynaptic signalling mechanisms in excitatory synapses ( Kirov et al . , 2008; Pocklington et al . , 2015; Fromer et al . , 2014; Fernández et al . , 2009a; Singh et al . , 2017 ) .", "The postsynaptic proteome of excitatory synapses is physically organised into multiprotein complexes of which the supercomplexes assembled by PSD95 ( Husi et al . , 2000; Frank et al . , 2016a; Frank and Grant , 2017; Frank et al . , 2017 ) play a major role in regulating cognitive functions ( Migaud et al . , 1998; Nithianantharajah et al . , 2013 ) and are disrupted by schizophrenia mutations ( Pocklington et al . , 2015; Fromer et al . , 2014; Fernández et al . , 2009a; Singh et al . , 2017; Kirov et al . , 2012; Purcell et al . , 2014 ) .", "Together these observations suggest there may be genetic mechanisms that account for the convergence between the many schizophrenia susceptibility genes , the postsynaptic proteome and the young adult brain .", "Transcriptome profiling of the brain at different ages has shown complex changes in expression levels across the lifespan ( Colantuoni et al . , 2011 ) .", "In early onset brain disorders , such as intellectual disability and autism , gene expression levels in fetal and early postnatal development correlate with enrichment in autism susceptibility genes ( Willsey et al . , 2013; Parikshak et al . , 2013 ) .", "However , correlation based approaches ( Willsey et al . , 2013; Parikshak et al . , 2013; Gulsuner et al . , 2013 ) ( e . g . weighted gene coexpression network analysis ) are unable to account for the full complexity of transcriptional changes that occur with age .", "Furthermore , although there has been extensive characterisation of cellular and anatomical maturation in neuronal , glial and synaptic subtypes ( Alexander and Goldman , 1978; Shaw et al . , 2006; Gogtay et al . , 2004 , 2006; Zehr et al . , 2006; Woo et al . , 1997; Huttenlocher , 1979; Anderson et al . , 1995; Bourgeois et al . , 1994; Kalsbeek et al . , 1988; Klingberg et al . , 1999 ) , the relevant transcriptome changes remain to be identified .", "To further understand the transcriptional events underlying brain development and ageing , we developed new tools that identify age-dependent gene regulatory events .", "These methods detect when gene expression trajectories change direction or plateau: we refer to these events as Transcriptome Trajectory Turning Points ( TTTPs ) ( Figure 1 ) .", "The timing of TTTPs has been shown to be an important feature of transcriptome trajectories during maize embryo development and the yeast cell-cycle: in both systems , genes with linked biological functions were found to turn/plateau at similar time points ( Lee et al . , 2002; Spellman et al . , 1998 ) .", "We have characterised TTTPs in the neocortex of humans and hippocampus of mice across their respective postnatal lifespans .", "This revealed a previously unknown , species conserved , gene regulatory program .", "These methods were also used with single-cell transcriptomes to define the age-dependent sequence of changes in neuronal , glial and endothelial cell-types .", "Our data suggest that the late onset of some psychiatric and neurological disorders is timed by mutations in this genetically programmed developmental sequence .", "Our findings also indicate that misregulation in the molecular maturation of synapse proteomes during a critical window in young adults is important for the onset of schizophrenia .", "These methods and findings open new areas of investigation into the genetic regulation of brain age and highlight their importance in the adolescent and young adult ." ], [ "As shown in the schematic diagram ( Figure 1a ) , genes can be categorised according to their age-dependent profile of expression into simple trajectories ( monotonic , upward or downward gradients ) or complex trajectories that contain a Trancriptome Trajectory Turning Point ( TTTP ) , marking both the direction of change and the age at which it occurs .", "Following a TTTP , the expression may reverse direction or plateau .", "We systematically identified gene expression trajectories by fitting cubic splines and marking TTTPs where the first derivative dE/dA , ( E = expression level and A = Age ) of the interpolated trajectories equals zero and changes sign .", "Next , to take into account the extent of expression changes prior to the TTTP ( ΔE ) , and thereby emphasise those genes for which the TTTP represents a significant regulatory event , we developed two complementary methods ( DeGeT , Decile-based Gene Turning; ALiGeT , Age-Linked Gene Turning ) .", "The DeGeT method depicted in Figure 1b is conceptually the simplest: the lifespan is divided into ten age groups within which an approximately equal number of TTTPs occur; each gene then receives a score for each age group ( ΔE if the gene turns within that age window , and zero otherwise ) .", "The ALiGeT method depicted in Figure 1c extends this to generate a score for each year of age , by decaying the contribution of ΔE the greater the distance between the TTTP and the scored year ( example trajectories and associated ALiGeT scores are shown in Figure 1—figure supplement 1 ) .", "The reason for using two scoring methods is that some age periods have many TTTPs and others have few: DeGeT controls for this by balancing the number of TTTPs within age groups ( and thereby has greater power to detect enrichments in earlier/later life stages ) , while ALiGeT scoring allows for the possibility that small time windows will have distinct molecular associations .", "Together , the TTTP , DeGeT and ALiGeT methods provide general purpose tools for exploring age-dependent gene regulation .", "We first applied these methods to the Braincloud dataset , which measured mRNA expression levels from 269 prefrontal cortex samples across the human lifespan ( 14th gestational week to 78 years ) .", "Although TTTPs were identified across the lifespan , they were sharply concentrated during early adulthood .", "Summing the number at each age shows a striking peak and a mean of 26 . 0 years in males and 27 . 5 years in females ( Wilcoxon signed rank test , p=0 , Figure 2a , example trajectories with different turning points shown in Figure 2—figure supplement 1 ) .", "Prominent peaks in early adulthood were confirmed with three regression methods ( cubic splines with three degrees of freedom , four degrees of freedom and Loess regression ) ( Figure 2b ) .", "Removing X-chromosome genes from the analysis had no effect on this sex difference ( p=0 ) .", "To determine whether this TTTP-peak was human specific , we performed an equivalent analysis using transcriptome data from the hippocampus of 186 mice of both sexes between 58 and 600 days of age ( Figure 2—figure supplement 2 ) : this also revealed a peak in the frequency of TTTPs with a mean of 156 days for male and 165 days for female mice ( p<1×10−323 , Figure 2c ) .", "Although there is a lack of previous research on the age equivalence of early adulthood between rodents and humans , our results are concordant with estimations made using the TranslatingTime species comparison model which suggests that p156 ( 5 months ) is equivalent to human early adulthood based on equivalent levels of cortical synaptic maturation ( Pinto et al . , 2015; Pinto et al . , 2013; Workman et al . , 2013 ) .", "Plotting the cumulative distribution of TTTPs across the lifespan further illustrates the TTTP-peak in young adults ( Figure 2d , e ) , and shows that 90% of TTTPs occur by 40 years of age , corresponding to the last stages of human brain development ( De Luca et al . , 2003; Lebel et al . , 2012; Wood et al . , 2004 ) and 7 months of age in mice .", "These data indicate that despite greatly differing lifespans , these two mammalian species share a lifespan program of brain gene expression with conserved features .", "To characterize the features of the human TTTPs in more detail , we focussed on those genes that showed the greatest changes .", "As shown in Figure 2f , the TTTPs for genes with the greatest expression changes prior to the TTTP were concentrated around the late-twenties , reinforcing the earlier finding that this is a significant period for switching in the trajectories .", "Next , we separated genes into those with upward or downward trajectories prior to the turning point: overall there were similar numbers in each category , although there was a skew toward upward inflecting genes being more common in young subjects ( <25 years ) and downward inflecting genes more common in older subjects ( >40 years ) ( Figure 2g ) .", "Finally , we examined the direction of the trajectories after the TTTP by dividing genes into those that established a stable plateau ( Post-Turn Plateau ) or reversed their direction ( Post-Turn Reversal ) ( Figure 2h ) .", "The vast majority of probes had plateaued by 30 years of age .", "While the exact ages at which TTTPs occur is sensitive to both the regression method and the dataset used , it is clear that the TTTP-peak reveals a major molecular reorganisation in young adults towards the end of development .", "Together these findings show that young adulthood is a crucial time for switching brain gene expression and establishing the set points of most genes for later life .", "Even though there are major changes during brain maturation in young adults , the complex trajectories were found throughout the lifespan , suggesting they could be used to predict the biological age of the brain .", "To test this , we used radial basis support vector machines and demonstrated that classifiers trained on partitioned subsets of the gene expression data ( training sets ) predicted age in the test sets with an accuracy ( defined as mean |AgeActual−AgePredicted| ) in humans of 5 . 5 years and 28 days in mice .", "Remarkably , they showed accurate age predictions across the entire range of ages in both species ( human , R2 = 0 . 88 , mice , R2 = 0 . 94 ) ( Figure 2i , j ) using only 40 probes in humans and 100 in mice ( Figure 2—figure supplement 3 ) .", "Thus , TTTPs and trajectories are highly characteristic features defining brain age across the lifespan in mice and humans .", "This indicates a ‘genetic lifespan calendar’ of transcriptome events is a conserved feature of mammals .", "To ascertain the biological processes affected by the TTTPs , we first sought to identify the cell types affected at each age .", "We asked if the TTTPs were enriched in the transcriptome of specific cell types using brain single-cell RNA-seq data and the Expression Weighted Cell-type Enrichment ( EWCE ) method ( Skene and Grant , 2016 ) .", "TTTPs were binned into approximately equal sized age-groups ( similar to the DeGeT method ) and then tested for cellular enrichments ( Figure 3a ) .", "We assumed that different biological processes could be associated with up/down-regulated genes and so performed enrichment analyses for each direction separately .", "To ensure the findings were robust , the analysis was performed on both the Braincloud ( Colantuoni et al . , 2011 ) dataset and an independent human prefrontal cortex transcriptome dataset ( Somel et al . , 2009 ) ( see Materials and methods ) .", "Significant enrichments were found for each cell-type tested and these were strongly correlated between the two datasets ( Figure 3a , median correlation of enrichments for each cell-type and direction with at least one significant change between the two datasets was 0 . 37 ) .", "The majority of significant enrichments were found amongst the Upward ( Figure 1a ) gene sets relative to the Downward trajectory gene sets .", "A striking sequence of events was observed where each cell type was regulated within distinct age-windows ( Figure 3a and summarised in Figure 7 ) .", "Early postnatal life was marked by TTTPs in endothelial cells ( pbraincloudBonferroni=0 . 025 , psomelBonferonni<0 . 00001 where ‘Bonferroni’ indicates a Bonferroni-adjusted p-value ) and oligodendrocytes ( psomelBonferroni=0 . 015 ) ; followed by microglial genes ( pbraincloudBonferroni<0 . 00001 , psomelBonferroni<0 . 00001 ) throughout adolescence; then pyramidal neuron genes ( pbraincloudBonferroni=0 . 0216 , psomelBonferroni<0 . 00001 ) in early adulthood ( corresponding to the peak in turning points ) ; then interneuron ( psomelBonferroni<0 . 00001 ) and oligodendrocyte genes through the late twenties and thirties ( pbraincloudBonferroni<0 . 00001 , psomelBonferroni<0 . 00001 ) .", "Astrocytes were found to have two periods of enrichment , the first during early adulthood ( psomelBonferroni=0 . 0028 ) and a second very late in life ( pbraincloudBonferroni=0 . 00675 , psomelBonferroni<0 . 00001 ) .", "These enrichments reveal the sequential maturation of cellular processes in the brain across the human lifespan .", "Moreover , most of these changes occurred prior to 35 years of age with prominent neuronal changes in young adults .", "We also performed this analysis on the mouse hippocampal dataset ( Figure 3b ) .", "As in the human neocortex datasets we found an early adulthood enrichment for upward-turns in pyramidal neuron genes ( pmouseBonferroni=0 . 008 ) , followed by a later enrichment for downward-turns in genes associated with both pyramidal and interneurons ( pmouseBonferroni=0 . 006 and , pmouseBonferroni=0 . 0086 respectively ) .", "Unlike in the human datasets , upward-turns in interneuron genes were found to be enriched at the same ages as those in pyramidal neurons ( pmouseBonferroni=0 . 025 ) .", "This may be because the earliest samples in the mouse ageing dataset were 56 days whereas the human datasets included fetal samples: correspondingly the early life enrichments for upward-turns in microglia and endothelial cells were not seen in mice .", "A later enrichment for upward-turns was seen in endothelial cells in mice ( pmouseBonferroni=0 . 048 ) .", "These findings show that the enrichment of neuronal genes in the early adult peak is conserved across species and brain regions .", "The young adult peak in TTTPs is a prominent landmark and we next sought to identify the key molecular mechanisms involved .", "We used ALiGeT to identify the top 10% of genes in both human and mouse in the year in which the largest number of TTTPs occurred ( and refer to this as the Peak Gene Turning ( PeGeT ) score ) ( Supplementary files 1a and 1b ) .", "The relevant biological processes in the genes with high PeGeT scores , conserved between mouse and human ( Supplementary file 1c ) , were first examined for enrichment in Gene Ontology terms: this revealed their role in synaptic transmission ( pFDR−adj=0 . 00024 where ‘FDR-adj’ indicates the p-value was adjusted for False Discovery Rate using the Benjamini-Hochberg method ) .", "Mammalian Phenotype Ontology annotations indicated their roles in behavioural ( p=7 . 5×10−05 ) and nervous system ( p=0 . 0002 ) phenotypes .", "To probe the synaptic role in more detail , we examined genes coding for proteins in the human postsynaptic density ( hPSD ) ( Bayés et al . , 2011 ) and the postsynaptic PSD95 supercomplexes ( Husi et al . , 2000; Frank et al . , 2017; Fernández et al . , 2009b; Frank et al . , 2016b ) , which are crucial in controlling synaptic plasticity and behaviour: both sets showed significantly higher PeGeT scores than expected by chance ( p<0 . 0009 and p=0 . 0019 respectively , Figure 4a , b , Supplementary file 1d ) .", "To establish the age window when synaptic genes showed TTTPs , we used a bootstrapping approach to test whether synaptic genes have higher ALiGeT scores in particular years in the Braincloud dataset .", "Each year between 22 and 33 years of age showed a significant increase in synapse-associated ALiGeT scores and we refer to this as the ALiGeThPSD window .", "A replication study ( Somel ) using a smaller human transcriptome dataset ( Somel et al . , 2009 ) gave an overlapping estimate for the synaptic ALiGeThPSD window at 17–22 years ( Figure 4c ) .", "We next applied the DeGeT method and found all five consecutive age sets from 24 to 36 years were significantly enriched in hPSD genes ( Figure 4d ) .", "This result was confirmed in two independent human frontal cortex datasets ( Somel et al . , 2009; Kang et al . , 2011 ) ( see Figure 4—figure supplement 1 ) .", "Similarly , we identified the ALiGeTmPSD window in mice at 126 to 151 days ( Figure 4e ) .", "These data show that transcripts encoding postsynaptic proteins were significantly enriched in TTTPs during early adulthood in all four datasets tested , spanning two species and two brain regions .", "Since these transcriptome results suggest that specific changes occur in the composition of components of the synapse proteome , we performed relative quantitation by mass spectrometry on 38 forebrain synaptosome samples between 1–5 months in mice .", "A total of 900 proteins were detected , of which 99 were found to show significant changes with age ( Supplementary file 1e , pFDR−adj<0 . 005 ) .", "We explored which functional classes of synapse proteins were affected by ageing during this period , and found that ion channel proteins and receptors showed increased likelihood of being differentially expressed ( pFDR−adj=0 . 00034 , expected = 7 , actual = 17 , Figure 4f ) .", "Amongst the affected channels were the majority of Ca2+- and Na+/K+-ATPases detected in our dataset , including three of the subunits of Atp2b ( often referred to as PMCA ) .", "This finding recapitulates and extends the previous reports that aged rodents have decreased Ca2+- and Na+/K+- ATPase activity ( Zaidi et al . , 1998; Tanaka and Ando , 1990 ) .", "These proteomic findings support the transcriptome findings that changes in synapse proteome composition occurs within the young adult age window .", "Prompted by previous results showing that postsynaptic proteins have been linked to the genetic susceptibility of schizophrenia ( Fromer et al . , 2014; Fernández et al . , 2009a; Nithianantharajah et al . , 2013; Kirov et al . , 2012; Purcell et al . , 2014 ) , we hypothesized that TTTPs may be relevant to the age of onset of schizophrenia .", "For these analyses we examined multiple sets of publically available genetic data including de novo and GWAS data ( see Materials and methods ‘Genes Lists’ , Supplementary file 1f ) and applied multiple analytical approaches .", "As a first step , we applied the same bootstrapping approach used earlier for the PSD genes on the pooled set of genes which have been associated with Schizophrenia using either GWAS or de novo approaches .", "Strikingly , this analysis showed the TTTPs in susceptibility genes predicted the known age windows for the onset of schizophrenia ( Figure 5 ) .", "The ALiGeTscz enrichment window spanned 22–26 years ( Figure 5a ) corresponding to the clinically reported age of onset defined by the first episode of psychotic symptoms and window of maximum vulnerability ( Kessler et al . , 2007; Häfner et al . , 1993 ) .", "Since males are reported to have an earlier disease onset than females ( Kessler et al . , 2007 ) , we tested males and females separately and found the ALiGeTscz enrichment window was significantly earlier in males than females ( males 20–26 years , females 26–28 years; wilcoxon p=4 . 7×10−14 , Figure 5b ) .", "To validate these results , we performed a series of technical control studies and biological replications .", "First , we used the DeGeT scoring system that showed the enrichment in schizophrenia peaked at 24–26 years ( Figure 5c ) .", "Secondly , we performed the DeGeT enrichment test in the two independent human frontal cortex datasets ( Somel et al . , 2009; Kang et al . , 2011 ) and confirmed that schizophrenia was significantly enriched ( confirming the Braincloud result ) ( Figure 5—figure supplement 1 ) .", "Thirdly , to confirm the results were not specific to a particular spline regression method , we demonstrate that enrichment window for schizophrenia was found using two alternate approaches to model fitting ( Figure 5—figure supplement 2 ) .", "While validating the occurrence of the disease enrichments , this analysis also revealed that the exact age at which the turning points/windows of enrichments occur depends on the regression model used ( Figure 5—figure supplement 2 ) .", "Fourth , we performed a down-sampling based sensitivity analysis to determine how the size of the gene set influences the enrichment: this indicated that the DeGeT enrichments are stronger for schizophrenia than for the hPSD ( using subsets of 650 genes , 95% of schizophrenia subsets were significant at 24—25 years but only 50% of hPSD subsets , Figure 5—figure supplement 3 ) .", "Fifth , we performed a variation of the bootstrapping analysis which accounts for transcript length and GC content ( both of which are known to affect the rate of de novo mutations ) and found no effect on the significance of the results ( Figure 5—figure supplement 4 ) .", "Sixth , we confirmed that varying the parameter in the ALiGeT scoring function , which controls the rate of decay with temporal distance , did not adversely affect the results ( Figure 5—figure supplement 5 ) .", "Finally , we dropped all fetal samples from the analysis , recalculated the TTTPs and confirmed that the major peak of TTTPs occurs in the early twenties , that it is delayed in females , that the later DEGET windows are enriched for PSD genes , and that schizophrenia shows discrete and significant enrichments using DEGET ( Figure 5—figure supplement 6 ) .", "To further validate our findings , we next examined different genetic datasets that have been used to identify schizophrenia susceptibility genes .", "The ALiGeTscz analysis results described above used a combined schizophrenia gene set from three orthogonal genome-wide methods: ( 1 ) an integrative analysis of Genome Wide Association Studies ( GWAS ) , expression analysis , copy number variants ( CNV ) and mouse models ( Ayalew et al . , 2012 ) ; ( 2 ) combined results of three exome-sequencing studies ( Fromer et al . , 2014; Xu et al . , 2012; Girard et al . , 2011 ) , and ( 3 ) the most recent GWAS results from the Schizophrenia Working Group of the Psychiatric Disease Consortium ( Schizophrenia Working Group of the Psychiatric Genomics Consortium , 2014 ) ( Supplementary file 1f ) .", "We therefore separately tested sets of susceptibility genes discovered with all three methods: all showed significantly increased PeGeT scores ( Integrative Analysis , pFDR−adj<0 . 0009 , de novo , pFDR−adj=0 . 0057 , GWAS , pFDR−adj=0 . 027 , Figure 5d–g ) .", "Interestingly , the stronger enrichment seen with de novo mutations may reflect that GWAS detects common variants that are assumed to have lower penetrance ( Schizophrenia Working Group of the Psychiatric Genomics Consortium , 2014 ) .", "We also validated the results using an additional set of de novo mutations ( Gulsuner et al . , 2013 ) : this also confirmed the adult enrichments , either when analysed independently or when combined with the combined sets described above ( Figure 5—figure supplement 7 ) .", "Much of the heritability for schizophrenia is associated with SNPs that have not reached genome-wide significance with current sample sizes ( Loh et al . , 2015 ) ( and were not included in our analysis thus far ) and the sample sizes for de novo studies are too small to determine whether any genes found are significantly associated with disease status .", "We therefore adapted our methods to include a greater fraction of the SNPs associated with schizophrenia heritability by using association statistics from all SNPs regardless of whether they are genome wide significant ( as defined in GWAS summary statistics files ) and explicitly modelling linkage disequilibrium ( based on results of the 1000 genomes project ) , such that disease association scores can be ascertained for each gene .", "The ALiGeT and DEGET approaches were extended to directly utilise the gene association scores generated by MAGMA ( Multi-marker Analysis of GenoMic Annotation ) ( de Leeuw et al . , 2015 ) based on schizophrenia GWAS summary statistics .", "Using this approach , schizophrenia showed a DEGET enrichment at 26 years in the Braincloud dataset ( pBonferroni=0 . 03135 , Figure 5h ) and at 15—16 years in the Somel dataset ( pBonferroni=0 . 0115 , Figure 5—figure supplement 8a ) and corresponding enrichment windows were found using ALiGeT ( Figure 5i , Figure 5—figure supplement 8b ) .", "Finally , we performed two additional sets of analyses where we examined mouse proteome datasets and single-cell transcriptome data .", "Consistent with the transcriptome results , the mouse synapse proteomic dataset showed that schizophrenia-associated proteins were enriched in the synapse proteins that changed prior to the 5 month peak ( p=0 . 018 , expected = 6 , actual = 11 ) , and 91% of those that change were found to be down-regulated .", "Next , we examined the role of specific cell types in schizophrenia by restricting the ALiGeT analysis to the subsets of genes defined by single cell transcriptome data ( see Materials and methods ) ( Zeisel et al . , 2015 ) .", "Schizophrenia associated genes showed a significant ( pBonferroni<0 . 05 ) window of enrichment in neuronal genes ( Figure 6a , Figure 6—figure supplement 1 ) .", "We were however unable to confirm this result using the summary statistics based method ( Figure 6b ) .", "We then examined the types of trajectories associated with schizophrenia and found genes were predominantly down-regulated prior to the TTTP ( Figure 6c ) .", "This result was confirmed using the summary statistics based method ( Figure 6d ) .", "Together these analyses show robust replication across multiple datasets , including different types of genetic variants , transcriptomes and proteomes , and several complementary analytical approaches .", "These data strongly suggest that the window of onset and sex difference in schizophrenia is timed by the regulation of susceptibility genes in young adults .", "In the mouse synapse proteome dataset , we noticed several proteins that cause adult onset monogenic disorders .", "We used the Human Phenotype Ontology age-of-onset annotations to identify these neurological disorders ( excluding neoplasms , and peripheral/autonomic disorders ) and from a total of 39 genes , six were detected in our samples , and we confirmed these were significantly enriched amongst the synapse proteins whose expression levels changed during maturation ( p=0 . 00008 , expected = 1 , actual = 4 , Figure 6—figure supplement 2 , Supplementary file 1e ) .", "The affected proteins/disorders included Atp2a2 ( Darier’s disease ) , Eef2 and Itpr1 ( Spinocerebellar ataxia ) , Atp1a3 ( Dystonia Parkinsonism ) .", "No equivalent enrichment was found for congenital/neonatal onset disorders ( p=0 . 45 , expected = 4 , actual = 4 ) .", "Further inspection ( because HPO annotations were incomplete ) found three additional , adult-onset haploinsufficiency disorders encoding by genes showing a 50–80% reduction in the maturing mouse synapse proteome ( Figure 6—figure supplement 2 ) : Vcp ( Frontotemporal Dementia ) ( fold change , FC = 0 . 48; p=0 . 002 ) ; Atl1 ( hereditary spastic paraplegia ) ( FC = 0 . 19 , p=0 . 00004 ) ; Dmxl2 , ( Polyendocrine-polyneuropathy syndrome ) ( fold change: 0 . 28 , p=0 . 00004 ) .", "These age-dependent reductions in levels of haploinsufficient disease genes is consistent with the model that their respective lifespan trajectories are relevant to their age of onset .", "The possibility exists that the lifespan trajectories are also relevant to the age of onset of other polygenic diseases with onset at different ages .", "To address this we compiled gene lists for six other major brain disorders with different age-windows of onset: onset during infancy ( autism and intellectual disabilities ) ; early adulthood ( multiple sclerosis ) ; and late adulthood ( Amyotrophic Lateral Sclerosis , Parkinson’s and Alzheimer’s ) ( corresponding gene sets shown in Supplementary file 1f ) .", "We applied the same bootstrapping approach used earlier for the PSD genes and none of the disorders showed significant results ( Figure 6—figure supplement 3 ) .", "Because these gene lists are not comparable ( different size , population sample size , obtained using different technical approaches etc . ) the relative importance of the genetic calendar to schizophrenia cannot be directly compared with these disorders ( see Discussion ) .", "In addition , because the transcriptome data is from prefrontal cortex and the primary pathology of several of these diseases is in other parts of the nervous system it cannot be assumed that the transcriptome trajectories in one part of the brain are the same as in others .", "Hence , the lack of any detectable age window does not preclude a role for gene regulation in the onset of these diseases ." ], [ "To date , there has not been a satisfactory mechanism or model that accounts for the following five central features of schizophrenia .", "First , the genetic susceptibility: the mechanism needs to account for the diverse sets of genes and the diverse types of mutations .", "Second , the age of onset: the mechanism needs to account for the age-window during which first-episode psychosis occurs , the heritability of this onset , as well as the earlier presence of prodromal cognitive symptoms ( Koutsouleris et al . , 2012 ) .", "Third , the sex difference: females have a later onset than males .", "Fourth , the cell biological mechanisms: there needs to be a common subcellular mechanism that incorporates the diverse classes of disease-relevant proteins , which include channels , receptors , synaptic adhesion proteins , scaffold proteins and signalling molecules .", "Fifth , the cognitive deficits: the molecular and cellular mechanisms need to play a key role in the relevant cognitive processes .", "A model that satisfies these criteria would also be expected to have explanatory power for other characteristics of schizophrenia .", "Our findings meet all five criteria and we propose the following model .", "A genetic program orchestrating transcriptome trajectories causes reorganisation of expression of synapse proteins in young adults .", "Mutations in these genes are functionally exposed in young adults because the reorganisation of expression produces inappropriate synapse signalling properties that result in abnormal behaviour .", "We refer to this as the genetic calendar model of schizophrenia .", "This model posits that schizophrenia is a genetic disorder targeting the mechanisms of brain aging during the young adulthood period of the lifespan .", "Our model offers a mechanistic explanation for the onset of first-episode psychosis and is consistent with prodromal cognitive impairments and the persistence of schizophrenia beyond the young adult years ( when many genes reach their plateau ) .", "Thus , the genetic calendar model can explain the onset and progression of schizophrenia .", "In addition to the robust association of schizophrenia genes with the TTTP-peak which was replicated across datasets including multiple types of genetic variants , transcriptomes and proteomes , and several complementary analytical approaches , our study identified specific molecules that further strengthens the mechanistic link between synaptic mechanisms and schizophrenia .", "PSD95 supercomplex proteins are enriched in schizophrenia genes ( Fromer et al . , 2014; Fernández et al . , 2009a; Singh et al . , 2017; Kirov et al . , 2012; Purcell et al . , 2014; Grant et al . , 2005 ) .", "Amongst the schizophrenia-susceptibility genes with the most prominent TTTPs were those with established synaptic functions , including Rgs4 , Snap25 , Kalrn , Htr2a and Nrg1 .", "The expression level of each of these genes has previously been shown to either influence , or be influenced by psychiatric symptoms ( Etain et al . , 2010; Guillozet-Bongaarts et al . , 2014; Yin et al . , 2013; Hill et al . , 2006 ) .", "Furthermore , altered expression of Snap25 , Htr2a and Nrg1 are noted to associate with earlier age of onset ( Etain et al . , 2010; Weickert et al . , 2012; Abdolmaleky et al . , 2011 ) .", "We found evidence that several mendelian neurological disorders with adolescent and young adult onset also involved proteins that were down-regulated in the TTTP-peak .", "Heterozygous mutations in Atp2a2 cause Darier’s disease and significantly increase the risk of many psychiatric disorders including mood disorders , depression , and schizophrenia ( Gordon-Smith et al . , 2010 ) .", "Mutations in Eef2 and Itpr1 cause spinocerebellar ataxia type 26 and 15/29 , respectively .", "Atp1a3 mutations cause rapid-onset dystonia Parkinsonism , which leads to Parkinson’s-like symptoms appearing during early adulthood , often with concurrent emergence of psychiatric symptoms ( Brashear et al . , 2012 ) .", "Vcp mutations cause a form of frontotemporal dementia with a mean age of onset in the mid-thirties ( Watts et al . , 2004 ) .", "One of the main causes of hereditary spastic paraplegia ( HSP ) are heterozygous mutations in Atl1 , which has an age of onset ~21 years ( McCorquodale et al . , 2011 ) .", "Interestingly , we found that the synaptic scaffold protein Dmxl2 ( haploinsufficiency causes Polyendocrine-polyneuropathy syndrome [Tata et al . , 2014] ) was reduced by ~70% ( fold change: 0 . 28 , p=0 . 00004 ) and studies in mice show heterozygous deletion of Dmxl2 in central neurons delayed the onset of puberty ( Tata et al . , 2014 ) .", "These findings support the view that the genetic lifespan calendar reduces expression below a critical threshold in young adults and is important for multiple neurological and psychiatric diseases .", "Our studies relied on human prefrontal cortex transcriptome data , which may have limited our ability to detect a role for the genetic lifespan calendar in the age-dependent onset of those diseases that are known to have primary pathology in other brain regions ( e . g . Parkinson’s disease ) .", "Given the evolutionary conservation between mouse hippocampus and human prefrontal cortex , we expect other human brain regions to show a calendar of transcriptome trajectories , but with different patterns and therefore different age windows of disease gene enrichments .", "Moreover , while our whole tissue transcriptome analysis appeared to be sensitive to neuronal changes , we expect that future single-cell transcriptome data will provide a more detailed insight into rarer cell types and potentially reveal mechanisms relevant to the age of onset of pathology in these cells .", "We do not expect that the age of onset of all brain diseases will be accounted for by the genetic calendar , as it will likely depend on the importance of cell autonomous processes and exogenous factors ( e . g . inflammatory processes involving microglia ) .", "It is also likely that high penetrance severe mutations will show an earlier onset and are less likely to show a dependence on the TTTPs .", "Weaker alleles may manifest with undetectable or subtle phenotypes at early ages and be exposed at later ages by the changes in gene expression .", "Thus , TTTPs would not necessarily account for the age of onset of intellectual disability or autism even though some of the same genes are involved with schizophrenia .", "The genetic lifespan calendar is an innate mechanism that regulates the levels of postsynaptic proteins and hence changes the physiological and behavioural properties of brain circuits .", "This indicates that the brain is not ‘hard wired’ but is continuously being modified by an evolutionary ancient and conserved genetic program .", "The postsynaptic proteins control innate and learned behaviours and thus the genetic calendar will modulate innate behaviours and the capacity to learn across the lifespan .", "The overarching biological function of this program could be to equip the animal for the challenges it faces at different ages .", "If there are mutations that interrupt this calendar of events , then the organism will not respond appropriately to environmental challenges .", "This may be relevant to schizophrenia where exogenous factors are thought to influence onset ( e . g . cannabis ) or behaviour ( e . g . smoking ) during young adulthood .", "Our model which posits a fundamental role for genetic and genomic mechanisms can therefore accommodate previous models that have considered exogenous factors in disease aetiology .", "Interestingly , environmental triggers of psychosis , such as cannabis and other drugs of abuse , are known to act on the synaptic signalling mechanisms ( Camp et al . , 2011; Abbas et al . , 2009 ) that are being reorganised in the TTTP-peak .", "Our model also has implications for those schizophrenia models that posit a ( non-genetic ) fetal insult as the predisposing factor for later onset ( Brown and Derkits , 2010 ) : the enrichments in schizophrenia susceptibility genes in young adults was present even when fetal samples were not included in the analysis supporting the notion that it is a disorder of postnatal brain ageing .", "Our genetic lifespan calendar model may also have implications for the development of pharmaceuticals for the adolescent and young adult onset psychiatric and neurological disorders .", "As an alternative to the ‘precision medicine’ approach which directs pharmacological treatments to each susceptible genotype , we suggest that therapeutics accommodating many genotypes might lie in drugs that modify the genetic lifespan calendar .", "The identification of the conserved TTTP-peak between mice and humans may also assist in refining animal models of adult-onset brain disorders .", "Our findings open a range of new approaches for understanding brain ageing and the mental disorders afflicting young adults ." ], [ "Hippocampi from 186 mice of both sexes and two background strains ( C57Bl/6 and 129s5 ) aged 58—600 days were used for the mouse microarray dataset ( Figure 2—figure supplement 2 ) .", "All animal experiments conformed to the British Home Office Regulations ( Animal Scientific Procedures Act 1986; Project License PPL80/2 , 337 ) , local ethical approval , and NIH guidelines .", "Animals were born and raised within the Research Support Facility at the Wellcome Trust Sanger Institute , and exposed to the same light/dark cycles and feed supply .", "Mice were exported to a holding facility three days prior to collection of brain samples , to allow time for stress response genes induced through movement to subside .", "All animals were sacrificed by cervical dislocation , and the hippocampi were dissected on ice , and snap frozen in liquid nitrogen .", "Mouse brain samples were homogenised using the Kontes Cordless Pellet Pestle system .", "Total RNA was extracted using the Qiagen miRNeasy kit , snap frozen using liquid nitrogen and stored at −80°C .", "Microarray processing was performed by the Wellcome Trust Sanger Institute’s Microarray facility .", "The Illumina TotalPrep-96 RNA Amplification kit was used for reverse transcription , amplification , and biotinylation of the RNA prior to hybridisation .", "The microarrays used were the Illumina MouseWG-6 v2 series .", "Hybridization , washing , and staining were performed according to standard Illlumina protocol .", "The microarrays were imaged using the Illumina BeadArray Reader .", "Images from the scanner were processed using the BeadStudio software .", "The microarray data is available to download from ArrayExpress ( accession number E-MTAB-3256 ) .", "The human samples were generated by the Braincloud project ( braincloud . jhmi . edu ) as described in their primary publication ( Colantuoni et al . , 2011 ) .", "In brief they used post-mortem human brains from the NIMH Brain Tissue Collection , National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders .", "RNA samples were described as being extracted from ~100 mg of tissue from BA46/9 , the dorsolateral prefrontal cortex .", "Custom-spotted two-colour oligonucleotide microarrays constructed from a set of oligos referred to as HEEBO7 ( Human Exonic Evidence Based Oligonucleotide ) were used .", "The preprocessed/normalized Braincloud expression data was downloaded from GEO ( GSE30272 , data contained within the Series Matrix file ) .", "No further normalisation was done beyond that which was already performed as described in the Braincloud publication .", "Low intensity probes and outliers had already been removed and expression levels were adjusted for sex , ancestry and a single surrogate variable using the sva package ( Leek et al . , 2012 ) .", "The ‘Somel’ dataset used to validate the increased PeGeT/DeGeT/ ALiGeT scores of schizophrenia genes , and EWCE enrichments , was downloaded from the GEO repository ( accession GSE11512 ) ( Somel et al . , 2009 ) .", "The dataset comprised samples taken from dorsolateral prefrontal cortex , which had been hybridised to Affymetrix Human Genome U133 +arrays .", "The dataset was read into R using the bioconductor ‘affy’ package , and processed using RMA .", "Probes with detection probabilities of less than 0 . 05 in over ten of the arrays were dropped .", "The BrainSpan RNA-Seq gene level dataset ( Kang et al . , 2011 ) was downloaded from the BrainSpan website ( brainspan . org ) on the 21st Jan 2016 .", "The data was read into R . Data from the following four regions were retained: dorsolateral prefrontal cortex , ventrolateral prefrontal cortex , orbital frontal cortex and anterior ( rostral ) cingulate ( medial prefrontal ) cortex .", "Ages were converted to numerical equivilents .", "Any transcripts whose summed expression over all retained samples was less than 0 . 1 were dropped .", "Because the oldest samples in the BrainSpan dataset are only 40 years of age we treated trajectories which did not turn as turning in final year of life .", "The mouse microarray data was read into R using the Bioconductor package Lumi ( Du et al . , 2008 ) ( RRID:SCR_012781 ) and re-annotated based on a published analysis ( Barbosa-Morais et al . , 2010 ) .", "The detectionCall function from the lumi package was used to drop probes undetected by the microarrays .", "Probes rated as having a ‘bad’ quality ( according to the re-annotation package ) were dropped , as were those with the coding zone given as ‘Transcriptomic ? ’ .", "A variance stabilizing transformation was applied followed by quantile normalization .", "Natural cubic splines with three degrees of freedom were fitted to the expression data using the R package splines .", "For the mouse expression data , age was modeled as the independent variable , with sex and background as covariates .", "For the human expression data , as variation attributed to the extraneous variables ( sex and ancestry ) had already been subtracted earlier using the sva package these terms were not included .", "The location of knots for the splines was determined by the quantiles of the data .", "For each species and gene , trajectories were then interpolated across the lifespan with extraneous variables ( sex and ancestry/background ) held constant .", "We refer to these interpolated spline trajectories as Brain Transcriptome Lifespan Trajectories ( BTLTs ) .", "Prior to detecting TTTPs , spline models were fitted to the data .", "To calculate where a TTTP occurred in a gene’s expression trajectory ( Ag ) , the derivative was approximated for each BTLT and the age of the TTTP was taken as the first point where dE/dA =0 and the derivative changed sign , where E is expression level in the spline model and A is age .", "If multiple turning points were found in a single spline , then only the first was used .", "Figure 2a–c show the number of TTTPs found across the transcriptome within a given year/month .", "To determine the TTTPs for each sex , all samples from the other sex were dropped , the splines remodeled ( without including sex as a covariate ) and TTTPs detected afresh .", "The numbers presented for the age of the TTTP peak in paragraph six are the mean age of TTTPs across all transcripts .", "To test for a difference in the age of TTTPs between the sexes , the Wilcoxon signed rank test was used .", "To test whether X-chromosome genes caused the sex difference , TTTPs in genes from that chromosome were dropped the same statistical test performed on the remaining set , and a similarly significant probably for difference was found .", "To analyse the properties of trajectories with TTTPs ( for generation of Figure 2g , h ) , linear models were fitted to the expression data before and after each TTTP , and tested for whether there is a significant change .", "A small fraction of probes which were detected as having TTTPs were not found to show significant differential expression prior to the TTTP , after multiple hypothesis correction with the Benjamini-Hochberg correction ( FDR = 0 . 05 ) and these were excluded from this analysis of TTTP characteristics .", "We classified genes as showing post-turn plateaus if there were not detected as differentially expressed after the TTTP with FDR = 0 . 05 .", "Those that were detected as showing significant differential expression after the TTTP , in the opposite direction to before the TTTP , are denoted as showing post-turn reversals .", "To generate Figure 2f we calculated the deciles of the ages at which TTTPs occur , and used these to split the data into ten groups with as closely matching sizes as possible .", "The Age-linked Gene Turning scores ( ALiGeTs ) were designed to represent two features: ( 1 ) proximity of the TTTP close to the target year , and ( 2 ) the changes in their expression level from the start of the dataset ( at 14th gestational week ) through to the age at which the TTTP occurred ( this change is denoted as ΔE ) .", "A human genes ALiGeT scores for a given age ( Sg , T ) can be calculated as:Sg , T=1 . 5−|Ag−T|∗|ΔE| Wherein g is the index of the gene of interest T is the year of age being queried ΔE is the change in expression level prior to the TTTP Ag is the age of the first TTTP in gene g For mice the equation is modified slightly to account for the difference in lifespan of the two species .", "To allow the ALiGeT window to have an approximately equal width across a given proportion of the mouse’s , we scaled the term | Ag−T | by 650/78 where 650 days represents old age for a mouse and 78 represents old age for a human .", "A mouse genes ALiGeT score for a given age can thus be calculated as:Sg , T=1 . 5−|Ag−T| ( 65078 ) ∗|ΔE| To penalize genes which are further from the peak of TTTPs , the distance between the age of turning point ( Ag ) and the target age ( T ) is used as the exponent in an exponential expression with base 1 . 5 .", "This function gives greatest emphasis to genes with D = 0 , with a rapid fall over neighbouring years such that a human gene with D = 6 years have scores 9% the size of those with equivalent pre-turn changes in expression level at D = 0 . The scoring function is graphically depicted in Figure 1—figure supplement", "1 . We show in Figure 5—figure supplement 5 that the results are stable to variation in the exponent base .", "The term PeGeT score is used to refer to the ALiGeT score for the year in which that species has the most TTTPs .", "To score genes using the DeGeT method ( represented in Figure 1b and used to generate Figure 4d and Figure 5c ) a similar scoring system was used .", "First ages were divided into ten groups based on the number of genes which turn/inflect within that age period , such that each age set has approximately equal size number of turning genes .", "Because there are many more TTTPs during early adulthood than in infancy/old age , the age windows span many years at start/end of the lifespan and as few as one year in during the twenties .", "For age set S , which covers ages { x , x+1 , … , y−1 , y } , the score was equal to the pre-turn expression change if the gene turns within the age window bounded by x and y .", "If the gene does not turn within within the age window bounded by x and y then the score for that gene is zero .", "Cell type enrichment analysis was performed on three datasets: Somel ( Somel et al . , 2009 ) , Braincloud ( Colantuoni et al . , 2011 ) and the mouse hippocampus .", "The EWCE ( 'Expression Weighted Celltype Enrichment' ) package from Bioconductor ( RRID:SCR_006442 ) was used to perform the enrichment with 100 , 000 bootstrap replicates used for each test .", "Age groups were assigned based on the frequency of TTTPs across the lifespan ( 0—10th , 11th—20th , … , 90th—100th percentile of all TTTPs ) .", "The genes which turn within each age window were sorted based on the size of pre-turn expression change .", "For each age window , the 10% of genes which turn within that window with largest positive ( upward ) and negative ( downward ) expression changes were assigned into two groups: we refer to these lists as the 'target' lists .", "A previously published single cell transcriptome ( SCT ) dataset containing cells from cortex and hippocampus was then loaded ( Zeisel et al . , 2015 ) .", "Any genes in the target lists which were not found to be expressed in the SCT dataset were dropped .", "For the analysis with Braincloud and Somel , the background gene set was taken as all genes with mouse orthologs that are also found in the SCT dataset for which a spline was fitted .", "For the mouse hippocampus analysis , the background set contained all genes for which a spline was fitted and which was also detected in the SCT dataset .", "For the directional analyses , the background set thus contains genes which change in both directions .", "Bonferroni correction was used to adjust for multiple testing .", "For both directions , for each cell type within each age group ( indexed by r , c and a∈A respectively ) , we calculated the mean ( μr , c , a ) and standard deviation ( μr , c , a ) of the bootstrap distribution , and used this to determine the distance ( in terms of standard deviations ) that the target list falls from the expected mean—we refer to this value as dr , c , a .", "Values were calculated separately for each dataset .", "The plots shown in Figure 3 show normalised values derived from dr , c , a by dividing by the maximal absolute value over all age windows .", "As a result the maximal absolute enrichment is either 1 or −1 .", "The values plotted on the x-axis each represent one of the age windows defined by the quantiles ( specifically , the values shown are the mean of the upper and lower bounds of the window ) .", "The age windows for Somel and Braincloud are not fully overlapped but were annotated with the central points of the Braincloud age windows to enable both datasets to be plotted against each other .", "To determine whether gene sets show larger ALiGeT scores than expected , a boot strapping test was performed .", "Genes in the target list , which were not detected as expressed on the arrays , were dropped from the analysis .", "Where multiple probes target the same gene , duplicated probes with smaller PeGeT inflection scores were dropped .", "For each target list being tested , ten thousand random gene lists of the same length were generated .", "The mean ALiGeT/PeGeT score for the target list and the random gene lists was calculated , and the proportion of random lists with smaller mean scores than the target list is taken as the probability .", "Where multiple lists were tested for significance against PeGeT scores , these were corrected using the Benjamini-Hochberg method ( FDR = 0 . 05 ) .", "The same method was used for the DeGeT set based analysis method as was used for the ALiGeT analysis , with bootstrapping being done with 10000 samples and correction for multiple testing over age sets done with the Bonferroni method .", "Figure 4a , b , Figure 5d–g , and Figure 6—figure supplement 3 represent the results of the PeGeT bootstrapping analysis in graph form .", "We sought to represent the extent to which enrichment results from high scores amongst either a small number of genes , or from a broad increase in scores throughout the list .", "As in the bootstrapping analysis we compare the score distribution in the target ( disease/synapse ) list to the scores in random lists of the same length .", "To keep the plot tidy we only represent data from 100 , rather than 10000 random lists .", "PeGeT scores for the target and random lists were sorted by numerical size .", "For each of the n genes in the target list , 100 dots were positioned with the y-axis determined by ith largest score in the target list , and the x-axis given by the ith largest score in each of the 100 random lists .", "To interpret the graph , note that if the majority of the random list scores fall above the red line , then the random lists have lower scores than the target list .", "It should be noted that the scales of the graph does bias the view towards the genes with largest scores .", "To perform a bootstrapping analysis which controls for gene size and GC content , we obtained those values from biomart .", "Where multiple transcript lengths were associated with a single HGNC gene we took the mean value .", "The deciles of gene size and GC content were calculated over the set of expressed genes .", "The two sets of decile values were used to define a grid , and each gene assigned to a position within the grid based on it’s transcript lengths and GC content .", "To run a bootstrap analysis on a particular target list , 10000 random lists were constructed with equal length to the target list .", "Gene i in each random list was selected from the same grid square as gene i in the target list .", "The disease gene lists are provided in Supplementary file 1f .", "The combined schizophrenia gene list ( results shown in Figure 5a–d , Figure 6a , b , Figure 5—figure supplement 1–5 , 7 and Figure 6—figure supplement 1 ) used genetic associations from three types of studies , which were also analysed individually: the integrative dataset contains 42 genes assembled using a translational convergent functional genomics approach ( Ayalew et al . , 2012 ) ; the de novo gene set comprised 609 genes pooled from three studies which used parent-child trios to detect de novo mutations ( Fromer et al . , 2014; Xu et al . , 2012; Girard et al . , 2011 ) ; the GWAS results are from the 2014 release of the Schizophrenia Working Group of the Psychiatric Genomics Consortium ( Abbas et al . , 2009 ) .", "Many SNPs from the GWAS study results were associated with multiple genes ( 349 genes were associated with 108 loci , with numerous loci associated with over twenty genes ) , and these were dropped leaving 62 genes to be used in our analyses ( not that this does not apply to the analyses which directly utilised the GWAS summary statistics files ) .", "The GWAS result remained significant if the alternative approach was taken and all genes associated with SNPs were used .", "The additional schizophrenia de novo gene set ( the ‘Gulsuner’ set ) came from exome sequencing of ‘quads and trios’ ( patients , their parents and unaffected siblings ) associated with 105 individuals with the disorder ( Gulsuner et al . , 2013 ) .", "Two autism lists were used , both based on finding de novo mutations through exome sequencing: the first contained 172 mutations ( Sanders et al . , 2012 ) , the second 358 ( Iossifov et al . , 2012 ) .", "The 78 intellectual disability genes were discovered through de novo sequencing of family groups ( de Ligt et al . , 2012 ) .", "The Multiple Sclerosis , Amyotrophic lateral sclerosis ( Lill et al . , 2011 ) , Parkinsons ( 23andMe Genetic Epidemiology of Parkinson's Disease Consortium et al . , 2012 ) and Alzheimer's ( Bertram et al . , 2007 ) lists come from the top results of the following websites: msgene . org ( 69 genes ) ; alsgene . org ( 17 genes ) ; pdgene . org ( 23 genes ) ; alzgene . org ( 10 genes ) .", "Gene list enrichments were evaluated across the lifespan by generating Sg , T for each gene , for each value of T between 1 through 78 ( Sg , T defined above ) .", "To test for disease enrichments the boot strapping method described above was applied .", "Enrichment of the gene sets were calculated at each age .", "The Bonferroni method was used to correct for the testing of the hypothesis at each of the 78 years of age .", "When running the ALiGeT for the eight diseases Bonferroni correction was applied across all the diseases as well as over each year of age .", "To obtain the sex specific datasets , the enrichments were calculated separately on the male subset of the data , and then again on the female subset .", "To test for sex differences in TTTP ages specific to disease gene sets , the age of TTTPs associated with those genes in the male and female data subsets were compared using the Wilcoxon signed-rank test .", "To control for transcript length , the maximum transcript length was determined for each gene using Biomart .", "As the distribution of transcript lengths is sharply peaked , ten quantiles of transcript length were used to group the genes for display of TTTP score distributions .", "To control for neuron specificity , the data on single cell transcriptomes from the Linnarsson/Hjerling-Leffler labs ( Zeisel et al . , 2015 ) was utilized ( data available at linnarssonlab . org/cortex/ ) .", "The cell-type specificity matrix was used to produce a metric for neuron vs glia enrichment .", "This was done by first calculating neuronal expression as the sum of expression in all cells they label as ‘pyramidal’ or ‘interneurons’ , whilst glial expression was the sum of expression in ‘astrocytes’ , ‘endothelial’ , ‘microglia’ or ‘oligodendrocytes’ .", "The ratio of these values was taken , and classic markers checked to ensure they were as expected ( Dlg4 = 15; Camk2a = 6; Map2 = 13; Gfap = 0 . 2; Mbp = 0 . 1; Aif1 = 0 . 2 ) .", "The 5000 genes most enriched for neurons were then used to perform an ALiGeT analysis for the combined Schizophrenia list .", "To confirm that the threshold used for how neuron-specific the genes are does not influence this result , we also show below a figure with PeGeT results using different thresholds between 1000 and 8000 ( Figure 6—figure supplement 1 ) .", "To perform the analyses restricted to up/down-regulating genes depicted in Figure 6c , d all probes which show a higher/lower expression level at the beginning of the spline , relative to the splines value at the TTTPs are dropped .", "Removal of duplicate probes targeting the same HGNC gene is performed after dropping the probes .", "Probes which do not have TTTPs are also dropped from from both analyses .", "For the comparisons of mouse and human genes with large PeGeT scores , orthologs were determined using the Biomart package , through querying which HGNC Genes are orthologs for a given MGI ID .", "To find GO terms enriched in this set of genes , the MGI symbols were analysed using DAVID ( Huang et al . , 2009 ) against a background of mouse genes , and the GO term for ‘synaptic transmission’ was found to be the most enriched ( the Benjamini-Hochberg corrected p-value is stated in the text ) .", "For the analysis of phenotypes from the Mammalian Phenotype Ontology ( Smith and Eppig , 2009 ) ( MPO ) , a full database of phenotypes was downloaded from ftp . informatics . jax . org .", "Any genes which were not detected as present by the microarrays were dropped from the MPO database , and a hypergeometric test for significance of enrichment performed on the remainder .", "Enrichment for ‘behavioural’ and ‘nervous system’ phenotypes was specifically tested for , and hence probabilities presented are not adjusted for multiple hypothesis testing .", "Gene association statistics were calculated from Genome Wide Association Study ( GWAS ) Summary Statistics using v1 . 05 of MAGMA ( Multi-marker Analysis of GenoMic Annotation ) ( de Leeuw et al . , 2015 ) .", "MAGMA enables disease/phenotypes association scores to be calculated for each gene , while accounting for linkage disequilibrium and the contributions from multiple SNPs .", "MAGMA takes GWAS summary statistics as input: these do not contain data on the individuals from the study and simply list the association p-values and z-scores/odds ratios for each SNP included in the study .", "The 1000 Genomes European panel was provided to MAGMA as reference data for calculating Linkage Disequilibrium .", "The schizophrenia summary statistics are associated with the GWAS analysis performed by the Psychiatric Genomics Consortium ( PGC ) which found 108 genome wide significant loci ( Schizophrenia Working Group of the Psychiatric Genomics Consortium , 2014 ) ; the file was downloaded from the PGC website ( https://www . med . unc . edu/pgc/results-and-downloads ) .", "DEGET enrichment was calculated by first grouping the genes into deciles based on the age of the turning point .", "DEGET groups for the MAGMA method are different from those calculated for gene list approaches for several reasons: firstly , sorting is performed using entrez gene IDs rather than HGNC gene symbols , secondly , all genes within the extended MHC region are removed from these analysis ( as their MAGMA gene associations cannot be properly assigned due to Linkage Disequilibrium in this region ) .", "DEGET scores were assigned to genes as they were done for the gene set based analysis .", "To determine whether a given GWAS trait is enriched at a particular age , the z-score calculated by MAGMA for each gene was multiplied by the DEGET score .", "Z-scores were then shuffled 20 , 000 times , multiplying at each iteration with the DEGET scores , and compared to the unshuffled value .", "The p-value is based on the frequency with which the sum of unshuffled value is greater than the shuffled values .", "The same approach ( multiplying ALIS scores with MAGMA z-scores , following by perturbations of z-scores ) is used to calculate ALIS enrichment probabilities .", "All of the human ( including fetal ) and all mouse samples were included in the age prediction analysis .", "Age predictions were performed with radial basis function Support Vector Machines through the e1071 package that provides an interface to libsvm in R ( Chang and Lin , 2011 ) .", "Two rounds of 10-fold partitioning were used to form training , validation and test sets .", "An initial round of random partitions separated test data from training/validation data .", "The combined set of training/validation data was then passed to the tune . svm ( ) function from the e1071 package which then uses 10-fold cross-validation to perform a parameter search .", "A first shallow grid search was performed for γ∈{−15 , −13 , … , 1 3 ) and cost∈{−5 , −3 , … , 13 , 15 ) , and the optimal pair of values selected as ( γ1 , cost1 ) .", "A second finer grid search was then performed over γ∈{γ1−1 . 5 , γ1−1 . 375 , … , γ1+1 .", "375 , γ1+1 . 5 ) and cost∈{cost1−1 . 5 , cost1−1 . 375 , … , cost1+1 .", "375 , cost1+1 . 5 ) .", "Probes were included in the model by first determining which probes are associated with age , as determined using a linear model .", "The linear modeling of age-associated probes was repeated for each of the partitioned sets of training/validation data; as such , data in the test set did not influence the selection of probes through the linear model .", "The probes were then ranked based on the level of association , and the top Ncutoff probes were used for training and testing .", "The reported accuracies were obtained using values of Ncutoff 40 for humans and 100 for mice .", "To determine the appropriate level of Ncutoff , a range of values were tested between 10 and 400 , and the optimal number manually selected on the basis of having optimal SSE without using an unnecessarily large number of probes .", "Crude PSD preparations were made from dissected mouse forebrain tissue from C57BL6/5J mice ages 1 month to 5 months .", "In brief , each forebrain was homogenized by performing 12 strokes with a Dounce homogenizer containing 5 mL of ice cold homogenization buffer ( 320 mM sucrose , 1 mM HEPES , pH = 7 . 4 ) containing 1X Complete EDTA-free protease inhibitor ( Roche ) and 1X Phosphatase inhibitor cocktail set II ( Calbiochem ) .", "Insoluble material was pelleted by centrifugation at 1000 x g for 10 min at 4˚C .", "The supernatant ( S1 ) was removed and the pellet was re-suspended in 2 mL of homogenization buffer and an additional six strokes were performed .", "Following a second centrifugation at 1000 x g for 10 min at 4˚C , the supernatant ( S2 ) was removed and pooled with S1 .", "The combined supernatants were then centrifuged at 18 , 500 x g for 15 min at 4˚C .", "The pellet was re-suspended in 5 mL of extraction buffer ( 50 mM NaCl , 1% DOC , 25 mM Tris-HCl , pH 8 . 0 ) containing 1X Complete EDTA-free protease inhibitor ( Roche ) and 1X Phosphatase inhibitor cocktail set II ( Calbiochem ) and incubated on ice for 1 hr .", "The resulting crude PSD extracts were centrifuged at 10 , 000 x g for 20 min at 4 ˚C and the resulting supernatant filtered through a 0 . 2 µm syringe filter ( Millipore ) .", "Protein concentration of PSD preparations was determined using 1X Quickstart Bradford assay ( BioRad ) .", "Thirty micrograms of PSD protein was prepared to contain 1X Novex NuPAGE LDS sample loading buffer ( Invitrogen ) with 100 mM DTT ( BioRad ) , boiled for 10 min at 100˚C and loaded into 1 well of a 10-well Novex NuPAGE 3–12% Bis-Tris gradient gel ( Invitrogen ) .", "Electrophoresis was performed under reducing conditions using the Novex NuPAGE SDS-PAGE system ( Invitrogen ) for 5 min .", "Gels were then stained with SimplyBlue SafeStain ( Invitrogen ) following the manufacturer’s instructions .", "Gel bands were excised and subjected to tryptic digestion using standard methods .", "Five microgram of tryptic digest was analysed by LC-MS/MS using a UPLC Dionex QExactive ( Thermo-Fisher , Waltham , Massachussets , USA ) .", "Protein identification was performed with MASCOT ( Matrix Sciences ) using Uniprot Mouse database ( downloaded on 2014 March 24th ) .", "Label-free quantitation analysis was then performed on all timepoints examined in the study using Progenesis ( Nonlinear Dynamics ) .", "The normalized Mass Spectroscopy dataset was read into R . Swissprot/Trembl ID’s for each detected protein was matched to a gene symbol .", "Orthologs were determined using the Biomart package , through querying which HGNC Genes are orthologs for a given MGI ID .", "The data was log2 transformed .", "A linear model was fitted to estimate protein expression based on age and sex using the bioconductor ‘lumi’ package .", "Significance of differential expression was corrected using the Benjamini-Hochberg method .", "Build #85 of the Human Phenotype Ontology was downloaded from http://compbio . charite . de/hudson/job/hpo . annotations . monthly/lastStableBuild .", "All diseases associated with the following HPO terms were extracted from the ontology using Phenexplorer ( compbio . charite . de/phenexplorer ) : ‘Neoplasm’ , ‘Abnormality of the Nervous System’ , ‘Abnormal peripheral nervous system morphology’ and ‘Abnormality of the autonomic nervous system’ .", "Using these lists all diseases associated with neoplasms , and peripheral/autonomic disorders , and which are not associated with nervous system disorders , were dropped from the HPO phenotype database .", "From the remaining dataset , congenital onset disorders were taken as those annotated with the following terms: ‘Congenital onset’ , ‘Infantile onset’ or ‘Neonatal onset’ .", "Adult onset disorders were annotated with either ‘Adult onset’ , ‘Young adult onset’ , ‘Schizophrenia’ or ‘Bipolar affective disorder’ .", "Schizophrenia enrichment was tested using the combined list described earlier .", "Enrichment analyses were performed using hypergeometric tests .", "Functional analysis was done by manually assigning a single functional category to each of the proteins detected in the dataset .", "Functional annotations were based on those from a previous paper on the synaptic proteome ( Emes et al . , 2008 ) and expanded based on Panther protein class .", "R code was used for all TTTP and age prediction analyses described herein ( see Bioconductor , ‘TurningPoints’ ) ." ] ]
[ "The genetic mechanisms regulating the brain and behaviour across the lifespan are poorly understood .", "We found that lifespan transcriptome trajectories describe a calendar of gene regulatory events in the brain of humans and mice .", "Transcriptome trajectories defined a sequence of gene expression changes in neuronal , glial and endothelial cell-types , which enabled prediction of age from tissue samples .", "A major lifespan landmark was the peak change in trajectories occurring in humans at 26 years and in mice at 5 months of age .", "This species-conserved peak was delayed in females and marked a reorganization of expression of synaptic and schizophrenia-susceptibility genes .", "The lifespan calendar predicted the characteristic age of onset in young adults and sex differences in schizophrenia .", "We propose a genomic program generates a lifespan calendar of gene regulation that times age-dependent molecular organization of the brain and mutations that interrupt the program in young adults cause schizophrenia ." ]
[ "In our lifetime , we go through many changes – physically and also intellectually .", "At certain ages , we are particularly vulnerable to develop psychiatric disorders , and the majority of mental conditions start to manifest in teenagers and young adults .", "The symptoms for schizophrenia , for example , a mental health disorder in which patients often experience hallucinations , delusion or changes in behavior , typically start in the mid-twenties .", "Schizophrenia tends to run in families and it is likely that different combinations of faulty genes that affect the connections between nerve cells increase the chance of having the disease .", "Until now , scientists have assumed that certain situations and environmental factors trigger the condition , but it was unknown if genes could influence the age at which the disease will begin .", "To explore whether genes in the brain change at certain time points , Skene et al . examined how the genes are turned on and off across the lifespan of healthy mice and humans .", "The results showed that in both mice and humans , a ‘genetic lifespan calendar’ controlled every cell type in the brain and directed the way they worked at different ages .", "The timing was so precise that it was possible tell the age of a mouse or a person simply by looking at the way the genes were expressed in a tissue sample .", "Skene et al . then studied how the genetic lifespan calendar controlled the genes damaged in schizophrenia , and found that the calendar caused a major reorganization of the genes at the time when the symptoms started .", "This suggests that the genetic lifespan calendar is a crucial factor that can determine at what age the disease will start .", "The next step will be to study how the genetic lifespan calendar programs changes throughout the brain and to explore if it could be manipulated to change how the brain ages .", "This could help to develop new types of treatments for schizophrenia and other conditions of the brain ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "immunology and inflammation" ]
Antigen presentation kinetics control T cell/dendritic cell interactions and follicular helper T cell generation in vivo
elife-06994-v2
[ [ "Following stimulation via the TcR , selective differentiation of T helper cell subsets is dependent on transcription factors , with Bcl-6 being responsible for T follicular helper ( Tfh ) cell differentiation ( Johnston et al . , 2009; Choi et al . , 2011 ) and antagonism of other T cell phenotypes ( Th1 , Th2 , Th17 ) .", "Gene knockout studies in mice have revealed the requirement for CD28 mediated-costimulation in the upstream control of Tfh differentiation ( Linterman et al . , 2009 ) , with subsequent signalling through inducible T cell costimulator ( ICOS ) involved in consolidation of differentiation ( Crotty , 2011; Xu et al . , 2013 ) .", "The mechanisms controlling the induction of this differentiation program are less clear , a role for the cytokines IL-6 and IL-21 and subsequent signalling via STAT3 have been implicated ( Nurieva et al . , 2008 ) , while other studies indicate that the strength and duration of the T cell receptor ( TCR ) signal itself is the major factor determining effector cell differentiation to Tfh ( Fazilleau et al . , 2009; Tubo et al . , 2013 ) .", "Using well defined and controlled conditions that preferentially induce Tfh responses in vivo , we have therefore adopted a reductionist approach to determine which of these factors are important in the differentiation of this subset in vivo .", "A variety of particle formulations , including alum , emulsions and liposomes have received sustained interest as vaccine adjuvants for over 80 years ( Brewer , 2006; Xiang et al . , 2006; De Temmerman et al . , 2011 ) .", "Their ability to affect the availability of antigen and antigen presentation has frequently been linked to their impact on the development of T cell responses ( Constant and Bottomly , 1997 ) , but causal evidence has been absent .", "More recently , studies have suggested that the use of nanoparticles as adjuvants can selectively induce Tfh responses ( Moon et al . , 2012 ) .", "Clearly selective differentiation of Tfh effector cells has implications in vaccine design and pathogen control as for the majority of vaccines the principal effector mechanism induced is production of high affinity , class switched antibodies by B lymphocytes .", "Here we employ defined antigen particles to selectively induce Tfh cells and antibody responses and show that increased duration of antigen presentation and subsequent T cell/dendritic cell ( DC ) interactions are associated with Tfh induction .", "The functional significance of these late stage ( 72 hr ) interactions in T cell differentiation was demonstrated by blockade of T/DC interactions in vivo .", "These studies demonstrate that by manipulating the form of antigen delivered late stage antigen presentation by DC and their interaction with T cells can be controlled to drive Tfh differentiation in vivo ." ], [ "In the present study , we employed polystyrene nanoparticles due to their inert nature , defined size , narrow polydispersity , and surface functionalisation allowing covalent linkage of antigen to their surface .", "Polystyrene nanoparticles do not induce ERK activity or subsequent activation of conventional inflammatory pathways in DC ( Karlson Tde et al . , 2013 ) .", "Our study focused on polystyrene nanoparticle sizes able to drain freely to the LN ( Manolova et al . , 2008 ) .", "Carbodiimide-mediated coupling of antigens was highly controlled , such that only particle size and physically dependent parameters ( surface area , antigen molecules per particle ) varied between immunisation conditions; all other conditions , antigen dose , mass of particles , and antigen density were consistent between 40 nm and 200 nm particle immunisations ( Table 1 ) .", "Consequently , immunisations with equivalent quantities of protein were not associated with any confounding difference in total antigenic mass and a single variable , particle size could be tested for its impact on antigen specific immune responses .", "Initial experiments investigating the effect of particle size on antibody responses revealed that immunisation of mice with Ag covalently linked to 200 nm particles induced significantly higher amounts of specific IgG1 and IgG2c than smaller particles or soluble Ag ( Figure 1A , B ) .", "1000 nm particles failed to induce an antibody response , potentially due to a failure in uptake by APCs and/or ability to drain to the LN .", "Subsequent studies thus focussed on the significance and processes by which the 200 nm particles were able to support this antibody response , in comparison to smaller particles ( 40 nm ) and soluble antigen that did not . 10 . 7554/eLife . 06994 . 003Table 1 . Comparison of particle conjugatesDOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 003Particle size40 nm200 nmAntigen dose/mouse100 μg100 μgAntigen molecules/mouse1 . 3 × 10151 . 3 × 1015Latex dose/mouse100 μg130 μgNumber of particles/mouse1 . 4 × 10122 . 9 × 1010Antigen molecules/particle100020 , 000Surface area/particle5024 nm2125 , 600 nm2Antigen density1 per 5 nm21 per 6 nm2Data shows an example of a typical carbodiimide-mediated conjugation of ovalbumin to either 40 or 200 nm .", "Various parameters relating to a typical s . c . immunisation with 100 μg of protein are shown . 10 . 7554/eLife . 06994 . 004Figure 1 . Immunisation with antigen loaded 200 nm particles induces elevated IgG and can provide protective immunity to influenza virus infection . Mice were immunised s . c . in the scruff with 100 μg of ovalbumin ( OVA ) alone or conjugated to 20 nm , 40 nm , 100 nm , 200 nm or 1000 nm particles .", "14 days later , serum was tested by ELISA for anti-OVA-IgG2c ( A ) and anti-OVA-IgG1 ( B ) .", "Data show the mean ( − ) absorbance ( 405 nm ) at a dilution of 1 in 200 with the five experimental animals per group ( ′ ) ***p < 0 . 001 comparing to the PBS group .", "To assess functional responses , C57BL/6 mice were immunised s . c . with 50 μg of hemagglutinin ( HA ) alone , conjugated to 40 or 200 nm particles , or emulsified with complete Freund's adjuvant ( CFA ) .", "Weight changes were then monitored following intranasal infection with 300 PFU of influenza A virus ( WSN ) ( C ) .", "Data shows the mean of five experimental animals per group ± standard deviation ***p < 0 . 001 comparing 200 nm group to 40 nm group . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 004 The functional importance of the observed antigen specific antibody response was evaluated in a murine influenza virus infection model where antibodies to the hemagglutinin ( HA ) component of influenza virus are important to protective immunity ( Murphy et al . , 1982; Gerhard , 2001 ) , blocking viral attachment and fusion of the viral and host cell membrane ( Knossow and Skehel , 2006 ) .", "Three weeks after immunisation with HA linked to 200 or 40 nm particles , emulsified in complete Freund's adjuvant ( CFA ) or as soluble protein , mice were challenged intranasally with influenza A virus ( H1N1; A/WSN/1933 [WSN] ) .", "Protection from infection was determined by weight loss ( Figure 1C ) .", "While mice immunised with 200 nm HA-particles were protected from virus induced pathology , significant weight loss was observed in groups immunised with 40 nm HA-particles , similar weight loss to that observed in unimmunised or soluble HA treated mice .", "To delineate the mechanisms that allow particle size to control antibody responses , we employed an established adoptive transfer model ( Garside et al . , 1998 ) .", "Antigen specific CD4+ T cells and B cells were co-transferred into congenic recipients then immunised with 130 μg of ovalbumin ( OVA ) -hen egg lysozyme ( HEL ) conjugate alone , in CFA or bound to 40 nm or 200 nm particles .", "Analysis of serum samples collected 10 days post immunisation revealed OVA-HEL conjugated to 200 nm particles induced significant levels of serum anti-HEL-IgMa , whereas antigen conjugated to 40 nm particles did not ( Figure 2A ) .", "Similarly , antigen conjugated to 200 nm particles induced greater expansion of antigen-specific IgMa+ MD4 B cells than challenge with 40 nm particles 7 days post immunisation ( Figure 2B ) .", "Enhanced expression of germinal centre ( GC ) markers ( GL7 and FAS ) following immunisation with 200 nm OVA-HEL particles compared with 40 nm OVA-HEL particles was also evident ( Figure 2C , D ) .", "Conventional GC responses are dependent on activated , antigen-specific CD4+ T cells ( Owens and Zeine , 1989; Nutt and Tarlinton , 2011 ) , suggesting that different particles could induce changes in the magnitude or phenotype of the resulting T cell response .", "However , immunisation with OVA-HEL conjugated to either 40 nm or 200 nm particles could equally increase the proportion and number of antigen specific OT-II T cells in vivo ( Figure 3A , B ) , consistent with a previous report ( Gengoux and Leclerc , 1995 ) .", "This implied that particle size induced a qualitative rather than quantitative difference in T cell response , favouring generation of the Tfh subset of T cells that have been defined as supporting B cell responses and GC formation ( Ma et al . , 2012 ) .", "Analysis of Tfh markers CXCR5 , PD-1 , ICOS and SLAM by adoptively transferred OT-II T cells demonstrated the percentage and number of antigen specific cells differentiating into Tfh cells was very low at day 5 post immunisation with soluble antigen or antigen conjugated to 40 nm particles ( Figure 3C–E ) .", "However , a significant proportion of OT-II T cells were CXCR5+PD-1+ following challenge with OVA-HEL conjugated to 200 nm particles ( Figure 3C–E ) .", "Changing a single physical characteristic of the immunising antigen selectively enhanced Tfh differentiation without impacting on the magnitude of T cell response , providing an excellent platform to dissect how external stimuli can contribute to Tfh development in vivo . 10 . 7554/eLife . 06994 . 005Figure 2 . Immunisation with antigen loaded 200 nm particles enhances B cell expansion and germinal centre ( GC ) B cell responses . To dissect the cellular responses in detail , MD4 BcR transgenic ( Tg ) B cells and OT-II TcR Tg T cells and were adoptively transferred into C57BL/6 recipients .", "Mice were then challenged with OVA-hen egg lysozyme ( HEL ) conjugated to 40 nm or 200 nm particles .", "Challenges with PBS , OVA-HEL alone or in CFA were included as negative and positive controls .", "Day 10 serum samples were assayed by ELISA to determine anti-HEL-IgMa levels ( A ) .", "Expansion of B220+IgMa+ MD4 B cells in popliteal LNs was assessed by flow cytometry 7 days post challenge ( B ) .", "Development of GC B cell responses was determined by examining GL7 and FAS expressing by MD4 B220+ cells ( C ) .", "Representative staining of GC markers GL7 and FAS are also shown ( D ) .", "Panels show mean value and five individual animals in a representative experiment of 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 00510 . 7554/eLife . 06994 . 006Figure 3 . 200 nm particles induce T follicular helper ( Tfh ) differentiation . MD4 BcR Tg B cells and OT-II TcR Tg T cells and were adoptively transferred into C57BL/6 recipients .", "Mice were then challenged with OVA-HEL conjugated to 40 nm or 200 nm particles .", "Challenges with PBS , OVA-HEL alone or in CFA were included as negative and positive controls .", "CD4+DsRed+ OT-II cell expansion in LNs 5 days post challenge ( A , B ) .", "These were defined as Tfh cells when being PD-1+ and CXCR5+ ( C , D ) .", "Cells were also confirmed as being both ICOS+ and SLAM+ ( E ) .", "Panels show mean value and five individual animals in a representative experiment of 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 006 Adjuvants are thought to influence the availability of antigen in vivo and the kinetics of antigen presentation influence consequent CD4+ T cell proliferative , effector and memory responses ( Obst et al . , 2005 , 2007; Celli et al . , 2007 ) .", "By covalently linking a traceable antigen , EαGFP ( Pape et al . , 2007 ) , to our particles we were able investigate the impact of particle size on peptide/MHCII presentation in vivo .", "Immunisation with 40 nm particles resulted in rapid presentation by both DCs and B cells within 6 hr of subcutaneous injection , consistent with published reports ( Itano et al . , 2003; Pape et al . , 2007 ) .", "Immunisation with antigen conjugated to 40 nm particles resulted in similar rapid presentation by DCs ( Figure 4A , B ) and B cells ( Figure 5A–C ) as previously observed with soluble antigen ( Itano et al . , 2003; Pape et al . , 2007 ) .", "An equivalent proportion of DCs also presented Eα/MHCII following immunisation with 200 nm particles .", "Percentages and numbers of Eα/MHCII positive DCs equivalent to soluble and 40 nm particle antigen formulations were detected at 24 and 48 hr .", "However , in contrast to soluble and 40 nm particle formulations , 200 nm particle challenge resulted in presentation of antigen beyond 48 hr , with 21 ± 3% of conventional DCs staining positive for Eα/MHCII at 72 hr post immunisation ( Figure 4C ) .", "Presentation of antigen conjugated to 200 nm particles by the B cell compartment was slower ( Figure 5A–C ) but did reach levels equivalent to soluble and 40 nm particle-bound antigen .", "The sustained presentation observed by DCs was not evident in the B cell compartment ( Figure 5D ) , with no significant differences in B cell presentation being detected beyond 24 hr ( Figure 5 ) .", "Thus , larger particles did not alter the magnitude of presentation but did prolong the duration of peptide/MHCII presentation by DCs . 10 . 7554/eLife . 06994 . 007Figure 4 . 200 nm particulate antigen challenge promotes sustained presentation of peptide/MHCII by dendritic cells ( DCs ) .", "C57BL/6 mice were immunized s . c . in the footpad with 100 μg of the model antigen EαGFP alone or covalently conjugated to 40 nm or 200 nm particles .", "Challenge with PBS was included as a negative control .", "The draining popliteal LNs were harvested after 1 , 6 , 24 , 48 and 72 hr post challenge .", "Flow cytometry was used to determine levels of presentation of the Eαpeptide/MHC class II complex using the monoclonal antibody YAe on CD11c+B220− conventional DCs .", "The percentage and number of conventional DCs cells staining YAe+ is shown in ( A ) and ( B ) respectively .", "Representative flow cytometry plots are shown for 72 hr post challenge ( C ) .", "Data shows the mean of three animals per time point ± standard deviation and are representative of ≥2 independent experiments .", "* 40 nm vs 200 nm p = 0 . 012 . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 00710 . 7554/eLife . 06994 . 008Figure 5 . Peptide/MHCII presentation by B cells . C57BL/6 mice were immunized s . c . in the footpad with 100 μg of the model antigen EαGFP alone or covalently conjugated to 40 nm or 200 nm particles .", "Challenge with PBS was included as a negative control .", "The draining popliteal LNs were harvested after 1 , 6 , 24 , 48 and 72 hr post challenge .", "Flow cytometry was used to determine levels of presentation of the Eαpeptide/MHC class II complex using the monoclonal antibody YAe on B220+CD11c− B cells .", "The percentage and number of conventional DCs cells staining YAe+ is shown in ( A ) and ( B ) respectively .", "Representative flow cytometry plots are shown for 6 and 72 hr post challenge ( C , D respectively ) .", "Data shows the mean of three animals per time point ± standard deviation and are representative of ≥2 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 008 The relevance of antigen presentation at 72 hr induced by antigen conjugated to 200 nm particles was questionable given evidence from intravital imaging studies .", "Such studies have demonstrated that T cell priming by DCs occurs in three successive stages ( Stoll et al . , 2002; Bousso and Robey , 2003; Mempel et al . , 2004; Miller et al . , 2004 ) ; stage 1 ( 0–8 hr ) characterised by rapid T cell motility and short T/DC interactions ( 2–3 min ) ; stage 2 ( 12–24 hr ) with long-term T/DC interactions associated with the induction of priming and; stage 3 ( 48–72 hr ) where T cells resume motility and short T/DC interactions .", "We therefore examined the impact of particle size on stage 3 T cell/DC interactions in vivo .", "CD11c-YFP recipients were adoptively transferred with OT-II DsRed T cells and challenged with OVA conjugated 40 nm or 200 nm particles 72 hr before lymph node imaging by MPLSM ( Video 1 and Figure 6A ) .", "Multiple short duration T cell-DC interactions ( 2 . 149 ± 0 . 139 min ) were observed with 40 nm OVA particles ( Figure 6B ) , comparable to that seen with naïve T cells and therefore consistent with an absence of cognate peptide/MHC and classic stage 3 motility ( Mempel et al . , 2004 ) .", "Notably , interactions longer than 10 min were seen following 200 nm particle challenge ( Figure 6B ) , implying that antigen driven cognate recognition was still occurring .", "This was further supported by the reduced T cell velocity observed in the 200 nm particle group ( Figure 6C ) and again in a lower T cell displacement rate ( Figure 6D ) .", "T cell migratory patterns within the LNs were not significantly different between challenges as evidenced by their equivalent meandering indices ( Figure 6E ) .", "Thus , the antigen presentation by DCs at 72 hr post challenge induced by antigen-conjugated 200 nm particles changed the dynamics of T cell/DC interactions , with stable , long-term interactions extending into the stage 3 time period , conventionally associated with transient interactions and rapid T cell motility ( Hugues et al . , 2004; Mempel et al . , 2004; Miller et al . , 2004; Zinselmeyer et al . , 2005 ) . 10 . 7554/eLife . 06994 . 009Video 1 . Imaging DC and T cell behaviour after challenge with 200 nm particulate antigen . DsRed OT-II T cells were adoptively transferred into CD11cYFP recipients and footpad challenged with 100 µg of OVA conjugated to 40 nm or 200 nm particles .", "Popliteal LNs were imaged at 72 hr . 2 hr prior to imaging , 200 nm challenged groups were given 500 μg mIgG2a or Y3P ( anti-mouse I-A ) .", "Data is representative of ≥3 individual animals and shows one of three separate areas imaged per lymph node .", "Scale bar represents 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 00910 . 7554/eLife . 06994 . 010Figure 6 . DC and T cell interactions persist after challenge with 200 nm particulate antigen . DsRed OT-II T cells were adoptively transferred into CD11cYFP recipients and footpad challenged with 100 µg of OVA alone , conjugated to 40 nm or 200 nm particles .", "Popliteal LNs were imaged at 72 hr .", "Representative images following challenge ( A ) correspond with Video 1 video file .", "The duration of OT-II T cell interaction with DCs was determined as intersection of DsRed and YFP signals ( B ) .", "The velocity ( C ) , displacement rate ( D ) and meandering index ( E ) for T cells were calculated .", "Data is representative of ≥3 individual animals and shows pooled tracks with mean values of three separate areas of a lymph node .", "Scale bars represent 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 010 Previous studies have demonstrated that differences in stage 2 interactions underlie the outcome of the developing immune response , with long-term ( >10 min ) and short-term ( <3 min ) interactions being associated with induction or priming and tolerance respectively ( Hugues et al . , 2004; Mempel et al . , 2004; Zinselmeyer et al . , 2005 ) .", "Significantly , the causal link between T cell behaviour and function was subsequently revealed through disrupting TcR/pMHCII interactions with a monoclonal antibody ( Y3P ) , which resulted in of loss long-term T/DC contacts , a return to rapid T cell motility , and a loss of T cell activation ( Celli et al . , 2007 ) .", "We hypothesised that the long-term T/DC interactions we observed at 72 hr post immunisation with 200 nm particles were responsible for selective Tfh differentiation .", "To test this hypothesis , we employed the approach described by Celli et al ( 2007 ) , using the Y3P monoclonal antibody against MHCII ( Celli et al . , 2007 ) administered at 72 hr post challenge .", "Disruption of TcR/pMHCII blocked the stable interactions between T cells and DCs ( Videos 1 , 2 , Figure 7A , B ) and increased T cell motility ( data not shown ) .", "Unlike blocking stage 2 T/DC interactions ( Celli et al . , 2007 ) , robust T cell proliferative responses were observed with Y3P treatment at 72 hr ( Figure 7C ) .", "However , blocking MHCII at this time point did significantly reduce the proportion of antigen specific cells co-expressing CXCR5 and PD-1 ( Figure 7D , E ) .", "Therefore , we conclude that while the sustained T/DC interactions at 72 hr induced by 200 nm particles do not increase the magnitude of the resulting immune response , these interactions specifically function to alter the quality of immune response by promoting Tfh differentiation . 10 . 7554/eLife . 06994 . 011Video 2 . Disruption of DC and T cell interactions using Y3P . As for Video 1 , DsRed OT-II T cells were adoptively transferred into CD11cYFP recipients and footpad challenged with 100 µg of OVA conjugated to 40 nm or 200 nm particles .", "Popliteal LNs were imaged at 72 hr . 2 hr prior to imaging , 200 nm challenged groups were given 500 µg mIgG2a or Y3P ( anti-mouse I-A ) .", "Videos were acquired using a 20×/1 . 0 NA water-immersion objective lens and 2× zoom .", "Areas of interaction are shown in grey .", "Scale bars represent 25 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 01110 . 7554/eLife . 06994 . 012Figure 7 . Blockade of stage 3 DC/T cell interactions following 200 nm particulate challenge prevents Tfh differentiation . Disruption of stage 3 DC/T cell interaction was demonstrated by imaging DsRed OT-II T cells adoptively transferred into CD11cYFP recipients .", "Mice were footpad challenged with 100 µg of OVA conjugated to 200 nm particles and popliteal LNs imaged at 72 hr . 500 µg isotype ( mIgG2a ) or Y3P ( anti-mouse I-A ) was given i . v . 2 hr prior to imaging .", "Representative video frames show areas of intersection in grey and highlighted by white arrows ( A , see Video 2 for video ) .", "Scale bars represent 25 μm .", "The duration of OT-II T cell interaction with DCs was determined as intersection of DsRed and YFP signals ( B ) .", "Next , CFSE OT-II TcR Tg T cells were adoptively transferred into C57BL/6 recipients .", "Mice were then challenged with PBS , OVA with CFA or OVA conjugated to 200 nm particles .", "Mice were treated with a single dose of Y3P or isotype antibody i . v . 72 hr post challenge with particles .", "Draining LNs were harvested on day 5 post challenge .", "( C ) Representative plots showing CFSE division of CD45 . 1+ OT-II TcR Tg T cells ( gated on CD4+ population ) .", "Tfh cells were defined as being PD-1+CXCR5+ ( D ) and confirmed as ICOS+ ( data not shown ) for plotting Tfh proportion as a percentage of the OT-II population ( E ) .", "Data is representative of two independent experiments with five animals per group . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 012 Analysis of antigen presentation kinetics following immunisation with 200 nm particles revealed that at the time of MHCII blocking ( 72 hr ) the major population of APCs were DCs .", "Our multiphoton studies further confirmed that T cells were continuing to interact with DCs at this time .", "However , interactions with antigen presenting B cells could not be ruled out as a contributing factor , especially given their critical role in affirming and maintaining the Tfh phenotype ( Johnston et al . , 2009; Crotty , 2011; Baumjohann et al . , 2013 ) .", "To establish whether the Tfh phenotype observed following immunisation with 200 nm particles was attributable purely to DC presentation at 72 hr we assessed Tfh differentiation in the absence of cognate pMHCII B cell presentation .", "Immunisation of MD4 mice with 200 nm OVA particles induced equivalent proportional increases in adoptively transferred OT-II T cells as seen in C57BL/6 mice ( Figure 8A ) .", "Interestingly , the Tfh phenotype induced following immunisation with 200 nm particles was not significantly altered in the absence of cognate T-B cell interactions ( Figure 8B ) .", "Inclusion of pMHCII blockade at 72 hr post immunisation in MD4 mice did not influence expansion of the OT-II population ( Figure 8A ) but did significantly reduce the proportion of cells co-expressing CXCR5 and PD-1 ( Figure 8B ) , reaffirming the importance of pMHCII recognition at this time .", "Taken together , the data implicates DCs rather than B cells as the APC population mediating this effect . 10 . 7554/eLife . 06994 . 013Figure 8 . Cognate interactions between B and T cells are not required for early Tfh differentiation . OT-II TcR Tg T cells and were adoptively transferred into C57BL/6 or intact MD4 BcR Tg recipients .", "Mice were then challenged with OVA conjugated to 200 nm particles and 500 µg isotype ( mIgG2a ) or Y3P ( anti-mouse I-A ) given i . v . 72 hr later .", "Challenges with PBS , OVA/CFA were included as negative and positive controls .", "Flow cytometry was used to assess CD4+CD45 . 1+ OT-II cell expansion in LNs 5 days post challenge ( A ) .", "Tfh were defined by PD-1 and CXCR5 co-expression ( B ) .", "Panels show mean value and five individual animals , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 06994 . 013" ], [ "We have provided , in unprecedented detail , the spatiotemporal sequence of events that underlie enhanced differentiation of Tfh cells , GC response and protective Ab production by antigen formulations in vivo .", "By combining highly defined antigen delivery systems , with trackable antigen , antigen-receptor transgenics ( Tgs ) and state of the art imaging techniques , we revealed that antigen size impacts on the duration of peptide/MHCII presentation and the maintenance beyond 48 hr of functional DC and T cell interactions in the draining LN .", "The functional relevance of longer DC-T cell interactions , associated with antigen conjugated to 200 nm particles , was dissected by specifically blocking later interactions , resulting in reduced Tfh induction , while the overall magnitude of the T cell response was unaffected .", "Thus , the temporal characteristics of T cell stimulation can determine their functional differentiation towards a Tfh phenotype , and this can be determined by the size of the particle upon which an antigen is delivered .", "Previous studies have investigated the impact of particle size on the immune response to antigen using a variety of formulations , for example lipid vesicles entrapping ( Brewer et al . , 2004; Moon et al . , 2012 ) antigens or antigens non-specifically adsorbed to the surface of inert particles ( Mottram et al . , 2007 ) .", "The inert nature , defined size and surface functionalisation of particles employed in the present study , allowed a single variable , size , to be tested for its impact on antigen immunogenicity .", "Initial studies simply altering particle size revealed 200 nm particles could induce antibody production following a single immunisation .", "The functional importance of this observation was startlingly clear , with 200 nm particles able to impart protective anti-HA humoral immunity to influenza infection .", "Starting with a functional outcome relevant to vaccine design , we sought to dissect the processes by which increasing particle size impacts on the humoral response .", "GC formation is central to development of high affinity antibody .", "GC structures support somatic hypermutation , selection of high affinity B cells and their differentiation into plasma and memory cells ( for a comprehensive review see Victora and Nussenzweig , 2012 ) .", "Immunisation with 200 nm particles enhanced this process , explaining our initial observation of increased antibody responses .", "Essential in this process is the cognate interaction between Ag-specific B and T cells .", "The nature of this interaction has been the focus of intense research in recent years , culminating in the identification of Tfh cells and the molecules ( surface and soluble ) involved in their differentiation and function ( Ma et al . , 2012 ) .", "While both sizes of particle could equally increase antigen specific T cell responses in vivo , we found that larger particles ( 200 nm ) induced greater Tfh differentiation than small ( 40 nm ) particles , consistent with their role in supporting GC responses .", "Even though the endogenous molecular cues governing the development of Tfh cells are multifactorial ( Crotty , 2011; Ma et al . , 2012 ) , understanding how external stimuli can influence T cell differentiation towards this phenotype is less well understood , yet has clear implications in vaccine design .", "In this case we have demonstrated that simply changing the size of the Ag can clearly impact on Tfh differentiation .", "A key event in the initiation of any CD4+ T cell response is the recognition of peptide/MHC class II complexes on DCs .", "The kinetics of presentation are known to influence the consequent CD4+ T cell proliferative , effector and memory responses induced ( Obst et al . , 2005 , 2007; Celli et al . , 2007 ) .", "By covalently linking a trackable antigen to the particles we were able to map temporally in vivo peptide/MHC class II presentation and its relation to particle size .", "Although 200 nm particle immunisation prolonged the duration of peptide/MHC class II presentation by DCs , significantly this did not impact upon the magnitude of the T cell response compared with the smaller 40 nm particle , consistent with a previous report ( Gengoux and Leclerc , 1995 ) .", "However , prolonged presentation was associated with the enhanced differentiation of Tfh cells .", "Antigen availability can dictate the magnitude of a Tfh response , with sustained presentation by B cells maintaining the Tfh phenotype ( Baumjohann et al . , 2013 ) .", "Notably , immunisation with 200 nm particles resulted in sustained antigen presentation by DCs with cognate interactions with B cells being dispensable for maintenance of a Tfh phenotype .", "Indeed , sustained antigen presentation associated with Tfh induction has been reported in the absence of cognate interactions with B cells ( Deenick et al . , 2010 ) .", "In the latter study , CD4+ T cells deficient for SAP , and therefore limited in their ability to interact with B cells , still adopted a Tfh phenotype when a second dose of soluble antigen was given 72 hr later .", "Presentation at this second challenge was attributed to DCs ( Deenick et al . , 2010 ) , consistent with our own findings .", "Indeed , DCs can induce Tfh development in the absence of cognate interactions with B cells ( Choi et al . , 2011 ) and likely represent the first step initiating Tfh differentiation ( Goenka et al . , 2011 ) .", "Imaging draining LNs 72 hr post challenge revealed that cognate T cell/DC interactions continued at this time .", "Importantly the current study adds mechanistic detail to the observational data above; temporal disruption of 72 hr ( stage 3 ) interactions , while not affecting clonal expansion , did inhibit 200 nm particle induced Tfh differentiation .", "Thus , increasing particle size , and sustaining presentation by DCs and their cognate interactions with T cells underpins the success of the larger particle in driving Tfh differentiation .", "It is unclear as to how particle size influences temporal availability of pMHCII on DCs .", "Particulates can impact on the mechanism of antigen uptake and therefore consequent endosomal targeting , processing and dynamics of presentation ( De Temmerman et al . , 2011; Zhao et al . , 2011; Ghimire et al . , 2012 ) .", "Thus the processing of antigen and loading of MHCII and/or its turnover at the cell surface may be contributing factors .", "In either case , increasing availability of TcR ligand and consequent TcR signal quantity may function in a similar way as increasing TcR dwell time in determining Tfh differentiation ( Tubo et al . , 2013; Tubo and Jenkins , 2014 ) .", "New delivery systems are required to generate safe and effective vaccines .", "This has resulted in increased focus on particle delivery systems .", "Our findings provide mechanistic insight as to why some formulations promote humoral immunity while others do not ( Mottram et al . , 2007; Moon et al . , 2012 ) .", "We have linked T cell behaviour to function , demonstrating the importance of stage 3 T/DC interactions in determining the selective differentiation of T cells towards a Tfh lineage .", "These late stage interactions could be selectively induced using immunisation with antigen conjugated to 200 nm but not 40 nm particles , rationalising approaches to vaccine design using nanoparticles for induction of Tfh , GC formation and high affinity , class switched protective antibody .", "Previous studies have demonstrated that in the absence of microbial factors , parameters such as the dose and persistence of antigen can affect T cell differentiation ( Constant and Bottomly , 1997 ) .", "The current data therefore explain why parameters such as the magnitude and duration of antigen presentation affect subsequent T/DC interactions and consequently determine T cell differentiation in the developing immune response ." ], [ "hCD2-DsRed mice ( gifted by D Kioussis and A Patel , National Institute for Medical Research , London ) were crossed with OVA specific OT-II ( Barnden et al . , 1998 ) TCR Tg mice .", "MD4 BcR Tg mice ( Mason et al . , 1992 ) on C57BL/6 backgrounds were used as a source of HEL specific B cells .", "6–8 week old C57BL/6J mice were used as recipients ( Harlan , Bicester , UK ) .", "CD11c-YFP mice ( Lindquist et al . , 2004 ) were used a recipients in multiphoton imaging experiments .", "All animals were specified pathogen free and maintained under standard animal house conditions at the University of Glasgow in accordance with UK Home Office Regulations .", "The recombinant HA protein used was expressed from DNA encoding the extracellular domain of the influenza A virus subtype H1N1 ( A/WSN/1933 ) HA ( Life Technologies , Paisley , UK ) .", "OVA-HEL conjugates were prepared using gluteraldehyde to couple the chicken OVA ( Worthington Biochemical , Lakewood , NJ , USA ) and HEL ( BBI Enzymes , Gwent , UK ) as published ( Garside et al . , 1998 ) .", "EαGFP was produced in house as detailed previously ( Rush and Brewer , 2010 ) .", "HA , OVA , OVA-HEL or EαGFP were covalently bound to Polybead Carboxylate Microspheres ( Park Scientific , Northampton , UK ) .", "Briefly , carboxylate-modified nanospheres were incubated with 2 . 5 mg/ml protein ( EαGFP , HA , OVA or OVA-HEL ) with 1 mM EDAC ( 1-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide; Sigma , Dorset , UK ) and 25 mM MES ( 2-N-morpholino ethane sulfonic acid; Sigma ) overnight , in line with manufacturers instructions .", "Conjugation mixes were then spun in an ultracentrifuge at 4°C for 40 min .", "Protein concentration in the supernatant was measured to allow the amount bound to the microspheres to be calculated .", "Preparations were stored at 4°C and used within 3 days of preparation .", "Mice were immunized subcutaneously ( s . c . ) in the scruff .", "Infections with influenza virus subtype H1N1 A/WSN/1933 ( WSN ) were carried out in mice anesthetized with isofluorane and 20 µl of PBS containing 300 PFU of WSN virus administered intranasally ( 5 mice per group ) .", "This dose was used as previous experiments showed that this dose led to approximately 20% weight loss in unvaccinated animals .", "Viral stocks were prepared and titred in MDCK cells .", "Adoptive transfers were performed as described previously ( Smith et al . , 2000 ) .", "Briefly , single cell suspensions of LNs and spleens were prepared from OT-II and MD4 mice .", "The proportion of Tg B and T cells was determined by flow cytometry and 2–3 × 106 Tg B cells and 2–3 × 106 Tg T cells adoptively transferred to age-matched C57BL/6J recipients via tail vein injection .", "For imaging studies , DsRed expressing CD4+ OT-II T cells were magnetically sorted from OT-II × hCD2-DsRed animals using mouse CD4+ T cell isolation kits ( Miltenyi Biotec , Surrey , UK ) .", "Mice were immunized via the footpad with 130 µg of OVA-HEL conjugate alone , with CFA ( Sigma , Dorset , UK ) , or covalently bound to carboxylate-modified microspheres .", "Injection with 50 μl of PBS was used as a vehicle control .", "Popliteal LNs were excised for ex vivo study .", "In MHCII blocking experiments , 500 μg Y3P ( BioXcell , New Hampshire , USA ) or isotype control ( mouse IgG2a; BioXcell ) was given via tail vein injection .", "In T cell/DC interaction studies , LNs were imaged 2 hr after antibody treatment .", "Single cell suspensions were stained using combinations of anti-CD11c-PE ( N418 , BD Biosciences , San Jose , CA , USA ) , anti-B220-PerCP ( RA3-6B2 , BD Biosciences ) , anti-I-Ab/Ea52-68-biotin ( YAe , eBioscience , San Diego , CA , USA ) , anti-CD4-eFluor450 ( RM4-5 , eBioscience ) , anti-ICOS-alexa-fluor-488 ( C398 . 4A , BioLegend , San Diego , CA , USA ) , anti-PD-1-PE-Cy7 ( J43 , eBiosciences ) , anti-SLAM-APC ( TC15-12F12 . 2 , BioLegend ) , biotinylated anti-CXCR5 ( 2G8 , BD Biosciences ) , anti-B220-eFluor450 ( RA3-6B2 , eBioscience ) , anti-GL-7-FITC ( BD Biosciences ) , anti-FAS-PE ( BD Biosciences ) and biotinylated anti-IgMa ( DM-1 , BD Biosciences ) .", "Biotinylated antibodies were detected by incubation with fluorochrome-conjugated streptavidin ( BD Biosciences ) .", "Appropriate isotype controls were used throughout .", "Samples were acquired using a MACSQuant analyzer ( Miltenyi Biotec , Surrey , UK ) and analysed using FlowJo software ( Tree Star Inc , Ashland , OR , USA ) .", "Multiphoton imaging was carried out using a Zeiss LSM7 MP system equipped with a 20×/1 . 0 NA water-immersion objective lens ( Zeiss UK , Cambridge , UK ) and a tunable Titanium: sapphire solid-state two-photon excitation source ( Chameleon Ultra II; Coherent Laser Group , Glasgow , UK ) and optical parametric oscillator ( OPO; Coherent Laser Group ) .", "Excised LNs were continuously bathed in warmed ( 37°C ) , oxygenated CO2 independent medium .", "A laser output of 820 nm and OPO signal at 1060 nm provided excitation of YFP CD11c+ and DsRed OT-II cells .", "Videos were acquired for 20 to 30 min with an X-Y pixel resolution of 512 × 512 in 2 μm Z increments .", "3D tracking was performed using Volocity 6 . 1 . 1 ( Perkin Elmer , Cambridge , UK ) .", "Values representing the mean velocity , displacement , and meandering index were calculated for each object .", "The intersection of DsRed and YFP objects was used to determine interaction between T cells and DCs respectively .", "Results are expressed as mean ± standard deviation .", "Gaussian distribution was confirmed by D'Agostino & Pearson omnibus normality test prior to significance being determined by one-way ANOVA .", "Significance in DC-T cell interaction experiments was confirmed by Mann Whitney .", "Values of p < 0 . 05 were regarded as significant ." ] ]
[ "The production of high affinity , class switched antibodies produced by B cells hinges on the effective differentiation of T follicular helper ( Tfh ) cells .", "Here we define conditions specifically enhancing Tfh differentiation and providing protection in a model of influenza infection .", "Tfh responses were associated with prolonged antigen presentation by dendritic cells ( DCs ) , which maintained T cell/DC interactions into stage 3 ( >72 hr ) of activation .", "Blocking stage 3 interactions ablated Tfh generation , demonstrating a causal link between T cell-DC behaviour and functional outcomes .", "The current data therefore explain how duration of antigen presentation affects the dynamics of T cell-DC interactions and consequently determine Tfh cell differentiation in the developing immune response ." ]
[ "The immune system protects the body from infections , cancer and other diseases .", "Invading microbes and cancerous cells exhibit proteins that are not normally found in the healthy cells of the body .", "Fragments of these molecules—known as antigens—may be detected by the immune system , which can then respond by producing antibodies and other responses that try to destroy the threat .", "Antibodies are produced by one type of immune cell ( known as B cells ) with the help of other cells called follicular helper T ( Tfh ) cells .", "During an immune response , Tfh cells form from ‘naïve’ T cells that have not encountered an antigen before .", "This process has several stages and is activated when the naïve T cells interact with antigens that are displayed on the surface of dendritic cells and other immune cells .", "However , it is not clear exactly how this process works .", "Here , Benson et al . studied the formation of Tfh cells in mice in response to antigens of different sizes .", "The experiments show that the dendritic cells displayed larger antigens for longer periods of time than they displayed the smaller antigens .", "Both the small and large antigens allowed dendritic cells to interact with T cells .", "However , only the dendritic cells that displayed the larger antigens maintained the interaction with T cells for longer periods of time ( into the last stage of Tfh cell formation ) .", "This enhanced the production of Tfh cells , which boosted the production of antibodies against the antigens to generate immunity to infection .", "Further experiments found that blocking the interaction between dendritic cells and T cells during the final stage of Tfh cell formation reduced the production of Tfh cells .", "Benson et al . 's findings show that the length of time that dendritic cells present antigens on their surface affects the production of Tfh cells and subsequent immune responses .", "Since Tfh cells are critical to the formation of long-lasting immunity against a virus , these findings could aid efforts to develop more effective vaccines against influenza and other diseases ." ]
2015
[ "Introduction", "Results and discussion", "Materials and methods" ]
[ "evolutionary biology" ]
Regulated aggregative multicellularity in a close unicellular relative of metazoa
elife-01287-v1
[ [ "Living organisms emerged from the integration of multiple levels of organization .", "These levels were shaped by both physiochemical constraints and historical circumstances , the latter being more important in more complex systems ( Jacob , 1977 ) .", "Therefore , it is important to identify the phylogenetic inertia ( sensu Burt , 2001 ) imposed by the raw starting material in order to properly understand major evolutionary transitions , such as the origin of metazoan multicellularity ( Knoll , 2011 ) .", "Examination of both the genetic repertoire ( King , 2004; Ruiz-Trillo et al . , 2007; Rokas , 2008 ) and the cell types present in the immediate unicellular relatives of metazoans can provide insights into this evolutionary transition , as they reveal the historical constraints in early metazoan evolution .", "In this regard , the analyses of unicellular holozoan genomes , that is choanoflagellates and filastereans , have shown that the genetic repertoire of the metazoan unicellular ancestor was much more complex than previously thought ( Abedin and King , 2008; King et al . , 2008; Sebé-Pedrós et al . , 2010; Suga et al . , 2013 ) .", "Multicellularity has been independently acquired multiple times during the evolution of eukaryotes , in more than 20 different lineages including animals , plants , fungi , slime molds , green and brown algae , and several other eukaryotes ( King , 2004; Parfrey and Lahr , 2013 ) .", "Multicellular organisms evolved through two major strategies: aggregation of different cells or clonal division of a single cell .", "Multi-level selection theory has proposed that the most complex multicellular organisms likely arose through clonal development rather than by aggregation of genetically diverse cells , since intra-organismal competition in the latter might be expected to be evolutionarily unstable ( Grosberg and Strathmann , 2007; Michod , 2007; Newman , 2012 ) .", "Accordingly , eukaryotic lineages that attained the most complex multicellular lifestyles ( i . e . , plants and metazoans ) arose through clonal cell division ( Grosberg and Strathmann , 2007 ) .", "In contrast to clonal multicellularity , aggregative cell behavior typically represents a transient life cycle stage .", "This type of multicellularity arose within several eukaryotic clades , including the dictyostelids ( Amoebozoa ) ( Schaap , 2011 ) , acrasid amoebas ( Heterolobosea , Discicristata , Discoba ) ( Brown et al . , 2011 ) , Guttulinopsis vulgaris ( Cercozoa , Rhizaria ) ( Brown et al . , 2012 ) , the genus Sorogena ( Ciliata , Alveolata ) ( Lasek-Nesselquist and Katz , 2001 ) , the holomycota Fonticula alba ( Opisthokonta ) ( Brown et al . , 2009 ) and the genus Sorodiplophrys ( Labyrinthulomycetes , Heterokonta ) ( Dykstra and Olive , 1975 ) ( Figure 1 ) . 10 . 7554/eLife . 01287 . 003Figure 1 . Phylogenetic position of Capsaspora owzarzaki within the eukaryotes . The Holozoa clade ( in yellow ) includes Metazoa and their closest unicellular relatives: Choanoflagellata , Filasterea , and Ichthyosporea .", "C . owczarzaki represents one of the two known filastereans taxa that form the sister-group of choanoflagellates and metazoans .", "Other major eukaryotic groups are shown .", "Within each group , species or clades with aggregative multicellularity ( see text ) are highlighted in red . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 003 Within the opisthokont clade that comprises Metazoa , Fungi and their unicellular relatives ( Cavalier-Smith , 2003; Steenkamp et al . , 2006; Ruiz-Trillo et al . , 2008 ) , so far only a single taxon has been described to have aggregative behavior , which is F . alba ( Brown et al . , 2009 ) , a close relative of Fungi .", "Moreover , among close unicellular relatives of Metazoa , clonal development is the only known multicellular behavior , as described in choanoflagellates and ichthyosporeans ( Dayel et al . , 2011; Suga and Ruiz-Trillo , 2013 ) .", "Within metazoans , which have largely clonal development , some cells show aggregative behaviors; for example , mesenchymal ( O’Shea , 1987 ) and germ line cells ( Savage and Danilchik , 1993 ) during development , sponge cells after cell dissociation ( Wilson , 1907 ) and arthropod blood cells through active amoeboid movement ( Loeb , 1903 , 1921 ) .", "To gain deeper insight into the possible transitions that arose during the emergence of metazoan multicellularity , we have performed a detailed examination of the life cycle and associated transcriptomic changes of Capsaspora owczarzaki , one of the closest known unicellular relatives of metazoans ( Figure 1 ) .", "Isolated decades ago as an endosymbiont of the fresh-water snail Biomphalaria glabrata ( Owczarzak et al . , 1980 ) , C . owczarzaki belongs to the clade Filasterea , the sister-group of Metazoa and choanoflagellates ( Torruella et al . , 2012 ) .", "Filasterea also includes a free-living marine unicellular species known as Ministeria vibrans ( Shalchian-Tabrizi et al . , 2008 ) .", "We analyzed the C . owczarzaki life cycle and its regulation using electron microscopy , flow cytometry and high-throughput RNA sequencing ( RNA-Seq ) .", "Through these analyses , we show that C . owczarzaki life cycle is tightly regulated at the level of gene expression and alternative splicing ( AS ) .", "Moreover , we demonstrate the existence of an aggregative multicellular stage in C . owczarzaki in which many orthologs of genes important for metazoan clonal multicellularity are upregulated ." ], [ "Under initial culture conditions ( ‘Materials and methods’ ) , C . owczarzaki differentiates into an amoeba that crawls over substrate ( Video 1 ) , surveying its environment with its filopodia ( Sebé-Pedrós et al . , 2013 ) .", "At this stage , active DNA replication occurs ( with >10% of the cells in S-phase ) and , within 48 hr , the cells enter an exponential growth phase ( Figures 2 and 3 ) .", "Subsequently , the cells start to detach from the surface and begin to retract their filopodia and encyst ( Figure 3B , C ) .", "After 8 days , no attached amoebas remain and growth is stabilized ( Figures 2 and 3 ) .", "This cystic stage may represent a dispersal resistance form .", "Strikingly , we observe an alternative path to this process involving the active formation of cell aggregates ( Videos 2 and 3 ) .", "In these aggregates , the cells attach to each other and produce cohesive extracellular material ( Figure 3D ) until a compact cell aggregate , in which cells no longer bear filopodia , is formed ( Figure 3E ) .", "Transmission electron microscopy demonstrates the presence of a thick , unstructured , extracellular material within the aggregates that appears to prevent direct contact between cells ( Figure 3F ) .", "Clusters of cells appear to occur at random under normal culture conditions . 10 . 7554/eLife . 01287 . 004Video 1 . C .", "owczarzaki filopodial amoeba stage cells crawling . Dark and refringent vesicles can be observed inside each cell .", "Up to nine different cells can be observed in the video .", "Also available on YouTube: http://youtu . be/0Uyhor_nDts . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 00410 . 7554/eLife . 01287 . 005Figure 2 . Flow-cytometry analysis of C . owzarzaki cell cycle .", "( A ) Total number of cells per day in each fraction ( adherent and floating , see ‘Materials and methods’ ) .", "( B ) Proliferation rate per day .", "( C ) Percentage of cells in S-phase per day and two examples of DNA-content profiles obtained from days 3 and 11 .", "Note the reduction in the number of G2/M cells ( second peak ) and the drastic reduction in S-phase cells ( the area between the two peaks ) .", "Data from adherent cells ( ‘Materials and methods’ ) is shown in green and data from floating cells in red .", "Experimental replicate 1 results are shown with solid lines and replicate 2 results with dashed lines . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 00510 . 7554/eLife . 01287 . 006Figure 3 . C .", "owzarzaki life cycle .", "( A ) Filopodial stage cells , amoebas with long filopodia .", "( B ) Transition from filopodial to cystic stage: cells retract filopodia .", "( C ) Cystic stage cells are rounded cysts without filopodia and slightly smaller than filopodial cells .", "( D ) Transition from filopodial to aggregative stage: cells attach to each other and an extracellular matrix appears .", "( E ) Mature aggregate .", "( F ) Transmission EM showing adjacent cells in the aggregate separated by extracellular matrix .", "Arrows indicate the observed stage inter-conversions .", "Scale bars = 1 μm , except in panel D = 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 00610 . 7554/eLife . 01287 . 007Video 2 . C .", "owczarzaki cells aggregation . Also available on YouTube: http://youtu . be/83HB8srWQw4 . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 00710 . 7554/eLife . 01287 . 008Video 3 . C .", "owczarzaki cells aggregation . Also available on YouTube: http://youtu . be/OvI6BvBucrc . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 008 To investigate whether C . owczarzaki cell clusters are formed through aggregation or clonal division , we first mixed two differentially stained populations of cells ( ‘Materials and methods’ ) and induced aggregate formation , resulting in dual-colored cell clusters ( Figure 4A ) .", "This indicates that cell clusters are not composed of daughter cells resulting from successive cell divisions , which would result in single-color cell clusters , but instead by aggregation of multiple cells .", "We also observed that aggregates could form efficiently even when cell division was blocked by two different inhibitors , hydroxyurea and aphidicolin ( Figure 4B ) .", "Finally , by flow cytometry , we observed that the proliferation rate of aggregative cells ( Figure 4—figure supplement 1 ) is extremely low ( compared with the observations in Figure 2 ) .", "Overall , these results show that C . owczarzaki cell clusters form by active cell aggregation , not by clonal cell division .", "This observation represents the first reported case of aggregative multicellularity in a close unicellular relative of Metazoa . 10 . 7554/eLife . 01287 . 009Figure 4 . C .", "owczarzaki cell clusters form by active aggregation , not clonal cell division . Aggregation was induced in different stained cell populations ( ‘Materials and methods’ ) .", "( A ) Left , population of cells stained with Lysotracker ( green ) , uniformly green aggregates are formed .", "Center , population of cells stained with Chormeo Mitochondrial Staining ( cyan ) , uniformly cyan aggregates are formed .", "Right , two independently stained populations of cells ( green or cyan ) are mixed and dual color aggregates are formed , indicating that cells from different origins aggregate to each other .", "( B ) Total number of cells per day in control cells , aphidicolin-treated cells and hydroxyurea-treated cells .", "Note that cell division is blocked by both aphidicolin and hydroxyurea .", "Aggregate formation was evaluated under each condition .", "All cells , even those treated with aphidicolin or hydroxyurea , developed aggregates .", "A representative aggregate is shown for each condition .", "Scale bars= 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 00910 . 7554/eLife . 01287 . 010Figure 4—figure supplement 1 . Proliferation rate per day of aggregative cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 010 The aggregative multicellularity observed in C . owczarzaki adds an additional cell behavior to those already known among extant close unicellular relatives of Metazoa ( i . e . , choanoflagellates , ichthyosporeans , and filastereans [Torruella et al . , 2012] ) .", "We can then infer that multiple cell types and behaviors ( including aggregative behavior , flagellar motility , amoeboid movement , clonal colony formation , etc ) were most likely present among the unicellular ancestors of metazoans .", "This range of cell behaviors may have provided the basis for the evolution of the diverse cell types seen in animals ( Arendt , 2008; Arendt et al . , 2009 ) .", "Interestingly , each one of the three known unicellular lineages closely related to Metazoa ( choanoflagellates , ichthyosporeans and filastereans ) has some kind of simple multicellularity .", "Moreover , the tight regulation observed in C . owczarzaki ( see below ) emphasizes that a regulated temporal cell differentiation was already in place among unicellular ancestor of animals .", "This reinforces the view that , during the transition to animal multicellularity , ancestral premetazoan cell types may have been integrated into a single multicellular entity by means of controlling cell differentiation spatially , rather than temporally ( Mikhailov et al . , 2009 ) .", "An alternative explanation is that some of the cell behaviors observed in extant unicellular relatives of Metazoa may had evolved independently in some particular unicellular holozoan lineages or species , and do not represent ancestral states .", "The limited taxon sampling in many of these poorly studied lineages makes it difficult to reliably assess whether these are derived or ancestral characters and this situation is especially dramatic in the case of filastereans , in which only two species have been described so far ( C . owczarzaki and the free-living M . vibrans ) .", "To investigate the molecular composition and regulation of the distinct life cycle stages of C . owczarzaki , we isolated filopodiated amoebae , aggregates , and cysts , and analyzed their transcriptomes using RNA-Seq .", "Of 8637 annotated genes , 4486 showed statistically significant differential regulation ( ‘Materials and methods’ ) in one or more pair-wise comparisons between life cycle stages , including 1354 changes between filopodial and aggregate stages , 3227 between filopodial and cystic stages , and 3096 between aggregate and cystic stages .", "Moreover , when performing one-versus-all comparisons , each cell stage had a specific transcriptomic profile ( Figure 5A ) , indicating tight regulation at the level of gene expression .", "Using both pairwise and one-versus-all comparisons , we identified significantly enriched gene ontology ( GO ) categories ( Figures 5 and", "6 ) and Pfam protein domains ( Figure", "7 ) in each set of differentially expressed ( both up and down-regulated ) genes ( p<0 . 01 for each significant category; Fisher’s exact test ) .", "Genes upregulated in the filopodial stage were enriched in signalling functions , such as tyrosine kinase activity and G-protein-coupled receptor activity , as well as in transcription factors , especially of the Basic Leucine Zipper Domain ( bZIP ) superfamily ( Figures 5 and 6 ) .", "Genes involved in protein synthesis and DNA replication were also significantly upregulated , consistent with the rapid cell proliferation at this stage observed by flow cytometry ( Figure 2 ) , and further suggesting a high metabolic rate . 10 . 7554/eLife . 01287 . 011Figure 5 . Differential gene expression in C . owczarzaki .", "( A ) Heatmap showing differential gene expression in the different biological replicates of each stage .", "Only genes with cRPKM ≥ 5 in at least one sample and with a 2-fold expression change in at least one pair-wise comparison are shown .", "( B ) Gene set enrichment analysis ( GSEA ) for the different cell stages ( ‘Materials and methods’ ) .", "Orange represents enrichment in the aggregative stage , blue in the cystic stage , and green in the filopodial stage , with color intensity proportional to enrichment significance .", "The node size is proportional to the number of genes associated to the GO category , and the width of the edges is proportional to the number of genes shared between GO categories .", "Groups of functionally related GOs are manually circled and assigned a label . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 01110 . 7554/eLife . 01287 . 012Figure 6 . GO enrichment in sets of differentially expressed genes . Pairwise ( Aggregative vs Filopodial , Cystic vs Aggregative and Cystic vs Filopodial ) and one-versus-all comparisons are indicated .", "The significantly overrepresented GO categories ( ‘Materials and methods’ ) are shown for sets of overexpressed ( green ) and downregulated ( red ) genes and for genes with differential intron retention ( gray ) .", "The number of genes included in each set is indicated with the same color code . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 01210 . 7554/eLife . 01287 . 013Figure 7 . Pfam protein domain enrichment in sets of differentially expressed genes . Pairwise ( Aggregative vs Filopodial , Cystic vs Aggregative and Cystic vs Filopodial ) and one-versus-all comparisons are indicated .", "Significantly overrepresented Pfam domains ( ‘Materials and methods’ ) are shown for sets of overexpressed ( green ) and downregulated ( red ) genes and for genes with differential intron retention ( gray ) .", "The number of genes included in each set is indicated with the same color code .", "Those Pfam domains mentioned in the text are shown in bold . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 013 When compared to filopodial and aggregative cells , cystic cells showed significant downregulation of genes associated with myosin transport , translation , DNA replication and metabolic activities ( especially mitochondrial energy production ) .", "However , genes involved in vesicle transport and autophagy were significantly upregulated at this stage ( Figure 5 ) .", "These differences may reflect recycling of intracellular components triggered by starvation or other adverse conditions , as has been observed under conditions of adaptive cell survival in other eukaryotes ( Kiel , 2010 ) .", "Protein domains involved in the ubiquitin pathway ( e . g . , UQ_con , zf-RING2 and Cullin domains ) and in synaptic cell–cell communication , such as SNARE , synaptobrevin and syntaxin , as well specific transcription factor families ( e . g . , bHLH transcription factors ) , were also significantly upregulated in the cystic cells ( Figure 7 ) .", "Altogether , these results suggest that major cytosolic rearrangement and protein turnover occur at the cystic stage .", "Remarkably , the aggregative stage showed strong upregulation of the components of the integrin adhesome and associated signalling and cell-adhesion proteins ( Figures 5 and 8A , B ) , such as the LamininG domain-containing protein CAOG_07351 ( which contains a N-terminal signal peptide sequence and therefore is likely to be secreted ) ( Figure 8C ) , the IPP complex ( ILK-PINCH-Parvin ) signalling module , G-protein α-13 ( Gong et al . , 2010 ) , several cytoplasmatic tyrosine kinases ( Hamazaki et al . , 1998 ) and two receptor tyrosine kinases ( which possess extracellular DERM [Lewandowska et al . , 1991] and fibronectin_3 domains , known to interact with integrins [Figure 8C] ) .", "These observations strongly suggest that the integrin adhesome and the likely associated tyrosine kinase signalling genes play an important role in the formation of the C . owzarzaki aggregates .", "Furthermore , we also observed upregulation of genes involved in mitosis and in the tubulin cytoskeleton ( e . g . , kinesins ) at the aggregative stage ( Figure 5 ) .", "These results indicate that a molecular repertoire associated with animal multicellularity , could function either in aggregative or in clonal multicellularity and in different phylogenetic contexts , in line with previous hypotheses ( Newman , 2012 ) . 10 . 7554/eLife . 01287 . 014Figure 8 . Expression of cell–ECM adhesion genes .", "( A ) Barplot of the expression values of each gene in the different stages , showing overexpression of most components in the aggregate stage ( orange ) .", "Asterisks indicate that the gene is significantly differentially expressed in both ( two asterisks ) or only one ( one asterisk ) pair-wise comparison ( agg . vs fil . and agg . vs cyst . ) .", "Bars show standard error .", "( B ) Schematic representation of the putative C . owczarzaki integrin adhesome and putative associated signalling proteins , colored according to overexpression in aggregates as shown in the barplot ( dark orange , two asterisks; light orange , one asterisk; and white , no differences in expression ) .", "( C ) Specific protein domain architectures for the fibronectin and DERM receptor tyrosine kinases ( CAOG_01676 and CAOG_06673 ) and for the laminin protein ( CAOG_07351 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 014 A hallmark feature of the evolution of metazoan multicellularity and cell type diversity is the expansion of AS complexity and regulation through exon skipping , which has entailed the formation of cell type-specific networks of co-regulated exons belonging to functionally related or pathway-specific genes ( Irimia and Blencowe , 2012 ) .", "In contrast , differential intron retention is the most widespread form of AS found in non-metazoan eukaryotic species ( McGuire et al . , 2008 ) .", "To assess the extent to which these forms of AS may contribute to gene regulation in C . owczarzaki , we systematically mapped reads from each life cycle stage to a comprehensive set of intron–exon and exon–exon junctions ( i . e . , formed by exon/intron inclusion and skipping ) to score their differential usage .", "Of 25 , 677 introns with sufficient RNA-Seq read coverage across the three life cycle stages , 2986 ( 11 . 6% ) showed ≥20% PSI ( Percent Spliced In , percent of transcripts from a given gene in which the intron sequence is present ) in at least one stage , and approximately a third of genes had at least one such intron retention event .", "Moreover , we observed marked differences in the extent to which detected intron retention is differentially regulated between the different life cycle stages ( Figure 9A ) .", "In particular , 797 retained introns ( in 441 genes ) and 259 retained introns ( in 232 genes ) display differential PSI ( dPSI ) values of 25% or more in the filopodial and cystic stages compared to the other stages , respectively ( Figure 9B ) .", "In contrast , no retained introns were found to be differentially spliced at the aggregative stage .", "Most ( 12 out of 15 , 80% ) of the analyzed cases of differentially retained introns were validated by RT-PCR ( Figure 9C and Figure 9—figure supplement 1 ) . 10 . 7554/eLife . 01287 . 015Figure 9 . Regulated alternative splicing in C . owzarzaki .", "( A ) Plot of percentage of intron inclusion by intron in rank order for the three studied cellular stages .", "Filopodial ( green ) and cystic ( blue ) stages show higher intron retention levels than the aggregative stage ( orange ) ( p<2 . 2e-16 , Wilcoxon Rank Sum test ) .", "( B ) Heatmap of PSIs of filopodial- and cystic-specific differentially retained introns across three replicates for each cellular stage .", "( C ) Examples of stage-specific intron retention .", "( D ) Intron length distributions for differentially retained introns in cystic ( blue ) , filopodial ( green ) , and weakly retained introns ( gray ) .", "( E ) Relationship between intron length and retention .", "Percentage of average intron retention in each of the three cellular stages for different bins of intron size .", "In the cystic stage , the percentage of intron retention increased with intron length . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 01510 . 7554/eLife . 01287 . 016Figure 9—figure supplement 1 . Intron retention validation ( see ‘Materials and methods’ ) .", "Genes with confirmed intron retention events are indicated using gene IDs .", "Each dot corresponds to the inclusion of one intron ( introns in C . owczarzaki usually have very similar lengths , and thus different combinations of the same size introns cannot be differentiated ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 016 GO enrichment analysis for the two sets of differentially retained introns showed distinct gene function enrichment ( e . g . , protein kinase activity and intracellular targeting in the filopodial stage , and histone modification and myosin complex in the cystic stage ) ( Figure 6 ) , implying that regulated intron retention plays different roles at these stages .", "A low fraction of read-through introns ( with length-multiples of three and no in-frame stop codons ) were found among the two sets of differentially retained introns , suggesting that most if not all of these retained introns act by reducing the levels of spliced mRNAs exported from the nucleus and translated into protein , as has been observed previously for regulated retained introns in metazoan species ( Yap et al . , 2012 ) .", "Moreover , we observe that multiple introns belonging to a gene can be retained in a stage-specific manner .", "For instance , >73% and >29% of multi-intronic genes with one differentially retained intron had at least one additional differentially retained intron at the filopodial or cystic-specific stages , respectively , and 22% and 5% of genes at these stages showed evidence of high retention for all introns in the same genes .", "Furthermore , RT-PCR analyses and mate information from paired-end read analyses suggested that multiple intron retention events often occur in a combinatorial manner ( Figure 9—figure supplement 1 ) , thereby increasing the potential impact of intron retentions on mRNA regulation .", "All of the above observations were highly consistent across three biological replicates ( Figure 9B ) , and not observed for neighbouring genes , ruling out contamination of genomic DNA .", "We analyzed different features of differentially retained introns that may account for their stage-specific regulation .", "First , we compared intron lengths .", "While filopodial-specific differentially retained introns have a similar length distribution to constitutive ( PSI less than 2% across all stages ) introns , cystic stage-specific introns were significantly longer ( p=1 . 7e−14 Wilcoxon rank sum test ) ( Figure 9D ) .", "In line with this observation , the average level of intron retention increased steadily with intron length only in the cystic stage ( Figure 9E ) .", "Furthermore , cystic stage-specific retained introns harbored significantly weaker canonical 5´ and 3´ splice site signals than other intron sets ( p<0 . 0013 Wilcoxon rank sum test for all comparisons ) .", "Collectively , these data suggest that differential intron retention in the cystic stage may be associated with suboptimal introns ( i . e . , long and with weak splice sites ) that are more efficiently spliced in the other cell stages .", "In the case of the filopodial-specific differentially retained introns , analyses of sequence motif enrichment with MEME show enrichment of a long T/G-rich motif that highly resembles a recently identified consensus binding site for Elav-like protein in mammals ( Ince-Dunn et al . , 2012 ) ( Figure 10 ) .", "Interestingly , the single ortholog for this gene in C . owczarzaki shows a highly-regulated expression pattern , with lowest expression in the filopodial stage ( Figure 10C ) .", "Therefore , it is tempting to speculate that Elav-like protein may negatively regulate filopodial-specific intron retention of some introns .", "Experimental depletion of Elav-like protein in C . owczarzaki will require the development of RNAi or gene-targeting methods in this species before this hypothesis can be tested . 10 . 7554/eLife . 01287 . 017Figure 10 . Possible role for an Elav-like ortholog in the negative regulation of filopodial stage-specific dRIs .", "( A ) Most significantly enriched motif in filopodial stage-specific dRIs , obtained by MEME .", "( B ) Consensus motif obtained by CLIP-Seq data for an Elav-like member in mammals by Ince-Dunn et al . ( 2012 ) and that closely resembles the motif in ( A ) .", "T∼U .", "( C ) Expression ( measured as cRPKMs ) of CAOG_02177 , a Elav-like ortholog from C . owczarzaki that shows lower expression in filopodial stage . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 017 Next , we investigated differential exon splicing and identified 191 cassette exons with PSIs <95% in at least one life cycle stage , 39 of which display PSIs <85% .", "29 of these exons showed a ≥15% PSI difference in pairwise comparisons between the cell stages , with lower PSIs typically associated with the filopodial stage ( Supplementary file 1 ) ; RT-PCR assays confirmed skipping for 7 out of 8 tested cases ( Figure 11A , and Figure 11—figure supplement 1 ) .", "Most ( ∼60% ) of these exons maintain an open reading frame when skipped .", "In contrast to previous reports demonstrating that differentially-regulated exons are significantly under represented in modular , folded domains in metazoans ( Romero et al . , 2006; Ellis et al . , 2012 ) , two thirds of the differently regulated exons in C . owczarzaki overlap annotated domains ( Supplementary file 1 ) .", "Furthermore , genes with differential exon skipping are statistically significantly enriched in protein kinase activity , impacting both tyrosine and serine/threonine kinases ( Figure 11B ) .", "This observation strongly suggests a role for coordinated exon skipping in the modulation of cell signaling in C . owczarzaki .", "To our knowledge , this represents the first example of a regulated exon network linked to a specific biological function in a unicellular organism . 10 . 7554/eLife . 01287 . 018Figure 11 . Regulated exon-skipping in C . owzarzaki .", "( A ) Examples of exon skipping .", "( B ) Gene set enrichment analysis ( GSEA ) of the genes containing cassette exons that are differentially-regulated among cellular stages showing high enrichment for protein kinase-associated activities . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 01810 . 7554/eLife . 01287 . 019Figure 11—figure supplement 1 . Exon Skipping validation by RT-PCR ( ‘Materials and methods’ ) .", "Note that DNA heteroduplexes between the two isoforms may be formed and appear as extra bands .", "Bars in the key correspond to the exons amplified: the alternative exon in red , and the adjoining constitutive exons in white . DOI: http://dx . doi . org/10 . 7554/eLife . 01287 . 019 In summary , our results offer new insight into the origin of metazoan multicellularity .", "In particular , the observation of an aggregative multicellular stage in C . owczarzaki represents the first example of such cellular behavior in a close unicellular relative of metazoans .", "This observation therefore adds to the repertoire of reported complex cellular behaviors among extant unicellular relatives of metazoans—including clonal colony formation in choanoflagellates and sporangia formation by hypertrophic syncytial growth in ichthyosporeans ( Dayel et al . , 2011; Suga and Ruiz-Trillo , 2013 ) , thus expanding the potential starting ‘raw material’ available for the evolution of animal multicellularity .", "We note that the current evolutionary framework on the opisthokonts , based on phylogenomic analyses ( Steenkamp et al . , 2006; Ruiz-Trillo et al . , 2008; Shalchian-Tabrizi et al . , 2008; Torruella et al . , 2012; Paps et al . , 2013 ) , discards the possibility that C . owczarzaki ( or choanoflagellates ) derives from a more complex multicellular lineage .", "The sister-group of opisthokonts is the unicellular biflagellates Apusozoa , and complex multicellularity has not yet been observed in any of the non-metazoan holozoan lineages .", "Furthermore , we show that the complex , metazoan-like genetic ‘toolkit’ of C . owczarzaki ( Sebé-Pedrós et al . , 2010 , 2011; Suga et al . , 2012 ) is dynamically deployed during its highly-regulated life cycle , with upregulation of integrin adhesome and signalling genes linked to multicellularity in metazoans during the aggregative stage .", "Extensive differential AS between the C . owczarzaki life cycle stages likely further contributes to the dynamic gene regulation observed in this species , with differential intron retention likely acting as an important mechanism in the control of transcript levels between life cycle stages , probably through triggering non-sense mediated decay ( NMD ) .", "Our discovery of an exon network associated with tyrosine kinase genes in C . owczarzaki further adds to the metazoan-like features of this species .", "Together with genes resembling those that function in metazoan multicellular processes , the emergence of an exon network that functions in conjunction with differentially-regulated intron retention may have provided a degree of proteomic and regulatory complexity that was key in the evolution of cell type complexity in metazoans ( Nilsen and Graveley , 2010 ) .", "Based on the collective results from our investigation of C . owczarzaki , it is intriguing to consider that the integration of regulatory innovations involving differential expression and splicing of metazoan-like genes set the stage for the evolution of cell specialization in the common ancestors of metazoans and C . owczarzaki ." ], [ "C . owczarzaki cells of the corresponding stage were fixed for 1 hr with 2 . 5% glutaraldehyde ( Sigma-Aldrich , St . Louis , MO , USA ) , and for another hour with 1% osmium tetroxide ( Sigma-Aldrich ) , followed by dehydration in a graded ethanol series ( 25% , 50% , 70% , 99% ) for 15 min per step , followed by three 15-min rinses in 100% ethanol .", "Samples were critical-point dried in liquid CO2 using a BAL-TEC CPD 030 critical-point drying apparatus .", "They were subsequently glued to SEM stubs with colloidal silver , sputter-coated with gold-palladium , and examined with a Hitachi S-3500N ( Hitachi High-Technologies Europe GmbH , Krefeld , Germany ) .", "Cell aggregates were loaded into the copper tubes and immediately cryoimmobilized using a Self-Pressurized Freezing System ( EM SPF ) ( Leica-Microsystems , Vienna , Austria ) .", "The cells were then stored in liquid nitrogen until further use .", "Peeled copper tubes were freeze-substituted in anhydrous acetone containing 2% osmium tetroxide and 0 . 1% uranyl acetate at −90°C for 72 hr and warmed to room temperature , following a 2°C increase per hour in five consecutive steps ( −60°C , −30°C , 0°C , 4°C , and room temperature ) with a total of 8 hr at each temperature and using an EM AFS ( Leica-Microsystems , Vienna ) .", "After several acetone rinses , samples were infiltrated with Epon resin during 7 days and embedded in resin and polymerised at 60°C during 48 hr .", "Ultrathin sections were obtained using a Leica Ultracut UC6 ultramicrotome ( Leica-Microsystems ) and mounting on Formvar-coated copper grids .", "The sections were stained with 2% uranyl acetate in water and lead citrate , and were observed in a Tecnai Spirit 120 kv electron microscope ( FEI Company , Eindhoven , Netherlands ) equipped with a Megaview III CCD camera .", "C . owczarzaki cells were grown axenically in 5-ml flasks with ATCC medium 1034 ( modified PYNFH medium ) in a 23°C incubator .", "Three biological replicates ( three independent cell lines ) were generated by subculturing from a single-founding cell and grown for 2 months .", "Adherent filopodiated cells were obtained by initiating a new 1/100 sub-culture ( from an approximately 5 × 106 cells/ml initial culture ) and , after 3–4 days , cells were scratched from the substrate .", "Aggregate formation was induced by initiating a new 1/250 sub-culture ( from an approximately 5 × 106 cells/ml initial culture ) and by gentle agitation at 60 rpm during 4–5 days .", "Finally , floating cystic cells were obtained from a 14-day-old culture , starting from a new 1/100 sub-culture ( from an approximately 5 × 106 cells/ml initial culture ) .", "Two different groups of cells ( from two different starting cultures , 5 ml flasks with a cell density of 106 cells/ml , consisting exclusively of adherent filopodial cells ) were stained either with 75 nM ( in PBS1x ) Lysotracker Green DND-26 ( Life Technologies , Carlsbad , CA , USA ) or with 1 μM ( in PBS1x ) Chromeo Live Cell Mitochondrial Staining Kit ( Active Motif Inc , Carlsbad , CA , USA ) .", "The cells were stained for 30 min at 23°C .", "After staining , 1/3 of the cells from the two differentially stained populations were mixed in a new culture flask and the remaining 2/3 of cells for each staining were kept as a control .", "All three cultures were grown for 2 hr and then the aggregate formation was induced ( see above , ‘Results and Discussion’ ) .", "After 8 hr , aggregates were visualized in poly-L-lysine covered ( Sigma-Aldrich , St . Louis , MO ) glass-bottom plates in a Leica TCS SP5 confocal microscope ( Leica-Microsystems ) .", "C . owczarzaki cell division was blocked using 100 mM hydroxyurea ( Sigma-Aldrich ) or 25 μg/ml aphidicolin ( Sigma-Aldrich ) .", "The effect of both drugs was evaluated by following cultures treated with each drug during 7 days , using Neubauer chamber .", "The cells were cultured in 16-multiwell plates and with an initial density of 5 × 104 cells/ml .", "Once these conditions were established , different cultures were treated with each drug for 1 day and , then , aggregate formation was induced ( see above ) .", "2 days later , the formation of aggregates was visualized in poly-L-lysine covered ( Sigma-Aldrich ) glass-bottom plates in a Leica TCS SP5 confocal microscope ( Leica-Microsystems ) .", "C . owczarzaki cells were grown in 5-ml flasks with ATCC medium 1034 ( modified PYNFH medium ) in a 23°C incubator .", "Total RNA from each cell stage ( and from three biological replicates from each stage ) was extracted using Trizol reagent ( Life Technologies ) .", "Nine libraries were sequenced over 2 lanes HiSeq 2000 instrument ( Illumina , San Diego , CA , USA ) , generating a total of 197M 76-base paired reads .", "Reads were aligned to the reference genome using Tophat ( Trapnell et al . , 2012 ) with default options and specifying that this was a strand-specific sequencing , rendering an average mapping of 90% .", "Significant differential expression was calculated by performing pairwise comparisons with DESeq ( Anders and Huber , 2010 ) ( threshold 1e-05 ) , EdgeR ( Robinson et al . , 2010 ) ( threshold 1e-05 ) , CuffDiff ( Trapnell et al . , 2012 ) ( threshold 1e-05 ) and NOISeq ( Tarazona et al . , 2012 ) ( threshold 0 . 8 ) and only genes that appear to be significant at least in three out of the four methods were considered as differentially expressed .", "Quality control analyses of the data were performed using cummeRbund R package ( Trapnell et al . , 2012 ) .", "These include count vs dispersion plot to estimate over-dispersion , density plot to assess the distributions of FPKM scores across samples and squared coefficient of variation plot to check for cross-replicate variability .", "A gene ontology of C . owczarzaki’s 8637 genes was generated using Blast2GO ( Conesa et al . , 2005 ) and GO enrichments of the different lists of differentially expressed genes ( see above ) were analyzed using Ontologizer ( Bauer et al . , 2008 ) using the Topology-Weighted method .", "A p-value threshold of 0 . 01 was used .", "The results from Ontologizer were loaded into Enrichment map cytoscape plug-in ( Merico et al . , 2010 ) to generate a network visualization .", "Pfam domains of all genes were analyzed using Pfamscan v26 with default Gathering Threshold , and counts were generated using custom Perl scripts .", "Fisher’s exact tests were performed using custom R scripts and a p-value threshold of 0 . 01 was used .", "Exon skipping and intron retention were analyzed as previously described ( Curtis et al . , 2012; Han et al . , 2013 ) .", "In short , for exon-skipping analyses , multifasta libraries of exon–exon junctions were built by combining all forward annotated splicing donors and acceptors .", "A minimum of eight base pairs was required at each boundary to assure specificity .", "Next , the number of effective mappable positions was calculated for each exon–exon junction , as previously described ( Labbé et al . , 2012; Barbosa-Morais et al . , 2012 ) .", "Then , RNA-seq reads ( previously trimmed to 50 nucleotides and combining each three replicates to increase read depth ) were aligned to these sequences using Bowtie , with−m 1 −v 2 parameters ( single mapping and two or fewer mismatches ) .", "Percentage of exon inclusion was calculated and a minimal read coverage was required , as previously described ( Khare et al . , 2012 ) .", "For intron retention , a similar approach was taken for each contiguous intron–exon and exon–exon junction , and percentage of intron inclusion ( PSI , Percent Spliced In , the percentage of transcript for a given gene that contain the intron ) was calculated as previously described ( Curtis et al . , 2012 ) .", "For comparisons among cellular stages , only events with enough read coverage in the three samples were considered ( either", "( i ) ≥10 reads in the exon–exon junction or", "( ii ) ≥10 reads in one intron–exon junction and ≥5 in the other ) , and introns showing >95% inclusion in the three samples were discarded .", "To assess whether differentially retained introns in the same genes were included in a coordinated or in a combinatorial manner , mate information of read pairs was used .", "If each end of a read mapped to two different intron retention events , each end may be providing support for retention of both introns or splicing of both introns ( coordinated regulation ) , or retention of one and splicing of another ( combinatorial intron retention ) .", "For the 555 pairs of retained introns that had read mate information , 196 ( 35 . 3% ) showed evidence for combinatorial regulation .", "Finally , for sequence motif enrichment analyses , full intron sequences were compared using MEME ( Bailey et al . , 2009 ) .", "To validate AS analysis predictions , the three stages were induced ( RNA-Seq and analysis sections ) and RNA was extracted using Trizol reagent ( Life Technologies ) .", "To eliminate genomic DNA , total RNA was treated with DNAse I ( Roche , Basel , Switzerland ) and purified using RNeasy columns ( Qiagen , Venlo , Netherlands ) .", "For each stage , cDNA was produced from 1 μg of total RNA using SuperScript III reverse transcriptase ( Life Technologies ) .", "Pairs of primers of similar melting temperature ( 60°C ) and spanning the putative alternatively spliced segments were designed using Geneious software .", "PCR was performed using ExpandTaq polymerase ( Roche ) .", "C . owczarzaki cells were grown for 10 to 15 days , sampling every day from both the supernatant ( to obtain floating cells , which after day 7 are completely encysted ) and the scratched flask ( to obtain filopodial adherent cells ) .", "Thus , two samples were obtained daily , for floating and adherent cells .", "For DNA-content analysis , a sample was fixed using 70% ethanol and stored at −20°C for one month .", "The samples were subsequently fixed and stained with Propidium Iodide ( as described in Darzynkiewicz and Huang , 2004 ) and DNA content estimated using FACScalibur flow cytometer ( Becton Dickinson , Franklin Lakes , NJ , USA ) .", "For cell counting , 1 ml of fresh sample ( one from the supernatant and one from the flask surface ) was mixed in a BD Trucount Tube ( Becton Dickinson ) , with a known number of beads , so absolute cell number counts could be calculated , using an LSR Fortessa flow cytometer ( Becton Dickinson ) .", "Two replicate experiments ( R1 and R2 ) were performed independently in order to confidently establish growth dynamics .", "Two measures were calculated from the DNA-content analysis .", "First , the proliferation rate , which indicates the proportion of number of cells in S and G2/M phases vs the number of cells in G0/G1 .", "Second , the percentage of cells in S-phase ." ] ]
[ "The evolution of metazoans from their unicellular ancestors was one of the most important events in the history of life .", "However , the cellular and genetic changes that ultimately led to the evolution of multicellularity are not known .", "In this study , we describe an aggregative multicellular stage in the protist Capsaspora owczarzaki , a close unicellular relative of metazoans .", "Remarkably , transition to the aggregative stage is associated with significant upregulation of orthologs of genes known to establish multicellularity and tissue architecture in metazoans .", "We further observe transitions in regulated alternative splicing during the C . owczarzaki life cycle , including the deployment of an exon network associated with signaling , a feature of splicing regulation so far only observed in metazoans .", "Our results reveal the existence of a highly regulated aggregative stage in C . owczarzaki and further suggest that features of aggregative behavior in an ancestral protist may had been co-opted to develop some multicellular properties currently seen in metazoans ." ]
[ "When living things made from many cells evolved from single-celled ancestors , it was a breakthrough in the history of life—and one that has occurred more than once .", "In fact , multicellular life has evolved independently at least 25 times , in groups as diverse as animals , fungi , plants , slime molds and seaweeds .", "There are broadly two ways to become multicellular .", "The most complex multicellular species , such as animals , will replicate a single cell , over and over , without separating the resultant cells .", "However , in species that are only occasionally multicellular , free-living cells tend instead to join together in one mass of many cells .", "Evolution is constrained by its raw materials; so looking at the living relatives of a given species , or group , can lead to a better understanding of its evolution because its relatives contain clues about its ancestors .", "To gain insights into how animal multicellular life might have began; Sebé-Pedrós et al . studied the life cycle of the amoeboid organism Capsaspora owczarzaki .", "Found within the bodies of freshwater snails , this single-celled amoeba is a close relative of multicellular animals and could resemble one of their earliest ancestors .", "At certain stages of the life cycle Sebé-Pedrós et al . noticed that individual amoebae gathered together to form a multicellular mass—something that had not been seen before in such a close relative of the animals .", "Moreover , the genes that ‘switched on’ when the amoebae began to aggregate are also found in animals; where , together with other genes , they control development and the formation of tissues .", "Sebé-Pedrós et al . suggest that the first multicellular animals could have recycled the genes that control the aggregation of single-celled species: in other words , genes that once controlled the changes that happen at different times in a life cycle , now control the changes that develop between different tissues at the same time .", "Sebé-Pedrós et al . also observed that alternative splicing—a process that allows different proteins to be made from a single gene—occurs via two different mechanisms during the life cycle of Capsaspora .", "Most of the time Capsaspora employs a form of alternative splicing that is often seen in plants and fungi , and only rarely in animals; for the rest of the time it uses a form of alternative splicing similar to that used by animal cells .", "The evolution of complex alternative splicing mechanisms is a hallmark feature of multicellular animals .", "The exploitation of two major forms of alternative splicing in Capsaspora could thus reflect an important transition during evolution that resulted in an increased diversity of proteins and in more complex gene regulation .", "Such a transition may ultimately have paved the way for the increased specialization of cell types seen in animals .", "This glimpse into the possible transitions in gene regulation that contributed to the birth of multicellular animals indicates that the single-celled ancestor of the animals was likely more complex than previously thought .", "Future analyses of the animals’ close relatives may further improve our understanding of how single-celled organisms became multicellular animals ." ]
2013
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cell biology", "physics of living systems", "tools and resources" ]
KymoButler, a deep learning software for automated kymograph analysis
elife-42288-v1
[ [ "Many processes in living cells are highly dynamic , and molecules , vesicles , and organelles diffuse or are transported along complex trajectories .", "Particle tracking algorithms represent powerful approaches to track the dynamics of such particles ( Jaqaman et al . , 2008; Sbalzarini and Koumoutsakos , 2005; Lee and Park , 2018 ) .", "However , in many scenarios particles follow a distinct pathway within cells and move much faster than the confounding cell .", "For example , molecules transported along neuronal axons , dendrites , or along cilia typically move along the structure’s long axis and do not show significant motion perpendicular to that path .", "Similarly , retrograde actin flow typically occurs along a single axis within the cell .", "Hence , when the cell is not moving significantly for the duration of imaging , one can define , for example manually draw , a so-called ‘stationary path’ ( Figure 1A ) along which particles move either forwards or backwards .", "In these cases , kymographs provide an elegant solution to the visualisation and analysis of particle dynamics .", "To generate a kymograph , the intensity profile along the manually drawn stationary path ( black dashed line in Figure 1A ) is extracted for each frame of a time-lapse movie , and then these profiles are stacked into individual rows of an image ( Figure 1A ) .", "In the resulting space-time image , each ( usually fluorescently ) labelled particle is shown as a line , whose slope , for example , represents the velocity of that particle ( Figure 1A ) .", "In many biological processes , multiple particles move along the same stationary path with little to no deviations , making kymographs a very useful representation of their dynamics .", "Hence , kymographs have been widely employed to visualise biological processes across different length scales , ranging from diffusion and transport of single molecules to whole cell movements ( Twelvetrees et al . , 2016; Barry et al . , 2015 ) .", "The analysis of these kymographs only requires tracing lines in 2D images , a rather simple task compared to the more general approach of particle tracking , where one has to identify the centre of the particles in each frame , and then correctly assign these coordinates to corresponding particles across frames .", "Publicly available kymograph analysis software simplifies the tedious and time-consuming task of tracing kymographs , but most of these solutions require manual supervision and/or high signal to noise ratios ( Neumann et al . , 2017; Mangeol et al . , 2016; Chenouard , 2010; Zala et al . , 2013 ) .", "These tools perform reasonably well when applied to particles with unidirectional motion and uniform velocities as , for example , growing microtubule +ends ( Figure 1C , example 2 ) and F-actin dynamics in retrograde actin flow ( Lazarus et al . , 2013; del Castillo et al . , 2015; Alexandrova et al . , 2008; Babich et al . , 2012 ) .", "In many other biological contexts , however , particles can stop moving , change velocity , change direction , merge , cross each other’s path , or disappear for a few frames .", "The kymographs obtained from these processes exhibit ‘bidirectional’ motion ( Figure 1C , example 1 ) ; this category includes cellular transport processes , for example molecular or vesicle transport in neuronal axons and dendrites ( Faits et al . , 2016; Tanenbaum et al . , 2013; Koseki et al . , 2017 ) .", "Thus , the problem of automatically and reliably tracking dynamic processes in kymographs still leaves substantial room for improvement , and given the limitations of currently available kymograph analysis software , most kymographs are still analysed by hand , which is slow and prone to unconscious bias .", "In recent years , Machine Learning ( ML ) , and particularly Deep Neural Networks , have been very successfully introduced to data processing in biology and medicine ( Mathis , 2018; Weigert , 2017; Florian , 2017; Guerrero-Pena , 2018; Falk et al . , 2019; Bates , 2017 ) .", "ML-based image analysis has several advantages over other approaches: it is less susceptible to bias than manual annotation , it takes a much shorter time to analyse large datasets , and , most importantly , it comes closer to human performance than conventional algorithms ( Mathis , 2018 ) .", "Most ML approaches to image analysis utilise Fully Convolutional Deep Neural Networks ( FCNs ) that were shown to excel at object detection in images ( Dai , 2016; Szegedy , 2014; LeCun et al . , 1989; Falk et al . , 2019 ) .", "Through several rounds of optimisation , FCNs select the best possible operations by exploiting a multitude of hidden layers .", "These layers apply image convolutions using kernels of different shapes and sizes , aiming to best match the output of the neural network to the provided training data labels , which were previously derived from manual annotation .", "This means that the network learns to interpret the images based on the available data , and not on a priori considerations .", "This approach has become possible due to the dramatic improvements in computation times of modern CPUs and the adoption of GPUs that can execute an enormous number of operations in parallel .", "Currently , the most successful architecture for biological and medical image analysis is the U-Net , which takes an input image to generate a binary map that highlights objects of interest based on the training data ( Ronneberger et al . , 2015 ) .", "Here we present KymoButler , a new stand-alone FCN software based on the U-Net architecture , to automatically and reliably extract particle tracks from kymographs .", "The software is packaged into an easy-to-use web interface ( http://kymobutler . deepmirror . ai ) and a downloadable software package , and it was benchmarked against traditional software and manual annotation on synthetic ( i . e . , ground truth ) and real ( biological ) data .", "We show that KymoButler performs as well as manual annotation on challenging bidirectional kymographs , where particles disappear , reappear , merge , cross each other’s path , move in any direction , change speed , immobilise , and reverse direction .", "KymoButler thus represents a substantial improvement in the automation of kymograph tracing , speeding up the experimental workflow , while preserving the accuracy of manual annotations ." ], [ "For our FCN-based kymograph analysis software , we implemented a customised architecture based on U-Net ( Ronneberger et al . , 2015 ) .", "This architecture comprises two segmentation networks ( ‘modules’ ) , one specialised on kymographs with exclusively unidirectional particle movements , the other one on bi-directional kymographs .", "These segmentation networks were trained to binarize the image into regions with particle tracks ( foreground ) and noise ( background ) .", "They take an input kymograph to generate 2D maps that assign a ‘trackness’ value between 0 and 1 to each pixel of the input image , with higher values representing a higher likelihood of pixels being part of a track ( see Materials and methods ) .", "Our training ( 95% ) and validation ( 5% ) data consisted of manually annotated tracks in 487 unidirectional and 79 bidirectional kymographs ( unpublished data from our group and other laboratories , see Materials and methods and Acknowledgements for details ) .", "Since no ground truth was available in the manually annotated kymographs , we also generated 221 synthetic unidirectional and 21 synthetic bidirectional kymographs that were used for training .", "The unidirectional segmentation module generates separate trackness maps for tracks with negative and positive slopes ( which could , for example , correspond to tracks of anterograde and retrograde transport processes , respectively ) , to remove line crossings from the output ( see Materials and methods ) .", "Since particles have uniform speeds , individual tracks can be extracted via binarization of the trackness map .", "In bidirectional kymographs , tracks show more complex morphologies , since they can change direction/speed and cross each other multiple times .", "The bidirectional segmentation module therefore generates a single trackness map , which needs to be further processed in order to obtain individual particle tracks .", "In particular , one has to resolve crossings between tracks .", "We did this by implementing a decision module which iterates through all crossings to find the most likely final segmentation ( see Materials and methods for details ) .", "We found the binarization thresholds for both modules to depend on the biological application and on the signal to noise ratio of the input image .", "However , we observed the best performance for both segmentation modules generally for values between 0 . 1–0 . 3 ( Figure 1—figure supplement 4 ) .", "Finally , our software has to decide whether to analyse a given kymograph with the unidirectional module or the bidirectional module .", "Therefore , we implemented a ‘classification module’ that classifies input kymographs into unidirectional or bidirectional ones .", "We linked the class module to the unidirectional and bidirectional segmentation modules as well as to the decision module and packaged them into KymoButler , an easy-to-use , drag and drop browser-based app for quick and fully automated analysis of individual kymographs ( http://kymobutler . deepmirror . ai ) .", "The only free parameter in KymoButler is the threshold for trackness map segmentation .", "The default threshold is set to 0 . 2 , but users can freely adjust it for their specific application .", "After the computation , which takes 1–20 s per kymograph ( depending on complexity ) , KymoButler generates several files including a dilated overlay image highlighting all the tracks found in different colours , a CSV file containing all track coordinates , a summary file with post processing data , such as average velocities and directionality , and preliminary plots of these quantities ( Figure 1B ) .", "KymoButler worked well on previously published kymographs from a variety of different biological data ( Figure 1C and Figure 1—figure supplement 1A ) and on unpublished data from collaborators ( Figure 2—figure supplement 1B and Figure 3—figure supplement 2 ) .", "We quantitatively evaluated the performance of KymoButler on unidirectional kymographs , that is particles that move with mostly uniform velocities and with no change in direction ( Figure 1C , Figure 2 , Figure 1—figure supplement 1A ) .", "The unidirectional module of KymoButler was compared to an existing kymograph analysis software , which is based on Fourier filters , and which provided the best performance among publicly available software in our hands ( KymographDirect package; Mangeol et al . , 2016 ) .", "Additionally , we traced kymographs by hand to obtain a control for the software packages .", "First , we generated 10 synthetic movies depicting unidirectional particle dynamics with low signal-to-noise ratio ( ~1 . 2 , see Materials and methods ) and extracted kymographs from those movies using the KymographClear ( Mangeol et al . , 2016 ) Fiji plugin .", "Each of the kymographs was then analysed by Fourier-filtering ( KymographDirect ) , KymoButler , and by hand , and the identified trajectories overlaid with the ground truth ( i . e . , the known dynamics of the simulated data ) ( Figure 2A ) .", "KymoButler typically took less than a minute to analyse the 10 kymographs while fourier filtering took about 10 min since thresholds had to be set individually for each image .", "Manual annotation by an expert took about 1 . 5 hr .", "To quantify the quality of the predicted traces , we first determined the best predicted track for each ground truth track ( in case several segments were predicted to cover the same track ) and then calculated the fraction of the length of the ground truth track that was correctly identified by that predicted track ( ‘track recall’ ) ( Figure 2B ) .", "Additionally , we determined the best overlapping ground truth track for each predicted track and then calculated the fraction of the length of the predicted track that was overlapping with the ground truth track ( ‘track precision’ ) .", "Examples of low/high precision and low/high recall are shown in Figure 2B .", "We then calculated the geometric mean of the average track recall and the average track precision ( the ‘track F1 score’ , see Materials and methods ) for each kymograph ( Figure 2E ) .", "The median F1 score of the manual control was 0 . 90 , KymoButler achieved 0 . 93 , while Fourier filtering achieved a significantly lower F1 score of 0 . 63 ( p=4⋅10-5 , Kruskal-Wallis Test , Tukey post-hoc: manual vs KymoButler p=0 . 6 , manual vs Fourier Filtering p=3⋅10-3 ) .", "Our synthetic data intentionally included gaps of exponentially distributed lengths ( see Materials and methods ) , allowing us to quantify the ability of KymoButler to bridge gaps in kymograph tracks ( Figure 2C , F ) , which are frequently encountered in kymographs extracted from fluorescence data ( Applegate et al . , 2011 ) .", "Both KymoButler and manual annotation consistently bridged gaps that belonged to the same trajectory , while Fourier filtering was less accurate ( 89% of all gaps correctly bridged by KymoButler , 88% by manual , and 72% by Fourier filter analysis; median of all 10 synthetic kymographs , p=10-4 , Kruskal-Wallis Test , Tukey post-hoc: manual vs KymoButler p=0 . 9 , manual vs Fourier Filtering p=2⋅10-3 , Figure 2F ) .", "We also quantified the ability of KymoButler to resolve track crossings ( Figure 2D ) .", "Again , both KymoButler and manual annotation performed significantly better than Fourier filtering ( 88% KymoButler , 86% manual , 60% Fourier filter; median percentage of correctly resolved crossings of all 10 synthetic kymographs , p=10-4 , Kruskal-Wallis Test , Tukey post-hoc: manual vs KymoButler p=0 . 9 , manual vs Fourier Filtering p=1⋅10-3 , Figure 2G ) .", "Remarkably , KymoButler was able to correctly pick up ~80% of all tracks at an SNR as low as 1 . 1 , where tracks are barely visible by eye ( Figure 1—figure supplement 5B , D ) , and at high particle densities ( ~70% of the kymograph image covered with signal ) ( Figure 1—figure supplement 5F , H ) .", "Manual annotation at such extremes performed similarly to KymoButler ( Figure 1—figure supplement 5C , D , G , H ) .", "Finally , we compared KymoButler’s overall performance on kymographs containing unidirectional traces with that of alternative analysis approaches .", "KymoButler performed similar to or better than manual annotation of synthetic data when analysing particle velocities , directionality , travel time , travel distance , and particle numbers , while the Fourier filter frequently deviated by more than 50% from ground truth averaged values ( Figure 2—figure supplement 1A ) .", "When testing KymoButler’s overall performance on real kymographs of our validation data set , we compared deviations from manual annotation as no ground truth was available .", "KymoButler deviated by less than 10% from most manual estimates ( but found ~30% more particles ) , while the Fourier filter approach deviated by up to 50% from the manually calculated values ( Figure 2—figure supplement 1B ) .", "In summary , KymoButler was able to reliably track particle traces in kymographs at low SNR and high particle densities in both synthetic and real data , and it clearly outperformed currently existing automated software , while being as consistent as manual expert analysis while being ~100 x faster .", "As in many kymographs obtained from biological samples trajectories are not unidirectional , we also tested the performance of KymoButler on complex bidirectional kymographs , that is of particles with wildly different sizes , velocities , and fluorescence intensities that frequently change direction , may become stationary and then resume motion again ( see Figure 1B , C , Figure 3A , Figure 1—figure supplement 1A for examples ) .", "Available fully automated software that relied on edge detection performed very poorly on our synthetic kymographs ( Figure 3—figure supplement 1 ) .", "Therefore , we implemented a custom-written wavelet coefficient filtering algorithm to compare our FCN-based approach to a more traditional non-ML approach ( Figure 3A , Figure 3—figure supplement 1 , Materials and methods ) .", "In short , the wavelet filtering algorithm generates a trackness map , similar to KymoButler , by applying a stationary wavelet transform to the kymograph to generate so-called ‘coefficient images’ that highlight horizontal or vertical lines .", "These coefficient images are then overlaid and binarized with a fixed value ( 0 . 3 ) , skeletonised , and fed into the KymoButler algorithm without the decision module , that is crossings are resolved by linear regression prediction .", "We generated 10 kymographs from our synthetic movies with the KymographClear package ( average signal-to-noise ratio was 1 . 4 , since any lower signal generally obscured very faint and fast tracks ) .", "Each of the kymographs was then analysed by wavelet coefficient filtering , KymoButler , and manual annotation , and the predicted traces overlaid with the ground truth ( Figure 3A ) .", "While the wavelet approach and KymoButler were able to analyse the 10 kymographs in less than 1 min , manual annotation by an expert took about 1 . 5 hr .", "Moreover , whereas the manual annotation and KymoButler segmentation overlaid well with the ground truth , the wavelet approach yielded numerous small but important deviations .", "Similarly to the unidirectional case , we quantified track precision and recall ( Figure 3B , E ) and calculated the resolved gap fraction ( Figure 3C , F ) and crossing fraction ( Figure 3D , G ) .", "The median of the track F1 scores per kymograph for manual annotation ( 0 . 82 ) was similar to KymoButler ( 0 . 78 ) , while the wavelet filter approach only gave 0 . 61 ( p=7⋅10-5 , Kruskal-Wallis Test , Tukey post-hoc: manual vs KymoButler p=0 . 3 , manual vs wavelet filtering p=10-4 , Figure 3E ) .", "While gaps were resolved by KymoButler and manual annotation in 86% and 95% of cases , respectively , only 63% were resolved by the wavelet algorithm ( median of all 10 synthetic kymographs , p=4⋅10-5 , Kruskal-Wallis Test , Tukey post-hoc: manual vs KymoButler p=0 . 2 , manual vs wavelet filtering p=10-5 , Figure 3F ) .", "Crossings were rarely resolved correctly by the wavelet algorithm ( 13% ) but much more reliably by KymoButler ( 59% ) and manual annotation ( 76% ) ( median of all 10 synthetic kymographs , p=3⋅10-5 , Kruskal-Wallis Test , Tukey post-hoc: manual vs KymoButler p=0 . 4 , manual vs wavelet filtering p=4⋅10-3 , Figure 3G ) .", "As bidirectional tracks varied in intensity , lower SNRs obscured faint tacks so that performance dropped slightly faster with decreasing SNR than for unidirectional tracks ( Figure 1—figure supplement 5A , D ) , and it decreased linearly for increasing particle densities ( Figure 1—figure supplement 5E , H ) .", "Manual annotation at high signal densities and low SNRs again yielded similar results as KymoButler ( Figure 1—figure supplement 5C , D , G , H ) .", "The analysis of quantity averages of 10 synthetic kymographs revealed that KymoButler was as accurate as manual annotation , while the wavelet filter deviated significantly more from the ground truth ( Figure 3—figure supplement 2A ) .", "We also tested KymoButler on our validation dataset .", "Results were similar to the performance on synthetic data ( ~10% deviation from manual annotation ) , and the wavelet filter performed significantly worse than KymoButler ( Figure 3—figure supplement 2B ) .", "Overall , these results showed that KymoButler performs well on both unidirectional and bidirectional kymographs , clearly outperforms currently available automated analysis of kymographs , and it performs as well as manual tracing , while being much faster and not prone to unconscious bias ." ], [ "In this work , we developed software based on Deep Learning techniques to automate the tracking of dynamic particles along a stationary path in a noisy cellular environment .", "Convolutional neural networks ( CNNs ) are nowadays widely applied for image recognition .", "Since tracking is a priori a visual problem , we built a modular software utilising CNNs for identifying tracks in kymographs .", "We deployed our networks as KymoButler , a software package that takes kymographs as inputs and outputs all tracks found in the image in a matter of seconds .", "The network outperforms standard image filtering techniques on synthetic data as well as on kymographs from a wide range of biological processes , while being as precise as expert manual annotation .", "The KymoButler software has only one adjustable detection parameter that is left to the user: a sensitivity threshold that , if low , allows more ambiguous tracks to be recognised , and if high discards them .", "For our synthetic data , the best value for the threshold lay between 0 . 1 and 0 . 3 ( Figure 1—figure supplement 4 ) , and we observed a similar range for a variety of kymographs from published data .", "However , the threshold depends on the SNR of the input images , so that the correct threshold has to be chosen based on each biological application and imaging conditions .", "Furthermore , the performance of both KymoButler and manual annotation decreased with decreasing SNR and increasing particle density ( number of crossings in the image , Figure 1—figure supplement 4 ) .", "Note that the particle density here also depends on the particle’s frequency of change in direction in dense kymographs as more bidirectional particles tend to cover larger proportions of the kymograph image .", "Hence , we strongly recommend to visually inspect the output of KymoButler for each new application , and to compare the output to manual annotation .", "Most of the publicly available kymograph analysis software requires manual labelling to extract quantitative data ( Chenouard , 2010; Neumann et al . , 2017; Zala et al . , 2013 ) .", "Some automated approaches have been published in the context of specific biological questions , but since these programs are currently not publicly available it is not clear how well they would perform on kymographs from other applications ( Mukherjee et al . , 2011; Reis et al . , 2012 ) .", "Other approaches do not extract individual tracks but only macroscopic quantities , as for example velocities ( Chan and Odde , 2008 ) .", "As KymoButler is fully automated and able to reliably analyze kymographs from a wide range of biological applications , it fills an important gap .", "Here we showed that KymoButler is able to quantify mitochondria movement in neuronal dendrites , microtubule growth dynamics in axons , and in vitro dynamics of single cytoplasmic dynein proteins ( Figure 1 and Figure 1—figure supplement 1 ) .", "The training and validation data for KymoButler comprised kymographs depicting the dynamics of microtubule +ends , mitochondria movement , molecular motor movements , and vesicle transport in neuronal processes ( example kymographs: Lazarus et al . , 2013; Cioni et al . , 2019; Hangen et al . , 2018; Gerson-Gurwitz et al . , 2011 ) .", "Hence , KymoButler will perform best on similar data .", "However , we predict that it can furthermore be applied to most other kymographs obtained from time-lapse fluorescence microscopy without the need of any modifications .", "KymoButler outperformed Fourier filtering , edge detection , and customised wavelet coefficient selection on synthetic kymographs .", "While Fourier filtering ‘only’ performed ~30% worse than KymoButler on synthetic unidirectional kymographs , edge detection on synthetic bidirectional kymographs suffered greatly from background fluctuations and low SNR to such an extent that the extracted data was unusable ( see Figure 3—figure supplement 1 for one example ) .", "Therefore , we designed a filtering algorithm based on wavelet coefficient image selection to analyse complex bidirectional kymographs specifically for our synthetic data .", "KymoButler still performed 20% better than this approach ( Figure 3 ) .", "The main problem with either filtering approach compared to KymoButler was their inability to bridge track gaps and resolve line crossings , both of which occur frequently in biological data ( Figures 2C , D and 3C , D ) .", "These challenges were met by KymoButler , which performed as well as expert annotation , but within a much shorter time ( Figures 2 and 3 ) .", "Consequently , KymoButler generated similar measures of averaged quantities ( average velocities , displacements , etc . ) as manual annotation ( Figure 2—figure supplement 1A and Figure 3—figure supplement 2A ) .", "Synthetic kymographs , however , will reproduce the complexity of real kymographs only to some degree , as they exhibit homogenous background , no artefacts , no varying particle intensity in time , and individual tracks can appear rather similar .", "Hence , we also benchmarked KymoButler for both the unidirectional and bidirectional ( real ) validation datasets .", "KymoButler calculated similar average quantities as obtained from manual annotation , such as average particle velocities and pausing times , with some minor deviations ( Figure 2—figure supplement 1B and Figure 3—figure supplement 2B ) .", "However , since values obtained from manual annotation deviated by up to 20% from ground truth values on synthetic data , deviations from manual annotation should not automatically be interpreted as an erroneous deviation ( Figure 2—figure supplement 1 and Figure 3—figure supplement 2 ) .", "Our results show that KymoButler is able to correctly identify individual full-length tracks in kymographs with an average track F1 score ( geometric mean of track precision and recall ) of 92% on unidirectional tracks and 78% on complex bidirectional tracks ( similar to manual annotation ) , without suffering from inconsistency , bias , and laborious tracing , that plague manual tracking .", "While KymoButler is already performing very well , we aim to significantly improve it over future iterations .", "Every time someone uses our webform , s/he has the option to anonymously upload the kymograph to our cloud .", "Once a large number of diverse kymographs is uploaded , these kymographs will be annotated by us and added to our training data , improving KymoButler even further .", "The ultimate challenge will be to expand our approach to 2D or even 3D tracking problems .", "Here , we defined a 1D region of interest in 2D time-lapse movies , extracted 2D ( space and time ) images ( kymographs ) , and finally tracked 2D lines in those images .", "A similar , albeit computationally heavier , approach could stack the frames of a 2D/3D movie on top of each other to generate a 3D/4D kymogram ( 2D space and time , or 3D space and time ) .", "Previously generated kymograms have led to intriguing results on whole-cell particle tracking problems with high SNR ( Racine et al . , 2007 ) .", "The use of higher dimensional FCNs in the future has great potential to yield human-like performance on any biological and medical tracking problems ." ], [ "The KymoButler software was implemented in Mathematica to take advantage of easy web form deployment and distribution .", "The workflow is shown in Figure 1—figure supplement 1B .", "Our approach was to first segment kymograph pixels that are part of particle tracks from pixels that were part of the background with our segmentation modules .", "From previous work we knew that kymographs that depict unidirectional movement only , can be filtered into tracks that have positive slope and those that have negative slope ( Chenouard , 2010 ) , while no such assumptions can be made about bidirectional kymographs .", "Hence , we decided to take advantage of this simplification of unidirectional kymograph analysis by training two modules: one that is specialised to segment unidirectional kymographs and another one that segments bidirectional ones .", "Note that the bidirectional module is able to analyze any kymograph , including unidirectional ones , but since it is not specialised it performs slightly worse than the unidirectional module on unidirectional kymographs .", "To further simplify software usability , we prepended a class module that classifies input kymographs as bidirectional or unidirectional , and then applies the corresponding segmentation module and , if bidirectional , decision module .", "Our downloadable software package on GitLab allows the user to call either segmentation module ( unidirectional/bidirectional ) directly , if they wish to do so .", "When the kymograph is classified as unidirectional by the class module , the unidirectional segmentation module generates two trackness score maps for particles with negative or positive slope ( Figure 1—figure supplement 1B ) .", "Since the particles move with roughly the same velocity , the resulting maps mostly do not exhibit any crossings .", "Thus , we binarize the maps with a threshold between 0 . 1–0 . 3 ( see benchmarking section for more information about the threshold ) .", "The resulting binary maps are then thinned iteratively so that each trace is only one pixel wide at any point and pruned so that branches that are shorter than three pixels are deleted .", "Subsequently , each trace is segmented and selected only if they are at least three frames long and three pixels large ( these values can be varied by the user if needed but were kept constant throughout this manuscript for unidirectional kymographs ) .", "In the final step , pixels that lie in the same row of the kymograph are averaged over so that the final track has only one entry per frame .", "For bidirectional kymographs the software generates a trackness map , applies a binarization threshold ( 0 . 1-0 . 3 , see benchmarking for more details ) , iterative thinning , and pruning ( minimum length 3 pixels ) .", "Similar to the unidirectional case , our software allows the selection of tracks with a minimum number of pixels and/or frames .", "However , since the resulting skeletonised map had a substantial number of crossings and could not be easily segmented to yield individual tracks , we implemented a further module in the software .", "First , all lines in the skeletonised map are shortened so that each white pixel at a track end only has neighbouring pixels in different rows ( time dimension ) .", "This was done so that we could detect track starting points ( ‘seeds’ ) with a Hit-Miss transformation with kernel: -1-1-1-11-1000 .", "Application of this kernel yielded a binary map with 0 everywhere except at track seeds ( Figure 1—figure supplement 1B , red dots ) .", "These seeds were then used to start tracing individual tracks in the kymograph by always advancing to the next white pixel .", "Once more than one potential future pixel is encountered , the decision module is called .", "The module generates three 48x48 crops of ( 1 ) the input kymograph , ( 2 ) the skeletonised trackness map , and ( 3 ) the skeleton of the current track and predicts a trackness map that has high values on the skeleton segment of the most likely future track ( Figure 1—figure supplement 1B ) .", "This map is binarized with threshold 0 . 5 and thinned .", "The precise threshold had little effect on the final output , so we fixed it at 0 . 5 for all applications .", "Users can vary this threshold as well in the source code on GitLab .", "Next , the largest connected component in the map is selected as the most likely future path and appended to the track if longer than 2 pixels .", "The average trackness value of this component ( from the decision module prediction ) is saved as a measure of decision ‘confidence’ .", "This process is repeated until no further possible pixels are found or no future path is predicted which is when the track is terminated .", "Once all seeds are terminated , the software subtracts all the found paths from the skeletonised trackness map and again looks for new seeds which are then again tracked in the full skeletonised image .", "The process is repeated until no further seeds are found , and then all tracks are averaged over their timepoints ( rows in the kymograph image ) .", "Subsequently the software deletes tracks that are shorter than 5 pixels or part of another track and assigns overlaps that are longer than 10 pixels to the track with the highest average decision confidence .", "Both the unidirectional and the bidirectional module output a coloured overlay in which each track is drawn in a different randomly assigned colour and dilated with factor one for better visibility ( see Figure 1B–C and Figure 1—figure supplement 1A ) .", "Additionally , the software generates one CSV file that contains all the track coordinates , a summary CSV file that gives derived quantities , such as track direction and average speed , and plots depicting these quantities .", "The software was deployed from Mathematica as a cloud based interface ( http://kymobutler . deepmirror . ai ) and a Mathematica package ( https://gitlab . com/deepmirror/kymobutler; copy archived at https://github . com/elifesciences-publications/KymoButler ) .", "Our networks were built from convBlocks ( a convolutional layer with 3 × 3 kernel size , padding , and arbitrary number of output channels followed by a batch normalisation layer and a ‘leaky’ ramp ( leakyReLU ) activation function ( leakyReLU ( x ) :=max ( x , 0 ) -0 . 1 max ( -x , 0 ) ) .", "Batch normalisation is useful to stabilise the training procedure as it rescales the inputs of the activation function ( leakyReLu ) , so that they have zero mean and unit variance .", "The leakyReLu prevents the so-called ‘dead ReLu’s’ by applying a small gradient to values below 0 .", "These building blocks were previously used for image recognition tasks in Google’s inception architecture and in the U-Net architecture ( Szegedy , 2014; Falk et al . , 2019 ) .", "The module architectures we settled on are shown in Figure 1—figure supplements 1–2 .", "All modules used the same core building blocks while having different input and output ports .", "The classification module takes a resized kymograph of size 64 × 64 pixels and generates two output values that correspond to the class probabilities for unidirectional/bidirectional kymographs ( Figure 1—figure supplement 2A ) .", "The unidirectional segmentation module takes one input kymograph and generates two output images that correspond to the trackness scores of particles with positive or negative slopes ( Figure 1—figure supplement 2B ) .", "The bidirectional segmentation module takes one input kymograph and generates one trackness score map highlighting any found particle tracks ( Figure 1—figure supplement 2C ) .", "Finally , the decision module takes three inputs of size 48 × 48 pixels to generate one trackness map ( Figure 1—figure supplement 2D ) .", "All modules share the same core network that is essentially a U-Net with padded convolutions and with 64 ( in the top level ) to 1024 ( in the lowest level ) feature maps .", "We experimented with more complex architectures ( parallel convolution modules instead of blocks , different number of feature maps ) but could only observe minor increase in accuracy at a large expense in computation time .", "Due to the U-Net architecture , each dimension of the inputs to the segmentation modules needs to be a multiple of 16 .", "Thus , inputs were resized when they did not match the dimension requirements , and then the binarized output images from the segmentation modules were resized to the original input image size before proceeding further .", "To train the networks we quantified the difference between their output o and the desired target output t through a cross entropy loss layer ( CEloss ( t , o ) =- ( t⋅ln ( o ) + ( 1-t ) ⋅ln ( 1-o ) ) .", "The loss was averaged over all output entries ( pixels and classes ) of each network .", "While we tried other loss functions , specifically weighted cross entropy loss and neighbour dependent loss as described in Bates ( 2017 ) , we persistently obtained higher track precision and track recall with the basic cross entropy loss above .", "Our training data comprised a mixture of synthetic data and manually annotated unpublished kymographs , kindly provided by the research groups mentioned in the acknowledgements .", "Most of the manual annotation was done by M . A . H . J . and A . D . In total , we used 487 ( +200 synthetic ) unidirectional , and 79 ( +21 synthetic ) bidirectional kymographs , with 95% of the data used for network training , and ~5% of retained for network validation .", "All network training was performed on a workstation , using a nVidia 1080 Ti or a nVidia 1070 GPU .", "The class module depicted in Figure 1—figure supplement 2A was trained with batches of size 50 ( with 25 unidirectional and 25 bidirectional kymographs to counter class imbalance ) with random image transformations that included image reflections , rotations , resizing , colour negation , gaussian noise , random noise , and random background gradients .", "The final input image was randomly cropped to 64 × 64 pixels ( see examples Figure 1—figure supplement 3A ) and the class module was trained using stochastic gradient descent ( ADAM optimiser , Kingma , 2017 , initial learning rate 0 . 001 ) , until the validation set error rate was consistently 0% .", "The unidirectional segmentation module ( Figure 1—figure supplement 2B ) was trained with batches comprising 20 randomly selected kymographs from our training set ( example in Figure 1—figure supplement 3B ) .", "We applied the following image transformations: Random reflections along either axis , random 180-degree rotations , random cropping to 128 × 80 pixels ( approximately the size of our smallest kymograph ) , random gaussian and uniform noise , and random background gradients .", "Note that we did not apply any resizing to the raw kymograph since that generally decreased net performance .", "Additionally , we added Dropout Layers ( 10–20% ) along the contracting path of our custom U-Net to improve regularisation .", "Each kymograph in this training set was generated by hand with KymographTracker ( Chenouard , 2010 ) , but to increase dataset variability we took the line profiles from KymographTracker and generated kymographs with a custom Mathematica script that applied wavelet filtering to the plotted profiles .", "The resulting kymographs have a slightly different appearance than the ones created with KymographTracker and are thus useful to regularise our training process .", "Several modules were trained until convergence and the best performing one ( according to the validation score ) was selected ( ADAM optimiser , initial learning rate of 0 . 001 , learning rate schedule = If[batch<4000 , 1 , . 5] ) .", "The bidirectional segmentation module ( Figure 1—figure supplement 2C , example data Figure 1—figure supplement 3C ) was trained in the same way as the unidirectional segmentation module , with the exception of a slightly different learning rate schedule ( If[batch<3000 , 1 , . 5] ) .", "Additionally , since we did not have access to many of the original movies from which the kymographs were generated , we could not generate kymographs with different algorithms as done for the unidirectional module .", "Training data for the decision module ( Figure 1—figure supplement 2D ) was obtained from the bidirectional ( synthetic +real ) kymographs by first finding all the branch points in a given ground truth or manually annotated image .", "Then , each track was separated into multiple segments , that go from its start point to a branching point or its end point .", "For each branchpoint encountered while following a track , all segments that ended within 3 pixels of the branchpoint were selected .", "Then , ( 1 ) a 48 × 48 pixel crop of the raw kymograph around the branchpoint , ( 2 ) a binary map representing the track segment upstream of the branching point ( centred with its end in pixel coordinates 25 , 25 , with image padding applied if the end was close to an image corner ) , and ( 3 ) the corresponding 48 × 48 pixel region in the binary image representing all possible paths were used as inputs to the decision module .", "The binary image representing the ground truth or annotated future segment downstream of the branchpoint was used as the target image ( see Figure 1—figure supplement 3D for an example training set ) .", "Thus , the training set comprised three input images and one output image which we used to train the decision module .", "To increase the module’s focus on the non-binary raw kymograph crop , we applied 50% dropout to the full skeletonised input and 5% dropout to the input segment .", "As explained above , we used random image augmentation steps like reflections , rotations , gaussian +uniform noise .", "Additionally , we employed random morphological thinning to the binary input/output images to simulate artefacts .", "Several networks were trained until convergence ( pixel wise cross entropy loss , ADAM optimiser , initial learning rate 0 . 001 , batch size 50 , learning rate schedule If[batch<8000 , 1 , . 5] ) , and the best performing one was selected .", "Synthetic data was generated by simulating individual particles on a stationary path of length 300 pixels for 300 frames to generate 300 × 300 pixel kymographs .", "To obtain unidirectional particles we seeded 30 + 30 particles with negative or positive slope at random timepoints/positions .", "Next , a random velocity between 1–3 pixels/frame was chosen for all particles in the movie , with a random noise factor to allow slight changes in velocity , and a particle PSF between 3–6 pixels .", "Each particle was assigned a survival time drawn from an exponential distribution with scale 0 . 01 , after which it would disappear .", "Gaps of random length ( exponentially distributed ) were subsequently assigned to each track individually .", "From these tracks we then generated a kymograph with gaussian noise , used for neural network training , and a 20 × 300 pixel movie with 300 frames for benchmarking .", "The resulting kymographs and movies had an average signal-to-noise ratio of 1 . 2 ( calculated as the average intensity of the signal , divided by the average intensity of the background ) .", "Finally , we removed tracks that overlapped for the whole duration of their lifetime .", "To obtain synthetic data of complex bidirectional particle movements , we generated datasets with either 15 tracks ( for benchmarking ) or 30 tracks ( for training ) per movie .", "The maximum velocity was set to three pixels/frame , as above this velocity it became hard to manually segment tracks from kymographs .", "Each movie was assigned a random velocity noise factor between 0 and 1 . 5 pixels/frame , a random switching probability between 0 and 0 . 1 ( to switch between stationary and directed movement ) and a random velocity flipping factor between 0 and 0 . 1 ( to flip the direction of the velocity ) .", "Individual particles were simulated by first calculating their lifetime from an exponential distribution with scale 0 . 001 .", "Then , a random initial state , moving or stationary , was selected as well as a random initial velocity and a particle size between 1–6 pixel .", "In the simulation , particles could randomly switch between different modes of movement ( stationary/directed ) , flip velocities and were constantly subjected random velocity noise ( movie specific ) .", "Finally , tracks that were occulted by other tracks were removed , and a movie ( used for benchmarking ) and a kymograph ( used for training ) were generated .", "The resulting kymographs and movies had an average signal-to-noise ratio of 1 . 4 .", "In order to benchmark the performance of software and manual predictions , we implemented a custom track F1 score which was calculated as the geometric mean of track recall and track precision .", "To calculate track recall , each ground truth track was first compared to its corresponding predicted track , and the fractional overlap between them was calculated .", "Since predicted tracks do not necessarily follow the exact same route through a kymograph , but frequently show small deviations from the ground truth ( see Figure 3 and Figure 3—figure supplement", "1 ) we allowed for a 3 . 2-pixel deviation from the ground truth ( two diagonal pixels ) .", "The maximum fractional overlap was then selected and stored as the track recall .", "The recall was thus one when the full length of a ground truth track was predicted , and 0 if the track was not found in the prediction .", "We would like to highlight that this criterion is very strict: if a ground truth track is predicted to be two tracks ( for example , by failing to bridge a gap along the track ) , the recall fraction would decrease by up to 50% , even if most of the pixels are segmented correctly and belong to predicted tracks .", "Track precision was calculated by finding the largest ground truth track that corresponded , that is had the largest overlap , to each prediction , and then calculating the fraction of the predicted track that overlapped to the ground truth track .", "Therefore , a track precision of 1 corresponded to a predicted track that was fully part of a ground truth track while a precision of 0 meant that the predicted track was not found in the ground truth .", "In general , increasing precision leads to a lower recall and vice versa , so that taking the track F1 score as the geometric mean between the two is a good measure of overall prediction performance .", "To quantify gap performance , we searched for track segments within 3 pixels of the gap for each frame , to allow for predictions that deviated slightly from the ground truth .", "Once each frame of the gap was assigned to a corresponding predicted segment , the gap was deemed resolved .", "If one or more frames of the gap had no overlapping segment to the prediction , the gap was labelled unresolved .", "Our synthetic tracks had 954 gaps in the 10 kymographs of unidirectional data , and 840 gaps in the 10 kymographs of bidirectional data , and the largest gap size was six pixels .", "For each kymograph , we then calculated the fraction of gaps resolved .", "To quantify KymoButler performance on crossings , we first generated binary images for each ground truth track and calculated overlaps with other ground truth tracks by multiplying those images with each other .", "The resulting images had white dots wherever two tracks crossed .", "Those dots were then dilated by a factor of 16 to generate circles and overlaid with the original single-track binary image to generate binary maps that contain segments of ground truth tracks that cross/merge with other tracks .", "Next , we generated dilated ( factor", "1 ) binary maps for each predicted track and multiplied them with each of those cross segments to obtain the largest overlapping track for each segment .", "We then visually inspected a few examples and determined that an overlap of 70% corresponds to a correctly resolved crossing and allowed for slight variations in predicted tracks when compared to ground truth .", "Finally , we calculated the fraction of crossings resolved per kymograph .", "Derived quantities were calculated as follows .", "For average velocities , we first calculated the absolute frame to frame displacement and from there the average frame to frame velocity per track and the average frame to frame velocity per kymograph .", "The absolute displacement was calculated as a sum of all absolute frame to frame displacements and then averaged to yield a measure per kymograph .", "The travel time was calculated as the absolute time a particle was visible in a given kymograph and averaged for each kymograph .", "Directionality per particle was calculated as the sign of the end to end displacement for unidirectional kymographs .", "For bidirectional kymographs , we first calculated the directionality of up to five frame long segments , which was +1 when all displacements in that segment were positive , −1 when all displacements were negative , and 0 otherwise .", "The sign of the sum of all segment directionalities was taken as a measure for the bidirectional track directionality .", "The pause time for each bidirectional particle was calculated as the number of segments with 0 displacement and averaged per kymograph .", "Finally , the percentage of reversing tracks was calculated by dividing the number of tracks that exhibit segments in both directions by all tracks .", "In Figure 2—figure supplement 1 and Figure 3—figure supplement 2 , we only show relative deviations from the manual annotation because we cannot disclose any data from the real , unpublished kymographs obtained from collaborators .", "All statistical analysis was carried out in MATLAB using either a Wilcoxon rank-sum test or a Kruskal Wallis test ( http://mathworks . com ) .", "To benchmark the unidirectional segmentation module of KymoButler , we generated 10 synthetic movies of the dynamics of particles that move with uniform speed and do not change direction as described in the section about synthetic data generation .", "We then imported these movies into ImageJ ( http://imagej . nih . gov ) via the Kymograph Clear package ( Mangeol et al . , 2016 ) , drew a profile by hand and generated kymographs from them .", "These kymographs were then imported into the KymographDirect software package ( also Mangeol et al . , 2016 ) , Fourier filtered and thresholded to extract individual particle tracks .", "This approach required manual selection of the threshold for each individual kymograph .", "We additionally traced the same kymographs by hand in ImageJ to compare software performance to expert analysis .", "To find a suitable range of binarization thresholds for our unidirectional segmentation module we calculated the track wise F1 score on the 10 kymographs for thresholds between 0 . 05 and 0 . 5 ( Figure 1—figure supplement 4 ) .", "We observed the highest scores between 0 . 1 and 0 . 3 for both our synthetic data and other unpublished kymographs and also deemed these thresholds best by visual inspection of predicted kymograph tracks .", "Hence , we chose 0 . 2 as the segmentation map threshold to benchmark our predictions at .", "In order to benchmark the bidirectional segmentation module and the decision module we generated 10 synthetic movies of the dynamics of complex bidirectional particles .", "These movies were imported into ImageJ with the KymographClear package and kymographs extracted .", "We subsequently tried to use the edge detection option in KymographDirect to extract individual tracks but were unable to obtain meaningful tracks ( Figure 3—figure supplement 1 ) .", "We also tried other options in the package but could not get good results on our synthetic data without substantial manual labour for each kymograph , defeating the goal of a fully automated analysis .", "Therefore , we wrote a custom script to carry out automated bidirectional kymograph analysis .", "We experimented with a few different approaches ( for example fourier-filtering and customised edge detection ) and settled on wavelet coefficient filtering as it gave the highest F1 score on our test dataset .", "This algorithm applied a stationary wavelet transformation with Haar Wavelets ( Mathematica wavelet package ) to each kymograph to decompose the image into different coefficient images that highlight different details ( for example vertical or horizontal lines ) .", "We then selected only those coefficient images that recapitulated particle traces in our synthetic kymographs .", "These images are overlaid and thresholded with an optimised threshold to generate binary maps that can be iteratively thinned to obtain a skeletonized ‘trackness’ map similar to the outputs of our segmentation modules .", "This map was then traced with the same algorithm as in our decision module .", "However , while the KymoButler decision module used a neural network to predict path crossings , the wavelet filtering algorithm performed simple linear prediction by taking the dilated ( factor", "1 ) binary segment of a track and rotating it by 180 degrees .", "Then the ‘prediction’ was multiplied with the skeletonized trackness map and the largest connected component selected as the future path .", "In contrast to the original decision module , this approach does not yield any information about decision ‘confidence’ .", "Thus , to resolve track overlaps at the end of the algorithm , we randomly assigned each overlap to one track and deleted them from the others .", "Note that the wavelet approach was heavily optimised on our synthetic kymographs and performed poorly on generic real kymographs .", "We also traced the same 10 kymographs by hand in ImageJ .", "To find a suitable range of binarization thresholds for our bidirectional segmentation module we calculated the track wise F1 score for thresholds between 0 . 05 and 0 . 5 ( Figure 1—figure supplement 4 ) and observed the same optimal range as the unidirectional segmentation module ( 0 . 1–0 . 3 ) for both our synthetic data and other unpublished kymographs .", "Hence , we chose 0 . 2 as the threshold score to benchmark our predictions ." ] ]
[ "Kymographs are graphical representations of spatial position over time , which are often used in biology to visualise the motion of fluorescent particles , molecules , vesicles , or organelles moving along a predictable path .", "Although in kymographs tracks of individual particles are qualitatively easily distinguished , their automated quantitative analysis is much more challenging .", "Kymographs often exhibit low signal-to-noise-ratios ( SNRs ) , and available tools that automate their analysis usually require manual supervision .", "Here we developed KymoButler , a Deep Learning-based software to automatically track dynamic processes in kymographs .", "We demonstrate that KymoButler performs as well as expert manual data analysis on kymographs with complex particle trajectories from a variety of different biological systems .", "The software was packaged in a web-based ‘one-click’ application for use by the wider scientific community ( http://kymobutler . deepmirror . ai ) .", "Our approach significantly speeds up data analysis , avoids unconscious bias , and represents another step towards the widespread adaptation of Machine Learning techniques in biological data analysis ." ]
[ "Many molecules and structures within cells have to move about to do their job .", "Studying these movements is important to understand many biological processes , including the development of the brain or the spread of viruses .", "Kymographs are images that represent the movement of particles in time and space .", "Unfortunately , tracing the lines that represent movement in kymographs of biological particles is hard to do automatically , so currently this analysis is done by hand .", "Manually annotating kymographs is tedious , time-consuming and prone to the researcher’s unconscious bias .", "In an effort to simplify the analysis of kymographs , Jakobs et al . have developed KymoButler , a software tool that can do it automatically .", "KymoButler uses artificial intelligence to trace the lines in a kymograph and extract the information about particle movement .", "It speeds up analysis of kymographs by between 50 and 250 times , and comparisons show that it is as reliable as manual analysis .", "KymoButler is also significantly more effective than any previously existing automatic kymograph analysis programme .", "To make KymoButler accessible , Jakobs et al . have also created a website with a drag-and-drop facility that allows researchers to easily use the tool .", "KymoButler has been tested in many areas of biological research , from quantifying the movement of molecules in neurons to analysing the dynamics of the scaffolds that help cells keep their shape .", "This variety of applications showcases KymoButler’s versatility , and its potential applications .", "Jakobs et al . are further contributing to the field of machine learning in biology with ‘deepmirror . ai’ , an online hub with the goal of accelerating the adoption of artificial intelligence in biology ." ]
2019
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
An insect-like mushroom body in a crustacean brain
elife-29889-v2
[ [ "Mushroom bodies are paired centers first identified in the brains of Hymenoptera ( Dujardin , 1850 ) .", "They have been defined anatomically as a discrete neuropil composed of an abundance of parallel fibers originating from densely packed cell bodies , called globuli cells or Kenyon cells ( Schürmann F-W , 1973 ) .", "Together , the parallel fibers form a distinctive peduncle that divides into two or more columnar tributaries termed lobes .", "Input and output neurons intersect parallel fibers down the length of each lobe , partitioning the lobe into discrete territories ( Mobbs , 1982; Li and Strausfeld , 1999 ) .", "In most neopteran insects ( those insects possessing wings that bend over the abdomen ) , parallel fibers provide systems of dendrites that form a distal cap- or cup-like domain called the calyx that receives afferents from olfactory neuropils and , in many species , from the optic lobes and gustatory centers as well .", "Calyces are absent in palaeopteran insects , such as odonates and Ephemeroptera ( dragonflies , darters , mayflies ) , as well as in secondarily aquatic Neoptera such as diving beetles ( Strausfeld et al . , 2009 ) .", "Additional characters conventionally attributed to mushroom bodies include the presence of inhibitory feedback pathways from proximal to more distal levels of the lobes and calyx ( Leitch and Laurent , 1996 ) .", "Mushroom bodies are further defined by enriched expression of proteins that are known to be essential for learning and memory functions in Drosophila ( Skoulakis et al . , 1993; Skoulakis and Davis , 1996; Wang et al . , 1998 ) .", "The exclusive location of the paired mushroom bodies is laterally in the protocerebrum ( forebrain ) , as they are in another mandibulate group , Myriapoda , which includes centipedes and millipedes ( Wolff and Strausfeld , 2015 ) .", "Mushroom body function has been attributed to learning and memory ( Heisenberg , 2003 ) .", "Currently , much of our knowledge about the roles of mushroom bodies comes from studies showing that convergence of populations of relay neurons from the antennal lobes onto sparse subsets of mushroom body Kenyon cells underlies multiple representations of odors ( Campbell et al . , 2013; Perez-Orive et al . , 2002; Lin et al . , 2014 ) ; that mushroom bodies mediate appetitive and aversive long-term memory ( Waddell , 2013 ) ; and that they support allocentric and sequential place memory such as during trap-line foraging and obligate parasitoidism ( Mizunami et al . , 1998; Montgomery et al . , 2016; Farris and Schulmeister , 2011 ) .", "Given that mushroom bodies have been identified in Myriapoda ( centipedes and millipedes ) and Chelicerata ( arachnids , horseshoe crabs , and sea spiders ) ( Wolff and Strausfeld , 2015 ) , it might be assumed that these centers would be ubiquitous across arthropods .", "However , that assumption is awkward because the paired higher centers in the lateral protocerebrum of crustaceans share few , if any , of the identifying characteristics of lobed mushroom bodies as described above .", "These paired centers are the domed hemiellipsoid bodies .", "Particularly in malacostracan crustaceans , such as crayfish and shrimps , hemiellipsoid bodies may correspond functionally to mushroom bodies .", "In many species the hemiellipsoid bodies are subdivided into discrete lateral domains and levels ( Sullivan and Beltz , 2004 ) .", "Their neurons originate from a dense population of minute cell bodies situated at the dorsal and dorsolateral surfaces of the lateral protocerebra , like the perikarya of globuli cells associated with the insect mushroom body .", "However , only in one group of crustaceans , the Anomura ( hermit crabs ) , has there been any description of enriched proteins involved in learning and memory that characterize insect mushroom body lobes .", "In the land hermit crab Coenobita clypeatus , these proteins are confined to discrete concentric layers arranged at intervals through the depth of the hemiellipsoid body .", "Planar arrangements of pre- and postsynaptic neurons in these strata are comparable to orthogonal networks observed in the mushroom body lobes of insects .", "Hence , the hemiellipsoid bodies of land hermit crabs are interpreted as homologues of insect mushroom bodies ( Brown and Wolff , 2012 ) .", "But the question remains whether such circuits have evolved convergently , particularly because marine hermit crabs show no evidence of orthogonal circuitry , nor do species of the related orders , Astacidea ( crayfish and lobsters ) and Brachyura ( true crabs ) .", "Nevertheless , crustaceans might be expected to have evolved insect-like mushroom bodies if , like insects , they engage in behaviors that demand high level sensory associations and memory ( Withers et al . , 2008 ) , such as trap-line foraging ( Montgomery et al . , 2016 ) , rational courtship ( Arbuthnott et al . , 2017 ) , individual recognition , establishment and defense of territories , and obligate predation by stealth ( Libersat and Gal , 2013; Berens et al . , 2017; Alcock and Bailey , 1997 ) .", "Stomatopoda ( mantis shrimps ) , which is the sister group of all eumalacostracan crustaceans ( Wolfe et al . , 2016 ) , engage in such behaviors .", "Stomatopods possess the most elaborate visual systems of any arthropod , where each eye moves independently of the other , and is endowed with binocular vision and numerous color- and polarization-sensitive channels , including channels for detecting circular polarization ( Marshall et al . , 2007 ) .", "Mantis shrimps frequent established hunting territories distant from their lair ( Cronin et al . , 2006 ) .", "Like insects , mantis shrimps form associative memories of kin and place through sensory integration employing the modalities of vision and olfaction ( Caldwell and Lamp , 1981 ) and can be trained in discrimination-learning paradigms using color and polarized light stimuli ( Marshall et al . , 1999 ) .", "Individuals remember conspecifics with which they have had aversive or non-aversive encounters ( Caldwell , 1992 ) .", "These behaviors require a brain equipped for memory of places , territories and individual recognition ( Marshall et al . , 1996 ) .", "Here we report the first comprehensive description of centers in Stomatopoda that correspond in detail to the mushroom bodies of insects .", "Their similarity of brain structures has suggested phylogenetic proximity of insects and malacostracan crustaceans ( Strausfeld and Andrew , 2011 ) .", "However , genomic evidence argues strongly that insects are instead most closely related to blind , morphologically simple cave-dwelling crustaceans , called remipedes ( Regier et al . , 2005; Oakley et al . , 2013; von Reumont et al . , 2012; Schwentner et al . , 2017 ) , which like all crustaceans other than stomatopods lack mushroom bodies .", "The identification of mushroom bodies in mantis shrimps sheds new light on insect-crustacean relationships .", "We show that , within more recently radiating eumalacostracan lineages , the neuronal composition and organization of mushroom body morphology appear to have undergone radical modification resulting in centers unrecognizable as mushroom bodies but retaining the function of a sensory integrator .", "The earlier identification of a hexapod-malacostracan-like brain in a fossil stem arthropod ( Ma et al . , 2015 ) together with the present results argues for an elaborate ancestral cerebral organization already present in the lower Cambrian ." ], [ "We have surveyed a broad range of Crustacea , particularly Malacostraca , for evidence of centers that would correspond to mushroom bodies identified in insects .", "Surprisingly , as outlined above , there have been very few criteria employed for identifying insect mushroom bodies .", "Those criteria are the presence of lobes , with or without an outer ‘calyx’ or ‘cap’ , arising from many hundreds of minute cell bodies clustered laterally in the forebrain ( Flögel , 1878 ) .", "Original descriptions of mushroom bodies emphasized a distal dendritic elaboration , called a calyx , although subsequent studies have shown that many insect groups lack this , as do Myriapoda and Chelicerata ( Wolff and Strausfeld , 2015 ) .", "Surprisingly , just these few characters have sufficed historically for claiming homology of lobed centers across Hexapoda .", "However they are hardly adequate to claim homology of neuropils in divergent arthropod lineages such as the two sub-phyla , Crustacea and Hexapoda , that comprise the clade Pancrustacea .", "Here we expand neuroanatomical criteria to unique characters that we view as necessary and sufficient to claim a center’s identity as a mushroom body and hence support possible phenotypic homology across species .", "Table 1 lists these characters and their occurrence in representative insects ( Drosophila and the cockroach Periplaneta americana ) and , within eumalacostracan crustaceans , Stomatopoda and five decapod species .", "Here Stomatopoda are represented by two species , Gonodactylus smithii ( Figure 1A , B ) and Neogonodactylus oerstedii .", "Examples of these characters are shown in Figure 1—figure supplement 1 .", "In stomatopods , as in most stalk-eyed crustaceans , the brain’s lateral protocerebra are contained within the distal eyestalks ( Figure 1C ) .", "These are exceptionally capacious in mantis shrimps due to the voluminous nested optic lobe neuropils , which serve the largest and most complex compound eyes yet described ( Figure 1A ) .", "The eyestalk also accommodates elaborate neuropils that in the literature have been collectively referred to as the ‘medulla terminalis . ’ These neuropils comprise the lateral protocerebrum , just as they do in insects ( Figure 1C , Figure 1—figure supplement 2 ) .", "In the mantis shrimp , the largest of these neuropils originates from a dorsal cell body cluster of about 170 , 000 ( in N . oerstedii ) minute and tightly clustered neuronal perikarya , which we here refer to as globuli cells .", "This population provides the neurons that form four columnar neuropils , each comprising thousands of parallel fibers .", "Only one of the four columns is surmounted by a large calyx-like neuropil ( Figure 1D ) composed of layers of dendritic arborizations .", "The four columns ( Figure 1E ) , which extend latero-caudally , together demarcate the proximal margin of the eyestalk neuropil , near its junction with the eyestalk nerve ( Figure 1C ) .", "Globuli cells provide one very large dome-like neuropil ( here termed the calyx , indicated Ca in Figure 1D , E ) from which one of the smaller columns originates ( Figure 1—figure supplement 3A ) .", "Globuli cells also provide parallel fibers constituting three additional columns ( Figure 1E; Figure 2B , C ) , one of which is equipped with a small calyx distally ( Figure 1—figure supplement 3B ) .", "The two other columns lack distal dendrites entirely .", "Neurites from globuli cells converge at the origin of each column , as they do at the pedunculus ( or initial stalk ) of an insect mushroom body ( Figure 1—figure supplement 3A , B ) .", "Each cell body neurite then increases in girth and extends for hundreds of microns as a thicker process decorated with varicosities and spines representing , respectively , pre- and postsynaptic sites ( Strausfeld and Meinertzhagen , 1998 ) .", "Like Kenyon cells in insect mushroom bodies , globuli cells in stomatopods lack a proper axon: each globuli cell’s parallel fiber serves as a local interneuron , corresponding in shape and decoration to the parallel fibers of the insect mushroom body lobes , exemplified by D . melanogaster ( Figure 1F ) .", "And by comparison to the Drosophila mushroom body lobe , each parallel fiber is assumed to contribute to local computational circuits within its column ( Cervantes-Sandoval et al . , 2017 ) .", "Comparing the columns of the stomatopod mushroom body and the mushroom body lobes of the cockroach Periplaneta americana ( Figure 1G , H ) further demonstrates that parallel fibers in both species are regularly intersected by the branched processes of efferent and afferent neurons .", "As in the insect mushroom body , in the stomatopod mushroom body orthogonally organized neurons and local circuits provided by parallel fibers ( Figure 1H ) offer many thousands of possible sensory associations ( Heisenberg , 2003; Cassenaer and Laurent , 2007; Huerta et al . , 2004; Aso et al . , 2014a ) .", "In insects , the packing density of globuli cells associated with a mushroom body exceeds the packing density of neuronal cell bodies elsewhere in the brain .", "Our measurements show that the packing density of globuli cells supplying the stomatopod mushroom body is comparable to that of globuli cells supplying the mushroom bodies of insects , here exemplified by P . americana ( Figure 2A ) .", "An important identifier of mushroom bodies in insects is the morphology of its Kenyon cells and their specializations; in particular , clawed specializations that partially wrap around large terminal boutons of sensory afferents in the calyces of flies and other insects ( Yasuyama et al . , 2002 ) .", "These are also identified in stomatopods , but only on dendrites contributing to the smaller of the two calyces ( Figure 2D ) .", "In the larger calyx , spined dendritic processes correspond to the spined dendrites of the mushroom body calyx of P . americana ( Figure 2A ) .", "However , the stomatopod calyx is deeper than is an insect calyx and is composed of four distinct levels , the second of which is characterized by hundreds of thousands of synaptic clusters comparable to microglomeruli identified in the calyces of insects ( Figure 3A ) .", "In insects , the processes of uniquely identifiable efferent and afferent neurons are arranged at specific loci along the length of a mushroom body’s lobe , giving the lobe the appearance of being segmented into discrete synaptic domains ( Li and Strausfeld , 1999; Ito et al . , 1998 ) .", "In Drosophila , such neurons at specific loci along the lobes have been shown to encode the adaptive values of sensory information associated with learned responses ( Aso et al . , 2014b ) .", "The same type of organization typifies the stomatopod mushroom body columns ( Figure 2E , F ) .", "And , as in the insect mushroom body , this arrangement is further demonstrated by the organization of aminergic neurons , the processes of which invade the columns at specific sites .", "In N . oerstedii GAD-immunoreactive neurons extending across the two largest columns are representative of centrifugal pathways back to the calyx ( Figure 3B , C ) .", "Other aminergic afferents are resolved using antisera against serotonin ( 5HT ) and tyrosine hydroxylase ( TH ) .", "These also demonstrate discrete territories along the length of the mushroom body columns ( Figure 3F , G ) .", "These arrangements of TH-immunoreactive neurons , first identified in the mushroom body lobes of the fly Calliphora erythrocephala ( Nässel and Elekes , 1992 ) , correspond to discrete dopaminergic territories in the mushroom body of D . melanogaster .", "Thermogenetics , optogenetics , and mutant analysis have demonstrated the pivotal role of these neurons in appetitive and aversive conditioning ( Waddell , 2013; Waddell , 2016 ) .", "Three-dimensional reconstructions of the stomatopod mushroom bodies confirm that only one of the four columns ( Clm 3 , Figure 2B ) originates from the extensive volume of dendritic processes that constitute the elaborate calyx ( Figure 2B ) .", "Golgi impregnations resolve a smaller column ( Clm 4 ) capped by a much smaller cluster of outer dendrites constituting a second calyx .", "The remaining two columns ( Clm 1 ,", "2 ) are the largest and are composed of ‘naked’ globuli cells entirely lacking distal dendrites ( Figure 1—figure supplement 3B ) .", "That all four columns are supplied by globuli cells is another correspondence with the organization of the insect mushroom body , which develops from four cell lineages , the clonal progeny of which do not remain separated but merge to form a single neuropil ( Ito et al . , 1997; Farris et al . , 2004 ) .", "In the mantis shrimp , four distinct columns originate from the same globuli cell population but remain separate from each other ( Figure 2B , Figure 1—figure supplement 1 ) .", "The absence of a cap or calyx crowning two of the stomatopod mushroom body columns is comparable to the organization of mushroom bodies in palaeopteran insects ( mayflies , damselflies ) , which entirely lack calyces ( Strausfeld et al . , 2009 ) .", "This ‘calyxless’ condition also typifies mushroom bodies in Myriapoda , another mandibulate taxon ( Wolff and Strausfeld , 2015 ) .", "Ancestral morphology is also suggested by mushroom body development in insects , where outgrowth of the lobes precedes the development of the calyx ( Ito et al . , 1997; Farris et al . , 2004 ) .", "To summarize , the combination of neuroanatomical characters that distinguish the insect mushroom body also distinguish the stomatopod mushroom body ( Table 1 ) .", "Furthermore , the stomatopod mushroom body columns and the insect mushroom body lobes are recognized at the molecular level by antibodies raised against three proteins required for learning and memory in Drosophila: ( 1 ) protein kinase A catalytic subunit alpha ( DC0 ) ( Skoulakis et al . , 1993 ) , ( 2 ) Leonardo ( Leo ) , the ortholog of mammalian 14-3-3ζ ( Skoulakis and Davis , 1996 ) , and ( 3 ) phosphorylated calcium/calmodulin-dependent protein kinase II ( pCaMKII ) ( Wang et al . , 1998 ) .", "In the cockroach lateral protocerebrum , all three antibodies selectively resolve the mushroom body lobes ( Figure 4F ) .", "Similarly , in the lateral protocerebrum of the stomatopod brain these three antibodies selectively resolve all four columns identified by reduced silver staining and Golgi impregnation ( Figure 4B–E ) .", "Mapping brain morphologies onto a representative genomic phylogeny of Pancrustacea ( Oakley et al . , 2013 ) demonstrates the absence of mushroom bodies in all taxa intervening between the clade Hexapoda+Remipedia and Eumalacostraca ( Figure 5A ) .", "To examine whether mushroom bodies may have evolved divergent arrangements within Eumalacostraca ( Figure 5A ) , we analyzed five species of more recently evolved groups of decapods ( Shen et al . , 2013 ) : Caridea ( including snapping or pistol shrimps ) , Stenopodidea ( cleaner shrimps ) , Astacidea ( including lobsters and crayfish ) , Anomura ( land hermit crabs ) , and Brachyura ( true crabs; Figure 5B ) .", "Alpheid caridids ( ‘pistol shrimps’ ) are the only group known to have evolved eusociality .", "Stenopid ( ‘cleaner shrimps’ ) hemiellipsoid bodies have been previously described , published images suggesting a short extension from a large hemiellipsoid body ( Sullivan and Beltz , 2004 ) .", "Comparing the DC0 immunocytology of these taxa with that of the Drosophila mushroom bodies ( Figure 5—figure supplement 1A ) identifies DC0-positive structures in two of the investigated species .", "In the banded coral shrimp Stenopus hispidus ( Stenopodidea ) , anti-DC0 resolves a pedestal-like extension from its domed hemiellipsoid body ( Figure 5—figure supplement 1B ) , which is divided into three distinct territories .", "Anti-DC0 also reveals prominent mushroom body-like centers in Alpheus bellulus ( Caridea ) .", "Golgi impregnations of A . bellulus show these prominently in the lateral protocerebrum where they are of comparable size to those of social or parasitoid hymenopteran insects ( Figure 5—figure supplement 1C , D ) .", "The land hermit crab C . clypeatus ( Anomura ) is distinct: its hemiellipsoid body consists of an enormous domed neuropil that is characterized by concentrically arranged DC0- and pCaMKII-immunoreactive layers ( Figure 5—figure supplement 2A ) .", "Each is a flat sheet comprising a planar arrangement of globuli cell processes that are orthogonally intersected by the arborizations of input and output neurons .", "Although these arrangements are peculiar to land hermit crabs , they demonstrate that the network organization within each layer corresponds to the three-dimensional networks that are defined by the sequential domains of input and output arborizations that extend across parallel fibers in the insect mushroom body ( Brown and Wolff , 2012; Wolff et al . , 2012 ) .", "Only in one group outside Malacostraca , is there a neuropil at all reminiscent of a mushroom body .", "This is in Cephalocarida , where an allantoid center , occupying an otherwise drastically reduced protocerebrum , receives inputs from the olfactory lobes on the same side of the brain , as occurs in insects ( Stegner and Richter , 2011 ) .", "In crayfish ( Astacidea ) , the dorsal globuli cell cluster provides a prominent domed center in the lateral protocerebrum .", "However this hemiellipsoid body does not show discernable levels of the proteins involved in Drosophila learning and memory ( Figure 5—figure supplement 2C ) .", "The hemiellipsoid bodies of the examined varunid crabs ( Brachyura ) are inconspicuous and they too lack elevated levels of those proteins ( Figure 5—figure supplement 2B ) .", "Together , with other neuroanatomical criteria ( Table 1 ) , these observations suggest that more recently evolved Eumalacostraca show a reduction or complete loss of mushroom body-like lobes ( columns ) , accompanied by accentuation and diversification of domed neuropils that have been historically referred to as hemiellipsoid bodies ( Figure 5B ) .", "Other elongated centers exist within eyestalk neuropils .", "One that is conspicuous in Stomatopoda , and which has an equivalent in the Varunidae ( shore crabs belonging to Brachycera ) , offers an interesting counterpoint to the identification of the stomatopod mushroom body .", "This is the reniform body , a prominent compound neuropil located near the inner margin of the optic lobes ( RB in Figure 1C ) , which in N . oerstedii arises from a dense cluster of about a thousand cell bodies .", "These provide a pedestal-like arrangement of smooth parallel neurites supplying four domains: an initial zone and a lateral , distal , and proximal zone .", "It is the latter three that define the center’s characteristic kidney shape .", "All four zones contain the dendritic processes of collaterals branching from the pedestal as well as numerous branched afferent processes that supply them ( Figure 6A , B ) .", "Antisera against serotonin ( Figure 6C ) and GAD amplify morphological distinctions amongst these domains , such as delineating glomerular divisions in just the distal zone ( dz in Figure 6D ) .", "Anti-DC0 immunocytology demonstrates expression of this antigen in all four zones , but at a much lower level than seen in mushroom body columns ( Figure 6E ) .", "The same center has been identified at the same location and orientation , with respect to the optic lobes , in the shore crabs Hemigrapsus nudus and H . oregonensis , again revealing domains corresponding to those of the reniform body in N . oerstedii .", "A cluster of cell bodies provides a short pedestal composed of about two hundred smooth undecorated parallel processes that branch to four discrete neuropils ( Figure 6F , G ) .", "However , in H . nudus , these and other discrete neuropils in the lateral protocerebrum either lack discernable DC0 or , if DC0 is expressed , it is at very low levels .", "An exception is a tract of axons leading from the optic lobes centrally ( Figure 6H ) .", "Superficially , the reniform body is suggestive of a mushroom body-like center due to the arrangement of parallel processes that make up its pedestal .", "It is this feature that has lead to its erroneous identification as a mushroom body homologue ( Maza et al . , 2016 ) .", "The rationale for rejecting that interpretation is provided in the Discussion ." ], [ "It is broadly accepted that insects originated from crustaceans .", "Molecular phylogenies favor blind , morphologically simple anchialine crustaceans , called remipedes as the closest relatives of the insects ( Regier et al . , 2005; Oakley et al . , 2013; von Reumont et al . , 2012; Schwentner et al . , 2017 ) .", "This view conflicts with neural cladistics , which resolves insect brain organization closely corresponding to that of phylogenetically distant malacostracan crustaceans ( e . g . , Decapoda ) ( Strausfeld and Andrew , 2011 ) .", "One source of conflict is that molecular phylogenies cannot provide reliable information about morphological changes that have occurred since related groups diverged .", "It is expected that through the course of evolution there will have arisen both genetic and structural novelties , as well as reversals and losses ( Chen et al . , 2013; Jenner , 2004 ) , the latter impossible to code in relational trees based on morphological characters ( Strausfeld and Andrew , 2011 ) .", "For example , branchiopod crustaceans , closely related to both hexapods and remipedes , lack most features that define the brains of those two groups , and possess only a pair of nested optic neuropils ( Figure 5A ) .", "Cephalocarids , a lineage closely related to Hexapoda and Remipedia , are also blind crustaceans , whose brains comprise predominantly olfactory centers and lack other cerebral neuropils common to hexapods and malacostracans ( Stegner and Richter , 2011 ) .", "Compared with Branchiopoda and Cephalocarida , the Remipedia midbrain shares more neural features with Malacostraca than it does with Hexapoda ( Fanenbruck et al . , 2004 ) .", "Similar cerebral arrangements in crustaceans and insects have frequently been ascribed to convergent evolution rather than to genealogical correspondence .", "This view ( Lozano-Fernandez et al . , 2016 ) can refer particularly to fundamental differences in insect and crustacean olfactory systems , even in crustaceans adapted to terrestrial life .", "In crustaceans , olfactory receptor neurons exclusively express ionotropic receptors , whereas in insects most olfactory receptor neurons are defined by their G-protein coupled receptors ( Stensmyr et al . , 2005; Corey et al . , 2013; Sato et al . , 2008 ) .", "A neuropil ascribed exclusively to crustaceans is the dome-like second-order olfactory center called the hemiellipsoid body , which is supplied by the axons of thousands of projection neurons from numerous subunits of the olfactory lobe and its satellite neuropils ( Sullivan and Beltz , 2004; Sullivan and Beltz , 2005; Schmidt and Mellon , 2011 ) .", "Hemiellipsoid bodies also integrate olfactory , haptic and visual information ( Schmidt and Mellon , 2011; Mellon et al . , 1992 ) .", "A comparable arrangement pertains to insects , with a few notable differences .", "Fewer and smaller relay neurons , most restricted to single glomerular subunits of the antennal lobe ( the primary olfactory lobe ) send axons to second-order centers , the mushroom bodies ( Galizia and Rössler , 2010 ) .", "These inputs relay information about odorants , airborne molecules that are detected by the ligand-gated olfactory receptors .", "In many species , including Drosophila , mushroom bodies receive other modalities , including vision and haptic information ( Vogt et al . , 2014; Paulk and Gronenberg , 2008 ) .", "However , because mushroom bodies are structurally so distinct from hemiellipsoid bodies , in that they are characterized by their extended lobes , they have been viewed as apomorphies of insects ( Stemme et al . , 2016; Farris , 2013; Sandeman et al . , 2016 ) .", "To summarize , the combination of neuroanatomical characters that distinguish the insect mushroom body also distinguishes the stomatopod mushroom body ( Table 1 ) .", "That hemiellipsoid bodies of only the closest eumalacostracan relatives of stomatopods possess some , but not all , mushroom body characters suggests that the hemiellipsoid body has derived from an ancestral mushroom body .", "Our analysis of five species of crown group Eumalacostraca show that only Alpheoidea , Stenopodidea ( Table 1 ) , and a single group of anomurans , the land hermit crabs , possess some mushroom body characters .", "There has been a reduction or complete loss of mushroom body columns in Anomura , Astacidea and Brachyura accompanied by an apparent elaboration of the ancestral calyx to provide a variety of morphologies of domed hemiellipsoid bodies , each characteristic of a genus ( Figure 5B ) .", "A central debate has been whether hemiellipsoid bodies , which support multisensory functions comparable to those supported by insect mushroom bodies , can be viewed as their homologues or as crustacean apomorphies .", "The abundance of divergent hemiellipsoid body morphologies described by Sullivan and Beltz across an even broader range of taxa ( Sullivan and Beltz , 2004; Sullivan and Beltz , 2005 ) , and their observations of taxon-specific innervation by the olfactory tract , support an interpretation of the hemiellipsoid body as a highly divergent mushroom body calyx , a suggestion originally entertained by a remarkable study by Giuseppe Bellonci in 1882 ( Bellonci , 1882 ) .", "Reported activity in an elongated volume of neuropil situated close to the inner margin of the lobula of the varunid crab Neohelice granulata ( Maza et al . , 2016 ) suggested its identity as a learning and memory center .", "Recordings from this center showed long-term habituation in response to repetitive visual stimuli .", "It was proposed that this center is homologous to the insect mushroom body on the basis of a cluster of small cell bodies located ventro-medially in the eyestalk neuropil providing a columnar domain showing elevated p-CAMKII-α ( Maza et al . , 2016 ) .", "Two territories populated by branched processes were referred to as vertical and medial lobes , adopting terms for insect mushroom body lobes .", "We have been unable to reconcile those neuroanatomical observations with characters defining the insect and stomatopod mushroom bodies .", "Parallel fibers comprising pedestal-like structures ( Figure 1E in Maza et al . , 2016 ) are shown as smooth , lacking spines and varicosities .", "They also lack efferent and afferent domains that would provide orthogonal networks typical of mushroom bodies .", "Dendrites described as clawed ( Maza et al . , 2016 , Figure 1F ) appear to be afferent terminals .", "Immunoreactivity to anti-pCaMKII shows elevated expression tightly constrained to the region of cell body neurites , after which its levels diminish in unspecific neuropil .", "In all respects , the described neuropil in N . granulata corresponds to the Hemigrapsus reniform body ( Figure 6F–I ) .", "Although more elaborate in Stomatopoda ( Figure 6A–E ) , its reniform body neuropils unambiguously correspond to those attributed to the N . granulata habituation center and to H . nudus .", "We are unable to show evidence for any lobed center that meets the relevant criteria for a mushroom body in varunids and suggest that the reniform body is an intriguing visual association center that may be unique to eumalacostracan crustaceans .", "Nothing comparable has yet been identified in the Drosophila lateral protocerebrum or in that of any other insect .", "Our survey across Pancrustacea ( Crustacea+Hexapoda ) identifies mushroom bodies in stomatopods and insects .", "No other eumalacostracan crustacean outside Stomatopoda has yet provided clear evidence of an insect-like mushroom body .", "Comparisons across this pancrustacean phylogeny ( Figure 5A ) thus suggest two interpretations .", "One is that mushroom bodies evolved convergently in insects and stomatopods .", "The other is that the mushroom body is an ancestral attribute of the pancrustacean brain that has been retained in just the hexapod and stomatopod lineages but modified or lost in all others .", "Fossil brains described from the lower Cambrian stem arthropod Fuxianhuia protensa suggest that optic lobe and neuromeric arrangements of the brain of this ancient stem arthropod typify those of extant malacostracans and insects: the presence of three nested visual centers arising from a cerebrum comprising a fused forebrain , midbrain and hindbrain , each neuromere denoted by its relationships with the compound eyes , antennules and paired post-antennular appendages ( Figure 5A , Figure 5—figure supplement 3A ) ( Ma et al . , 2015; Ma et al . , 2012 ) .", "Although fossils cannot reveal the organization of centers within the brain , those preserved features described above are cardinal indicators of an ancestral ground pattern that originated more than half a billion years ago at the onset of the lower Cambrian .", "Subsequent lineages originating at an estimated time of 487 million years ago provide today’s crown Pancrustacea ( Collette and Hagadorn , 2010 ) .", "Taking neuropils that have been identified as common to Pancrustacea and superimposing these onto the ancestral ground pattern ( Figure 5—figure supplement 3B ) suggests a plausible ancestral arrangement in such a brain ( Figure 5—figure supplement 3C , D ) .", "That mushroom bodies have been demonstrated in Myriapoda , the sister group of Pancrustacea , and Chelicerata the sister group to all mandibulate arthropods , further supports a very ancient origin of this center ( Wolff and Strausfeld , 2016 ) .", "Attendant to such considerations is that many elements of the ancestral ground pattern have been lost or drastically reduced in numerous crustacean lineages , even to just a few neurons such as in Cirripedia and Cephalocarida ( Callaway and Stuart , 1999 ) .", "Simplification from genealogically ancient complexity is common in nature at many phylogenetic levels ( Collin et al . , 2009 ) .", "It should come as no surprise to find examples of simplification or independent retentions of past complexity in the neuromorphological landscape .", "The demonstration that phenotypically identical organs in widely separated species can be ascribed to convergent genomic evolution ( Pankey et al . , 2014 ) suggests caution in claiming homology of identical phenotypes .", "Thus , although corresponding morphologies in stomatopods and insects suggest mushroom body homology , transcriptomic evaluation must , eventually , be the arbiter of whether these centers indeed correspond genealogically .", "At present , the strongest argument against is that the sister group of all hexapods ( Remipedia ) and the sister group of all Malacostracans ( Leptostraca ) possess simple hemiellipsoid bodies , not mushroom bodies ( Fanenbruck et al . , 2004; Kenning et al . , 2013 ) .", "Convergent evolution would imply that mushroom bodies in insects and stomatopods evolved independently in response to comparable selective constraints .", "The most obvious would be the appearance of biotopes requiring superior vision .", "Stomatopods and certain insects possess superior optics and underlying optic lobe circuitry that enable the detection and discrimination of visual flow fields , chromatic features , patterns and structures .", "However , speaking against this are archaeognathan insects , the sister group of all other insects ( Misof et al . , 2014 ) , which have a more elaborate visual system than those of apterygote Zygentoma , the sister group of all winged insects ( Misof et al . , 2014 ) .", "Yet archaeognathans lack mushroom bodies .", "Lepisma saccharina , which is a typical zygentoman insect , has diminutive eyes and reduced optic lobes but possesses robust mushroom bodies equipped with calyces and elaborate lobes ( Farris , 2005 ) .", "Amongst crustaceans , many eumalacostracan species possess prominent eyes surmounting elaborate visual centers .", "Shore crabs , for example , have excellent vision and highly elaborated optic lobes ( Sztarker et al . , 2005; Berón de Astrada et al . , 2001 ) , but they lack mushroom bodies .", "Given these contradictions , what aspects of behavior specific to stomatopods and insects might be relevant to mushroom body evolution ?", "One proposed driver of the evolution of large mushroom bodies is the requirement to recall the exact locations and properties of places from which to obtain nourishment .", "Heliconid butterflies maintain their enlarged mushroom body lobes when allowed repeated visits to distributed foraging sites ( Montgomery et al . , 2016 ) .", "The large mushroom bodies of social hymenopterans are suggested to have evolved in conjunction with the evolution of parasitic behavior , where individuals learn the many locations of potential hosts ( Farris and Schulmeister , 2011 ) .", "It may be significant , therefore , that the only eumalacostracan groups in addition to stomatopods that evidence memory of exact locations are cleaner shrimps , pistol shrimps , and land hermit crabs , all of which have mushroom body-like attributes ( Figure 5B – Figure 5—figure supplement 1B–D , 2A ) .", "Cleaner shrimps are renowned for their fidelity to specific locations visited by the fish they clean ( Limbaugh et al . , 1961 ) .", "Pistol shrimps are the only crustaceans to have evolved eusociality ( Duffy , 1996 ) , an attribute requiring memory of position both outside and within the community .", "And land hermit crabs are known for their navigational skills and memory of sites at which they socially interact ( Rotjan et al . , 2010 ) .", "There are , then , valid grounds to suppose convergent evolution of mushroom bodies in certain crustaceans and hexapods .", "Stomatopod and insect mushroom bodies also pass three crucial tests for phenotypic homology ( Patterson , 1988 ) .", "These are their similarity , the exclusion of competing structures that might suggest a similar interpretation ( conjunction ) , and the co-existence of additional homologies ( congruence ) .", "The present results have demonstrated structural identicalities .", "Reniform bodies , proposed as a homologue of the insect mushroom body ( Maza et al . , 2016 ) , are excluded because in stomatopods they coexist with mushroom bodies .", "The third condition , that structures in two species are more likely to be homologues if they share other homologies , is supported by the locations of other corresponding brain centers in insects and stomatopods ( Figure 1—figure supplement 2 ) .", "Specific examples are the fan-shaped central complex neuropils in the midbrain of stomatopods and insects connected to identical suites of satellite neuropils ( Thoen et al . , 2017 ) .", "We are obliged to conclude that there is , as yet , no definitive conclusion .", "Phenotypic and genomic homology would support the hypothesis that mushroom bodies evolved very early in pancrustacean or even in panarthropod evolution ( Wolff and Strausfeld , 2016 ) .", "That identical centers of such stunning complexity may have evolved convergently in stomatopods and insects is just as fascinating and should lead to a greater understanding of the overarching significance of mushroom bodies in arthropod behavior ." ], [ "One hundred and eight mature stomatopods ( both sexes ) , Neogonodactylus oerstedii , were obtained commercially from waters off the coast of Florida .", "Forty-seven Gonodactylus smithii were obtained from designated areas overseen by the Lizard Island Research Station , Australia ( GBRMPA Permit no . G12/35005 . 1 , Fisheries Act no . 140763 ) .", "Five Stenopus hispidus ( Caribbean ) and 25 Alpheus bellulus ( Caribbean ) were obtained from LiveAquaria . com , Rhinelander , WI , USA .", "Animals were maintained isolated in small perforated transparent ‘Ziplock’ containers immersed in running artificial seawater .", "Drosophila melanogaster and Periplaneta americana were reared in laboratory cultures at the University of Arizona , Tucson and Washington University , Seattle .", "Five specimens of Coenobita clypeatus were purchased locally and maintained in a vivarium .", "Thirty Hemigrapsus nudus and H . oregonensis were collected from designated sites on San Juan Island , WA , USA .", "Six Procambarus clarkii were purchased from domestic suppliers ( Carolina Biological Supply Company , Burlington , NC ) .", "Golgi impregnations were performed on 72 N . oerstedii and 40 G . smithii .", "Each immunocytological method was performed identically on at least 3 individuals .", "Bodian and Golgi silver staining was performed on 6 N . oerstedii .", "Cell body counts were obtained from two mushrooms bodies from N . oerstedii and P . americana .", "A monoclonal antiserum against α-tubulin ( DSHB Cat# 12G10 anti-alpha-tubulin , RRID:AB_1157911 ) used at a concentration of 1:100 , was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa , Department of Biology ( Iowa City , IA ) .", "Monoclonal antiserum against synapsin ( anti-SYNORF1 , DSHB Cat# 3C11RRID:AB_528479 ) obtained from the same source were used at a concentration of 1:100 .", "Polyclonal antiserum raised against serotonin ( ImmunoStar , Hudson , WI , Cat# 22941 , RRID:AB_572268 ) was used at a concentration of 1:500 .", "Anti-DC0 ( D . Kalderon , Columbia University; New York; USA Cat# DC0 , RRID:AB_2314291 ) , a generous gift from Dr . Daniel Kalderon , was used at a concentration of 1:250 for immunohistochemistry .", "Anti-Leonardo , a generous gift from Dr . Ronald Davis was used at a concentration of 1:500 and anti-pCaMKII ( Santa Cruz Biotechnology Cat# sc-12886-R , RRID:AB_2067915 ) was used at a concentration of 1:100 for immunohistochemistry .", "Each antiserum was from aliquots demonstrated for specific affinity to Drosophila mushroom body lobes .", "As pioneered by studies on remipede brains ( Stemme et al . , 2016 ) , a polyclonal antibody raised against of the synthesizing enzyme glutamic acid decarboxylase ( GAD ) ( Sigma-Aldrich Cat# G5163 , RRID:AB_477019 ) was used to detect putative GABAergic neurons .", "A monoclonal antibody against tyrosine hydroxylase ( TH ) raised in rats ( ImmunoStar , Hudson , WI Cat# 22941 , RRID:AB_572268 ) which had been previously shown to specifically label dopaminergic neurons in the lobes of insect mushroom bodies was used on intact stomatopod and cockroach brains .", "In preparation for immunocytology , animals were immobilized by refrigeration at 4°C and the brains were dissected free in cold ( 4°C ) fixative containing 4% paraformaldehyde in PBS , pH 7 . 4 ( PBS from Sigma , St . Louis , MO ) .", "10% sucrose was added to the fixative solution for marine organisms .", "Holes were cut in the retina and in cuticle covering eyestalk neuropils .", "The eyestalks were then severed and immersed in fixative and placed in a microwave adjusted to 18°C for two cycles of 2 min with power , and then 2 min under vacuum .", "Next , the brains were left in fresh fixative overnight at 4°C ( but see below for TH immunostaining ) .", "The following day brains were washed three times , 10 min each , in PBS and then embedded in albumin gelatin .", "Embedded tissue was cut into 60 µm serial sections using a vibratome ( Leica , Nussloch , Germany ) .", "Individual sections were placed in wells of a well plate ( 1 ml capacity ) for further processing .", "Sections were washed six times over 20 min in PBS containing 0 . 5% Triton X-100 ( Electron Microscopy Supply , Fort Washington , PA , Cat . no . 22140; PBS-TX ) .", "Then 50 µL normal serum ( Jackson ImmunoResearch Labs Cat# 017-000-121 , RRID:AB_2337258 ) was added to each well containing 1000 µL PBS-TX .", "After 1 hr , primary antibody or antiserum was added to each well and the well plate was left on a slow agitating shaker overnight at room temperature .", "The next day , sections were washed six times over 3 hr in PBS-TX .", "Then 1000 µL aliquots of PBS-TX were placed in tubes with 2 . 5 µL of secondary Cy2 , Cy3 , or Cy5 conjugated IgGs ( Jackson ImmunoResearch , West Grove , PA , respectively Cat# 715-225-150 , RRID:AB_2340826 , 715-165-150 , RRID:AB_2340813 , and 715-175-150 , RRID:AB_2340819 ) and centrifuged at 13 , 000 rpm for 15 min at 4°C .", "A 900 µL aliquot of this solution was added to each well .", "The well plate was left on a shaker to gently agitate the sections overnight at room temperature .", "Tissue sections were next washed six times in PBS over 3 hr , embedded on glass slides in a medium of 25% polyvinyl alcohol , 25% glycerol and 50% PBS , for imaging using confocal microscopy .", "Where applicable , sections were rinsed in 0 . 01M tris-HCl buffer ( pH 7 . 5; Sigma , T1503 ) and incubated in the fluorescent nuclear stain Syto-13 ( Life Technologies , Grand Island , NY ) at a concentration of 1:2000 prior to mounting on glass slides .", "For labeling F-actin , preparations were left to incubate in phalloidin conjugated to Alexa Fluor 546 ( Thermo Fisher Scientific Cat# A-22283 , RRID:AB_2632953 ) following secondary antibody incubation at a concentration of 1:40 in 0 . 5% PBST for three days at room temperature on a gentle shake .", "For tyrosine hydroxylase ( TH ) immunocytology , eyestalks were fixed at 4°C for 30 min in 0 . 01M phosphate buffered 4% paraformaldehyde containing 10% sucrose .", "The tissue was then dissected free , and whole eyestalk neuropils were soaked for 2 hr in PBS containing 1% Triton X-100 , before being transferred to a vial containing 1% PBS-TX and 5% normal donkey serum ( Jackson ImmunoResearch Labs Cat# 017-000-121 , RRID:AB_2337258 ) .", "After 2 hr , primary antibody diluted 1:250 was added to the container , which was left on a slow agitating shaker for 48 hr at room temperature .", "Tissue was then washed several times in 0 . 5% PBS-TX over 3 hr before treatment with Cy5 conjugated IgGs , as above .", "Tests using an antiserum against dopamine ( Polyclonal Rabbit anti Dopamine ( Abcam Cat# ab8888 , RRID:AB_306841 ) resolved identical neurons but with poor resolution of fine processes and twigs , hence the exclusive use of TH for this study .", "Secondary antibody treatment lasted for 24 hr followed by several washes in PBS-TX , cleared in glycerol and then mounting in welled glass slides under glycerol for confocal microscopy .", "Tissue was subjected to the two cycle microwave treatment described above once every 24 hr while the tissue was incubating in either primary or secondary antibody .", "When labeling tissue with a second primary antibody , such a serotonin , in conjunction with TH , after whole tissue labeling the specimen was again fixed as if it were fresh , for an additional 12 hr at 4°C in 4% paraformaldehyde in PBS .", "The next day , the tissue was embedded and sectioned as described above , and subjected to the relevant immunohistochemical treatment .", "Bodian's original ( Bodian , 1936 ) method was used on brains fixed in AAF ( 16 ml 80% ethanol , 1 ml glacial acetic acid , 3 ml 37% formaldehyde ) , dehydrated , cleared in terpineol , and embedded in Paraplast Plus ( Sherwood Medical , St . Louis , MO ) .", "Stomatopods were briefly immobilized by covering with granulated ice .", "The head and eyestalks were removed and neural tissue dissected and desheathed in 1 part 25% glutaraldehyde and 5 parts 2 . 5% potassium dichromate adjusted with sucrose ( Thoen et al . , 2017 ) .", "After a suitable incubation period , tissue was washed in potassium dichromate before immersion in a 0 . 01% osmium tetroxide carried in 2 . 5% potassium dichromate for 12 hr before a 24 hr immersion in 0 . 75% silver nitrate .", "These last two steps were then repeated before embedding tissue in Durcupan plastic and serial sectioning at 40 µm .", "Images through depths of 50–100 µm were obtained using serial step-through focusing and reconstitution using Adobe Photoshop .", "Immunohistological images were collected using a Zeiss Pascal or 880 confocal microscope .", "Light microscopy images were obtained with a Leitz Orthoplan microscope .", "Step focus series of stitched images were reconstructed using the software Helicon Focus ( Helicon Soft , Kharkov , Ukraine; RRID:SCR_014462 ) .", "Three-dimensional reconstruction of the hemiellipsoid body and its lobes employed 220 stacked 12 µm-thick serial silver-stained and interpolated profiles processed using ImageJ ( RRID:SCR_003070 ) .", "Sections were prealigned with reference to 5 fiducial landmarks provided by non-neural elements , such as blood vessels and neural sheath extending vertically through the section series .", "Bodian-stained brains of N . oerstedii and P . americana were used for estimating densities of globuli cells , which provide mushroom body parallel fibers .", "Serial photographs at 1 µm intervals through a depth of 10 µm captured images of perikarya , their nuclei and nucleoli .", "Counts of the latter were made within 50 × 50 × 10 ( 25 , 000 µm3 ) volumes .", "Averages were calculated for globuli cell packing densities within 1 mm3 , arriving at 3 . 48 × 106/mm3 for N . oerstedii and 4 . 8 × 106/mm3 for P . americana .", "The total volume of the globuli cell population was calculated from serial reduced-silver sections .", "For N . oerstedii images were taken at 1173 × 846 pixels at a resolution of 0 . 5068 pixels per micron .", "Voxel size was calculated as 1 . 9730 × 1 . 9730 µm3 , with 12-µm-thick sections , resulting in 6 . 08 pixels per z-level .", "P . americana was imaged at 2080 × 1542 pixels at a resolution of 2 . 02 pixels per micron .", "Voxel size was calculated as 0 . 4950 µm3 12-µm-thick sections resulting in 24 . 24 pixels per z-level .", "Sections were aligned as described above and the regions containing the globuli cell population were segmented using the TrakEM2 plugin in Fiji ( Cardona et al . , 2012; Schindelin et al . , 2012 ) using the TrakEM2 measuring tool to calculate the total volume .", "Depiction of the fossilized brain of Fuxianhuia protensa ( Figure S6 ) was obtained by superimposing the imaged brain of specimen YKLP 11357 onto the montaged part and counterpart of YKLP 15006 .", "Original data for these specimens is published in Ma et al . , 2015 .", "Terms and abbreviations follow the recommendations of the insect brain consortium that has devised terms applicable to both insect and crustacean ( Pancrustacea ) central nervous systems .", "These are provided and comprehensively explained in Ito et al . ( 2014 ) ." ] ]
[ "Mushroom bodies are the iconic learning and memory centers of insects .", "No previously described crustacean possesses a mushroom body as defined by strict morphological criteria although crustacean centers called hemiellipsoid bodies , which serve functions in sensory integration , have been viewed as evolutionarily convergent with mushroom bodies .", "Here , using key identifiers to characterize neural arrangements , we demonstrate insect-like mushroom bodies in stomatopod crustaceans ( mantis shrimps ) .", "More than any other crustacean taxon , mantis shrimps display sophisticated behaviors relating to predation , spatial memory , and visual recognition comparable to those of insects .", "However , neuroanatomy-based cladistics suggesting close phylogenetic proximity of insects and stomatopod crustaceans conflicts with genomic evidence showing hexapods closely related to simple crustaceans called remipedes .", "We discuss whether corresponding anatomical phenotypes described here reflect the cerebral morphology of a common ancestor of Pancrustacea or an extraordinary example of convergent evolution ." ]
[ "With more than four million species , arthropods are the largest and most diverse group of animals on the planet and include , for example , crustaceans , insects and spiders .", "They are defined by their segmented bodies , hard outer skeletons and jointed limbs .", "All arthropods share a common ancestor that lived more than 550 million years ago .", "Exactly how this ancestral arthropod gave rise to the myriad species that exist today is unclear but we know that at some point the arthropod family tree split into branches , one of which went on to become the crustaceans .", "The crustacean branch then split again , giving rise to a line of descendants that would become the insects .", "But although insects evolved from crustaceans , the brains of insects possess structures that those of crustaceans do not .", "Known as mushroom bodies , these structures help to form and store memories .", "Their absence in crustaceans has therefore been an enduring mystery .", "Wolff et al . now add a piece to the puzzle by showing that one group of modern-day crustaceans , the mantis shrimps , does in fact possess mushroom bodies .", "By visualizing cells and pathways within the brains of mantis shrimps , and also a number of closely related species , Wolff et al . show that only these shrimps possess true mushroom bodies .", "However , some of the mantis shrimp’s close relatives possess a few attributes of these structures .", "This suggests that mushroom bodies are evolutionarily ancient structures that arose in a common ancestor of insects and crustaceans , before being lost or radically modified in most of the crustaceans .", "So why did this happen ?", "Mantis shrimps are top predators with excellent vision that hunt over considerable distances , requiring them to evaluate and memorize complex features of their environment .", "These cognitive demands , which might not be shared by other crustaceans , may have led to the mantis shrimps retaining their mushroom bodies .", "Further research into the brains and behavior of the mantis shrimp may provide insights into how mushroom bodies construct memories of a complex sensory world ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "cancer biology" ]
Bladder-cancer-associated mutations in RXRA activate peroxisome proliferator-activated receptors to drive urothelial proliferation
elife-30862-v3
[ [ "Bladder cancer is the sixth most common cancer in the US and is predicted to cause ~17 , 000 deaths in 2017 [The website of the National Cancer Institute ( https://www . cancer . gov ) ] .", "In contrast to other common malignancies , bladder cancer management has not yet benefited from molecularly targeted therapies .", "Early phase clinical data with agents targeting FGFR3 or ERBB family members have shown encouraging activity in the minority of patients with oncogenic versions of these kinases ( Nogova et al . , 2017; Choudhury et al . , 2016 ) , but identification of new targetable genomic drivers is needed to expand the armamentarium .", "Nuclear receptors are amongst the most successful therapeutic targets in oncology , with small molecule inhibitors of estrogen receptor ( ER ) and androgen receptor ( AR ) improving survival in breast and prostate cancers ( Fisher et al . , 1996; Beer et al . , 2014 ) .", "The established ability to modulate nuclear receptors with drugs makes them an attractive class of targets for other cancer types when there is compelling evidence supporting their role as tumor drivers .", "RXRA is a nuclear receptor that regulates transcription as a homodimer or as an obligate heterodimerization partner for 14 other nuclear receptors , including the three peroxisome proliferator-activated receptors PPARA , PPARD , and PPARG ( Evans and Mangelsdorf , 2014 ) .", "Exome analysis of bladder cancer samples across three independent cohorts identified mutations at RXRA S427 in 5–8% of cases , always leading to an amino acid substitution with an aromatic amino acid , phenylalanine ( ~5% ) or tyrosine ( ~1% ) ( Cancer Genome Atlas Research Network , 2014; Guo et al . , 2013; Van Allen et al . , 2014 ) .", "Reported analysis from TCGA found evidence of up-regulated PPAR pathway activity in RXRA hot-spot mutant cases ( Cancer Genome Atlas Research Network , 2014 ) .", "Copy number analysis identified PPARG as amplified in 17% of bladder cancer cases ( Cancer Genome Atlas Research Network , 2014 ) , with PPARG amplified cases being highly enriched for PPARG mRNA expression ( q value = 2 . 6 × 10-9 ) ( Cerami et al . , 2012 ) .", "These data raised the possibility that hyperactive PPAR signaling , either due to gene-amplification-driven hyper-expression or RXRA hot-spot mutation may drive 20–25% of bladder cancers .", "Here , we characterize structural and biological consequences of the RXRA hot-spot mutations ." ], [ "To establish a causal role of RXRA hot-spot mutations in hyper-activation of PPAR singling , we used retroviral transduction to introduce wild-type or mutant RXRA into two bladder cancer cell lines , JMSU-1 and 575A .", "Equivalent RXRA expression levels were confirmed by qPCR and western blot ( Figure 1A ) .", "RNA-seq was used to compare transcriptomes in the wild-type and S427F RXRA expressing cells .", "Gene transcripts robustly up-regulated by the mutation ( twofold , FDR <0 . 05 ) were analyzed using over-representation analysis ( ORA ) to identify enriched pathways .", "In both cell lines , the PPAR signaling pathway ( KEGG-hsa03320 ) was the top scoring hit .", "When the ORA was limited to genes up-regulated by the mutation in both cell lines , again PPAR signaling was the top hit ( Figure 1B , Figure 1—source data 1 ) .", "We also compared transcriptome changes induced by the RXRA mutation to those induced after treating RXRAwt expressing cells with the PPARG agonist pioglitazone .", "There was robust correlation in both JMSU-1 and 575A ( Pearson r = 0 . 72 , p=1 . 4 × 10−34 or 3 . 1 × 10−105 ) , confirming that the mutation drives expression changes similar to agonist-induced PPAR activation ( Figure 1C ) .", "Mutation-driven hyper-activation of PLIN2 and FABP3 , two genes found up-regulated in our RNA-seq analysis , also occured in the context of RXRAS427Y in JMSU-1 cells ( Figure 1D ) , confirming gain-of-function with either aromatic substitution .", "Previous analysis of human bladder cancer specimens identified a correlation between the presence of RXRAS427F/Y and both the up-regulation of PLIN2 expression and PPAR signaling pathway activity ( Cancer Genome Atlas Research Network , 2014 ) .", "Our data establishes a causal role of the RXRA mutations in driving these expression changes .", "Canonical RXRA-mediated gene regulation involves direct engagement of promoter/enhancers of target genes in the context of homodimers or heterodimers .", "Specific partner-pairs preferentially bind to direct repeats ( DR ) with defined spacer lengths .", "RXR homodimers and RXR/PPAR heterodimers both show preference for DRs with a single nucleotide spacer ( DR1 ) ( Evans and Mangelsdorf , 2014; Nielsen et al . , 2008 ) .", "Using ChIP-seq data for RXRA and acetylated H3K27 ( H3K27ac ) , we identified all active enhancer/promoters bound by RXRAwt and/or RXRAS427F in JMSU-1 ( ‘RXR-bound Active Enhancer/promoters’ or RAEs ) .", "Differential mean peak height of the H3K27ac signal was used as an indicator of relative activation status in the wild-type and mutant condition at each RAE .", "~12% of RAEs showed significant up-regulation in the mutant condition ( FDR <0 . 05 ) ( Figure 1E , Figure 1—source data 2 ) .", "HOMER motif finding was then performed to identify motifs enriched at these hyper-activated RAEs , relative to the background of all RAEs .", "Consistent with activation of PPAR pathway genes , the canonical RXR/PPAR ( DR1 ) motif was the only known motif found to be enriched with significant q-value .", "The most statistically robust motif identified by de novo discovery is shown in Figure 1E .", "Comparing the de novo identified motif to known motifs found it to be most similar to a PPAR response element ( DR1 ) .", "These data demonstrate that hyper-activation of enhancer/promoters driven by the RXRA hot-spot mutation occurs preferentially at canonical RXR/PPAR response elements .", "Using transient transfection of a DR1-driven luciferase reporter , we confirmed relative hyper-activation of DR1 in the JMSU-1 cells expressing mutant RXRA compared to wild-type ( Figure 1F ) , with a degree of induction similar to that observed after pioglitazone treatment .", "Having established that mutant RXRA up-regulates PPAR target gene expression , we next tested if this was dependent on PPARs .", "TCGA data were first queried to determine which of the three PPARs are most highly expressed in bladder cancer specimens with mutant RXRA ( Cerami et al . , 2012 ) .", "Both PPARG and PPARD were significantly more highly expressed than PPARA , but the relative proportion of PPARG and PPARD varied amongst the samples ( Figure 2A , B ) .", "Using siRNA , we acutely knocked-down PPARG and PPARD in the JMSU-1 and 575A cells transduced with wild-type or mutant RXRA and used RT-qPCR to query the expression of PLIN2 and FABP3 ( Figure 2C ) .", "In the non-targeting control , both genes were again found up-regulated by mutant RXRA .", "PPARD knock-down alone appeared to increase PPARG expression suggesting receptor cross talk and loss of negative feedback regulation .", "However , expression of neither PPAR target gene was significantly reduced by PPARD knock-down alone .", "PPARG knock-down partially inhibited PLIN2 expression in both cell lines , but did not have significant impact on FABP3 .", "Combined knock-down of PPARD and PPARG , however , strongly inhibited RXRAS427F-driven hyper-expression of both genes , to an extent greater than knockdown of PPARG alone .", "Thus , both PPARD and PPARG contribute to mutant RXRA-mediated transcriptional hyperactivity in human bladder cancer cells and appear to have redundant function .", "Since hyperactivity of mutant RXRA was dependent on PPAR expression , we reasoned that no hyperactivity would be observed in the DR-1 luciferase reporter assay in a bladder cancer cell line with low endogenous expression of PPARs .", "Treatment of UM-UC-3 with agonists to either PPARG or PPARD did not have meaningful impact on reporter activity when RXRA was transfected without a PPAR ( Figure 2D ) , indicating a lack of relevant endogenous expression in this cell line .", "Similarly , RXRAS427F/Y did not show increased activity relative to RXRAwt ( Figure 2D ) .", "Since RXRA can drive transcription as a homodimer with a preference for the DR1 motif , these data indicate that the mutation does not confer gain-of-function in the homodimer context .", "We then examined reporter activity with co-transfection of PPARG or PPARD .", "Expression of either PPAR was sufficient to elicit mutant RXRA associated hyperactivity to an extent that approximated agonist induced activation of the PPAR in the wild-type RXRA condition ( Figure 2D ) .", "Similar to what was observed in the JMSU-1 stable cells , RXRAS427F appeared to have somewhat stronger activity than RXRAS427Y , but both were hyperactive relative to RXRAwt ( Figure 2D ) .", "Lastly , we tested reporter activity with co-transfection of retinoic acid receptor alpha ( RARA ) , which belongs to another family of RXRA heterodimerization partners .", "Both the DR1 reporter and a reporter with the preferred RXR/RAR binding motif , DR5 , were induced by the RAR agonist all-trans-retinoic acid , but mutant RXRA did not elicit greater activity than wild-type RXRA ( Figure 2—figure supplement 1 ) .", "These data confirmed that RXRA hot-spot mutations confer gain-of-function selectively in the context of PPAR expression .", "Nuclear receptors share a conserved domain structure and are prototypically activated by agonist binding to the ligand-binding domain ( LBD ) .", "The activator function two domain ( AF2 ) is then stabilized in a conformation favoring recruitment of co-activators , initiating transcriptional regulation .", "In the context of RXRA/PPAR heterodimers , activation can be induced with agonists to either receptor ( Evans and Mangelsdorf , 2014 ) .", "Solved crystal structures of the RXRA/PPARG heterodimer reveal that RXRA S427 resides in helix 10 at a heterodimerization interface ( Figure 3A ) ( Chandra et al . , 2008; Gampe et al . , 2000 ) .", "Based on its location , we hypothesized that the RXRA mutation directly activates the PPARG AF2 , independent of PPAR ligand binding .", "To gain evidence , we determined the inducibility of PPARG Q286P , a previously characterized substitution in the ligand-binding pocket ( Figure 3A ) that prevented ligand activation by a panel of PPARG agonists ( Walkey and Spiegelman , 2008 ) .", "The Q286P mutation completely blocked pioglitazone-driven receptor activation , but RXRA S427F-driven receptor hyperactivity was maintained ( Figure 3B ) .", "In contrast , the PPARG E471A mutation previously characterized to diminish the transactivation activity of the PPARG AF2 ( Chen et al . , 2000 ) , strongly reduced both pioglitazone and RXRA S427F-driven activity ( Figure 3B ) .", "The analogous AF2 mutation in RXRA , E453A , diminished activation by the RXRA agonist SR11237 , but had no impact on mutation-driven hyperactivity ( Figure 3C ) .", "These data support a model in which RXRAS427F hyperactivity relies primarily on the PPAR AF2 , independent of PPAR agonist binding .", "To gain insight into how RXRAS427F/Y regulates the PPARG AF2 , we performed long-time scale molecular simulations of RXRA/PPARG heterodimers with wild-type RXRA or after the S427F substitution .", "The starting structure was the agonist-bound crystal structure of the RXRA/PPARG LBDs ( PDB: 1FM6 ) ( Gampe et al . , 2000 ) ; however , the agonist compounds were deleted to reduce subsequent computational complexity and to enable drift toward the inactivated state .", "We next constructed Markov State Models ( MSMs ) following the procedure from Hart et al . ( 2016 ) to quantify the thermodynamics and kinetics of each heterodimer .", "First , conformations adopted by backbone-heavy atoms near the mutated site in RXRA and the PPARG AF2 ( RXRA 425–429 and PPARG 445–477 ) from both wild-type and mutant simulations were clustered in the same state space .", "An MSM was then constructed for each variant based on how often the corresponding simulations transitioned between every pair of clusters .", "The wild-type and mutant largely populate different clusters , consistent with our hypothesis that mutant and wild-type RXRA differentially regulate the PPARG AF2 region ( Figure 3—figure supplement 1A ) We next inspected the three most frequently occupied clusters for wild-type and mutant by superimposing the PPARG residues 445–459 on helix 11 as an anchor , enabling us to visualize relative displacement of the AF2 region located in helix 12 ( Figure 3D ) .", "In the RXRA mutant condition , PPARG E471 more closely approximated the agonist confirmation , in agreement with our mutational studies showing the importance of this residue .", "The terminal tyrosine of PPARG ( Y477 ) also appeared in distinct spaces in the two conditions .", "Comparing the 5% most frequently occupied clusters for wild type and mutant , we observed a significant difference in inter-residue distance between RXRA 427 and PPARG 477 ( Figure 3E ) .", "These data raised the possibility that an aromatic interaction between RXRA S427F/Y and the terminal tyrosine found in all PPARs may underlie activation of PPAR ( Figure 3F ) .", "To test this , the terminal tyrosine was either deleted or mutated to serine in both PPARG ( Y477 ) and PPARD ( Y441 ) and expression of the mutant PPARs was confirmed by western blot ( Figure 3—figure supplement 1B ) .", "Inducibility by RXRAS427F or agonist was then determined ( Figure 3B and G ) .", "In all cases , inducibility by agonist was maintained , but RXRA S427F-mediated hyperactivity was eliminated .", "We conclude that S427F/Y initiates an allosteric relay via an aromatic interaction with the terminal tyrosine on PPAR , leading to PPAR AF2 activation .", "No other RXR dimerization partners have an aromatic amino acid at the corresponding position , providing a structural basis for the selective activation of PPARs ( Figure 3F ) .", "We next sought to determine if PPAR activation drives proliferation of urothelial cells .", "To culture primary urothelial cells , we developed a mouse urothelial organoid culture system .", "Similar organoid systems have proven successful at identifying pro-tumorigenic phenotypes driven by cancer-associated mutations in multiple tissue types ( Sachs and Clevers , 2014; Karthaus et al . , 2014; Drost et al . , 2015; Hwang et al . , 2016; Boj et al . , 2015 ) .", "With our urothelial approach , hollow spherical epithelial structures can reliably be grown and passaged from primary mouse bladder epithelium , utilizing epidermal growth factor ( EGF ) as the primary growth factor ( Figure 4—figure supplement 1A ) .", "Because growth-factor-independent growth is a classical hallmark of transformed cells , we asked if stimulation with PPAR agonists could confer growth in the absence of EGF .", "As expected , there was no growth of three independently derived wild-type organoids in the absence of growth factor .", "Treatment with the PPARD agonist GW0742 or the PPARG agonist pioglitazone had no significant effect on growth in these conditions , suggesting that PPAR activation is not sufficient to drive growth in normal urothelial organoids ( Figure 4A ) .", "Bladder cancer genomes typically harbor mutations in several genes recognized as recurrently altered in bladder cancers ( Cancer Genome Atlas Research Network , 2014 ) .", "We speculated that pro-tumorigenic activities of PPAR signaling may only be apparent in the context of tumor suppressor loss .", "For our analysis , we focused on TP53 and KDM6A which are amongst the most frequently mutated tumor suppressors in bladder cancer , mutated in ~50% and~25% of bladder cancer cases , respectively ( Cancer Genome Atlas Research Network , 2014; Guo et al . , 2013; Van Allen et al . , 2014 ) .", "Of 16 patient samples identified with an RXRA hot-spot mutation across three cohorts , four had mutations in both TP53 and KDM6A , five had mutations in TP53 only , three had mutations in KDM6A only , and four had mutations in neither ( Cerami et al . , 2012 ) .", "To determine if loss of tumor suppressor function would enable pro-tumorigenic activities of PPAR activation , we generated conditional knock-out bladder organoids from mice with the following three genotypes: ( 1 ) Trp53F/F , ( 2 ) Kdm6aF , and ( 3 ) Trp53F/F;Kdm6aF .", "Three independent organoid lines ( each from a distinct mouse bladder ) were generated with each genotype .", "These organoid lines were then all infected with Adeno-cre and complete deletion of the conditional alleles was confirmed by genotyping PCR ( not shown ) .", "The wild-type organoids utilized in Figure 4A were generated from the littermates of these mice and also were infected with Adeno-Cre to control for Cre exposure .", "We then determined growth of the organoids in EGF deplete media after treatment with PPAR agonists ( Figure 4B , Figure 4—figure supplement 1B ) .", "No genotype showed consistent positive growth in the vehicle condition .", "Elimination of Trp53 , but not Kdm6a , was sufficient for GW0742 to drive proliferation .", "With combined knock-out of Trp53 and Kdm6a , growth induction by GW0742 was even more robust than with Trp53 knock-out alone .", "These data suggest that loss of tumor suppressors creates a context permissive for PPARD-driven proliferation .", "Even though responsiveness to GW0742 was consistent across all of the dual knock-out organoids , we reasoned that utilizing a clonal sub-line might minimize variation due to random clonal skewing in later experiments involving retroviral infection and drug selection .", "Therefore , we established DKO 431 . A from a single organoid picked from the DKO 431 line and assessed its responsiveness to PPAR agonists .", "DKO 431 . A showed strong , dose-dependent , enhanced growth when treated with GW0742 , but not pioglitazone ( Figure 4C ) .", "To assess transcriptional activation by the same PPAR agonists , DKO 431 . A organoids were plated in standard media for 7 days and then treated with two doses of GW0742 or pioglitazone for 48 hr .", "Expression of two PPAR target genes , Plin2 and Fabp4 was then determined by RT-qPCR .", "GW0742 strongly induced both genes , while pioglitazone and little to no effect ( Figure 4D , Figure 4—figure supplement 1C ) .", "These findings confirm that the ability of PPAR agonists to induce target genes in DKO 431 . A correlates with their ability to promote growth-factor-independent growth .", "The apparent lack of response to PPARG agonist in these experiments is likely simply due to its lower expression in these mouse organoids relative to 575A and JMSU-1 , the two cells lines in which we had established induction of PPAR target genes with pioglitazone in Figure 1 ( Figure 4—figure supplement 1D ) .", "We then asked if RXRAS427F would promote growth-factor-independent growth .", "DKO 431 . A was infected with retrovirus bearing empty vector , RXRAwt , or RXRAS427F and similar expression levels of RXRA between the wild-type and mutant condition was confirmed by western blot and qPCR ( Figure 4E ) .", "Growth of the organoids in EGF deplete media was then assessed .", "Mutant RXRA-expressing organoids grew significantly faster than empty vector or wild-type expressing organoids ( Figure 4F ) .", "However , growth was also observed in the wild-type condition .", "To ensure that the difference between mutant and wild-type growth was robust , we passaged wild-type and mutant-expressing organoids with tyrpsinization weekly , always plating an identical number of cells and then counting the number of cells present at time of each passage .", "The total number of doublings over six passages was then determined .", "Mutant RXRA organoids showed consistent growth throughout the experiment , while the wild-type condition appeared to plateau ( Figure 4G ) .", "Comparing the number of doublings per passage between the two conditions , we found the difference to be highly significant ( paired t test p=0 . 0003 ) , confirming gain-of-function with the mutant allele .", "Having established that RXRAS427F phenocopies GW0742 induced growth in the organoid growth assay , we next looked for transcriptional evidence that RXRAS427F was activating PPARD .", "Organoids were cultured in standard media for seven days and then treated with PPAR antagonists for an additional two days .", "We then determined mRNA expression levels of two PPAR target genes by RT-qPCR .", "Comparing vehicle-treated samples , Plin2 and Fabp4 were found significantly up-regulated in the mutant condition compared to the empty vector or wild-type organoids , confirming that mutant RXRA up-regulates expected PPAR targets in the organoid system ( Figure 5A ) .", "Treatment of the mutant expressing organoids with either of two PPARD inverse agonists , ST247 and GSK0660 , blunted up-regulation of the target genes ( Figure 5A , Figure 5—figure supplement", "1 ) ( Naruhn et al . , 2011; Shearer et al . , 2008 ) .", "The PPARG antagonist T0070907 did not show any inhibitory activity .", "These findings confirm that mutant RXRA drives PPARD hyper-activity in the organoid system .", "We next sought to determine if mutant RXRA-driven growth could be reversed with PPAR inhibition .", "When treated with either of the two PPARD antagonists , the mutant expressing organoids showed significant , dose-dependent growth inhibition ( Figure 5B . ) To determine if the proliferative effects of PPARD inhibitors was specific to the RXRA-mutant-driven proliferation , we utilized an organoid line we established from a carcinogen-induced bladder tumor , called MCB6C , which shows robust growth in EGF deplete media , but does not harbor the RXRA mutation ( not shown ) .", "Treatment of MCB6C with the PPARD inhibitors in EGF deplete media did not inhibit their growth across the identical doses ( Figure 5C ) .", "As expected based on the gene expression analysis , the PPARG antagonist T0070907 also had no antiproliferative effects on 431 . A . RXRAS427F ( Figure 5D ) .", "Lastly , 431 . A . RXRwt organoids in EGF-containing media were treated with the same PPAR antagonists , none of which significantly inhibited growth of this organoid line ( Figure 5E ) , confirming PPAR dependence is specific to mutant RXRA-driven proliferation ." ], [ "A role for PPAR activity in promoting bladder cancer is supported by the presence of a PPARG gene amplification in 17% of cases ( Cancer Genome Atlas Research Network , 2014 ) .", "Intriguingly , pioglitazone use in patients with diabetes appears to increase bladder cancer risk , further hinting that PPARG activation can promote bladder cancer growth ( Lewis et al . , 2011 ) .", "Our studies identified RXRA point mutations as a second genomic mechanism by which PPARs can be hyperactivated .", "These data , along with our direct functional evidence that PPAR activation by agonist or mutant RXRA drives urothelial proliferation , build on a growing body of data credentialing PPARs as bladder cancer drivers .", "Importantly , human bladder cancer samples appear to express both PPARD and PPARG and our data suggest both are activated by mutant RXRA and play a role in transcriptional hyperactivity in human bladder cancer cells .", "In order to guide drug development , future studies will be needed with a broad panel of human-derived tissue to determine if one PPAR isoform is of greater clinical relevance to RXRA mutation-driven disease .", "We speculate co-targeting of PPARD and PPARG will be necessary , perhaps even in PPARG amplified cases , as functional redundancy of homologous nuclear receptors or kinases is known to be exploited by tumors , in some cases underlying treatment resistance to therapeutics targeting only one homologue ( Arora et al . , 2013; Juric et al . , 2015 ) .", "Heyman and colleagues showed two decades ago that RXR agonists can induce activation of RXR/PPAR heterodimers , independent of the RXR AF2 , by inducing conformational changes in PPAR ( Schulman et al . , 1998 ) .", "The RXRA hot-spot mutations appear to co-opt a similar , although mechanistically distinct , activation principle , relying on the introduction of an aromatic interaction that drives ligand-independent activation of PPARs .", "While this manuscript was under preparation , Korpal et al . reported biophysical data characterizing the mutant RXRA/PPARG heterodimer ( Korpal et al . , 2017 ) .", "Those in vitro findings align with our modeling predictions , which we further functionally validated with cell-based mutational studies , both with PPARG and PPARD .", "This allosteric activation mechanism of the PPAR AF2 by mutant RXRA raises unique challenges with regard to inhibitor design .", "In our study , we utilized inverse agonists which bind the LBD and stabilize interactions with co-repressors .", "While they are effective at reversing mutant RXRA-driven activity , we suspect chemical screens using assays in which PPARs are activated through the mutation-based mechanism may identify optimal inverse agonists capable of more complete target inhibition .", "Inverse agonists binding the PPAR LBD remain the most characterized strategy to inhibit PPAR activity , but may not prove to be the most effective approach for blocking ligand independent activity driven by RXRA mutations .", "A potentially more effective strategy might be through the identification of small molecules that induce PPAR degradation .", "Supporting the concept , ligands that induce destabilizing conformations in ER have been identified ( Wu et al . , 2005 ) , and the clinical utility of the ER degrader fluvestrant has been validated in breast cancer clinical trials ( Howell et al . , 2002; Osborne et al . , 2002 ) .", "Similar to ER , PPARs undergo agonist-induced degradation , suggesting therapeutic ligand-induced degradation of PPARs might also be feasible ( Hauser et al . , 2000 ) .", "Perhaps , a more rational approach to inducing PPAR degradation would be to engineer PPAR ligands linked to a chemical tag ( e . g . PROTAC ) that targets proteins to the proteasome .", "Such approaches have been used in preclinical systems to degrade other nuclear receptors ( Rodriguez-Gonzalez et al . , 2008 ) , although the clinical utility of the approach is not yet established .", "Lastly , the identification of biologically active small molecules that disrupt co-regulator binding to the ER AF2 raises yet another possible paradigm for targeting ligand-independent activation through the identification of small molecules that recognize the activated PPAR AF2 ( Raj et al . , 2017 ) .", "Our work here should encourage such approaches with the goal of identifying inhibitors that effectively overcome PPAR tumorigenic activity due to PPARG amplification and RXRA mutation .", "The organoid system provided us with a unique capability to assay epithelial autonomous effects of PPAR activity in defined genetic contexts , including normal urothelium .", "In contrast to classical growth factors such as EGF which stimulate proliferation of wild-type organoids , we found that PPARD activation is only sufficient to drive growth with concurrent tumor suppressor loss .", "One possible explanation is that tumor suppressor loss alters PPARD-driven transcriptional regulation .", "Studies with AR in prostate cancer suggest that the AR cistrome is broadly reprogrammed during tumorigenesis ( Pomerantz et al . , 2015 ) , supporting a model in which nuclear receptors take on de novo target regulation in transformed cells .", "Alternatively , tumor suppressor loss may alter the cellular response to PPARD activation and/or provide complementary pro-tumorigenic signals .", "These possibilities will be explored in future studies and should help elucidate the cellular mechanisms by which PPAR signaling can drive urothelial proliferation .", "Regardless of the underlying mechanisms , however , our findings , along with recent work from others demonstrating PPARG orchestrates an immunosuppressive micro-environment to enhance tumor growth in vivo ( Korpal et al . , 2017 ) , provide an increasingly compelling rationale for the development of a new class of molecularly targeted drugs with potential relevance to at least a quarter of bladder cancer cases ." ], [ "Animals were handled and housed according to protocols approved by the Washington University School of Medicine Institutional Animal Care and Use Committee .", "Kdm6aF mice were generated by Dr . Lukas Wartman ( Washington University School of Medicine ) with ES cells obtained from EUCOMM with the Kdm6atm1a ( EUCOMM ) Wtsi allele ( manuscript in preparation ) and maintained on the C57BL6/J background .", "B6 . 129P2-Trp53tm1brn/J ( Trp53Flox ) mice were purchased from Jackson Laboratory ( stock # 008462 ) .", "Mice were genotyped via PCR ( Kdm6a 5’ CGAGAAAGGAAATGTGAGAGCAAGG 3’ and 5’ CTGGCAGGATATGATAGCAATGTG 3’; Trp53 5’ GGTTAAACCCAGCTTGACCA 3’ and 5’ GGAGGCAGAGACAGTTGGAG 3’ ) .", "Experimental animals resulted from crosses between Kdm6aFlox/+ and Trp53Flox/+ mice .", "To generate Trp53-/- , Kdm6a- , and Trp53-/-;Kdm6a- organoids , Trp53Flox/Flox , Kdm6aFlox , and Trp53Flox/Flox;Kdm6aFlox organoids were infected with Ad5CMVCre-eGFP adenovirus ( University of Iowa Viral Vector Core ) in vitro .", "Organoids were trypsinized into single cells .", "500 , 000 cells were pelleted in a 1 . 5-mL tube and re-suspended in 100 µL of organoid medium supplemented with a final concentration of 5 µg/mL polybrene ( TR-1003-G Millipore ) with or without virus at a MOI of 50 .", "Cells were incubated at 32°C with shaking at 700 rpm on an Eppendorf ThermoMixer for 1 hr .", "Cells were then incubated at 37°C without shaking for approximately 2 . 5 hr .", "Cells were pelleted and re-suspended in 650 µL Matrigel to make twelve 50 µL Matrigel tabs .", "Tabs were hardened at 37°C for 15 min and organoid medium was added .", "To verify recombination of floxed alleles , DNA was isolated from organoid cell pellets using the DNeasy Blood and Tissue Kit ( 69506 Qiagen , Germany ) and genotyped via PCR using the primers listed in Key Resources .", "Wild-type human RXRA was expressed from pBABE puro RXRA ( plasmid #11441 Addgene ) .", "Point mutations were introduced via the QuikChange Site-Directed Mutagenesis protocol ( Agilent Technologies ) .", "pBABE puro empty vector was generated by digesting pBABE puro RXRA with EcoRI to remove RXRA and by ligating the plasmid ends together with T4 DNA ligase ( M0202S New England BioLabs ) .", "Wild-type human PPARG1 was expressed from pCMV6-XL4 PPARG ( SC108192 Origene ) .", "Point mutations were introduced via the QuikChange Site-Directed Mutagenesis protocol .", "pCMV6-XL4 empty vector was generated by digesting pCMV6-XL4 PPARG with NotI to remove PPARG and by ligating the plasmid ends together with T4 DNA ligase .", "Human PPARD was cloned from JMSU-1 epithelial bladder cancer cells .", "Cells were harvested upon reaching 80–90% confluency and total RNA was isolated using the RNeasy Mini Kit .", "JMSU-1 cDNA was generated using a First-Strand cDNA Synthesis Kit ( 27-9261-01 GE Healthcare Life Sciences ) with random hexameric primers and 5 µg total RNA .", "Primers targeting the 5’ and 3’ UTRs were used to amplify PPARD .", "PPARD was inserted into the pCMV6-XL4 expression vector using the In-Fusion HD Cloning System ( 639646 Clontech ) .", "Point mutations were introduced via the QuikChange Site-Directed Mutagenesis protocol .", "See Key Resources for primer sequences .", "JMSU-1 , 575A , and UM-UC-3 were obtained from Dr . David Solit ( MSKCC ) .", "293 Lenti-X cells were obtained from Clonetech .", "Cells were cultured in recommended medium supplemented with 10% fetal bovine serum ( FBS; FB-11 Omega Scientific ) , 1% penicillin-streptomycin ( 15140122 Gibco ) , and 1x GlutaMAX Supplement ( 35050061 Gibco ) .", "Cells were grown at 37°C in 5% CO2 .", "Retrovirus was produced by using LipoD293 DNA In Vitro Transfection Reagent ( SL100668 SignaGen Laboratories ) to transiently transfect 293 Lenti-X cells with 0 . 45 µg VSVG , 2 . 27 µg pCL-ampho , and 2 . 27 µg pBABE puro empty vector , RXRA WT , RXRA S427F , or RXRA S427Y in a 100 mm TC-treated tissue culture dish .", "Medium was replaced with recommended culturing medium approximately 6 hr post-transfection .", "Retrovirus was collected 48 hr post-transfection and was filtered through a 0 . 45-µM filter .", "Stable cells lines were generated by seeding cells in a 100 mm TC-treated tissue culture dish 18–24 hr before adding virus so that cell confluency was 50–70% when virus was added .", "The next day , medium was removed and retrovirus diluted 1:4 in the recommended culturing medium and supplemented with a final concentration of 8 µg/mL polybrene ( TR-1003-G Millipore ) was added to the cells .", "After approximately 24 hr , the virus-containing medium was replaced with fresh medium .", "Cells were cultured for an additional 36 hr , at which time puromycin ( P8833 Sigma-Aldrich ) was added at a predetermined concentration to select for transduced cells .", "To generate stable organoid lines , organoids were trypsinized into single cells .", "500 , 000 cells were pelleted in a 1 . 5-mL tube and resuspended in 500 µL of 1:1 organoid medium:retrovirus mixture supplemented with a final concentration of 8 µg/mL polybrene .", "Cells were incubated at 32°C with shaking at 600 rpm on an Eppendorf ThermoMixer for 1 . 5 hr .", "Cells were then incubated at 37°C without shaking for approximately 5 hr .", "Cells were pelleted and resuspended in enough Matrigel to make three 50 µL Matrigel tabs .", "Tabs were hardened at 37°C for 15 min and organoid medium was added .", "48 hr after infection , puromycin was added at a predetermined concentration to select for transduced cells .", "Cells were seeded in a 12-well cell culture plate 18–24 hr before transfection so that cell confluency was 50–70% at time of transfection .", "PepMute siRNA Transfection Reagent ( SL100566 SignaGen Laboratories ) was used to transiently transfect cells with ON-TARGETplus Non-targeting Pool ( D-001810-10-20 Dharmacon ) , ON-TARGETplus Human PPARG siRNA ( L-003436-00-0005 Dharmacon ) , and/or ON-TARGETplus Human PPARD siRNA ( L-003435-00-0005 Dharmacon ) .", "PepMute/siRNA complex-containing medium was replaced with recommended culturing medium approximately 24 hr post transfection .", "Cells were collected 72 hr post transfection .", "Cells and organoids were lysed in M-PER ( 78501 Thermo Scientific ) supplemented with 1x Halt Protease Inhibitor Cocktail ( 87786 ThermoFisher Scientific ) and 0 . 45 M NaCl for 10 min on ice with periodic vortexing .", "Lysates were cleared by centrifugation at 18 , 000 x g for 15 min .", "Protein concentration was determined with Pierce BCA Protein Assay ( 23224/23228 ThermoFisher Scientific ) .", "Protein lysates were denatured by boiling in 1x Bolt LDS Sample Buffer ( B0008 Invitrogen ) and 1x NuPAGE Sample Reducing Reagent ( NP0004 Invitrogen ) for 10 min .", "Proteins were separated on Bolt 4–12% Bis-Tris Plus Gels ( NW04122BOX Invitrogen ) , transferred onto Immobilon-P Membrane , PVDF , 0 . 45 µM ( IPVH00010 Millipore ) , and blocked according to antibody specifications .", "Blots were incubated with primary antibody in blocking solution overnight at 4°C .", "Blots were washed with 1x TBS plus 0 . 1% Tween and primary antibodies were detected with HRP-conjugated secondary antibodies .", "Amersham ECL Prime western blotting detection reagent ( RPN2232 GE Healthcare Life Sciences ) or Clarity Western ECL Substrate ( 170–5060 Bio-Rad ) was used for chemiluminescence and luminescence was detected with the Bio-Rad ChemiDoc XRS + System .", "Antibodies and concentration list in Key Resources RNeasy Mini Kit ( 74106 Qiagen ) was used to isolate RNA from organoid and cell pellets .", "iScript cDNA Synthesis Kit ( 1708891 Bio-Rad ) was used to synthesize cDNA from 0 . 5 to 1 µg of RNA .", "RT-qPCR was performed according to package instructions for SsoFast EvaGreen Supermix with low ROX ( 172–5211 Bio-Rad ) on the Applied Biosystems QuantStudio 3 Real-Time PCR system .", "See Key Resources for primer sequences used .", "Cells were seeded in a 48-well cell culture plate 18–24 hr before transfection so that cell density was approximately 70% at time of transfection .", "LipoD293 DNA In Vitro Transfection Reagent ( SL100668 SignaGen Laboratories ) was used to transiently transfect cells with a reporter plasmid , expression vectors , and a plasmid expressing Renilla luciferase .", "LipoD293/DNA complex-containing medium was replaced with recommended culturing medium supplemented with 1% FBS 16–18 hr post-transfection .", "Drugs were added to cells 48 hr post transfection .", "Drug-containing medium was removed 16 hr later and cells were washed with 1x PBS .", "Luciferase activity was assayed using Dual-Glo Luciferase Assay System ( E2940 Promega ) and in accordance with the Dual-Glo protocol except that a 1:1 mixture of 1xPBS:Dual-Glo Reagent was added to the cells .", "Luminescence was measured on the SpectraMax i3 Platform ( Molecular Devices ) .", "See Key Resources for list of plasmids .", "Organoids were plated at 1000 or 1500 cells per tab and cultured in normal or EGF deplete organoid medium , respectively , for 8–10 days .", "Drugs or DMSO were added to medium at time of plating .", "Medium was changed every 4 days post-plating .", "Cell viability was measured day 1 ( baseline measurement ) and at indicated days post-plating using CellTiter-Glo ( G7571 Promega ) .", "Briefly , tabs were homogenized in 1:1 mixture of 1xPBS:CellTiter-Glo reagent and incubated at room temperature for 10 min with shaking .", "Homogenate was transferred to a white , 96-well plate and luminescence was measured on the SpectraMax i3 Platform .", "For organoid counting assay , organoids were digested into a single-cell suspension with trypsin and the counted in duplicate using a Bio-rad TC20 .", "Cells were seeded in biological triplicate in 6-well tissue culture plates at 300 , 000 cells ( JMSU-1 ) or 125 , 000 ( 575A ) cells per well .", "48 hr later , 0 . 1% DMSO or 1 µM pioglitazone ( E6910 Sigma-Aldrich ) was added to cells .", "RNA was isolated with the RNeasy Mini Kit ( 74106 Qiagen ) 16–18 hr after addition of drugs .", "RNA library prep and sequencing were done by the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine .", "Briefly , Ribosomal RNA was removed by poly-A selection using Oligo-dT beads .", "mRNA was then fragmented and reverse transcribed to yield double stranded cDNA using random hexamers .", "cDNA was blunt ended , had an A base added to the 3’ ends , and then had Illumina sequencing adapters ligated to the ends .", "Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags .", "Fragments were sequenced on an Illumina HiSeq-2500 or HiSeq-3000 using single reads extending 50 bases .", "RNA-seq reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2 . 0 . 4b .", "Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1 . 4 . 5 .", "Transcript counts were produced by Sailfish version 0 . 6 . 3 .", "Sequencing performance was assessed for total number of aligned reads , total number of uniquely aligned reads , genes and transcripts detected , ribosomal fraction known junction saturation and read distribution over known gene models with RSeQC version 2 . 3 .", "All gene-level and transcript counts were then imported into the R/Bioconductor package EdgeR and TMM normalization size factors were calculated to adjust for samples for differences in library size .", "Genes or transcripts not expressed in any sample were excluded from further analysis .", "The TMM size factors and the matrix of counts were then imported into R/Bioconductor package Limma and weighted likelihoods based on the observed mean-variance relationship of every gene/transcript and sample were then calculated for all samples with the voomWithQualityWeights function .", "Performance of the samples was assessed with a spearman correlation matrix and multidimensional scaling plots .", "Gene/transcript performance was assessed with plots of residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals .", "Generalized linear models were then created to test for gene/transcript level differential expression .", "Differentially expressed genes and transcripts were then filtered for FDR adjusted p-values less than or equal to 0 . 05 .", "Go-Elite version 1 . 2 was used to identify over-represented pathways curated by KEGG .", "JMSU-1 cells stably expressing RXRAwt or RXRAS427F were seeded in biological triplicate in 150 mm TC-treated culture dish and collected when 70–90% confluency was reached .", "Crosslinking and ChIP were performed as described for adherent cells in the ENCODE experiment summary for ENCSR000BJW ( https://www . encodeproject . org/experiments/ENCSR000BJW/ ) .", "Chromatin was sonicated using the Diagenode Pico Bioruptor .", "DNA-protein complexes were precipitated with antibodies against RXRA ( PP-K8508-00 R and D Systems ) and H3K27Ac ( ab4729 Abcam ) .", "Ovation Ultralow System V2 ( 0344–32 NuGen ) was used to generate the sequencing library .", "The library was sequenced by the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine .", "Reads were aligned to hg19 using NovoAlign version 3 . 04 . 06 with the command novoalign -r None -l 30 -e 100 -i 230 140 –H .", "For the RXRA and H3K27ac IPs , peaks were called for each phenotype using the IDR ( Li et al . , 2011 ) pipeline described here ( Kundaje , 2012 ) with MACS version 2 . 1 . 1 ( Zhang et al . , 2008 ) as the peak caller .", "RAE regions were defined for each phenotype by running bedtools intersect on the H3K27ac and RXRA IDR peak lists .", "Differential binding analysis was performed on the H3K27ac signal using DiffBind ( Ross-Innes et al . , 2012 ) comparing mutant to wild-type with the IDR peak lists as input and an FDR cutoff of 0 . 05 .", "Motif analysis of the RAE regions was performed using HOMER version 4 . 9 ( Heinz et al . , 2010 ) with the command findMotifsGenome . pl –h –bg <Background> <Peaklist> hg19 .", "Peaklist was the RAE regions found by DiffBind to have higher H3K27ac binding in mutant .", "Background was the list of all RAE regions minus the ones found in peaklist .", "The published structure of complexed RXRA/PPARG LBDs bound to agonist ( PDB: 1FM6 ) ( Gampe et al . , 2000 ) was used as the starting conformation for simulations .", "To reduce subsequent computational complexity and enable drift towards an inactive state during simulations , the respective agonist structures binding RXRA and PPARG were removed .", "The small peads were aligned to hg19 usptide nuclear receptor coactivator 1 ( NCOA1 ) bound to both RXRA and PPARG AF2 domains was also removed with the same purpose .", "The Chimera molecular visualization tool ( Pettersen et al . , 2004 ) was used to mutate RXRA serine 427 to phenylalanine ( RXRA S427F ) , creating wild-type and mutant structures for subsequent simulations .", "The protocol described by Hart et . al . was used for molecular dynamics simulations ( Hart et al . , 2016 ) .", "Briefly , simulations were performed using the molecular dynamics package GROMACS 5 . 1 . 3 ( Abraham et al . , 2015 ) .", "The Amber03 force field ( Duan et al . , 2003 ) was used and hydrogen atoms in each structure were replaced with virtual sites .", "The protein structure was solvated within a dodecahedron whose border was at least 10 Å away from the protein in all directions .", "Chlorine counterions were added to neutralize the system’s overall positive charge .", "To ensure the system contains no steric clashes or inappropriate geometry , energy minimization was used to relax the system below a 1000 kJ/mol/nm threshold .", "To equilibrate solvent and ions around the structure , the system underwent position-restrained molecular dynamics simulation for one nanosecond using a step size of 4 fs .", "After relaxation and equilibration , each system was subjected to long-timescale molecular simulations .", "Simulations took place using the NPT ensemble at 1 bar and 300 K . Parrinello-Rahman pressure coupling ( Parrinello and Rahman , 1981 ) and the V-rescale thermostat ( Bussi et al . , 2007 ) were used during simulations .", "The LINCS method ( Hess , 2008 ) constrained hydrogen bonds and allowed the use of virtual sites .", "The cutoffs for electrostatic and van der Waals interactions was 9 Å .", "Periodic boundary conditions were applied during simulation and the particle-mesh Ewald summation reconstituted any long-distance electrostatic interactions .", "Each simulation lasted 100 nanoseconds with conformations stored every 10 picoseconds .", "Ten simulations were run to produce an aggregate 1 microsecond of simulation for both wild-type and mutant conditions .", "MSMBuilder 2 . 8 ( Beauchamp et al . , 2011 ) was used to analyze conformations adopted during molecular simulation .", "Wild-type and mutant structures were clustered in overlapping state space using the hybrid k-centers/k-medoids technique .", "Clustering was based on the RMSD of between backbone-heavy atoms from PPARG 445–477 and RXRA 425–429 .", "The inter-cluster distance cutoff was 1 Å and 50 iterations were performed to refine cluster assignment .", "A microstate Markov state model ( MSM ) was constructed using the Transpose method for symmetric counts matrix estimation and without applying an ergodic trim .", "The 5% most frequently occupied microstates during wild-type and mutant conformations were selected .", "Distance between alpha carbons of RXRA 427 and PPARG 477 was calculated for comparison between microstate structures from wild-type and mutant simulations .", "The inter-residue distances were normalized to the respective distance in the agonist-bound crystal structure .", "The Chimera molecular visualization tool was used to generate representative figures .", "All statistical tests are as indicated in figure legends .", "Analysis was done in GraphPad Prism with the exception of pearson coefficients which were calculated using Microsoft Excel .", "All statistical comparisons are two-tailed .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 ." ] ]
[ "RXRA regulates transcription as part of a heterodimer with 14 other nuclear receptors , including the peroxisome proliferator-activated receptors ( PPARs ) .", "Analysis from TCGA raised the possibility that hyperactive PPAR signaling , either due to PPAR gamma gene amplification or RXRA hot-spot mutation ( S427F/Y ) drives 20–25% of human bladder cancers .", "Here , we characterize mutant RXRA , demonstrating it induces enhancer/promoter activity in the context of RXRA/PPAR heterodimers in human bladder cancer cells .", "Structure-function studies indicate that the RXRA substitution allosterically regulates the PPAR AF2 domain via an aromatic interaction with the terminal tyrosine found in PPARs .", "In mouse urothelial organoids , PPAR agonism is sufficient to drive growth-factor-independent growth in the context of concurrent tumor suppressor loss .", "Similarly , mutant RXRA stimulates growth-factor-independent growth of Trp53/Kdm6a null bladder organoids .", "Mutant RXRA-driven growth of urothelium is reversible by PPAR inhibition , supporting PPARs as targetable drivers of bladder cancer ." ]
[ "Bladder cancer is the sixth most common type of cancer in the United States .", "At the moment , treatment options for advanced bladder cancer are limited to chemotherapy and immunotherapy , both of which benefit only some patients .", "Many other types of cancer can be treated with drugs that are specific to genetic mutations found in those cancer cells , often making the treatments more efficient with fewer side effects .", "Between 5–8% of people with bladder cancer have a mutation in the gene that produces a protein called RXRA .", "This protein partners with itself or with other proteins to control gene activity .", "However , it was not clear what mutant RXRA proteins do in bladder cancer cells .", "Halstead et al . studied the RXRA mutation in human bladder cancer cells and “mini-bladders” grown in the laboratory from mouse bladder cells .", "Biochemical experiments showed that the mutant RXRA protein causes abnormally high activity in one group of its partner proteins , called peroxisome proliferator-activated receptors ( PPARs ) .", "The PPARs , in turn , switch on genes that help cancer cells to grow and multiply .", "Computational simulations of the mutant RXRA binding to PPARs revealed , at a molecular level , how this activation occurs .", "Lastly , Halstead et al . used chemicals that block the activity of PPARs to stop the growth of cells in the mouse mini-bladders that contained the RXRA mutation .", "These findings suggest that bladder cancer patients with the RXRA mutation may benefit from therapies that inhibit PPARs .", "Such therapies could also benefit the approximately 15–20% of people with bladder cancer who do not have the RXRA mutation but who do have over-active PPARs .", "Although there are chemicals that block the activity of PPARs , more research is needed to refine them before they can be used to treat cancer ." ]
2017
[ "Introduction", "Results", "Discussion" ]
[ "neuroscience" ]
Endocannabinoid dynamics gate spike-timing dependent depression and potentiation
elife-13185-v2
[ [ "Bidirectional long-term plasticity of synaptic strength ( LTD and LTP ) underlies multiple forms of learning and memory ( Citri and Malenka , 2008; Nabavi et al , 2014 ) .", "Bidirectionality is of paramount functional importance since it allows LTP and LTD to reverse each other with time at a single synapse , thus enabling adaptive changes of the synaptic weight .", "Endocannabinoids ( eCBs ) have emerged as a major actor in learning and memory because of their powerful influence on synaptic plasticity ( Chevaleyre et al . , 2006; Heifets and Castillo , 2009; Kano et al . , 2009; Katona and Freund , 2012 ) .", "The eCB system is mainly composed of active biolipids ( notably 2-arachidonylglycerol , 2-AG and anandamide , AEA ) synthesized and released on-demand acting as retrograde neurotransmitters on presynaptic type-1 cannabinoid receptor ( CB1R ) and postsynaptic transient receptor potential vanilloid-type-1 ( TRPV1 ) ( Piomelli , 2003; Piomelli et al . , 2007; Di Marzo , 2008; Alger and Kim , 2011 ) .", "The major neurotransmitter systems , glutamate and GABA , allow bidirectional synaptic plasticity ( Citri and Malenka , 2008 ) , i . e . the same signaling pathway in the same cell gates the neuron towards potentiation or depression depending on the activity pattern .", "In contrast , eCBs have been widely described as a powerful unidirectional system that depresses neuronal communication on a short or long timescale .", "However , recent reports challenge this view and indicate that eCBs could also act as a bidirectional system for synaptic plasticity .", "We recently reported the existence of an eCB-mediated spike-timing dependent LTP in the dorsal striatum induced by a low number of paired stimulations and dependent on the activation of CB1R and TRPV1 ( Cui et al , 2015 ) .", "We found that few coincident pre- and post-synaptic spikes ( 5–15 ) were sufficient to increase synaptic efficacy through a signaling pathway that relies on the activation of CB1R and TRPV1 and on 2-AG elevations .", "The latter are triggered by coupled postsynaptic rises of calcium and DAG lipase α ( DAGLα ) activity mediated by type-5 metabotropic glutamate receptors ( mGluR5 ) , muscarinic M1 receptors and voltage-sensitive calcium channels ( VSCCs ) ( Cui et al , 2015 ) .", "In addition , it has been reported an indirect role of eCBs in promoting LTP at mixed ( chemical and electrical ) synapses of the goldfish Mauthner cell via intermediary dopaminergic neurons ( Cachope et al . , 2007 ) or at hippocampal CA1 synapses via a GABAA receptor-mediated mechanism ( Lin et al . , 2011; Xu et al . , 2012 ) .", "Likewise , facilitation of LTP in the hippocampus via eCB-induced presynaptic depression of GABAergic transmission ( Carlson et al . , 2002; Chevaleyre and Castillo , 2004; Zhu and Lovinger , 2007 ) , and mediation of heterosynaptic short-term potentiation via intermediary astrocytes ( Navarrete and Araque , 2010 ) have been reported .", "There exists a growing body of evidence that paves the way for a bidirectional action of eCBs in synaptic plasticity depending on the activity pattern on either side of the synapse .", "In the case of glutamate , the principal mechanism put forward to account for bidirectionality is the calcium-control hypothesis , which states that postsynaptic calcium levels and/or time courses decide the outcome of plasticity ( LTP or LTD ) ( Shouval et al . , 2002; Graupner and Brunel , 2012 ) .", "However , how eCBs induce both LTD and LTP remains to be elucidated .", "Here , combining experimental and computer modeling approaches , we show that the bidirectionality of eCB-dependent STDP in striatum is controlled by eCB-levels: moderate level and prolonged release of eCB lead to LTD while brief releases of high eCB concentration yield LTP .", "In this aspect , MAG-lipase appears as a key controller of synaptic plasticity .", "Our results considerably enlarge the spectrum of action of eCBs since they show that eCBs not only promote depression but also potentiation , i . e . they act as a bidirectional system , depending on the regime of activity pattern on either side of the synapse ." ], [ "STDP is a major synaptic Hebbian learning rule ( Sjöström et al . , 2008; Feldman , 2012 ) in which synaptic weight changes depend on the time delay ΔtSTDP between presynaptic and postsynaptic paired stimulations: ΔtSTDP<0 when post-synaptic stimulation occurs before the paired pre-synaptic one ( post-pre pairings ) , whereas ΔtSTDP>0 when pre-synaptic stimulation occurs before the post-synaptic one ( pre-post pairings ) .", "Corticostriatal synapses are known to exhibit a bidirectional eCB-dependent STDP in which tLTP or tLTD can be obtained depending on the spike timing ( ΔtSTDP ) but also on the number of pairings ( Npairings ) ( Fino et al . , 2005; Shen et al . , 2008; Pawlak and Kerr , 2008; Fino et al . , 2010; Paillé et al . , 2013; Cui et al . , 2015 ) .", "In agreement with those reports , we obtained a bidirectional plasticity when we induced STDP with 100 pairings in medium-sized spiny neurons ( MSNs ) : post-pre pairings ( -30<ΔtSTDP<0 ms ) induced tLTP ( mean value of the EPSC amplitude recorded 50 min after STDP protocol: 156±15% , p=0 . 0015 , n=19 ) , while pre-post pairings ( 0<ΔtSTDP<+30 ms ) induced tLTD ( 76±8% , p=0 . 0051 , n=11 ) ( Figure 1A , B , C1-2 and D1-2 ) .", "Note that this STDP displays an anti-hebbian polarity in accordance with previous reports ( Fino et al . , 2005; Fino et al . , 2010; Schulz et al . , 2010; Paillé et al . , 2013; Cui et al . , 2015 ) but not with other studies ( Pawlak and Kerr , 2008; Shen et al . , 2008 ) at corticostriatal synapses ( Fino and Venance , 2010 ) .", "We have previously shown that GABA acts as an Hebbian/anti-Hebbian switch ( Paillé et al . , 2013 ) , so polarity of the corticostriatal STDP depends on whether GABAA receptor antagonists are applied ( Hebbian STDP; Pawlak and Kerr , 2008; Shen et al . , 2008 ) or not ( anti-Hebbian STDP; Fino et al . , 2005; Fino et al . , 2010; Fino and Venance , 2010; Cui et al . , 2015; this study ) .", "Examples of tLTP and tLTD induced by 100 post-pre and 100 pre-post pairings are shown in C1 and D1 , respectively , and the experiment summary in C2 and D2 .", "tLTP was NMDAR-mediated since blocked by the selective NMDAR blocker D-AP5 ( 50 μM ) ( 99±3% , p=0 . 7998 , n=4 ) ( Figure 1C2 ) .", "while tLTD relied on eCBs because pharmacological inhibition of CB1R with AM251 ( 3 μM ) impaired this plasticity ( 102±7% , p=0 . 8108 , n=4 ) ( Figure 1D2 ) .", "As recently reported ( Cui et al . , 2015 ) , lowering the number of pairings down to 10 yields tLTP for post-pre pairings ( 163±12% , p<0 . 0001 , n=25 ) ( Figure 1E with an example of LTP induced by 10 post-pre pairings in E1 and the experiment summary in E2 ) and a lack of significant plasticity for pre-post pairings ( 97±11% , p=0 . 3844 , n=8 ) .", "tLTP induced with 10 post-pre STDP pairings was CB1R-mediated since treatment with AM251 ( 3 μM ) resulted in an absence of significant plasticity ( 88±11% , p=0 . 3073 , n=5 ) ( Figure 1E2 ) .", "Based on this eCB-dependence , we refer to the tLTP triggered by 10 post-pre pairings as eCB-tLTP . 10 . 7554/eLife . 13185 . 003Figure 1 . Bidirectional endocannabinoid-mediated STDP depends on the number of pairings .", "( A ) Whole-cell recording from the dorsal striatum with the stimulation electrode placed in layer 5 of the somatosensory cortex in horizontal rat brain slice .", "( B ) Experimental design .", "Extracellular stimulation evoked EPSCs monitored at RMP .", "During pairings , recordings were switched to current-clamp to allow postsynaptic MSN to fire single action potentials paired with single cortical extracellular stimulations .", "MSN and cortical stimulation were repeated N times ( 10 or 100 ) at 1 Hz .", "ΔtSTDP indicates the time delay between pre- and post-synaptic stimulations .", "-30<ΔtSTDP<0 ms and 0<ΔtSTDP<+30 ms refers to post-pre and pre-post pairings , respectively .", "( C ) 100 post-pre pairings induced NMDAR-mediated tLTP .", "( C1 )", "Example of tLTP induced by 100 post-pre pairings .", "Top , EPSC strength before and after pairings ( before pairings: 91±3 pA; 45–55 min after pairings: 169±2 pA; increase of 87% ) .", "Bottom , time courses of Ri ( before , 132±1 MΩ; after , 134±2 MΩ; change of 2% ) and injected current ( Iinj ) ( before , -2±1 pA; after , -12±2 pA; change of 6% of baseline EPSC amplitude ) for this cell .", "( C2 )", "Summary of tLTP induced by 100 post-pre pairings .", "15/19 cells showed significant tLTP .", "Inhibition of NMDAR with D-AP5 ( 50 μM , n=4 ) prevented the induction of tLTP; 4/4 cells showed no significant plasticity .", "The normality of D-AP5 data was assumed ( test not passed ) .", "( D ) 100 pre-post pairings induced CB1R-mediated tLTD .", "( D1 )", "Example of tLTD induced by 100 pre-post pairings .", "Top , EPSC strength before and after pairings ( before pairings: 134±2 pA; 45–55 min after pairings: 82±2 pA; decrease of 39% ) .", "Bottom , time courses of Ri ( before , 156±2 MΩ; after , 157±1 MΩ; change of 1% ) and injected current ( Iinj ) ( before , 14±1 pA; after , 20±1 pA; change of 5% ) for this cell .", "( D2 )", "Summary of tLTD induced by 100 post-pre pairings .", "7/11 cells showed significant tLTD .", "Inhibition of CB1R with AM251 ( 3 μM , n=4 ) prevented the induction of tLTD; 4/4 cells showed no significant plasticity .", "The normality of AM251data was assumed ( test not passed ) .", "( E ) 10 post-pre pairings induced CB1R-mediated tLTP .", "( E1 )", "Example of tLTP induced by 10 post-pre pairings .", "Top , EPSC strength before and after pairings ( before pairings: 112±4 pA; 45–55 min after pairings: 213±4 pA; increase of 90% ) .", "Bottom , time courses of Ri ( before , 171±2 MΩ; after , 167±1 MΩ; change of 2% ) and injected current ( Iinj ) ( before , 10±1 pA; after , 12±1 pA; change of 2% ) for this cell .", "( E2 )", "Summary of tLTP induced by 10 post-pre pairings .", "21/25 cells showed significant tLTP .", "Inhibition of CB1R with AM251 ( 3 μM , n=5 ) prevented the induction of tLTP; 5/5 cells showed no significant plasticity .", "Normality was assumed for the ctrl 10x post-pre data ( test not passed ) .", "( F-H ) eCB-LTP is maintained by a mechanism located downstream of CB1R activation in the presynaptic terminals .", "( F ) Representative EPSCs and summary bar graphs ( n=14 ) of paired-pulse cortical stimulations ( 50 ms interstimulus interval ) illustrate a decrease of facilitation after 10 post-pre pairings .", "This indicates a presynaptic locus of the eCB-tLTP .", "( G ) Mean variance analysis ( CV-2 , n=17 ) indicates a presynaptic locus of the eCB-tLTP maintenance .", "( H ) Summary of tLTP induced by 10 post-pre pairings with application of CB1R inhibitor just after the pairings ( AM251* ) ( 7/8 cells showed significant tLTP ) .", "This treatment did not prevent tLTP , indicating that the maintenance of eCB-tLTP does rely on the signaling downstream of CB1R .", "Normality was assumed for the data og CV-2 after STDP protocol ( test not passed ) .", "Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "Error bars represent s . d . *p<0 . 05 .", "ns: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 00310 . 7554/eLife . 13185 . 004Figure 1—figure supplement 1 . NMDAR-tLTP relies on CaMKII activity . NMDAR-mediated tLTP induced by 100 post-pre pairings is CaMKII-activation dependent .", "Summary of tLTP induced by 100 post-pre pairings in control conditions ( n=19 ) and the absence of plasticity observed with KN62 ( 3 μM , n=6 ) ; 15/19 and 1/6 cells showed significant tLTP , respectively .", "Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "Error bars represent s . d . *p<0 . 05 .", "ns: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 00410 . 7554/eLife . 13185 . 005Figure 2 . Mathematical model predicts NMDAR-tLTP with large numbers of post-pre pairings .", "( A ) Scheme of the modeled signaling network .", "The synaptic weight Wtotal is the product of pre- and postsynaptic weights Wpre and Wpost .", "The NMDAR-based pathway sets Wpost as the phosphorylation state of postsynaptic CaMKIIα .", "In the second pathway , coincident activation of phospholipase Cβ by postsynaptic mGluR and calcium entry via VSCC and TRPV1 induces the production of 2-AG and AEA .", "2-AG , and to a lower extent AEA , activates CB1R ( xCB1R is the fraction of non-desensitized CB1R ) , which then modulates the presynaptic weight , Wpre .", "Color code: glutamate receptors: dark blue; PLC pathway: yellow; IP3 pathway: powderblue; calcium pathways: green ( green disks indicate calcium-dependent steps ) ; DAGLα pathway: lavander; AEA pathway: light blue; CB1R pathway: blue .", "Abbreviations: PIP2: phospatidylinositol 4 , 5-biphosphate; DAG: diacylglycerol; IP3: inositol-1 , 4 , 5-triphosphate; PLCβ/δ: phospholipase C β/δ; DAGK: diacylglycerol kinase; IP-5P: inositol polyphosphate 5-phosphatase; IP3K: IP3-kinase; DAGLα: diacylglycerol lipase α;B/BCa: free/bound endogeneous calcium buffer; IP3R: IP3-receptor channel; SERCA: sarcoplasmic/endoplasmic reticulum calcium ATPase; CaER: calcium in the endoplasmic reticulum; ( Ca ) 4CaM: fully bound calmodulin; CaN: calcineurin aka PP2B; PKA: protein kinase A; I1p/I1: phosphorylated/unphosphorylated protein phosphatase 1 inhibitor 1 ( DARPP-32 in striatal output neurons ) ; PP1: protein phosphatase 1; pCaMKII/CaMKII: phosphorylated/unphosphorylated CaMKII; DAGK: diacylglycerol kinase; MAGL: monoacylglycerol lipase; the '…' sign indicates transformation into products that are considered not to interfere with the other interactions of the model .", "( B ) Corresponding changes in the levels of active CaMKII starting from the down ( non-activated ) state .", "The number of pairings , Npairings , is indicated for 1 Hz pairings at spike-timing ΔtSTDP=-15 ( B1 ) or +15 ( B2 ) ms . ( C ) Intracellular calcium changes for the first pairing in post-pre ( C1 ) or pre-post ( C2 ) pairing protocols .", "The colorcode shows the corresponding amount of calmodulin activation according to the colorbar . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 005 Location of CB1R at the presynaptic terminals of the corticostriatal pathway ( Katona and Freund , 2012 ) suggests that the locus of eCB-tLTP maintenance would likely be presynaptic .", "First , we applied presynaptic paired pulses with 50 ms interpulse interval , known to induce a significant EPSC paired-pulse facilitation ( PPF ) in MSNs , ( Goubard et al . , 2011 ) before and after STDP pairings .", "We observed a significant decrease of the PPF after the STDP pairings ( PPFplasticity/baseline=0 . 872±0 . 044 , p=0 . 0470 , n=14 ) ( Figure 1F ) , which indicates a presynaptic locus of eCB-tLTP .", "Second , using the mean variance analysis of EPSCs , we found a CV-2 value of 3 . 6 ± 0 . 6 ( p=0 . 0008 , n=17 ) , which confirmed a presynaptic maintenance of eCB-tLTP ( Figure 1G ) .", "To further distinguish between induction and maintenance loci , we performed experiments in which we applied the CB1R antagonist AM251 just after the STDP pairings , and we still observed significant tLTP ( 146±12% , p=0 . 0092 , n=8 ) ( Figure 1H ) whereas AM251 applied during the protocol prevented tLTP ( Figure 1E2 ) .", "This indicates that eCB-tLTP is maintained by a mechanism located downstream of CB1R activation in the presynaptic terminals .", "We then questioned how eCBs could mediate either potentiation or depression , depending on the activity pattern of either side of the synapse .", "To address this question , we built a realistic mathematical model of the molecular mechanisms of corticostriatal synaptic plasticity ( Figure 2A ) .", "Our model is based on the two signaling pathways involved in corticostriatal STDP induced by 100 pairings: NMDAR- and CB1R-signaling ( Pawlak and Kerr , 2008; Shen et al . , 2008; Fino et al . , 2010; Paillé et al . , 2013 ) .", "NMDAR-tLTP is CaMKII-dependent since we found that pharmacological inhibition of CaMKII with KN62 ( 3 µM ) blocked NMDAR-tLTP ( 88 ± 11% , p=0 . 3324 , n=6 ) ( Figure 1—figure supplement 1 ) .", "We thus combined in the model a first signaling pathway leading from NMDAR to calmodulin and CaMKII with a second , distinct one that assembles mGluR and cytosolic calcium to eCB production and the resulting activation of CB1R ( Figure 2A ) .", "Most of the parameter values were restricted by previous experimental measurements ( Supplementary file 1 ) .", "In the model , the total synaptic weight ( Wtotal ) is given by the product of presynaptic ( Wpre ) and postsynaptic ( Wpost ) contributions ( see Methods ) .", "The postsynaptic contribution to the synaptic weight , Wpost is taken proportional to the amount of CaMKII activated by the NMDAR pathway .", "This part of our model ( from Graupner and Brunel , 2007 ) exhibits bistable dynamics between a down state where CaMKII is inactive and an up state where CaMKII is highly activated ( Figure 2B1 ) .", "Transitions between those two states therefore emulate transitions between no plasticity ( down state ) and NMDAR-tLTP ( up state ) .", "The time scale of CaMKII dephosphorylation after a pairing being larger than the period between two successive pairings ( 1 sec ) , the amounts of activated CaMKII progressively accumulates with the number of pairings .", "Importantly , the level of activated CaMKII needs 50–60 post-pre pairings ( with ΔtSTDP=-15 ms ) to reach the threshold between the up and down states ( Figure 2B1 ) .", "As a result , Wpost converges to the up state ( potentiation ) only when Npairings>50 post-pre pairings , thus emulating the experimental observations of NMDAR-dependent LTP and its dependence on the number of pairings ( Cui et al . , 2015 ) .", "For pre-post pairings , the calcium response after each pairing activates less of the CaMKII-activating calmodulin ( Figure 2C ) so the amount of activated CaMKII never reaches the threshold for the up state ( Figure 2B2 ) .", "Thus , the model predicts no NMDAR-dependent for pre-post pairings ( 0<Npairings<100 at 1 Hz , Figure 2C2 ) , in agreement with experimental observations ( Cui et al . , 2015 ) .", "Within a wide parameter range , the amplitude of the calcium peaks triggered by each paired stimulation shows a peculiar biphasic envelope ( Figure 3A ) : calcium first increases for the first 10–20 pairings then decreases afterwards , until it reaches constant amplitude after 50 pairings .", "During the first 10–20 pairings , repeated activation of mGluRs progressively increases the quantity of IP3 , which contributes an extra influx of calcium from the endoplasmic reticulum .", "This boost of cytoplasmic calcium however progressively disappears when Npairings increases further because the concentration of calcium in the endoplasmic reticulum decreases .", "Moreover , after each pairing , the width of the postsynaptic calcium peak in the model is larger with post-pre than pre-post pairings ( Figure 3B ) .", "As a consequence , the fraction of calcium-activated DAGLα is significant only for small values of |ΔtSTDP| ( <25 ms ) and larger for post-pre than pre-post pairings .", "As a result , the biphasic envelope of the calcium peak amplitude with Npairings ( first increase , then decrease ) is transmitted to the amplitude of eCB transients and , ultimately , to CB1R activation ( yCB1R ) .", "The biphasic envelope is even more marked at the level of CB1R activation because of CB1R desensitization that amplifies the decay above 20 pairings .", "Figure 3C illustrates the dynamics of CB1R activation in the model .", "In all cases , the amplitude of the CB1R activation peaks first increases for the first 10–20 pairings , then decreases to converge to constant amplitude .", "yCB1R reaches large values only for short post-pre pairings ( ΔtSTDP around -15 ms ) while even short pre-post pairings ( 0<ΔtSTDP<10 ms ) do not give rise to such large amplitude peaks . 10 . 7554/eLife . 13185 . 006Figure 3 . Spike-timing dependence in the endocannabinoid-signaling part of the model .", "( A-C )", "Predicted dynamics of cytoplasmic calcium and CB1R activation for post-pre ( first column ) or pre-post pairings ( second column ) : in our model , the postsynaptic calcium peaks ( A , B ) are slightly more width at large calcium values with post-pre ( A1-B1 ) than pre-post pairings ( A2-B2 ) .", "As a consequence , DAGLα activation ( color-coded in B ) and the resulting CB1R activation , yCB1R ( C ) is larger too .", "The biphasic envelope of the calcium peak amplitude with the number of pairings ( A ) is amplified as a marked biphasic envelope for the amplitude of yCB1R .", "( C ) As a result , whatever the stimulation , the amplitude of the yCB1R peaks first increases for the first 10–20 pairings , then decreases to converge to roughly constant amplitude .", "But yCB1R reaches large values only for short post-pre pairings ( C1 ) .", "This particular dynamics of yCB1R during the stimulations suggests a possible explanation to the bidirectional characteristics of eCB-dependent plasticity , where the presynaptic contribution to the synaptic weight Wpre depends on the magnitude of yCB1R .", "( D ) Wpre decreases ( LTD ) when yCB1R reaches an intermediate range whereas it increases ( LTP ) if yCB1R overcomes a LTP threshold .", "The corresponding thresholds and ranges are reported in C1-2 as dashed lines and hashed boxes , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 006 This peculiar dynamics of yCB1R brings a plausible explanation to the bidirectional features of eCB-dependent plasticity .", "Under this scenario , Wpre depends on the magnitude of yCB1R so that whenever yCB1R reaches moderate amounts – i . e . when it is located between two threshold values , ΘLTDstart and ΘLTDstop- Wpre drops ( LTD ) ; whereas Wpre rises ( LTP ) if yCB1R is larger than a third threshold , ΘLTPstart ( see the dashed lines in Figure 3C1 and 3C2 and summary in Figure 3D ) .", "Wpre remains unchanged outside those ranges , i . e . if yCB1R<ΘLTDstart or if θLTDstop<yCB1R<ΘLTPstart .", "Combining this mechanism with the shape of yCB1R evolution upon Npairings explains the main characteristics of corticostriatal STDP .", "With short pre-post pairings ( 10<ΔtSTDP<40 ms ) , yCB1R reaches the LTD range ( between ΘLTDstart and ΘLTDstop Figure 3C2 ) during most of the 100 pairings: each pairing reduces Wpre .", "Since pre-post pairings do not alter Wpost ( Figure 2B2 ) , the net result is a progressive reduction of Wtotal , i . e . the expression of eCB-tLTD .", "The situation is different for post-pre pairings .", "The amplitude of yCB1R peaks overcomes ΘLTPstart for 5 to 30 post-pre-pairings , resulting in an increase of Wpre .", "Since more than 50 post-pre pairings are needed to alter Wpost ( Figure 2B2 ) , this Wpost increase results in eCB-tLTP ( Figure 3C1 ) .", "Above 30 post-pre pairings , the amplitude of yCB1R transients gets back below ΘLTPstart so that the Wpre increase is no more triggered , thus explaining why eCB-tLTP is not expressed for Npairings>30 .", "Finally , when Npairings>50 , Wpost is predicted to trigger the rise of Wtotal , thus reflecting NMDAR-tLTP .", "In conclusion , the mechanism proposed by our mathematical model to account for eCB-STDP is the following: eCB-tLTD requires moderate levels of CB1R activation , which can be reached with pre-post pairings; eCB-tLTP demands higher levels of CB1R activation that are reached only with 5–30 post-pre pairings , where every component of the model contributes maximally to CB1R activation ( maximal cytosolic calcium influx from NMDAR , VSCC , TRPV1 and maximal calcium efflux from internal stores , combined with a minimal CB1R desensitization ) .", "Beyond 30 post-pre pairings , calcium efflux from the internal calcium stores decreases while in parallel CB1R desensitization increases .", "CB1R activation becomes insufficient to maintain the elevation of the synaptic weight , so that eCB-tLTP vanishes .", "We then tested whether the model generated correct qualitative predictions in agreement with experimental data for the plasticity outcome when both ΔtSTDP and Npairings were varied .", "The changes of the total synaptic weight for the whole range of ΔtSTDP and Npairings are illustrated in Figure 4A by the model-generated color-coded map .", "The outcome of plasticity according to the model is split along three domains: a first LTP domain for -3<ΔtSTDP<-25 ms and 3<Npairings<40 , a second LTP domain for -10<ΔtSTDP<-25 ms and Npairings>50 , and a LTD domain for 10<ΔtSTDP<25 ms and Npairings>20 .", "Note that the model correctly accounts for a plasticity gap for 40–60 post-pre pairings that isolates the two LTP domains in agreement with experimental observations ( Cui et al . , 2015 ) and that the expression of plasticity does not change when Npairings>100 ( Figure 4—figure supplement 1A ) .", "To compare model prediction and experimental data on a quantitative basis , Figure 4A2 and A3 also show the average weight change predicted for -25<ΔtSTDP<-10 ms or 10<ΔtSTDP<25 ms . Even quantitatively , model predictions ( full lines ) are in agreement with the experimental data ( full circles ) .", "Likewise , Figure 4A4 and A5 show the weight change predicted for STDP protocols featuring 10 or 100 pairings , with ΔtSTDP ranging from -40 to 40 ms , i . e . cross-sections of the color-coded map along the vertical dashed lines .", "Again , model prediction ( full lines ) matches experiments ( full circles ) .", "Quantitative agreement is found for the amplitude and sign of plasticity , as well as for the dependence of plasticity on spike timing .", "To our knowledge , the present model is the first mathematical model able to account for the outcome of the plasticity when both ΔtSTDP and Npairings are varied .", "We ran simulations of model variants where parts of the signaling pathways were removed ( in-silico knock-out ) .", "In the NMDAR signaling knockout , we removed the whole signaling pathway downstream of NMDAR , i . e . calmodulin and CaMKII .", "Since Wpost relies entirely on CaMKII activation , the NMDAR signaling knockout corresponds to a situation where the contribution of Wpost is absent and only Wpre contributes to Wtotal .", "As expected , the post-pre NDMAR-dependent LTP is absent in this NMDAR signaling knockout model , but pre-post tLTD and post-pre tLTP ( observed with low numbers of pairings: 5<Npairings<35 ) are conserved ( Figure 4B ) .", "Comparison with experimental data where NMDAR signaling was blocked with D-AP5 or CaMKI with KN62 confirms the match between model and experiments ( Figure 4B ) .", "Simulations of the CB1R in-silico knockout model , where CB1R activation remains null whatever eCB levels are shown in Figure 4C .", "Because Wpre depends on CB1R activation , the CB1R in-silico knockout model actually reflects the case were only Wpost contributes to Wtotal .", "In this case , the only remaining plasticity domain is the LTP expressed for post-pre pairings ( Npairings>50 ) .", "Again , averaging over -25<ΔtSTDP<-10 ms and 10<ΔtSTDP<25 ms with 10 or 100 pairings evidences the match of the model with experimental data in which CB1R was inhibited with AM251 ( Figure 4C ) . 10 . 7554/eLife . 13185 . 007Figure 4 . The mathematical model matches the experimental data .", "( A ) Changes of the total synaptic weight Wtotal ( LTP and LTD ) when Npairings and ΔtSTDP vary .", "( A1 )", "Color-coded changes of Wtotal in the ( Npairings , ΔtSTDP ) space .", "The color bar indicates the color code .", "The background map shows the simulation results whereas the color-coded points ( same color-code as the simulations ) are experimental results .", "The average changes with Npairings of Wtotal integrated over short positive or short negative ΔtSTDP are shown in ( A2 ) and ( A3 ) , respectively .", "Cross-sections of the two-dimensional map ( A1 ) along the N-axis are shown as changes of Wtotal with ΔtSTDP , for Npairings=10 ( A4 ) or 100 ( A5 ) pairings at 1 Hz .", "In ( A2-A5 ) , full black lines represent the simulation results whereas the full black circles show experimental results .", "( B , C )", "Corresponding results obtained with variants of the mathematical model where NMDAR-signaling ( B ) or eCB-signaling ( C ) were knocked-out in silico .", "The 2D maps ( B1 , C1 ) use the same color code and symbols as ( A1 ) .", "The average changes of Wtotal over short positive or short negative spike timings ΔtSTDP ( B2 , C2 ) and ( B3 , C3 ) , respectively , use the same symbols as ( A1-2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 00710 . 7554/eLife . 13185 . 008Figure 4—figure supplement 1 . Robustness of the model .", "( A ) Output of the model for more than 100 pairings and ( upper panel ) the sharp threshold mechanism for eCB-plasticity given by Equation 1 The behavior observed with 100 pairings is conserved .", "( B ) Output of the model when the sharp eCB plasticity ( Equation", "1 ) is replaced by a smooth function ( upper panel , given by eq . S1-S2 of Supplementary file 2 , here used with kS = 2 ) .", "Even without changing any of the model parameters outside the threshold function , the model output with such smooth thresholds is very similar to the one obtained with the sharp threshold of Equation 1 ( compare with Figure 4A1 ) .", "Other values of kS essentially lead to the same conclusion .", "( C ) Sensitivity analysis of the model parameters .", "On the y-axis , the parameters are ranked according to their standardized linear-regression coefficient ( SRC , see Materilas and methods ) that measures the sensitivity of the model output to variations of the parameter . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 008 We then analyzed how much the model outcome was sensitive to variations of the parameters .", "First , we changed the sharp thresholds for eCB-dependent plasticity into smooth thresholds .", "To this end , we replaced function Ω in Equation 1 above by a smooth equivalent function whose graph is depicted in Figure 4—figure supplement 1B ( the corresponding equation is given in Supplementary file 2 , eq . S1-S2 ) .", "In spite of the smooth thresholds , the model output is very similar to that obtained with sharp thresholds ( compare the color map of Figure 4—figure supplement 1B with that of Figure 4A1 ) .", "Therefore , our choice of a sharp thresholding for eCB-dependent plasticity is not crucial for the model output .", "We further undertook sensitivity analysis of the model ( Figure 4—figure supplement 1C ) .", "As expected , the most sensitive parameters were those related to reactions that are known from pharmacological experiments to be indeed crucial to STDP: the total amount of Calmodulin or CaMKII ( Figure 1—figure supplement 1 ) , post-synaptic calcium buffering ( Fino et al . , 2010; Cui et al . , 2015 ) , TRPV1 and NMDA channels ( Fino et al . , 2010; Cui et al . , 2015 ) , DAGLipase activity ( Cui et al . , 2015 ) or FAAH and MAGLipase activity ( see below ) .", "The model was also found sensitive to the dynamics of CB1R desensitization , in agreement with the importance of CB1R desensitization in the decay of eCB-LTP above 15–20 post-pre stimulations .", "The model was also sensitive to the value of the threshold for eCB-LTP induction ( whether smooth or sharp ) .", "We suspect that this could explain the dispersion of the amplitudes of eCB-tLTP ( Figure 4A4 ) .", "More surprising is the sensitivity of the model to the dynamics of glutamate in the synaptic cleft ( decay rate τG ) .", "Alterations of the dynamics of glutamate release and uptake can thus be expected to play an important role in the control of STDP at the corticostriatal synapse .", "In addition to spike timing and number of pairings , STDP is also known to be dependent on the pairing frequency .", "All our above results were obtained at 1 Hz .", "We now test the frequency dependence of plasticity induced by a low number of pairings .", "Figure 5A shows the prediction of the model for Npairings=10 .", "When frequency increases above 1 Hz , the eCB-tLTP triggered by post-pre stimulations ( ΔtSTDP<0 ) persists and is even observed for an increasingly large ΔtSTDP range .", "The model also predicts the expression of another tLTP , triggered by 10 pre-post stimulations ( ΔtSTDP>0 ) for frequency larger than 2 Hz . 10 . 7554/eLife . 13185 . 009Figure 5 . Frequency dependence of eCB-tLTP .", "( A ) Color-coded changes of Wtotal in the ( ΔtSTDP , frequency ) parameter space for 10 pairings .", "Except the pairing frequency , all parameters are the same as in Figure 4 ( values given in Supplementary file 1A-C ) .", "For 10 post-pre pairings ( ΔtSTDP<0 ) , tLTP disappears quickly below 1 Hz but is maintained above 1 Hz , within an even enlarged ΔtSTDP range .", "For 10 pre-post pairings ( ΔtSTDP>0 ) , a new tLTP emerges for frequencies larger than 2 Hz .", "( B-D )", "Summary graphs of STDP occurrence for 10 pairings at 0 . 1 Hz ( B ) , 1 Hz ( C ) , 2 . 5 Hz ( D ) and 4 Hz ( E ) ; each grey empty circle represent the synaptic efficacy changes 45–50 min after pairings protocols for a single neuron; the black circles represent the averages of plasticity .", "tLTP was induced with 10 post-pre pairings at 0 . 1 Hz ( 7/10 cells showed significant tLTP ) and 1 Hz ( 21/25 cells showed significant tLTP ) ; no significant plasticity was observed for pre-post pairings .", "For 10 pairings at 2 . 5 and 4 Hz , symmetric Hebbian plasticity ( tLTP for post-pre and pre-post pairings ) was observed in an enlarged ΔtSTDP; at 2 . 5 Hz for post-pre and pre-post pairings , 18/23 and 10/20 cells displayed significant tLTP; at 4 Hz for post-pre and pre-post pairings , 18/22 and 7/10 cells showed significant tLTP .", "Normality was assumed for the post-pre pairings data ( test not passed ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 00910 . 7554/eLife . 13185 . 010Figure 5—figure supplement 1 . Both CB1R and NMDAR are involved in symmetric hebbian plasticity induced with 10 pairings at 4 Hz .", "( A ) Summary bar graphs illustrate that symmetric Hebbian plasticity ( tLTP ) induced with post-pre and pre-post were not prevented by AM251 ( 3 μM ) or D-AP5 ( 50 μM ) ( except for pre-post pairings ) but were precluded by the application of both antagonists AM251+D-AP5; with AM251 , 6/6 and 6/7 cells showed significant tLTP with 10 post-pre and pre-post pairings , respectively; with D-AP5 , 8/11 and 3/5 cells showed significant tLTP with 10 post-pre and pre-post pairings , respectively; with AM251+D-AP5 , 8/9 and 3/3 cells showed an absence of significant plasticity with 10 post-pre and pre-post pairings , respectively .", "Error bars represent SEM .", "*p<0 . 05 .", "ns: not significant .", "( B ) The mathematical model predicts similar behavior for Npairings = 15 ( at 4 Hz ) .", "Except the pairing frequency , all parameters are the same as in Figure 4 ( values given in Supplementary file 1A-C ) .", "With post-pre pairings ( ΔtSTDP<0 ) , removing CB1R- or NDMAR-signaling ( see Figure", "4 ) separately only partially impairs tLTP , while no plasticity can be obtained when both are removed .", "Comparable results are obtained with pre-post pairings ( ΔtSTDP>0 ) , with the exception that tLTP is much more dependent on CB1R- signaling .", "Note that the additional NMDAR component of the post-pre tLTP emerges in the model at 4 Hz for Npairings>12 . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 010 To test the validity of these model predictions , we explored experimentally 10 pairings STDP for 0 . 1 , 2 . 5 and 4 Hz ( besides 1 Hz ) .", "10 post-pre pairings at 0 . 1 Hz were able to induce tLTP ( 133±14 , n=10 , p=0 . 0386 ) ( Figure 5B ) , which was not significantly different from eCB-tLTP induced with 10 pairings at 1 Hz ( p=0 . 1538 ) ( Figure 5C ) .", "This result is not predicted by the model , for which the tLTP induced by 10 post-pre pairings vanishes quickly below 1 Hz .", "At frequencies >1 Hz , we observed tLTP for 10 post-pre pairings at 2 . 5 Hz ( 161±15 , n=23 , p=0 . 0004 ) and 4 Hz ( 165±14 , n=22 , p=0 . 0001 ) , but also for pre-post pairings at 2 . 5 Hz ( 130±14 , n=12 , p=0 . 0490 for ΔtSTDP<+50 ms; 119±9 , n=20 , p=0 . 060 for ΔtSTDP<+100 ms ) and 4 Hz ( 139±13 , n=10 , p=0 . 0150 ) .", "Moreover , the ΔtSTDP range for tLTP induction was considerably enlarged for post-pre pairings: from -30<ΔtSTDP<0 ms at 1 Hz to -100<ΔtSTDP<0 ms at 2 . 5 or 4 Hz .", "Note that for pre-post pairings , tLTP could be observed for ΔtSTDP<+50 ms ( Figure 5D and E ) .", "Therefore , when we increased the frequency of the pairings to 2 . 5 or 4 Hz , our experimental results show a very good match with the prediction of the model: we observed first a symmetric Hebbian plasticity , i . e . the induction of tLTP not only for post-pre but also for pre-post pairings , and , secondly an enlargement of the range of ΔtSTDP in which plasticity was observed .", "We then investigated the signaling pathways involved in those two tLTP ( Figure 5—figure supplement 1A ) .", "We observed that for 2 . 5 and 4 Hz STDP , post-pre tLTP was not prevented with AM251 ( 3 μM ) ( 150±11 , n=6 , p=0 . 0069 ) or with D-AP5 ( 50 μM ) ( 135±12 , n=11 , p=0 . 013 ) but was precluded with a mixture of both AM251 and D-AP5 ( 96±3 , n=9 , p=01800 ) .", "Similarly , for pre-post pairings at 2 . 5 and 4 Hz , tLTP was still observed with AM251 ( 149±15 , n=7 , p=0 . 0178 ) but was prevented with D-AP5 ( 134±27 , n=5 , p=0 . 2684 ) or a mixture of AM251 and D-AP5 ( 88±11 , n=3 , p=0 . 4090 ) .", "The mathematical model with Npairings=10 does not show such a mixed NMDAR- and eCB-LTP ( both tLTP are purely eCB-dependent ) .", "Remarkably , however , the tLTP in the model starts becoming mixed with Npairings>12 .", "For 15 pairings ( at 4 Hz ) , for instance ( Figure 5—figure supplement 1B ) , the post-pre LTP in the model depends both on CB1R and NMDAR .", "Therefore , model predictions and experiments provide converging suggestion that at frequencies above 1 Hz , the tLTP triggered by 10–15 post-pre or pre-post pairings becomes both eCB and NMDAR-dependent .", "Based on the ability of our mathematical model to reproduce our experimental data , we explored further the biochemical mechanisms of eCB-dependent plasticity using a model-guided experimental strategy .", "Our strategy was to use the model to propose experiments that would question the role of the amplitude of CB1R activation to determine eCB-STDP polarity ( LTP or LTD ) .", "We then systematically carried out the experiments necessary to test the validity of the model prediction .", "We first tested experimentally the main prediction of the model: different levels of released 2-AG , low or high , would orientate the plasticity toward , respectively , eCB-tLTD or eCB-tLTP .", "For this purpose , we directly applied brief puffs ( 300 ms duration ) of 2-AG ( at low , 20 μM , or high , 100 μM , concentrations ) either 100 or 10 times at 1 Hz , thus with the same total duration as the 100 and 10 pairings STDP protocol at 1 Hz .", "First , we tested a low [2-AG] ( 20 μM ) by delivering 100 and 10 puffs .", "We observed that in the absence of STDP protocol , 100 puffs of 2-AG were able to induce a significant LTD ( 65±5% , p=0 . 0009 , n=6 ) ( Figure 6A1 and 6A2 ) with magnitude similar to the tLTD induced by 100 pre-post pairings ( FigFigure 1D2 ) ( p=0 . 9340 ) .", "When we delivered 10 puffs of low [2-AG] ( 20 μM ) , no significant plasticity was detected ( 95±11% , p=0 . 6931 , n=6 ) ( Figure 6B1 and 6B2 ) .", "We then increased [2-AG] five-fold ( i . e . 100 μM ) .", "After applying 100 puffs of 100 µM 2-AG , a potent LTD was observed ( 61±7% , p=0 . 0021 , n=7 ) ( Figure 6C1 and 6C2 ) with magnitude similar to the tLTD induced by 100 pre-post pairings ( Figure 1D2 ) ( p=0 . 6676 ) .", "We verified that this LTD was CB1R-mediated by preventing plasticity with AM251 ( 3 μM ) ( 94±5% , p=0 . 2817 , n=5 ) ( Figure 6C2 ) .", "Strikingly , 10 puffs of high[2-AG] ( 100 μM ) induced a potent LTP ( 168±29% , p=0 . 0106 , n=5 ) ( Figure 6D1 and 6D2 ) with magnitude similar to the tLTP induced by 10 post-pre pairings ( Figure 1E2 ) ( p=0 . 0106 ) .", "This LTP was CB1R-mediated because when CB1R was inhibited with AM251 ( 3 μM ) , 10 puffs of [2-AG] ( 100 μM ) did not induce significant plasticity ( 92±4% , p=0 . 1542 , n=5 ) ( Figure 6D2 ) .", "Therefore , 100 puffs of low or high [2-AG] induce LTD while only high [2-AG] succeeds to trigger LTP , thus validating the model prediction .", "To further substantiate the causal role of the amplitude of 2-AG transients in bidirectional eCB-plasticity , we boosted the endogenous levels of 2-AG during STDP protocols .", "Indeed , if the amplitude of CB1R activation controls the expression of eCB-STDP , the outcome of a given STDP protocol should change if one modifies the amount of CB1R activated by this very same STDP protocol .", "For this purpose , we inhibited the MAG lipase ( MAGL ) , the major enzyme responsible for 2-AG degradation ( Piomelli , 2003 ) , to increase the endogenous level of 2-AG .", "We took advantage of the model to select three scenarios in which it should be possible in silico , by inhibiting MAGL , to", "1 ) increase the magnitude of an existing eCB-tLTP ,", "2 ) induce an eCB-tLTP for a paradigm which normally exhibits neither eCB-tLTP nor NMDAR-LTP ( i . e . 50 post-pre pairings; Figure 4A and Cui et al . , 2015 ) and", "3 ) convert an eCB-LTD ( induced with 100 presynaptic stimulations without postsynaptic simulations ) into eCB-LTP .", "First , we tested the possibility to increase the eCB-LTP magnitude by inhibiting MAGL .", "For this purpose , we chose the minimal pairing protocol for which we detected eCB-LTP , which is five post-pre pairings ( Figure", "7 ) ( Figure 4A3 and see Figure 6 in Cui et al . , 2015 ) .", "5Five pairings appearas the lowest number of pairings needed to induce significant eCB-tLTP as illustrated by the representative and average STDP ( 134±13% , p=0 . 0190 , n=17 ) ( Figure 7B and D ) ; Note that the model also faithfully predicted eCB-tLTP for such number of pairings ( Figure 4A and 7A ) .", "In the model , we introduced noncompetitive inhibition of the MAGL by decreasing its maximal rate rMAGL ( Supplementary file 1C ) .", "Simulation of the model with 5 post-pre pairings under MAGL inhibition predicts that such an inhibition increases the net level of 2-AG produced during the protocol and the amplitude of eCB-LTP ( Figure 7A ) .", "As predicted by the model , inhibition of the MAGL with JZL184 ( 1 . 5 μM ) significantly increased the magnitude of eCB-tLTP ( 182±17% , p=0 . 0048 , n=6; p=0 . 0294 when compared to 5 post-pre pairings in control conditions ) ( Figure 7: with an example of LTP induced by five 5 post-pre pairings at ΔtSTDP=-19 ms in B , with an example of LTP induced by five post-pre pairings at ΔtSTDP=-18 ms with JZL184 in C and the experiment summary in D ) .", "We confirmed that this amplification was CB1R-mediated since no plasticity was observed when CB1R were blocked by AM251 ( 3 μM ) ( 96±8% , p=0 . 6123 , n=5 ) ( Figure 7D ) .", "We also ensured that bath-applied JZL treatment in the absence of STDP pairings did not induce significant plasticity ( 96±7% , p=0 . 5943 , n=5 ) .", "It should be noted that the occurrence of eCB-tLTP was also higher with MAGL inhibition: in control , five post-pre pairings yielded 60% of eCB-tLTP ( 10/17 cells showed significant tLTP ) while with MAGL inhibition , 100% of the recorded cells displayed eCB-tLTP ( 6/6 cells displayed significant tLTP ) . 10 . 7554/eLife . 13185 . 011Figure 6 . 2-AG level and duration control the eCB-plasticity polarity .", "( A ) Repeated ( 100 times ) brief application of 2-AG ( at a low concentration , 20 μM ) induces LTD .", "A series of 100 2-AG puffs ( 20 μM , 300 ms duration each ) delivered at 1 Hz at the vicinity ( 50–100 μm ) of the recorded striatal neuron , induced LTD in the absence of any STDP protocol ( n=6 ) .", "( A1 )", "Example of LTD induced with 100 puffs of 20 μM 2-AG .", "Top , EPSC strength before and after 2-AG puffs ( before 2-AG puffs: 223±3 pA; 45–55 min after 2-AG puffs: 144±3 pA; decrease of 35% ) .", "Bottom , time courses of Ri ( before , 118±1 MΩ; after , 111±1 MΩ; change of 6 . 2% ) and injected current ( Iinj ) for this cell .", "( A2 )", "Summary of LTD induced with 100 puffs of 20 μM 2-AG; 6/6 cells showed significant LTD . ( B ) Repeated ( 10 times ) brief application of 2-AG ( at a low concentration , 20 μM ) failed to induce plasticity .", "( B1 )", "Example of absence of plasticity observed with 10 puffs of 20 μM 2-AG .", "Top , EPSC strength before and after 2-AG puffs ( before 2-AG puffs: 165±3 pA; 45–55 min after 2-AG puffs: 143±2pA; change of 14% ) .", "Bottom , time courses of Ri ( before , 82±1MΩ; after , 93±1MΩ; change of 13% ) and injected current ( Iinj ) for this cell .", "( B2 )", "Summary of absence of plasticity observed with 10 puffs of 20 μM 2-AG; 2/6 cells showed no significant plasticity .", "( C ) Repeated ( 100 times ) brief application of 2-AG ( at a high concentration , 100 μM ) induces LTD . ( C1 ) Example of LTD induced with 100 puffs of 100 μM 2-AG .", "Top , EPSC strength before and after 2-AG puffs ( before 2-AG puffs: 95±3 pA; 45–55 min after 2-AG puffs: 77±1 pA; decrease of 19% ) .", "Bottom , time courses of Ri ( before , 92±1 MΩ; after , 78±1 MΩ; change of 15% ) and injected current ( Iinj; before , 16±1 pA; after , 26±1 pA; change of 12 . 8% ) for this cell .", "( C2 )", "Summary of LTD induced with 100 puffs of 100 μM 2-AG; 7/7 cells showed significant LTD .", "This 2-AG-mediated LTD was prevented by AM251 ( 3 μM , n=5 ) ; 5/5 cells showed no significant LTD . ( D ) Repeated ( 10 times ) brief application of 2-AG ( at a high concentration , 100 μM ) induces LTP .", "( D1 )", "Example of LTP induced with 10 puffs of 100 μM 2-AG .", "Top , EPSC strength before and after 2-AG puffs ( before 2-AG puffs: 171±4 pA; 45–55 min after 2-AG puffs: 331±6 pA; increase of 94% ) .", "Bottom , time courses of Ri ( before , 119±1 MΩ; after , 109±1 MΩ; change of 8 . 4% ) and injected current ( Iinj ) ( before , 26±1 pA; after , 18±1 pA; change of 4 . 7% ) for this cell .", "( D2 )", "Summary of LTP induced with 10 puffs of 100 μM 2-AG; 4/5 cells showed significant LTP .", "This 2-AG-mediated LTP was prevented by inhibition of CB1R with AM251 ( 3 μM , n=5 ) ; 5/5 cells showed no significant plasticity .", "Example recording monitoring EPSCs ( at 0 . 1 Hz ) ( A1 , B1 , C1 and D1 ) before and after 2-AG puffs , together with the time course of Ri and of the injected current ( Iinj ) .", "Summary ( A2 , B2 , C2 and D2 ) show global average of experiments with error bars representing s . d . Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "*p<0 . 05 .", "ns: non-significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 01110 . 7554/eLife . 13185 . 012Figure 7 . MAGL inhibition increases eCB-tLTP magnitude induced by 5 pairings .", "( A ) Model prediction for eCB-LTP amplitude induced by Npairings=5 post-pre pairings with ΔtSTDP=-15 ms . ( A1 ) In control ( full black line ) , the synaptic weight increases during the 5 s stimulation protocol ( white background ) and stabilizes afterwards ( gray background ) to a moderate tLTP .", "When eCB production is amplified in the model by MAGL inhibition and DAG-Kinase activity ( full red line ) , the amplitude of the tLTP resulting from the same stimulation is drastically amplified .", "In the model , MAGL and DAG-Kinase inhibition were simulated by fixing the value of the maximal rates of each enzyme to 0 and 5% , respectively , of their default values listed in Supplementary file 1C .", "( A2 )", "Summary bar graph of the tLTP amplitude predicted by the model for Npairings=5 post-pre pairings , ΔtSTDP=-15 ms . ( B ) Corresponding example of experimental tLTP induced by 5 post-pre pairings .", "Top , EPSC strength before and after pairings ( before pairings: 145±4 pA; 45–55 min after pairings: 247±5 pA; increase of 70% ) .", "Bottom , time courses of Ri ( before , 165±1 MΩ; after , 170±1 MΩ; change of 3 . 0% ) and injected current ( Iinj ) ( before , 2±0 . 2 pA; after , 3±0 . 1 pA; change of 0 . 7% ) for this cell .", "( C ) MAGL inhibition by JZL184 ( 1 . 5 μM ) led to an increase of tLTP magnitude .", "Example of tLTP induced by 10 post-pre pairings with bath-applied JZL184 .", "Top , EPSC strength before and after pairings ( before pairings: 180±6 pA; 45–55 min after pairings: 412±7 pA; increase of 129% ) .", "Bottom , time courses of Ri ( before , 116±1 MΩ; after , 126±1 MΩ; change of 8 . 6% ) and injected current ( Iinj ) ( before , -13±1 pA; after , -8±1 pA; change of 2 . 8% ) for this cell .", "( D ) Summary of tLTP induced by 5 post-pre pairings in control conditions and with JZL184 treatment .", "10/17 and 6/6 cells showed significant tLTP in control and in JZL184 , respectively .", "Normality was assumed for the ctrl 5x post-pre data ( test not passed ) .", "( E ) Summary bar graph illustrates that tLTP magnitude was increased by MAGL inhibition ( JZL184 ) while prevented by CB1R inhibition ( JZL184 1 . 5 μM +AM251 3 μM ) .", "Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "*p<0 . 05 .", "ns: non-significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 012 Second , we tested the possibility to induce eCB-tLTP by inhibiting MAGL .", "Indeed , our model predicts that MAGL inhibition may turn a STDP protocol that yields no plastic change in control conditions into eCB-tLTP .", "For this purpose , we chose a STDP pairing for which we detected no plasticity in control conditions: i . e . 50 post-pre pairings ( Figure 4A3; Cui et al . , 2015 ) .", "Figure 4A3 and Figure 8 illustrates this 'plasticity gap' ( the zone between 40 and 60 pre-post pairings that separates the two LTP domains ) .", "In silico the control STDP protocol ( 50 pairings with ΔtSTDP=-15 ms ) does not trigger any plasticity but when MAG lipase is inhibited , eCB-tLTP emerges ( Figure 8A ) .", "Experimentally , as previously reported ( Cui et al . , 2015 ) , STDP protocols with 50 post-pre pairings failed to induce any plasticity in control conditions as illustrated by the representative and average STDP ( 101±7% , p=0 . 9030 , n=13 ) ( Figure 8B and D; with an example of an absence of plasticity for 50 post-pre pairings at ΔtSTDP=-20 ms in B and the experiment summary in D ) .", "As predicted by the model , we found that 50 post-pre pairings under inhibition of MAGL with JZL184 ( 1 . 5 μM ) induced tLTP ( 139±15% , p=0 . 0248 , n=9 ) ( Figure 8C and D; with an example of tLTP induced by 50 post-pre pairings at ΔtSTDP=-16 ms with JZL184 and the experiment summary in D ) .", "This tLTP was eCB-mediated since suppressed by AM251 ( 3 μM ) ( 93±4% , p=0 . 3365 , n=5 ) ( Figure 8D2 ) .", "Therefore , by acting on the 2-AG levels , we were able to trigger eCB-tLTP for an activity pattern , which does not generate LTP in control conditions . 10 . 7554/eLife . 13185 . 013Figure 8 . MAGL inhibition unveils eCB-tLTP expression with 50 pairings .", "( A ) Model prediction for the plasticity induced by Npairings=50 post-pre pairings .", "( A1 )", "In control ( full black line ) , the synaptic weight is unchanged by 50 post-pre pairings for 0>ΔtSTDP>-25 ms . Amplified eCB production due to MAGL inhibition ( full red line ) , uncovers a large-amplitude tLTP .", "In the model , MAGL inhibition was emulated by setting its maximal rate to 40% of its default value ( Supplementary file 1C ) .", "( A2 )", "Summary bar graph of the tLTP amplitude predicted by the model for Npairings=50 post-pre pairings at ΔtSTDP =-15 ms . ( B ) 50 post-pre pairings did not induce significant plasticity .", "Example of the absence of plasticity observed when 50 post-pre pairings were applied .", "Top , EPSC strength before and after pairings ( before pairings: 108±3 pA; 45–55 min after pairings: 106±2 pA; change of 2% ) .", "Bottom , time courses of Ri ( before , 92±1 MΩ; after , 91±1 MΩ; change of 0 . 1% ) and injected current ( Iinj ) ( before , 2±0 . 2 pA; after , 2±0 . 1 pA; no detectable change ) for this cell .", "( C ) 50 post-pre pairings induced tLTP with MAGL inhibition .", "Example of tLTP induced by 50 post-pre pairings with bath-applied JZL184 ( 1 . 5 μM ) .", "Top , EPSC strength before and after pairings ( before pairings: 170±4 pA; 45–55 min after pairings: 243±4 pA; increase of 43% ) .", "Bottom , time courses of Ri ( before , 110±1 MΩ; after , 113±1 MΩ; change of 2 . 7% ) and injected current ( Iinj ) ( before , 4±0 . 3 pA; after , 3±0 . 2 pA; change of 0 . 6% ) for this cell .", "( D ) Summary of synaptic weight along time induced by 50 post-pre pairings in control conditions and with JZL184 treatment .", "4/13 and 8/9 cells showed significant tLTP in control and in JZL184 , respectively .", "( E ) Summary bar graph illustrates that MAGL inhibition allowed tLTP to be expressed , which was CB1R-mediated since prevented by AM251 ( 3 μM ) .", "Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "*p<0 . 05 .", "ns: non-significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 013 Our third model prediction is that amplifying 2-AG production during STDP may even eliminate the need for a coincidence between presynaptic and postsynaptic activity to express eCB-LTP .", "In silico , pre-post pairing coincidence is needed for the model to express plasticity .", "Indeed , a protocol with 100 presynaptic stimulations only ( i . e . in the absence of postsynaptic stimulation ) , does not change Wtotal in the model ( Figure 9A ) .", "However , if we decrease the maximal rates of MAGL and DAG kinase activity ( the major source of DAG consumption in the model ) , we obtain a robust eCB-tLTP , even in the absence of any postsynaptic stimulation .", "Experimentally , 100 presynaptic stimulations ( without postsynaptic pairing ) induced LTD ( 76±9% , p=0 . 0337 , n=8 ) , which was CB1R-mediated since prevented with AM251 ( 3 μM ) ( 102±7% , p=0 . 8108 , n=4 ) ( Figure 9B; with an example of LTD induced by 100 pre stimulations in B1 and the experiment summary in B2 ) ; note that this LTD was not predicted by the model .", "In agreement with the model , when 2-AG levels were amplified by MAGL inhibition with JZL184 ( 1 . 5 μM ) , 100 pre-synaptic stimulations triggered LTP ( 143±17% , p=0 . 0299 , n=11 ) instead of LTD in control conditions ( Figure 9C; with an example of tLTP induced by 100 pre stimulations with JZL184 in C1 and the experiment summary in 9C2 ) .", "This tLTP was eCB-mediated since it was prevented when JZL184 was co-applied with AM251 ( 3 μM ) ( 93±4% , p=0 . 1509 , n=5 ) ( Figure 9C2 ) . 10 . 7554/eLife . 13185 . 014Figure 9 . MAGL inhibition shifts eCB-LTD into eCB-LTP , induced by 100 presynaptic stimulations .", "( A ) Model prediction for the changes in synaptic weight induced by 100 presynaptic stimulations .", "( A1 )", "In the absence of postsynaptic activity , 100 presynaptic stimulations in the model do not change the synaptic weight in control conditions ( full black line ) , but MAGL inhibition and DAG-Kinase activity ( full red line ) generates eCB amounts that are large enough to trigger LTP .", "MAGL and DAG-Kinase inhibition were obtained by fixing the value of the maximal rates of each enzyme to 0 and 5% , respectively , of their default values listed in Supplementary file 1C .", "( A2 )", "Summary bar graph of the LTP amplitude predicted by the model for 100 presynaptic stimulations in the absence of postsynaptic stimulations .", "( B ) Experimentally , 100 presynaptic stimulations alone ( i . e . without paired postsynaptic stimulation ) induced significant LTD . ( B1 ) Example of LTD induced by 100 presynaptic stimulations .", "Top , EPSC strength before and after pairings ( before pairings: 118±2 pA; 45–55 min after pairings: 78±1 pA; decrease of 34% ) .", "Bottom , time courses of Ri ( before , 187±1 MΩ; after , 189±1 MΩ; change of 1 . 1% ) and injected current ( Iinj ) ( before , 2±0 . 6 pA; after , 3±0 . 4 pA; change of 0 . 8% ) for this cell .", "( B2 )", "Summary of LTD induced with 100 presynaptic stimulations; 6/8 cells showed significant LTD .", "This 2-AG-mediated LTD was prevented by AM251 ( 3 μM , n=7 ) ; 6/7 cells showed no significant plasticity .", "( C ) MAGL inhibition by ZZL184 ( 1 . 5 μM ) shifts eCB-LTD , induced by 100 presynaptic stimulations , into eCB-tLTP .", "( C1 )", "Example of LTP induced by 100 presynaptic stimulations with MAGL inhibition .", "Top , EPSC strength before and after pairings ( before pairings: 143±2 pA; 45–55 min after pairings: 224±2 pA; increase of 57% ) .", "Bottom , time courses of Ri ( before , 131±1 MΩ; after , 131±2 MΩ; no significant change ) and injected current ( Iinj ) ( before , -3±1 pA; after , -12±1 pA; change of 6 . 3% ) for this cell .", "( C2 )", "Summary of LTP induced with 100 presynaptic stimulations; 7/11 cells showed significant LTP .", "This 2-AG-mediated LTD was prevented by AM251 ( 3 μM , n=5 ) ; 5/5 cells showed no significant plasticity .", "Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "*p<0 . 05 .", "ns: non-significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 014 To summarize , manipulating the activity of the MAGL was sufficient to ( 1 ) control the magnitude of eCB-tLTP , ( 2 ) induce eCB-tLTP or ( 3 ) even to reverse eCB-LTD into eCB-tLTP .", "These experimental validations of the model predictions thus support our model hypothesis that 2-AG levels control eCB plasticity in a bidirectional way , with large 2-AG levels yielding eCB-tLTP and lower levels eCB-tLTD .", "We next aimed at identifying which molecular actors are responsible for the modification of the presynaptic weight that is controlled by CB1R activation .", "In previous reports of eCB-dependent plasticity , Wpre was found to rely on the phosphorylation state of a yet unknown target protein involved in glutamate exocytosis , controlled by PKA and calcineurin ( CaN ) ( Heifets and Castillo , 2009 ) .", "In particular , PKA and CaN inhibition upon CB1R activation is thought to be involved in eCB-LTD induced with high-frequency stimulation protocol ( Heifets and Castillo , 2009 ) .", "We thus first tested the implication of PKA and CaN in eCB-tLTD .", "Inhibition of PKA by bath-applied inhibitor KT5720 ( 1 μM ) during a STDP protocol that triggers eCB-tLTD in control condition ( 100 pre-post pairings ) did not affect the expression of eCB-tLTD ( 74±10% , p=0 . 020 , n=5; p=0 . 8752 compared to control tLTD ) ( Figure 10A ) .", "We then tested the involvement of the phosphatase CaN activity in eCB-tLTD expression .", "We found that CaN inhibition by cyclosporin A ( 1 μM ) prevented eCB-tLTD ( 122±18% , p=0 . 2560 , n=6 ) ( Figure 10A2 and 10B ) Note that cyclosporin A being cell-permeant , we cannot distinguish from those results the location ( pre- or post-synaptic ) of the implicated CaN . 10 . 7554/eLife . 13185 . 015Figure 10 . eCB-tLTP maintenance relies on presynaptic PKA activation .", "( A-B ) eCB-tLTD is CaN-dependent .", "( A1 )", "Summary of plasticity induced by 100 pre-post pairings with bath applied KT5720 ( 1 μM ) or with cyclosporin ( 1 μM ) .", "eCB-tLTD was prevented with CaN ( cyclosporin A ) inhibition but unaffected with PKA inhibition ( KT5720 ) ; 0/6 and 4/5 cells showed LTD with cyclosporin A and KT5720 , respectively .", "( A2 )", "Summary bar graphs illustrate that eCB-LTD maintenance involves the activation of presynaptic CaN by a CB1R-triggered signal and was independent of the activation of presynaptic PKA .", "( B ) Main conclusion scheme: eCB-LTD is triggered by moderate prolonged levels of CB1 activations and requires active CaN .", "Normality was assumed for the cyclosporine A & KT5720 data ( test not passed ) .", "( C-D ) eCB-tLTP is PKA-dependent .", "( C1 )", "Summary of plasticity induced by 10 post-pre pairings with bath applied KT5720 ( 1 μM ) or with i-PKI6-22 ( 20 μM ) .", "eCB-tLTP was prevented with bath-applied KT5720 but unaffected with i-PKI6-22 , a cell-impermeant PKA inhibitor applied intracellularly in the postsynaptic neuron; 0/4 and 6/7 cells showed LTP with KT5720 and i-PKI6-22 , respectively .", "( C2 )", "Summary bar graphs illustrate that eCB-tLTP depends on presynaptic PKA activation since it was prevented by bath-applied KT5720 but unaffected when KT5720 or PKI6-22 , was applied intracellularly in the postsynaptic neuron ( i-KT5720 , i-PKI6-22 ) .", "Cyclosporin A had no effect on eCB-tLTP , showing that it was CaN-independent .", "Thus , eCB-tLTP maintenance involves the activation of presynaptic PKA by a CB1R-triggered signal .", "Normality was assumed for the ctrl & KT5720 data ( test not passed ) .", "( D ) Main conclusion scheme: eCB-LTP is triggered by large levels of short duration of CB1R activation and requires presynaptic active PKA .", "Representative traces are the average of 15 EPSCs during baseline ( black traces ) and 50 min after STDP protocol ( grey traces ) .", "Error bars represent SD .", "*p<0 . 05 .", "ns: not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 01510 . 7554/eLife . 13185 . 016Figure 10—figure supplement 1 . Schematic of a possible mechanism for PKA-CaN control of eCB-dependent bidirectional STDP . CB1R is a Gαi GPCR which activation by eCB reduces the activity of PKA .", "In the striatum , CB1R activation also inhibits presynaptic VSCC , which is expected to reduce presynaptic calcium and , potentially , calcium-activated calcineurin .", "Therefore , CB1R activation may reduce both PKA and CaN activity .", "The top panel gives a schematic illustration of this effect in a case where the kinetic parameters are such that the ratio between PKA and CaN activity changes when CB1R activation increases: CaN/PKA > 1 for intermediate CB1R activation and switches to CaN/PKA < 1 for large activation .", "Our experimental data for eCB-dependent STDP and its control by CB1R activation ( bottom ) are compatible with the hypothesis that eCB-LTD would be expressed when CaN/PKA > 1 whereas CaN/PKA < 1 leads to eCB-LTP . DOI: http://dx . doi . org/10 . 7554/eLife . 13185 . 016 We then tested the involvement of PKA and CaN on eCB-tLTP induced with 10 post-pre pairings .", "Bath-applied CaN inhibitor cyclosporin A did not preclude the expression of eCB-tLTP ( 154±17% , p=0 . 0305 , n=5 ) ( Figure 10C ) .", "Inhibition of PKA with KT5720 ( 1 μM ) prevented plasticity with 10 post-pre pairings ( 98±2% , p=0 . 3203 , n=4 ) ( Figure 10C ) demonstrating that PKA activity is critically involved in eCB-tLTP .", "We then aimed at determining which pools of PKA ( pre- and/or postsynaptic ) were involved in eCB-tLTP .", "For this purpose , we restricted PKA inhibition to the postsynaptic neuron with intracellular application ( through the patch-clamp pipette ) of KT5720 ( i-KT5720 , 1 μM ) or a cell-non-permeant PKA inhibitor PKI 6–22 ( i-PKI 6–22 , 20 μM ) .", "Both treatments did not significantly affect eCB-tLTP ( with i-KT5720: 137±8% , p=0 . 0108 , n=5; with i-PKI 6–22: 163±29% , p=0 . 03249 , n=7 ) ( Figure 10C ) .", "We therefore conclude that the activity of presynaptic PKA is critical for the expression of eCB-tLTP .", "Together , these results suggest that the expression of eCB-tLTD at the corticostriatal synapse depends on the activity of CaN ( Figure 10B ) ( and possibly on PKA inhibition ) , whereas the expression of eCB-tLTP is conditioned by the activity of presynaptic PKA ( Figure 10D ) .", "Therefore , intermediate levels of CB1R activation trigger eCB-tLTD through a combination of PKA inhibition and CaN activity , whereas high levels of CB1R activation leads to eCB-tLTP through the reverse combination: PKA activity combined to CaN inhibition ." ], [ "Long-term synaptic changes at corticostriatal synapses provide fundamental mechanisms for the function of the basal ganglia in action selection and in procedural learning ( Yin et al . , 2009 ) in which eCB plasticity have emerged as the major form underlying long-term synaptic strength changes ( Mathur et al . , 2012 ) .", "We describe here a paired-activity dependent tLTP and tLTD , wherein eCB dynamics tightly control both the induction/maintenance and polarity of synaptic weight changes .", "Due to their on-demand intercellular signaling modus operandi ( Alger and Kim , 2011 ) , eCB biosynthesis and release are evoked by precisely timed physiological stimuli .", "Our study demonstrates that STDP , an important physiological form of Hebbian plasticity , efficiently triggers eCB signaling and that eCB signaling controls the STDP polarity in a bidirectional manner depending on the activity pattern .", "Since its discovery , STDP has been attracting a lot of interest in computational neuroscience because it is based on the patterns of spike timing .", "Computational models of STDP can be clustered into two families .", "A first group of models aims at predicting the consequences of STDP on e . g . neuronal receptive fields or network dynamics ( Clopath et al . , 2010; Costa et al . , 2015 ) .", "In those models , the function describing weight changes with spike timing is usually given as hypothesis of the model .", "A second group of models starts from the signaling pathways implied in STDP and aims at understanding how the function describing weight changes with spike timing emerges from those signaling pathways ( see e . g . Graupner and Brunel , 2010 for a review ) .", "In a number of models in this second group , intracellular signaling is actually restricted to cytoplasmic calcium variation , thus implementing calcium-control hypothesis ( Shouval et al . , 2002 ) .", "The mathematical models that consider signaling downstream of calcium usually account for a single intracellular signaling pathway ( i . e . a single coincidence detector ) , most often NMDAR-CAMKII ( Rubin et al , 2005 , Graupner and Brunel , 2007; Urakubo et al . , 2008 ) .", "Noticeable exceptions are for instance Karmarkar and Buonomano ( 2002 ) , Evans et al . , 2012 or Paillé et al . ( 2013 ) , where the calcium pool entering via NMDAR controls tLTP whereas the calcium pool entering though VSCCs controls tLTD , thus implementing two coincidence detectors .", "However those models do not consider the signaling pathways beyond calcium entry through NMDAR and VSCC .", "Our mathematical model belongs to the latter group .", "To our knowledge , this is the first model to incorporate two detailed signaling pathways to account for STDP: NMDAR-CAMKII ( with calmodulin , PKA , CaN and PP1 ) for tLTP and the eCB system for tLTD and tLTP .", "In the model , the eCB system comprises mGluR5 , PLCβ , DAGL , MAGL , DAG-Kinase , calcium-induced calcium release ( IP3R channels , SERCA pumps ) , IP3 dynamics ( PLCδ , PI3K ) , VSCC , TRPV1R and CB1R ( Figure 2A ) .", "Thank to this very fine grain description , the present mathematical model is able to predict the weight change when any of the STDP parameters is varied , that is , not only spike timing ΔtSTDP , but also Npairings and frequency .", "This capacity has allowed us to explore a novel form of plasticity , eCB-LTP , induced by a low number of post-pre pairings at 1 Hz ( Cui et al . , 2015; the present study ) .", "Our mathematical model features 36 ordinary differential equations and roughly 150 parameters , among which more than one half is constrained by experimental data .", "A classical view assesses that with enough parameters , one can fit any data set , whatever the equations used .", "This view however does not apply in our case because we constrained the model with experimental data embedded in a three-dimensional parameter space ( ΔtSTDP , Npairings and frequency ) .", "In these conditions of strong constraining by experimental data , our parameter estimations systematically converged to a unique scenario that features two fundamentals:", "( i ) eCB-transients allow bidirectional eCB plasticity whereby tLTD is triggered by moderate levels of eCB while high-amplitude eCB transients yield LTP and", "( ii ) large eCB-transients are obtained for low numbers of pairings ( for 5<Npairings<20 at 1 Hz ) while for larger number of pairings ( Npairings>40 pairings at 1 Hz ) reduced calcium influx from internal stores and/or CB1R desensitization curtails the amplitude of the eCB-transients .", "We confirmed this prediction experimentally by two means: eCB puffs with different concentrations and durations , and MAGL inhibition in various conditions .", "Highly concentrated short puffs of 2-AG indeed yielded LTP in the absence of any electrical stimulation whereas less concentrated prolonged 2-AG puffs produced LTD .", "By decreasing MAGL activity , thus favoring high 2-AG levels , we could increase the magnitude of eCB-tLTP ( 5 post-pre pairings ) , induce eCB-tLTP at 50 post-pre pairings or even switch eCB-LTD to eCB-tLTP ( 100 presynaptic stimulations ) .", "Therefore , under MAGL inhibition , the temporal coincidence between pre- and postsynaptic spikes is not mandatory anymore for the induction of long-term plasticity .", "This discovery may have far-reaching consequences , since it means that the manipulation of MAGL activity may bootstrap synaptic plasticity in synapses where the postsynaptic neuron is silent , thus rescuing possible pathological situations or waking up new local circuits .", "Further work is however needed to check the realism of this implication in vivo but it is noteworthy that recently , nerve growth factor ( NGF ) signaling in cholinergic projection neurons of fetal mice has been shown to control MAGL degradation in vivo and in vitro in a spatially specific way ( Keimpema et al . , 2013 ) .", "In light of our findings , these results suggest that the regulation of MAGL activity may indeed be a potent mechanism to control synaptic plasticity and thus learning and memory .", "Our experimental results showing that short puffs of highly concentrated 2-AG yields LTP in the absence of any electrical stimulation is a strong argument in favor of the model-derived hypothesis that eCB can support tLTP in addition to tLTD .", "Both the amplitude ( ≃165% ) and the pharmacology ( suppression by CB1R antagonist AM251 ) of the LTP observed with these puffs are identical to those observed for tLTP triggered by 10 post-pre pairings .", "Moreover , it is important to note that when we applied 2-AG in the form of prolonged puffs ( x100 ) , we observed LTD instead of LTP .", "According to our mathematical model , this behavior would be due to desensitization of the CB1R that becomes prominent with prolonged puffs .", "2-AG puff application is therefore generally expected to give rise to depression except if the puffs are of short duration , where potentiation can be observed .", "This feature may account for the widespread observation that prolonged applications of 2-AG systematically yielded to LTD and might explain why eCB-dependent potentiation has proven difficult to observe experimentally .", "Our mathematical model in general shows very good agreement with experimental data , even for conditions for which the model was not fitted ( stimulation frequency >1 Hz , alteration of MAGLipase activity ) .", "Some mismatches are however notable .", "Our focus here has mostly been on the signaling part of the system .", "In comparison , our modeling of the synaptic machinery and the electrophysiological response of the MSN neurons have been less sophisticated .", "For instance , Figure 9B shows that 100 presynaptic stimulations at 1 Hz , in the absence of post-synaptic stimulation , are sufficient to trigger eCB-LTD experimentally .", "This is likely due to the production of eCB resulting from the postsynaptic depolarization triggered by the 100 presynaptic stimulations ( depolarization-induced LTD ) .", "In the model however , the postsynaptic depolarization triggered by 100 presynaptic stimulations at 1 Hz is not large enough to allow the entry of the minimal amount of calcium that is needed to overcome the eCB-LTD threshold , so no plasticity is observed .", "The dynamics of glutamate release and binding are expected to be crucial for the expression of STDP , as illustrated by our sensitivity analysis ( Figure 4 ) .", "Several parts of the glutamate release and binding machinery at the corticostriatal synapse are known to display nontrivial frequency-dependence , including presynaptic glutamate release , uptake by transporters and receptor activation because of desensitization of AMPAR ( Goubard et al . , 2011 ) .", "Since our mathematical model features none of these frequency dependencies , we cannot expect a precise quantitative match between experiments and model when frequency is varied .", "However , it is remarkable that the model still yields correct predictions of the main qualitative trends observed in the experiments .", "For instance , 10 post-pre pairings at very low frequency ( 0 . 1 Hz ) are sufficient to trigger tLTP in the experiments , while the model predicts no plasticity at those frequencies ( Figure 5 ) .", "At 4 Hz , the experimental pharmacology profile with 10 pairings is not matched by the model with 10 pairings , but with 15 pairings ( Figure 5 ) .", "Nevertheless , the model predictions that increased frequency should witness", "i ) an enlarged range for the expression of tLTP with post-pre 10 pairings and", "ii ) the emergence of a new tLTP with 10 pre-post pairings turned out to be generally correct .", "In the hippocampus , theta-burst stimulation induces eCB-dependent LTD at the synapse between inhibitory interneurons and pyramidal cells that is blocked when presynaptic CaN is inhibited ( Heifets et al . , 2008 ) .", "Similarly , we found that eCB-tLTD triggered at the corticostriatal synapse also needs CaN activity .", "In our experiments , we could not estimate the localization of the CaN that mediated eCB-LTD ( pre or postsynaptic ) but it is likely that in analogy with theta-burst STDP in the hippocampus , the implied CaN would be presynaptic .", "The implication of PKA in eCB-LTD is less clear .", "In the hippocampus ( Chevaleyre et al , 2007 ) and nucleus accumbens ( Mato et al . , 2008 ) , the expression of frequency-dependent eCB-LTD is blocked when cAMP levels are increased by the adenylyl-cyclase activator forskolin .", "Since PKA is activated by cAMP , this indicates that reduced levels of PKA are necessary for eCB-LTD .", "In both cases however , direct inhibition of presynaptic PKA actually blocks the expression of eCB-LTD , thus showing that the implication of PKA in eCB-dependent plasticity is a complex and subtle phenomenon .", "In the case of tLTD in the dorsolateral striatum , we found that direct PKA inhibition does not obliterate eCB-tLTD .", "Our result is , therefore , in line with the notion that the expression of eCB-tLTD requires PKA inhibition .", "Strikingly , the eCB-tLTP triggered at the corticostriatal synapse by STDP protocols had the exact inverse dependence on PKA and CaN compared to eCB-tLTD .", "Indeed , we found that PKA inhibitors block eCB-tLTP expression whereas CaN inhibition has no effect .", "Therefore , our study of STDP protocols at the corticostriatal synapse shows that the expression of eCB-tLTD needs CaN ( but not PKA ) activity whereas the expression of eCB-tLTP demands PKA ( but not CaN ) activity .", "Whether the same or two separate pathways support eCB-tLTD and eCB-tLTP is still a pending question which resolution is rendered highly challenging by the difficult access to the molecular mechanisms occurring in the presynaptic compartment of the corticostriatal synapses .", "However , we can propose a simplified schematic mechanism ( Figure 10—figure supplement 1 ) .", "CB1R activation inhibits PKA activity but also inhibits presynaptic VSCCs ( Mato et al . , 2008 ) , which are expected to hamper calcium influx in the presynaptic compartment .", "Such a decrease in calcium levels reduces the CaN activity .", "CB1R activation is expected to reduce both CaN and PKA activity , although the shapes ( kinetics parameters ) of the decays of PKA and CaN activity with increasing CB1R activation are not necessarily identical .", "For example , in Figure 10—figure supplement 1 , PKA activity dominates CaN when CB1R activation is large , whereas CaN dominates for a range of intermediate CB1R activations .", "Considering our experimental data , this putative mechanism suggests that eCB-STDP is gated by the ratio between PKA and CaN activities: large CB1R activation would produce high values of the PKA/CaN ratio yielding eCB-tLTP whereas intermediate CB1R activations would result in low values of the PKA/CaN ratio and , consequently , in eCB-tLTD .", "Future experimental investigation is needed to test the validity of this proposed mechanism .", "The bidirectionality of synaptic plasticity is a key parameter since it allows LTP and LTD to reverse each another with time at a single synapse ( probably at the same presynaptic side ) , thus enabling adaptive changes of the synaptic weight .", "Altogether , our results show that eCB bidirectional plasticity constitutes a versatile system , which operation may underlie a complex repertoire of learning abilities , depending on activity pattern at corticostriatal circuits and on the behavioral context ." ] ]
[ "Synaptic plasticity is a cardinal cellular mechanism for learning and memory .", "The endocannabinoid ( eCB ) system has emerged as a pivotal pathway for synaptic plasticity because of its widely characterized ability to depress synaptic transmission on short- and long-term scales .", "Recent reports indicate that eCBs also mediate potentiation of the synapse .", "However , it is not known how eCB signaling may support bidirectionality .", "Here , we combined electrophysiology experiments with mathematical modeling to question the mechanisms of eCB bidirectionality in spike-timing dependent plasticity ( STDP ) at corticostriatal synapses .", "We demonstrate that STDP outcome is controlled by eCB levels and dynamics: prolonged and moderate levels of eCB lead to eCB-mediated long-term depression ( eCB-tLTD ) while short and large eCB transients produce eCB-mediated long-term potentiation ( eCB-tLTP ) .", "Moreover , we show that eCB-tLTD requires active calcineurin whereas eCB-tLTP necessitates the activity of presynaptic PKA .", "Therefore , just like glutamate or GABA , eCB form a bidirectional system to encode learning and memory ." ]
[ "Learning and memory depend on processes that alter the connections – or synapses – between neurons in the brain .", "For example , molecules called endocannabinoids can alter synapses to decrease the influence that one neuron has on another neuron’s activity .", "This “synaptic depression” is an important mechanism through which the brain can adapt to an experience .", "However , recent research also suggests that endocannabinoids might also increase the influence one neuron has on another neuron’s activity by strengthening the synaptic connection between neurons .", "This opposite process is known as synaptic potentiation , and is also important for learning from experience .", "But how do endocannabinoids manage to produce opposing effects ?", "Using a combination of electrophysiological recording experiments and mathematical modeling , Cui et al . have now deciphered the molecular mechanisms that govern the action of endocannabinoids at key synapses in rat brain slices .", "This revealed that both the levels and timing of endocannabinoid release control changes in the strength of the synaptic connections .", "Electrical stimulations that produced moderate amounts of endocannabinoids over a prolonged period led to synaptic depression .", "However , stimulation that produced short but large endocannabinoid peaks caused synaptic potentiation .", "The enzymes that control endocannabinoid levels thus play a crucial role in determining whether a given stimulation leads to the strengthening or weakening of a synaptic connection .", "In the type of synapses studied by Cui et al . , changes to synaptic strength also depend on another chemical called dopamine .", "Abnormal dopamine production is implicated in a number of disorders , including Parkinson’s disease and addiction .", "Future work will therefore investigate how dopamine controls endocannabinoid-dependent changes to the strength of synapses ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics" ]
Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis
elife-09532-v2
[ [ "Deducing the native conformation of a protein provides insight into its biological function .", "X-ray crystallography , NMR and more recently Cryo-EM ( Bartesaghi et al . , 2015; Cheng , 2015 ) are the major techniques used to accomplish this at atomic resolution .", "However , these require soluble purified protein at high concentration .", "In a few cases ( Garbuzynskiy et al . , 2005 ) , the structures solved by different methods do not agree with each other , for various reasons .", "In many cases , the folded conformation is believed to be at a global free energy minimum ( Anfinsen , 1973 ) .", "This can be used as a guide to either deduce the structure ( Havel et al . , 1979 ) or fold a protein in silico ( Das et al . , 2009; Das and Baker , 2008; Pande et al . , 2003 ) .", "The structural and functional integrity of a protein requires maintenance of specific interactions during the course of evolution .", "Evolution allows either conservation of these interacting pairs of residues or mutation at interacting positions in a correlated manner ( Godzik and Sander , 1989; Melero et al . , 2014 ) .", "The fitness cost of most amino acid substitutions depends on the genetic context in which they occur .", "Substitutions beneficial in one background can be detrimental in a different background ( Breen et al . , 2012; Shah et al . , 2015 ) .", "Interestingly , some sites are conserved not because a given amino acid is functionally irreplaceable , but rather because the right context is not available for its evolution ( Wellner et al . , 2013 ) .", "This provides further evidence of correlation amongst mutations , that is epistatic interactions .", "Often , the detrimental effects of a mutation can be alleviated by a second ( suppressor ) mutation which compensates for defects in stability , packing or function caused by the first mutation .", "Such correlated substitutions have been experimentally identified in attempts to screen for second-site suppressors ( Araya et al . , 2012; Hecht and Sauer , 1985; Machingo et al . , 2001; Pakula and Sauer , 1989; Sideraki et al . , 2001 ) .", "Correlated mutations have also been employed to identify protein interaction sites ( Melamed et al . , 2015 ) .", "Two other recent studies have described a large number of second-site suppressors for the RRM2 domain of the yeast poly ( A ) -binding protein ( Pab1 ) and the IgG-binding domain of protein G ( GB1 ) , respectively ( Melamed et al . , 2013; Olson et al . , 2014 ) .", "While there have been past efforts to identify compensatory mutations , a comprehensive method to explicitly identify suppressors and to distinguish suppressors that are spatially proximal from those that are distal to the site of the original inactive mutation is lacking .", "In the current work , we attempt to address these issues .", "In addition to experimental studies to identify correlated mutations , several computational methods also exist which detect coevolution of residues in homologous sequences .", "Various statistical approaches which identify correlated mutations have been used to decode such evolutionary information contained in multiple homologous sequences of a protein ( Göbel et al . , 1994 ) .", "Progressive improvement in this area has resulted in more accurate strategies such as use of a Bayesian network model ( Burger et al . , 2010 ) , maximum entropy in DCA ( Morcos et al . , 2011 ) and EVfold ( Marks et al . , 2011 ) , sparse inverse covariance estimation in PSICOV ( Jones et al . , 2012 ) and a pseudo-likelihood based approach in GREMLIN ( Kamisetty et al . , 2013 ) to predict residue-residue contacts to computationally build protein 3D structures ( Marks et al . , 2011; Jones et al . , 2012; Ovchinnikov et al . , 2014; Sulkowska , et al . , 2012 ) .", "A sparse network of co-evolving residues of a protein constraining its structure , specificity and function has been examined by statistical coupling analysis of evolutionarily rich sequence data in protein families ( Halabi et al . , 2009 ) .", "Although these methods show great promise in the area of macromolecular structure determination , the fidelity of the predictions is questionable for candidates with small protein families , where the total number of sequences is less than five times the length of the protein in case of GREMLIN ( Kamisetty et al . , 2013 ) or less than 1000 for some of the other methods ( Morcos et al . , 2011 ) .", "Further , stringent constraints present during natural evolution limit the number and nature of sequences that are sampled .", "We propose alternative experimental methodology termed saturation suppressor mutagenesis to complement and extend residue contact information obtained from computational analyses of correlated mutations in a multiple sequence alignment .", "The test protein in this study , Controller of Cell Division or Death B ( CcdB ) is the toxin component of the Escherichia coli CcdA-CcdB antitoxin-toxin system .", "It is a globular , dimeric protein with 101 residues per protomer , involved in maintenance of F plasmid in cells by a mechanism involving its binding to and poisoning of DNA Gyrase ( Dao-Thi et al . , 2005 ) .", "CcdB has two primary ligands with overlapping binding sites .", "These are its cognate antitoxin , CcdA and its cellular target , DNA Gyrase .", "CcdB has ~350 homologs in its protein family , which is less than five times the length of the protein .", "Therefore , computational methods cannot be reliably employed to deduce correlated mutations from a multiple sequence alignment of CcdB homologs .", "Using CcdB , we identify spatially proximal residues by doing an exhaustive search for suppressor mutations for a given inactive mutant ( Figure 1 ) .", "We subsequently use this information for protein model discrimination and structure prediction .", "We have previously constructed a saturation mutagenesis library of CcdB in which each residue was individually randomized .", "The library was screened at multiple expression levels in E . coli and relative activities of ~1430 individual mutants were inferred by deep sequencing ( Adkar et al . , 2012 ) .", "It was observed that the mutational sensitivity of each residue of CcdB is related to its depth from the protein surface .", "Residue depth is defined as the distance of any atom/residue to the closest bulk water ( Chakravarty and Varadarajan , 1999; Tan et al . , 2011 ) .", "An empirical parameter ( RankScore ) related to mutational sensitivity was defined for each residue , which could be reliably used as a proxy for the residue’s depth , provided the residue was not part of the active-site .", "Active-site residues could be deduced solely from mutational sensitivity data .", "A brief description of the screening procedure and definitions of mutational sensitivity score and RankScore are provided in the Materials and methods section .", "In the present study , residues likely to be present at the core of CcdB were identified by their mutational sensitivity in the single mutant library ( Adkar et al . , 2012 ) .", "Inactive mutants at five such putative buried residues were identified .", "Next , exhaustive second-site saturation mutagenesis libraries constructed in the background of each inactive mutant were displayed on the surface of yeast ( Chao et al . , 2006 ) and screened for binding to the CcdB ligand , DNA Gyrase , to identify suppressors .", "Distal and proximal suppressors could readily be distinguished based on the RankScore of the suppressor site in the original single-site saturation mutagenesis library ( Adkar et al . , 2012 ) .", "The residue proximity information thus obtained , was used to generate spatial constraints , which were in turn able to capture the protein’s native structural features and recovered >98% of native-like models ( with backbone RMSD within 2 . 5 Å of the native structure ) from a pool of decoys .", "Thus , high-throughput , exhaustive application of this approach , combined with ways to disentangle proximal from distal suppressors would be a useful way to identify a substantial number of distance constraints , which can be used for protein structure prediction ( Marks et al . , 2011 ) .", "Diacylglycerol kinase A ( DgkA ) is a homotrimeric integral transmembrane protein ( 121 residues per protomer ) in E . coli , catalyzing the phosphorylation of diacylglycerol to phosphatidic acid .", "Gram-negative bacteria use this reaction product to shuttle water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in their cell envelope ( Van Horn and Sanders , 2012 ) .", "The protein has been captured in two distinct conformations by X-ray crystallography ( PDB id 3ZE5 [Li et al . , 2013] ) and NMR ( PDB id 2KDC [Van Horn et al . , 2009] ) , respectively .", "The two structures are significantly different from each other , with ‘domain swapping’ being the key feature of the NMR model .", "Each structure is characterized by several unique residue contacts .", "It is important to identify whether one or both these structures are present in-vivo .", "We therefore isolated inactive mutants at residues involved in differential contacts in the two structures .", "Next , we experimentally identified suppressors of these inactive mutants .", "The residue contact pairs identified via these suppressors were consistent only with the contacts found in the crystal structure , thereby suggesting that the X-ray structure represents the native , active conformer of this membrane protein in-vivo ." ], [ "In the current work , we aimed at identifying suppressors for buried inactive non-active site residues of CcdB .", "Since WT CcdB is toxic to E . coli only cells transformed with inactive mutants will survive , thus providing a facile selection for such mutants .", "Relative activities of ~1430 single-site mutants of CcdB have been obtained previously from phenotypic screening of a single-site saturation mutagenesis library at multiple expression levels in E . coli .", "Deep sequencing was used to determine the identities of all surviving ( inactive ) mutants at each expression level ( Adkar et al . , 2012 ) .", "An empirical parameter ( RankScore ) was defined for each residue ( see Materials and methods ) .", "It is related to the average mutational sensitivity ( see Materials and methods ) at each position .", "It was observed that for non-active site positions , RankScore is proportional to residue depth .", "The sequencing data from the above library was analyzed to select five inactive mutants; V5F , V18W , V20F , L36A and L83S ( hereafter referred to as parent inactive mutants ) at buried , non-active site residues .", "Several buried , non-active site residues were identified from sequencing data .", "Parent inactive mutants were chosen so as to include different kinds of mutations , namely large→small , small→large and hydrophobic→polar .", "Patterns of mutational sensitivity ( MSseq ) for mutants at a position were analysed to distinguish between active-site and buried mutations as described below .", "Both buried and active-site residue positions possess high RankScores and high average mutational sensitivity ( MSseq ) values .", "Active-site residues can be distinguished from buried ones based on the pattern of mutational sensitivity .", "Mutational sensitivity ( MSseq ) scores were determined from the sequencing analysis of the single-site saturation library of CcdB and refer to the expression level at which partial loss of function mutants show an active phenotype ( methodology described in [Adkar et al . , 2012] ) .", "Patterns of MSseq values for active-site , buried and exposed positions differ from each other .", "Representative plots are shown in Figure 1—figure supplement 1 . At buried positions , typically most aliphatic substitutions are tolerated; hence they have low MSseq values .", "Polar and charged residues are poorly tolerated at buried positions , thus showing high MSseq values ( Figure 1—figure supplement 1A ) .", "By contrast , mutations to aliphatic residues are often poorly tolerated at active-site residues ( which are typically exposed ) and have higher MSseq values ( Figure 1—figure supplement 1B ) .", "Polar and charged residues are sometimes tolerated and the average mutational sensitivity for active-site residues is typically higher than for buried residues .", "By analyzing the mutational sensitivity patterns at all positions , Q2 , F3 , Y6 , S22 , I24 , N95 , W99 , G100 and I101 can be identified as putative active-site residues .", "These residues were identified based solely on mutational data ( unpublished results ) .", "Similar mutational patterns are seen for at least two other proteins for which extensive mutational data exist , the PDZ domain ( PSD95pdz3 ) ( McLaughlin et al . , 2012 ) and the IgG-binding domain of protein G ( GB1 ) ( Olson et al . , 2014 ) ( unpublished results ) . 10 . 7554/eLife . 09532 . 003Figure 1 . Strategy adopted to determine proximal residue pairs .", "( A ) Parent inactive mutant binds cognate ligand with lower apparent affinity than Wild-Type protein ( WT ) .", "( Parent inactive mutant , Suppressor mutant ) pair binds ligand with higher affinity than the parent inactive mutant .", "Refer to Figure 1—figure supplement 3 for the PCR strategy to introduce a parent inactive mutant into the single-site saturation mutagenesis ( SSM ) library of CcdB .", "Proximal and distal suppressors can be discriminated based on the mutational sensitivity of the corresponding single mutants .", "( B ) Yeast Surface Display is used as the screening system in case of CcdB .", "( C ) Suppressor mutants are identified using Fluorescence Activated Cell Sorting ( FACS ) of the mutant library using yeast surface display .", "Active-site residues can be distinguished from buried ones based on the pattern of mutational sensitivity ( MSseq ) .", "Putative active-site , buried and exposed residue positions were identified from the MSseq data .", "Representative plots are shown in Figure 1—figure supplement 1 . Possible mechanisms responsible for reduced activity of a mutant protein are described in Figure 1—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 00310 . 7554/eLife . 09532 . 004Figure 1—figure supplement 1 . Barplots depicting mutational sensitivity ( MSseq ) values of all available mutants at representative positions of CcdB . A value of zero indicates that the mutant was not obtained in the library .", "( A ) MSseq values at two buried positions .", "At buried positions , typically most aliphatic substitutions are tolerated ( low MSseq ) .", "Polar and charged substitutions are poorly tolerated ( high MSseq ) .", "( B ) MSseq values at two active-site positions .", "Very few substitutions are tolerated at active-site positions .", "Aliphatic residues are also poorly tolerated ( high MSseq ) .", "( C ) MSseq values at two exposed , non-active site positions .", "At exposed non-active site positions , almost all substitutions are tolerated and have low MSseq values with the possible exceptions of G and P . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 00410 . 7554/eLife . 09532 . 005Figure 1—figure supplement 2 . Possible mechanisms for reduced activity of a mutant protein .", "( 1 ) Folding and binding equilibrium for the Wild-Type ( WT ) protein which has low values of equilibrium dissociation constant Kd and equilibrium unfolding constant Ku .", "( 2 ) Some mutations do not affect the folding or activity .", "( 3 ) If the mutation is present at the ligand binding site , then the protein is rendered inactive because of an increase in Kd while Ku is unchanged .", "( 4 ) If the mutation at a buried site leads to the formation of a misfolded protein , then both Kd and Ku increase .", "( 5 ) In case of a destabilizing mutation that does not perturb the native structure , Kd is unchanged but Ku increases .", "In both ( 4 ) and ( 5 ) , an increase in Ku results in an increase in the population of the unfolded state which is prone to aggregation and degradation , thereby resulting in a decrease in the level of folded , functional protein in vivo . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 00510 . 7554/eLife . 09532 . 006Figure 1—figure supplement 3 . Strategy to introduce an inactive mutant into all members of a Single-site Saturation Mutagenesis ( SSM ) library of CcdB cloned in a yeast surface display ( YSD ) vector ( pPNLS ) .", "Step I . CcdB gene cloned in the yeast surface display vector ( pPNLS ) is amplified in two segments ( Set A and Set B ) at the position of introduction of parent inactive mutation ( PIM ) , using two sets of primers ( arrows shown in black and red ) .", "The forward primer of Set B ( arrow in red ) and reverse primer of Set A ( arrow in black ) contain the inactive mutant codon in the middle of the primer sequence and are complementary to each other .", "The regions of the vector ( shown in dashed black and red lines ) complementary to the outside primers ( dashed black and red arrows ) are shown .", "These outside primers bind the vector >200 bases upstream and downstream of the ccdb gene and result in amplicons ( A_Lib_PIM or B_Lib_PIM or A_WT_PIM or B_WT_PIM ) of reasonable size ( >200 bases ) , irrespective of the point of introduction of parent inactive mutant .", "Single-site saturation mutagenesis library ( left ) and WT CcdB ( right ) cloned in pPNLS vector are used as template ( 1–5 pg ) .", "Step II .", "( A_Lib_PIM and B_WT_PIM ) or ( A_WT_PIM and B_Lib_PIM ) are recombined with the linearized vector by three fragment homologous recombination in Saccharomyces cerevisiae EBY100 at regions complementary to each other that is Vector – ( Amplicon from Set A ) – ( Amplicon from Set B ) – Vector , to assemble the CcdB mutant library .", "50% of the members of the assembled library have the parent inactive mutant only , while the rest of the members carry parent inactive mutant and a single mutant ( obtained from single-site saturation mutagenesis library used as template ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 006 Recent work ( Melamed et al . , 2015 ) suggests that saturation mutagenesis in combination with evolutionary conservation data can also be used to identify residues at interaction sites .", "In addition to differences in mutational sensitivity patterns which are employed here to discriminate between active-site and buried residues , an important difference between active-site and buried mutations is that the former typically affect specific activity and not the level of properly folded protein , while the latter primarily affect the level of properly folded protein ( Bajaj et al . , 2008 ) .", "Thus , measurements of protein levels , and possibly sensitivity of mutant activity to chaperone overexpression ( Tokuriki and Tawfik , 2009 ) , can also be used to distinguish between active-site and buried mutants .", "The average hydrophobicity and hydrophobic moment ( Varadarajan et al . , 1996 ) are supplementary parameters that can help distinguish between exposed , active-site and buried residues .", "Mutations at the selected non-active site positions perturb activity by reducing the amount of functional , folded protein in vivo ( Figure 1—figure supplement 2 ) ( Bajaj et al . , 2008 ) .", "In order to identify residues which can compensate for the parent inactive mutant , the selected parent inactive mutations described above were individually introduced into the single-site saturation mutagenesis library constructed previously ( Adkar et al . , 2012 ) .", "The resulting double mutant saturation-suppressor libraries were cloned into a yeast surface display vector ( pPNLS ) by three fragment recombination in Saccharomyces cerevisiae ( Figure 1—figure supplement 3 ) .", "Recombination yielded ~105 transformants for each library .", "Yeast surface display ( Chao et al . , 2006 ) and Fluorescence Activated Cell Sorting ( FACS ) were used as screening tools to isolate populations exhibiting", "( i ) enhanced binding to the ligand DNA Gyrase , and", "( ii ) increased surface expression relative to the corresponding parent inactive mutant .", "The ligand concentration was decreased in subsequent rounds of sorting to increase the stringency in selection of true suppressors ( Supplementary file 1 ) .", "The methodology used to generate the library along with the use of very low ( pg ) amount of template DNA for PCR was effective in introducing the parent inactive mutant into all members of the single-site saturation mutagenesis library .", "This is important as the presence of even a small fraction of WT residue at the position of the parent inactive mutant will rapidly lead to its selection , resulting in a high amount of false positive data .", "Following multiple rounds of sorting ( typically 3–4 ) , over 10% of the population was shown to bind significantly better to DNA Gyrase than the parent inactive mutants .", "Data for the L83S mutant library is shown ( Figure 2 ) .", "At this stage , 96 individual clones for each library were sequenced by Sanger sequencing to identify 1–3 potential suppressors for each parent inactive mutant ( Table 1 ) . 10 . 7554/eLife . 09532 . 007Figure 2 . Enrichment of second-site suppressor population for L83S CcdB inactive mutant following multiple rounds of Fluorescence Activated Cell Sorting ( FACS ) . 62 pM of Gyrase-3xFLAG was used in all cases for analysis .", "4 nM , 1 nM and 0 . 25 nM Gyrase-3xFLAG was used for the three rounds of sort respectively .", "The population gated in green shows the parent inactive mutant L83S , while that in magenta shows the sorted population which has higher surface expression and tighter binding to the ligand than L83S .", "Sort details for different libraries of CcdB are summarized in Supplementary file 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 00710 . 7554/eLife . 09532 . 008Table 1 . Experimentally determined ( Parent inactive mutant , suppressor ) pairs for CcdB occur at both spatially proximal and distal residues . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 008Parent inactive mutant ( X ) Suppressor Mutant ( Y ) ASA ( % ) Depth ( Å ) RankScoreaShortest distanceb ( Å ) Centroid-centroid distancec ( Å ) V5Fd06 . 854L36Md , e07 . 3833 . 95 . 5A81Gd , e32 . 43 . 9262 . 84 . 2V18W09 . 359M63Te0 . 18 . 1464 . 25 . 8I90Ve0 . 17 . 4604 . 85 . 9V20F08 . 660E11Rf112 . 73 . 4118 . 422 . 4E11Kf112 . 73 . 4118 . 422 . 4L36A07 . 383M63Le0 . 18 . 1463 . 86 . 4R10Gf76 . 63 . 6111 . 618 . 1E11Pf112 . 73 . 4111 . 716 . 8L83S1 . 55 . 842V54Le0 . 45 . 7283 . 94 . 5aRankScore for residues X or Y estimated from phenotypic screening of single-site saturation mutagenesis library of CcdB and deep sequencing ( Adkar et al . , 2012 ) bShortest distance between residues X and YcDistance between side chain centroids of residues X and YdThe suppressors ( Y ) were identified as a triple mutant with the parent inactive mutant ( X ) V5F , that is V5F/L36M/A81GeSuppressor residues spatially proximal to parent inactive mutantfSuppressor residues distal from the parent inactive mutantHeavy atoms of the residues are considered for calculating distances using the crystal structure of CcdB , PDB id 3VUB ( Loris et al . , 1999 ) .", "For a pair of residues in contact , it is likely that a destabilizing substitution at one residue can be suppressed by a substitution with complementary properties at the other residue .", "For example , a cavity formed by a large→small substitution of a residue may be compensated by a small→large substitution of the partner residue in contact with it .", "Consequently , while each of the individual mutations will be destabilizing in the WT background , the pair will have increased stability and activity relative to their corresponding single mutants , thus demonstrating positive sign epistasis .", "However , suppressors can be either spatially proximal or distal from the site of the original inactive mutation .", "In contrast to proximal suppressors , distal suppressors will typically be on the surface of protein ( Bank et al . , 2015 ) and , hence , the individual suppressor mutation is expected to be neutral in the WT background .", "Further , unlike proximal suppressors no complementarity in amino acid property relative to the parent inactive mutant is expected for a distal suppressor .", "In the present study , we use RankScore values to discriminate between proximal and distal suppressors .", "The approach is validated by mapping proximal and distal suppressors onto the known crystal structure of CcdB .", "We have previously shown that the RankScore parameter correlates with residue depths derived from the crystal structure of the protein ( Figure 4B of [Adkar et al . , 2012] ) .", "The value for RankScore ranges between 1 to 100 .", "For a given non-active site residue , a higher value of RankScore indicates that the residue is likely to be buried in the protein structure .", "All the parent inactive mutants were chosen such that they are non-active site and have a high RankScore .", "These residues are predicted to be buried and indeed are found to be buried in the crystal structure .", "It is therefore expected that positions at which local suppressors occur will also have high depth , high RankScores and high average mutational sensitivity in the single-site saturation mutagenesis library ( Figure 1A ) .", "All the positions with low RankScores are found to be exposed on the protein structure .", "Hence , suppressors with RankScore=1 were classified as distal suppressors ( Table 1 ) .", "A RankScore of 1 is a conservative cutoff for distal suppressors , probably a cutoff of five or ten would yield similar results .", "The RankScore cutoff of 25 which we have chosen for proximal suppressors , clearly identifies only buried residues with depth > 5 . 5 Å .", "All these residues have accessibility < 1 . 5% .", "The PCR strategy adopted ( Figure 1—figure supplement 3 ) to construct the second-site suppressor mutagenesis library for screening suppressors against the parent inactive mutants of CcdB was designed to generate", "( i ) 50% double mutants with each member containing the parent inactive mutant and a single mutant , and", "( ii ) 50% parent inactive mutants .", "Sequencing data obtained was analyzed to identify proximal and distal suppressors ( as discussed above ) .", "We largely obtained double mutants containing the parent inactive mutant and a suppressor , and a small fraction of the triple mutant ( V5F/L36M/A81G ) ( Table 1 ) .", "Occurrence of the triple mutant was due to selection and enrichment of an additional mutation introduced into the double mutant library likely due to PCR based errors .", "The selected triple mutant contained the parent inactive mutant V5F and two proximal suppressors ( L36M and A81G ) .", "Overall , six residue pairs were identified , which contained the parent inactive mutant and a putative proximal suppressor ( Table 1 ) .", "The shortest distance between individual members of the identified ( parent inactive mutant , proximal suppressor ) pairs in the structure of WT CcdB was 2 . 8–4 . 8 Å ( PDB id: 3VUB ( Loris et al . , 1999 ) , Table 1 , Figure 3 ) .", "This validated the methodology described above .", "Two residues , R10 and E11 ( in the pairs L36A/R10G , L36A/E11P , V20F/E11R and V20F/E11K ) were identified as locations for distal suppressors based on their low RankScore values in the single-site saturation mutagenesis library .", "Mutations at E11 were observed to suppress parent inactive mutants at multiple residue positions .", "The shortest distances between L36-R10 and V20-E11 are 11 . 6 Å and 18 . 4 Å , respectively .", "As expected , the proximal suppressors are clustered together in the protein interior while the distal ones are present on an exposed loop region ( Figure 3A , Video 1 ) . 10 . 7554/eLife . 09532 . 009Figure 3 . Experimentally obtained ( Parent inactive mutant , suppressor ) pairs mapped onto the crystal structure of CcdB ( PDB id 3VUB [Loris et al . , 1999] ) .", "Only the Wild-Type ( WT ) residues of each member of the pair are shown as structures of the mutants are not available .", "( A ) The two monomers of the dimeric CcdB protein are shown in ribbon ( dark grey ) with the distal suppressors ( R10 ( dark blue ) and E11 ( light blue ) mapped on an exposed loop region while the proximal suppressors ( light brown ) and parent inactive mutants ( dark red ) are present in the core of the protein .", "One of the monomers of the dimeric CcdB protein has been shown in B-F for ease of visualization of the mapped parent inactive mutants and corresponding proximal suppressors .", "( B ) Parent inactive mutant V5 and its proximal suppressors L36 and A81 , ( C ) Parent inactive mutant V18 and its proximal suppressors M63 and I90 , ( D ) Parent inactive mutant L36 and its proximal suppressor M63 , ( E ) Parent inactive mutant L83 and its proximal suppressor V54 are shown .", "Dotted lines indicate the shortest distance between the two residues in each pair .", "( F ) ( Parent inactive mutant , proximal suppressor ) pairs are clustered in the core of the protein forming an interconnected network , with side chains facing towards each other .", "Residues common to multiple ( Parent inactive mutant , suppressor ) pairs , for example L36 and M63 , provide additional information about the network of interactions .", "The figure has been prepared using the UCSF Chimera package ( developed by Resource for Biocomputing , Visualization , and Informatics at the University of California , San Francisco [Pettersen et al . , 2004] ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 00910 . 7554/eLife . 09532 . 010Video 1 . Experimentally obtained ( Parent inactive mutant , suppressor ) pairs mapped onto the crystal structure of CcdB ( PDB id 3VUB ) , related to Figure 3 .", "Parent inactive mutant is abbreviated as PIM in the video . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 010 The ability to suppress parent inactive mutants at multiple positions is indicative of the suppressor being a global suppressor .", "The most reliable way to identify these would be to confirm that the same putative distal suppressor is able to suppress multiple parent inactive mutants , preferably the ones which are not in contact with each other .", "We treated both the distal suppressors ( R10 and E11 ) as likely global suppressors and performed experiments described in the following section to test this hypothesis .", "Identified CcdB suppressors individually conferred improved binding and thermal stability to the corresponding parent inactive mutants ( Figure 4 , Figure 4—figure supplement 1 , Figure 4—figure supplement 2 , Supplementary file 2 ) .", "An increased affinity for DNA Gyrase for the ( Parent inactive mutant , suppressor ) pair relative to the parent inactive mutant alone was observed in all cases except for ( L36A , M63L ) and ( L36A , R10G ) pairs ( Figure 4C ) , which exhibited similar affinity towards Gyrase as the parent inactive mutant L36A .", "However , the ( Parent inactive mutant , suppressor ) pairs showed higher thermal stability ( Tm ( L36A , M63L ) =55 ± 0 . 8°C , Tm ( L36A , R10G ) =55 . 1 ± 0 . 4°C , Tm ( L36A ) =47 . 1 ± 0 . 3°C ) and increased surface expression ( Figure 4A , D ) .", "Surface expression levels of proteins displayed on the yeast surface have previously been found to correlate with the protein’s stability ( Shusta et al . , 1999 ) .", "Increased surface expression was observed for all ( Parent inactive mutant , suppressor ) pairs ( including the distal suppressor ( L36A , R10G ) pair ) relative to the parent inactive mutant , except for the ( V20F , E11R ) pair ( Figure 4A , B ) .", "This distal suppressor pair exhibited slightly lower expression than its parent inactive mutant , V20F , but displayed enhanced activity in terms of its binding to Gyrase ( Figure 4B , C ) . 10 . 7554/eLife . 09532 . 011Figure 4 . Restoration of defects in CcdB parent inactive mutants by suppressors . Proteins displayed on the yeast surface were monitored by their surface expression ( abscissa ) and Gyrase binding ( ordinate ) signals in terms of Mean Fluorescence Intensity ( MFI ) by Fluorescence Activated Cell Sorting ( FACS ) .", "In order to express the mutant of interest prior to sorting/analysis , yeast cells were induced at ( A ) 30°C for more stable parent inactive mutants and their suppressor pairs or ( B ) at 20°C for less stable parent inactive mutants and corresponding suppressor pairs .", "Suppressor mutations result in enhanced ligand binding and surface expression .", "In each ( Parent inactive mutant , suppressor ) pair , parent inactive mutants and corresponding proximal suppressors are denoted with the same color .", "Distal suppressors R10G and E11R are colored in blue in ( A ) and ( B ) .", "MFI values averaged over two independent experiments are shown .", "Suppressor mutations result in increased ( C ) ligand affinity and ( D ) thermal stability .", "( C ) Kd for Gyrase measured using yeast surface display titrations .", "Refer to Figure 4—figure supplement 1 for yeast surface display titrations of parent inactive mutants and ( Parent inactive mutant , suppressor ) pairs .", "( D ) Thermal melting temperatures ( Tm ) for parent inactive mutants in the absence and presence of second-site suppressor mutations measured using a thermal shift assay ( see Materials and methods ) with purified proteins .", "Refer to Figure 4—figure supplement 2 for the thermal unfolding profiles of parent inactive mutants and ( Parent inactive mutant , suppressor ) pairs .", "Error bars represent standard deviation from at least two independent experiments .", "The results have been summarized in Supplementary file 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01110 . 7554/eLife . 09532 . 012Figure 4—figure supplement 1 . Yeast surface display titrations of parent inactive mutants and ( Parent inactive mutant , suppressor ) pairs to determine Kd ( dissociation constant between CcdB displayed on the yeast surface and purified Gyrase ) .", "Yeast surface display titrations of ( A ) Wild Type ( WT ) , ( B ) R10G , ( C ) V5F , ( D ) V5F/L36M/A81G , ( E ) V18W , ( F ) V18W/M63T , ( G ) V18W/I90V , ( H ) V20F , ( I ) V20F/E11R , ( J ) L36A , ( K ) L36A/M63L , ( L ) L36A/R10G , ( M ) L83S , ( N ) L83S/V54L are shown .", "Kd values are mentioned in each sub-plot . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01210 . 7554/eLife . 09532 . 013Figure 4—figure supplement 2 . Thermal stabilities of purified CcdB Wild Type ( WT ) , R10G , parent inactive mutants and ( Parent inactive mutant , suppressor ) pairs measured by thermal shift assay ( TSA ) .", "Thermal unfolding profiles of 4 µM of purified WT and mutant proteins have been shown in absence ( filled circles showing the melt data and smooth line of the same color showing the fitted melt data ) and in presence ( open circles showing the melt data and dashed line of the same color showing the fitted melt data ) of the CcdA peptide ligand ( residues 46–72 ) ( 20 µM ) ( described in Materials and methods ) .", "The thermal unfolding profiles for the same mutant in absence and presence of CcdA peptide ( residues 46–72 ) are shown in the same color .", "Thermal unfolding curves of ( A ) WT ( in black ) , V18W ( in cyan ) and V18W/M63T ( in orange ) , ( B ) WT ( in black ) , R10G ( in red ) , L36A ( in cyan ) , L36A/M63L ( in green ) and L36A/R10G ( in orange ) , ( C ) WT ( in black ) , L83S ( in cyan ) and L83S/V54L ( in orange ) are shown .", "Values of thermal melting temperatures ( Tm in °C ) for the mutants are shown in Supplementary file 2 and Figure 3 .", "The ( V18W , M63T ) pair shows enhanced stability relative to the parent inactive mutant V18W only in the presence of CcdA .", "However the remaining pairs show enhanced stability relative to the parent inactive mutant both in the presence and absence of CcdA . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 013 R10G is a distal suppressor .", "The ability of the R10G mutation to suppress defects at other positions was examined by constructing the corresponding double mutants .", "Increased surface expression of R10G paired with each of V5F , V18W , L36A and L83S was seen relative to the individual parent inactive mutants ( Figure 4A , B ) .", "This demonstrates that R10G likely acts as a global suppressor .", "E11 suppresses activity of two parent inactive mutants , L36A and V20F ( L36A/E11P , V20F/E11R , V20F/E11K ) and is anticipated to play a role similar to R10G .", "The presence of the CcdB ligand , CcdA during thermal denaturation of CcdB , shifts the unfolding equilibrium towards the folded fraction of CcdB , resulting in an increased Tm than when monitored in its absence ( Supplementary file 2 , Figure 4—figure supplement 2 ) .", "This increase in Tm was observed for all mutants except R10G .", "L36A/R10G showed an increase of only 9oC in the presence of CcdA while other mutants showed an increase of >20oC .", "These observations indicated a decrease in affinity of R10G CcdB for CcdA .", "The distal suppressors R10G and E11R are present on an exposed loop of CcdB and contact CcdA in the crystal structure of the CcdB-CcdA complex , PDB id 3G7Z ( De Jonge et al . , 2009 ) .", "R10 forms a hydrogen bond ( 2 . 9 Å ) with N69 of CcdA .", "Hence , mutations at R10 and E11 are likely to destabilize binding of CcdB to CcdA , consistent with our results .", "The suppressor mutant , R10G has an increased stability relative to WT ( R10G ( Tm ( R10G ) =74 . 8 ± 0 . 2oC , Tm ( WT ) =66 . 8 ± 1 . 0oC , Supplementary file 2 , Figure 4D ) .", "However , the compromised ability to bind to its antitoxin CcdA results in increased toxicity in native contexts where CcdB and CcdA are both present .", "Thus , to maintain homeostasis in the system , evolutionary pressure defines a trade-off between function and stability of the protein ( Schreiber et al . , 1994 ) , settling on an optimally stable wild type protein rather than a maximally stable one .", "This explains why the R to G mutation is not found in naturally occurring CcdB homologs .", "The screen employed in this work identifies stabilizing variants which are functionally competent to bind to only one of the binding partners , DNA Gyrase , explaining the identification of the R10G like mutation .", "To understand the molecular mechanism ( s ) by which R10G rescues L36A present at the core , we examined if any long range functional interaction was predicted between the sites by the program SCA ( Halabi et al . , 2009 ) .", "No interaction was seen although this analysis was limited by low sequence diversity of CcdB .", "Experimentally , we observed that R10G stabilized the WT protein and other parent inactive mutants while the E11R substitution stabilized the parent inactive mutant V20F .", "The large conformational flexibility of glycine ( in case of R10G ) might stabilize the loop harboring residue 10 by accessing conformations not accessible to other residues .", "Further experiments need to be done to understand the mechanism of stabilization of the parent inactive mutant V20F by E11R and to determine if E11R , like R10G also functions as a global suppressor .", "A decoy set of 10 , 659 models ( Adkar et al . , 2012 ) ( doi:10 . 5061/dryad . 3g092 ) of CcdB was used to probe the utility of the experimentally obtained contact information in model discrimination .", "The decoy set contained models ranging from 1 . 9–20 . 4 Å ( backbone RMSD relative to the crystal structure , PDB id 3VUB [Loris et al . , 1999] ) .", "The models were scored based on ContactScore , which was defined as the number of times the experimentally identified residue contact pairs ( 6 pairs ) are within a cutoff distance of 7 Å of each other in a given model ( Figure 5A ) ( see Materials and methods ) .", "ContactScore is an integral value ranging from 0 to 6 , as there are six ( Parent inactive mutant , proximal suppressor ) pairs .", "Proximal suppressor mutants are likely to have their side chains facing towards the corresponding parent inactive mutant ( Figure 3B–F ) , and hence , the side chain centroids of the pair are likely to be closer than their corresponding Cα atoms .", "Results for other choices of distances are shown in Figure 5—figure supplement 1 . The distribution of recovery of models ( defined as the percentage of models selected by the metric within a specified RMSD range , in a pool of models ) with respect to their backbone RMSD relative to the crystal structure ( Figure 5—figure supplement 2 ) shows the sensitivity of ContactScore and its relevance as a metric for model discrimination .", "Models satisfying all the experimental constraints that is ContactScore=6 are distributed in the RMSD bin <4 Å .", "The distribution progressively shifts towards a higher RMSD range with decrease in the number of constraints being satisfied ( ContactScore<6 ) .", "This emphasizes the sensitivity and selectivity of the metric .", "The correlation coefficient of a plot of RankScore as a function of residue depth in a model , rdepthRankScore ( or ) rdepthscore has been previously used as a model discriminator ( Adkar et al . , 2012 ) .", "rdepthscore=0 . 6 for the native structure of CcdB .", "Thus , models with rdepthscore≥ 0 . 6 were selected as 'correctly folded models' when rdepthscore was used as the metric .", "A comparison of the two metrics shows that ContactScore performs significantly better than rdepthscore , recovering 100% , 98% and 80% structures in backbone RMSD ranges 1 . 5–2 Å , 2–2 . 5 Å and 2 . 5–3 Å , respectively , while the latter recovered 0% , 7% and 12% in the above ranges ( Figure 5C ) .", "ContactScore and rdepthscore identified 585 and 67 models respectively with RMSD range <4 Å from the decoy set ( Figure 5A , B ) .", "Thus , ContactScore is able to recover 66% of native-like models ( RMSD <4 Å ) from the dataset as compared to only 8% by rdepthscore . 10 . 7554/eLife . 09532 . 014Figure 5 . Experimentally determined 'ContactScore' and rdepthRankScore ( Adkar et al . , 2012 ) as model discriminators . Distribution of ( A ) ContactScore ( CSc ) and ( B ) rdepthRankScore as a function of backbone RMSD with respect to the crystal structure for 10 , 659 models of CcdB .", "CSc values are integers ranging from 0 to 6 .", "Thus , models with different RMSD values ( abscissa ) can have the same CSc ( ordinate ) .", "The Csc values for such points are randomly incremented by upto 0 . 7 units from their actual integral CSc value for clearer visualization .", "( C ) Black and red bars represent recovery for CSc and rdepthRankScore respectively .", "CSc performs significantly better than rdepthRankScore , recovering a much higher fraction of low RMSD models .", "The total RMSD range was divided into 0 . 5Å bins .", "Cutoff values of CSc and rdepthRankScore used were 6 and 0 . 6 respectively .", "Refer to Figure 5—figure supplement 1 for recovery of CcdB models using Csc=6 shown for various choices of cut-off distances used to define contact .", "Refer to Figure 5—figure supplement 2 for recovery of CcdB models as a function of different Csc values .", "These two plots demonstrate that a distance of <7 Å and Csc=6 are optimal for model recovery in this system . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01410 . 7554/eLife . 09532 . 015Figure 5—figure supplement 1 . Recovery of CcdB models using a ContactScore ( CSc ) value of Csc=6 shown for various choices of cut-off distances used to define contact . RMSD bin size=0 . 5 Å .", "Upper limit of each bin is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01510 . 7554/eLife . 09532 . 016Figure 5—figure supplement 2 . Recovery of CcdB models as a function of different ContactScore ( Csc ) values using a fixed cut-off contact distance value of <7 Å .", "High Csc values recover only low RMSD models .", "With decreasing CSc values , the fraction of low RMSD models decreases .", "RMSD bin size is 0 . 5 Å .", "Upper limit of each RMSD bin is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 016 We compared the decoy discrimination efficiency of rdepthscore and ContactScore with another method which uses a simple scoring function based on residue accessiblity in globular proteins ( Bahadur and Chakrabarti , 2009 ) .", "The function ( Rs ) evaluates the deviation from the average packing properties of all residues in a polypeptide chain corresponding to a model of its three-dimensional structure ( Bahadur and Chakrabarti , 2009 ) .", "The parameter Rs was calculated ( see Materials and methods ) for the CcdB decoy set .", "Since Rs estimates deviation from the average Accessible Surface Area ( ASA ) , the native structure should ideally possess the lowest value of Rs .", "However , when the CcdB decoy set was sorted according to the Rs values , the native structure was ranked 934th and the correlation between RMSD and Rs was seen to be only 0 . 3 .", "These data demonstrate that both rdepthscore and ContactScore parameters derived from mutational data perform better than simple solvent accessibility based correlations such as the one observed above .", "The structures of the integral membrane protein , DgkA solved by X-ray crystallography ( Li et al . , 2013 ) and NMR ( Van Horn et al . , 2009 ) are different from each other in important respects .", "The NMR structure appears to be in a domain swapped conformation relative to the crystal structure . Several pairs of differential contacts serve to discriminate the two structures , including contacts made by residues V62 , M66 , I67 , V68 and W112 ( Table 2 , Supplementary file 3 , Figure 6A , B , Figure 6—figure supplement 1 , see Materials and methods ) .", "Consequently , residues in proximity to each of these residues in the X-ray structure ( PDB id 3ZE5 [Li et al . , 2013] ) are distant in the NMR structure ( PDB id 2KDC [Van Horn et al . , 2009] ) .", "Thus by constructing parent inactive mutants at the above positions and isolating corresponding suppressors , it should be possible to determine which of the two structures represents the functional conformation in-vivo .", "It has previously been shown ( Raetz and Newman , 1978; 1979 ) that cells deleted for dgkA do not grow under conditions of low osmolarity , providing a facile screen for both parent inactive mutants and their corresponding suppressors . 10 . 7554/eLife . 09532 . 017Figure 6 . Screening of suppressors for the parent inactive mutant I67V DgkA . I67 ( ball and stick in black ) and inter-helical residues in contact with it in either ( A ) crystal ( PDB id 3ZE5 [Li et al . , 2013] ) or ( B ) NMR ( PDB id 2KDC [Van Horn et al . , 2009] ) structures of the homotrimeric DgkA are shown .", "Each monomer of the trimer is shown in a different color .", "The residues in proximity to I67 in a specific structure are shown in green , while the ones in contact in the alternate structure are highlighted in red .", "Differential contact pairs for other parent inactive mutants of DgkA have been shown in Figure 6—figure supplement 1 . The figure has been prepared using the UCSF Chimera package ( developed by Resource for Biocomputing , Visualization , and Informatics at the University of California , San Francisco [Pettersen et al . , 2004] ) .", "Second-site suppressor libraries of the parent inactive mutant were constructed by randomizing each of the residue partners present in either of the two structures of the protein .", "Active mutants were screened on selective media at decreasing NaCl concentrations in an E . coli strain knocked out for dgkA .", "( C ) and ( D ) show the phenotype of the putative suppressors isolated from the libraries at ( C ) 0 . 15% NaCl and 0 . 01% arabinose and ( D ) 0% NaCl and 0 . 01% arabinose .", "Phenotypes of suppressors corresponding to other parent inactive mutants have been shown in Figure 6—figure supplement 2 . ( E ) A representative plate showing the location of the variants and the controls .", "' ( X ) ' and ' ( N ) ' indicate the structure from which the residue partner has been chosen that is either X-ray or NMR structure , respectively .", "Parent inactive mutant I67V , WT DgkA and the empty vector pBAD-33 act as reference , positive and negative controls , respectively .", "The true suppressors are anticipated to grow on the plate with low salt concentration while the parent inactive mutant and ( Parent inactive mutant , non-suppressor ) pairs fail to grow .", "Suppressors ( I67V , I103L ) and ( I67V , A104T ) , which are in spatial proximity in the crystal structure restore the growth defect of the parent inactive mutant I67V , whereas the ( I67V , E34L ) pair which is in proximity only in the NMR structure , fails to restore the growth defect . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01710 . 7554/eLife . 09532 . 018Figure 6—figure supplement 1 . Differential contact residue pairs mapped onto the structures of DgkA . Parent inactive mutant positions V62 ( A , B ) , M66 ( C , D ) , V68 ( E , F ) and W112 ( G , H ) ( shown in ball and stick in black ) and their corresponding inter-helical contacting partners in the crystal and NMR structures have been mapped onto the ( A , C , E , G ) X-ray ( PDB id 3ZE5 [Li et al . , 2013] ) and ( B , D , F , H ) NMR ( PDB id 2KDC [Van Horn et al . , 2009] ) structures of the homotrimeric DgkA protein ( shown in ribbon with the three monomers in three different colors ) .", "The residues in proximity of the parent inactive mutants in a specific structure are shown in green , while the ones in contact in the alternate structure are highlighted in red .", "The figure has been prepared using the UCSF Chimera package ( developed by Resource for Biocomputing , Visualization , and Informatics at the University of California , San Francisco [Pettersen et al . , 2004] ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01810 . 7554/eLife . 09532 . 019Figure 6—figure supplement 2 . Screening for suppressors of parent inactive mutants of DgkA . Second-site suppressor libraries of the parent inactive mutants V62Q , M66L , M66S and V68G were constructed by individually randomizing each of the partners in both the NMR and X-ray structures of the protein .", "Suppressors were identified by screening on selective media at decreasing NaCl concentrations in an E . coli strain deleted for dgkA .", "( A , B ) , ( D , E ) , ( G , H ) and ( J , K ) show the phenotype of the suppressors isolated from the libraries of V62Q , M66L , M66S and V68G , respectively at ( A , D , G , J ) 0 . 15% NaCl , 0 . 01% arabinose , ( B , K ) 0%NaCl , 0 . 01% arabinose and ( E , H ) 0 . 03% NaCl , 0 . 01% arabinose .", "( C , F , I , L )", "Representative plate photograph showing the location of the variants and the controls for the screening of suppressors of the parent inactive mutants V62Q ( A , B ) , M66L ( D , E ) , M66S ( G , H ) and V68G ( J , K ) , respectively .", "' ( X ) ' and ' ( N ) ' indicate the structure from which the residue partner has been chosen that is either X-ray or NMR structure , respectively .", "Parent inactive mutant , Wild-Type ( WT ) DgkA and the empty vector pBAD-33 act as reference , positive and negative controls , respectively .", "The true suppressors are anticipated to appear before the parent inactive mutant on the plate when grown in low salt conditions .", "Suppressors ( V62Q , A41G ) , ( M66L , V38A ) , ( M66S , G35A ) , ( V68G , A100V ) , which are in spatial proximity in the crystal structure restore the growth defect of the parent inactive mutants .", "A similar analysis for the parent inactive mutant I67V has been shown in Figure 6C–E .", "Overall , suppressors were only found at positions proximal to the parent inactive mutant in the X-ray structure of DgkA . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 01910 . 7554/eLife . 09532 . 020Table 2 . Experimentally determined ( Parent inactive mutant , Suppressor ) pairs for DgkA are spatially close only in the corresponding crystal structure . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 020Parent inactive mutant positionContact partnersa in X-ray structurebContact partnersa in NMR structurecExperimentally identified ( Parent inactive mutant , Suppressor ) pairsV62A41 , 108 , W112L102 , I103 ( V62Q , A41G ) M66F31 , G35 , V38A99 ( M66L , V38A ) ( M66S , G35A ) I67A100 , I103 , A104F31 , E34 ( I67V , I103L ) ( I67V , A104T ) V68A100 , V101 , A104F31 , G35 ( V68G , A100V ) W112A41 , I44 , L58 , S61-daInter-helical contact pairs ( see Materials and methods for details ) bX-ray structure of DgkA ( PDB id 3ZE5 [Li et al . , 2013] ) cNMR structure of DgkA ( PDB id 2KDC [Van Horn et al . , 2009] ) dNo suppressors for this parent inactive mutant could be isolated , probably because the large volume change in the parent inactive mutant ( W112V ) is difficult to compensate by a single suppressor mutation .", "-No inter-helical contacts Each of the above five residues was individually randomized and the resulting small libraries were screened for inactive mutants .", "Charged and aromatic substitutions were excluded from the finally selected parent inactive mutants as these are likely to be highly destabilizing , making it difficult to isolate suppressors for such mutations .", "Indeed , in the case of CcdB it was more challenging to find suppressors for the aromatic parent inactive mutants ( V18W , V20F ) , relative to other parent inactive mutants ( such as L36A and L83S ) ( Supplementary file 1 ) .", "V62Q , M66S , M66L , I67V , V68G and W112V were identified as parent inactive mutants from screening of single-site saturation mutagenesis libraries constructed at these positions .", "For these mutants , colonies appeared on plates only at high osmolarity ( NaCl concentrations of 0 . 15% , 0 . 15% , 0 . 15% , 0 . 03% , 0 . 15% and 0 . 15% respectively ) after 12 hr of incubation at 37oC , as opposed to cells expressing WT DgkA which grew even at 0% NaCl .", "Second-site suppressor mutagenesis libraries in which each residue in contact with the parent inactive mutants in both NMR and crystal structures was individually randomized , ( Table 2 ) were screened for growth under low salt conditions .", "At all selected parent inactive mutants , suppressors were found only at those positions in contact with the parent inactive mutant in the X-ray structure ( i . e . V62Q/A41G , M66L/V38A , M66S/G35A , I67V/I103L , I67V/A104T and V68G/A100V ) , ( Figure 6 , Figure 6—figure supplement 2 , Table 2 , Video 2 ) .", "The only exception was for the parent inactive mutant W112V , where no suppressors were experimentally identified , possibly due to the large change in volume for the parent inactive mutant relative to the WT residue .", "The data reported here are consistent results obtained from more than five independent experiments for each mutant .", "The results strongly suggest that the crystallized conformation is the native , functional conformation in-vivo . 10 . 7554/eLife . 09532 . 021Video 2 . Experimentally obtained ( Parent inactive mutant , suppressor ) pairs mapped onto the two structures of DgkA ( NMR PDB id 2KDC and X-ray PDB id 3ZE5 ) exhibit spatial proximity only in the corresponding crystal structure .", "Parent inactive mutant is abbreviated as PIM in the video . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 021 In the recent past there have been several computational efforts to identify residues in contact , involving analysis of correlated substitution patterns in a multiple sequence alignment of a protein .", "DCA ( Morcos et al . , 2011 ) , PSICOV ( Jones et al . , 2012; Nugent and Jones 2012 ) , GREMLIN ( Kamisetty et al . , 2013 ) , SCA ( Halabi et al . , 2009 ) and EVfold ( Marks et al . , 2011 ) analyze co-variation matrix data from a multiple sequence alignment to deduce residues in contact .", "The methods rank residue pairs based on a co-variation or correlation score specific to each method .", "The top ranked pairs which typically lie in the top L/2 pairs , where L is the length of the protein sequence ( Jones et al . , 2012 ) are predicted to be in contact .", "The methods perform well when the size of the multiple sequence alignment is large , that is >5L ( Kamisetty et al . , 2013 ) .", "DgkA ( 121 residues per protomer ) exhibits large sequence diversity ( 4175 sequences in the multiple sequence alignment ) .", "Putative contact predictions by the above methods were analyzed by calculating sidechain-sidechain centroid distances between the predicted pairs using both X-ray and NMR structures of DgkA .", "Some high scoring , co-varying pairs predicted by DCA , GREMLIN , PSICOV and EVfold were found to be true contacts ( centroid-centroid distance <7 Å , Figure 7 ) when mapped onto the crystal structure .", "There were a few high scoring pairs which were either far apart in the X-ray structure ( predictions by PSICOV ) or were in proximity when analyzed with the NMR structure ( predictions by GREMLIN , PSICOV and EVfold ) .", "However , overall sequence co-variation data are more consistent with the X-ray structure , in agreement with conclusions from suppressor mutagenesis .", "Of the six contacts identified from our suppressor analyses ( Table 2 ) , three ( 62–41 , 67–104 , 68–100 ) were predicted in the top L/2 co-varying pairs by GREMLIN , PSICOV and EVfold ( Figure 7 ) , only 67–104 was predicted by DCA and none by SCA .", "This suggests that natural sequence co-variation and suppressor mutagenesis can provide complementary information . 10 . 7554/eLife . 09532 . 022Figure 7 . Computational analyses of co-varying residues for DgkA and comparison with experimentally determined contact residue pairs . Co-variation analyses using ( A ) DCA , ( B ) GREMLIN , ( C ) PSICOV , ( D ) SCA and ( E ) EVfold are shown .", "Residue pair side-chain centroid distances calculated from DgkA crystal ( PDB id 3ZE5 ) and the NMR 1st pose structure ( PDB id 2KDC ) are shown in left ( filled circles ) and right ( open circles ) panels respectively .", "'au' denotes arbitrary units .", "Blue lines parallel to the X-axis indicate the co-variation score of the L/2th residue pair ( when arranged in descending order of the score ) , where L is the length of the protein ( L=121 for DgkA ) .", "Blue lines parallel to the Y-axis indicate a sidechain–sidechain centroid distance of 7 Å between the predicted co-varying residue pairs in the corresponding structure .", "Experimentally determined spatially proximal ( Parent inactive mutant , suppressor ) contact pairs are shown in cyan .", "Although several computationally predicted contact pairs in the top L/2 predictions ( top panel ) are proximal to each other in the crystal structure , there are several predicted pairs which are far apart .", "Computational analyses of co-varying residues for DgkA and comparison with experimentally determined contact residue pairs using Cα-Cα distances instead of sidechain-sidechain centroid distances are shown in Figure 7—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 02210 . 7554/eLife . 09532 . 023Figure 7—figure supplement 1 . Computational analyses of co-varying residues for DgkA and comparison with experimentally determined contact residue pairs using Cα-Cα distances instead of sidechain-sidechain centroid distances . Co-variation analyses using ( A ) DCA , ( B ) GREMLIN , ( C ) PSICOV , ( D ) SCA and ( E ) EVfold are shown .", "Residue pair Cα distances calculated from DgkA crystal ( PDB id 3ZE5 ) and NMR 1st pose structure ( PDB id 2KDC ) are shown in left ( filled circles ) and right ( open circles ) panels respectively .", "'au' denotes arbitrary units .", "Blue lines parallel to the X-axis indicate the co-variation score of the L/2th residue pair ( when arranged in descending order of the score ) , where L is the length of the protein ( L=121 for DgkA ) .", "Blue lines parallel to the Y-axis indicate a distance of 7 Å between the predicted co-varying residue pairs in the corresponding structure .", "Experimentally determined spatially proximal ( Parent inactive mutant , suppressor ) contact pairs are shown in cyan . DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 023 The co-variation prediction methods use a multiple sequence alignment as input .", "The predictions therefore are not specific to the identities of the side-chains of the residues present in the sequence of interest at the predicted contact positions .", "Therefore , we also analyzed the predictions by calculating the Cα-Cα distances between the predicted pairs using both X-ray and NMR structures ( Figure 7—figure supplement 1 ) .", "No side chain information is involved in these calculations .", "Similar results were obtained as when using the sidechain-sidechain centroid distances .", "Co-variation prediction becomes increasingly challenging for proteins with very few homologs .", "CcdB ( 101 residues per protomer ) has only 350 sequences ( <5 L , where L is the length of the protein ) in the multiple sequence alignment .", "Therefore , co-variation predictions for CcdB were not included .", "There is considerable interest in accurate prediction of mutational effects on the free energy of folding ( Guerois et al . , 2002; Kellogg et al . , 2011; Shen and Sali , 2006 ) .", "We therefore examined whether ΔΔG calculations could be used to rationalize the identity of the experimentally observed local suppressors .", "To this end the difference in stability between the ( Parent inactive mutant , suppressor ) pair and the parent inactive mutant for CcdB mutants was calculated .", "△△Gfolding ( △GfoldingDouble mutant-△GfoldingParent inactive mutant ) was calculated using Rosetta v3 . 3 ( Kellogg et al . , 2011 ) .", "Putative proximal suppressors were considered to arise at residues within 7 Å ( sidechain–sidechain centroid distance ) of the parent inactive mutant .", "Many stable substituents were predicted ( △△Gfolding<0 , Figure 8 ) .", "However , amongst the six experimentally identified stable compensatory pairs , only L36A/M63L ( −3 . 7 kcal/mol ) was predicted to be stable .", "The remaining five contact pairs were predicted to be either marginally stable or unstable .", "Several other mutations besides the experimentally determined ones were predicted to be stabilizing for example V5F/L16G , V18W/I90A , V20F/I90A , L36A/V54I and L83S/V18I .", "These might be present in the earlier rounds of sorting but are lost in later rounds due to stringent sort conditions .", "A marked bias for aromatic substitutions was observed in the predictions ( Figure 8 , substitutions underlined in magenta ) though such aromatic substitutions were not observed experimentally .", "Aromatic substitutions are rigid and were found to over pack the cavity created by the parent inactive mutants in the models generated using Rosetta .", "Further , several of the mutations that were computationally predicted to be highly stabilizing are unlikely to be so as they are not complementary in size to the original parent inactive mutant , for example L36A/W61F , V5F/L16Y , V18W/I90F and V20F/I90F .", "If aromatic substitutions are excluded , Rosetta predictions using ΔΔG values are in reasonable qualitative agreement with experiment . 10 . 7554/eLife . 09532 . 024Figure 8 . Heatmaps showing calculated values of ∆∆Gfolding using Rosetta for double mutants of CcdB . All 19 mutations ( X-axes ) were made at positions ( Y-axes ) whose side chain centroids are within 7 Å of the side chain centroid of the corresponding parent inactive mutant .", "The parent inactive mutants are indicated in bold on the top of each heatmap .", "For example , the bottom left corner of the first panel represents the ∆∆G value for the ( V5F , L83G ) ( Parent inactive mutant , suppressor ) pair .", "Position 199 ( 98+101 ) in the V20F heatmap refers to residue 98 of the other protomer .", "Wild type residues are shown in dashed boxes in single letter code .", "Experimentally obtained suppressors are indicated by thick black boxes .", "Double mutants showing negative values of ∆∆Gfolding ( blue shades ) are ones where the putative suppressor mutation is predicted to have a stabilizing effect on the inactive mutant .", "Aromatic substitutions are underlined in magenta .", "Residues on X-axis are grouped into the following classes , separated by thick vertical lines: ( G , P ) , aliphatic ( A , C , V , L , I , M ) , aromatic ( H , F , Y , W ) , polar ( S , T , N , Q ) and charged ( D , E , K , R ) .", "Similar analysis was performed using FoldX ( Figure 8—figure supplement 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 02410 . 7554/eLife . 09532 . 025Figure 8—figure supplement 1 . Heatmaps showing calculated values of ∆∆Gfolding using FoldX for double mutants of CcdB . All 19 mutations ( X-axes ) were made at positions ( Y-axes ) whose side chain centroids are within 7 Å of the side chain centroid of the corresponding parent inactive mutant .", "The parent inactive mutants are indicated in bold on the top of each heatmap .", "Position 199 ( 98+101 ) in the V20F heatmap refers to residue 98 of the other protomer .", "Wild type residues are shown in dashed boxes in single letter code .", "Experimentally obtained contact pairs are indicated by thick black boxes .", "Double mutants showing negative values of ∆∆Gfolding ( blue shades ) are ones where the putative suppressor mutation is predicted to have a stabilizing effect on the inactive mutant .", "Aromatic substitutions are underlined in magenta .", "Residues on the X-axis are grouped into the following classes , separated by thick vertical lines: ( G , P ) , aliphatic ( A , C , V , L , I , M ) , aromatic ( H , F , Y , W ) , polar ( S , T , N , Q ) and charged ( D , E , K , R ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09532 . 025 A similar analysis was done using FoldX ( Guerois et al . , 2002 ) ( Figure 8—figure supplement 1 ) .", "However , these predictions were in poorer agreement with the experimental results , compared to those of Rosetta .", "Thus , in addition to their use in protein structure prediction , results from such suppressor analyses can also be used to benchmark and improve computational approaches to predict mutational effects on protein stability ." ], [ "Interactions at the protein core are important in determining its structure and stability .", "The saturation-suppressor mutagenesis methodology described here ( Figure 1 , Figure 1—figure supplement 3 ) enabled identification of 8 residues at the hydrophobic core and their pairwise interactions ( Figure 3A , F ) placing important constraints on packing of the model protein CcdB .", "Encouragingly , proximal suppressors could be obtained for four of the five parent inactive mutants in the case of CcdB as well as DgkA .", "There may be mutations in the core which allow the protein to fold but are functionally defective ( Figure 1—figure supplement 2 ) ( Roscoe et al . , 2013 ) .", "We eliminated such mutations as the CcdB screen required variants to both fold into a stable conformation and be functionally active to bind to Gyrase .", "The experimentally identified residue pairs for CcdB are in physical contact and have suppressor positions common to each other ( Table 1 , Figure 3B–F , Video 1 ) .", "This interlinked network of residues restricts the conformational space during folding .", "The ContactScore metric defined above selects models satisfying the identified spatial constraints .", "A histogram of recovery of models with respect to backbone RMSD of the models selected by this metric shows a maximum of 100% for models with backbone RMSD <2 Å , gradually decreasing to 66% for models <4 Å and subsequently plateauing to 0% for models >5 Å , reflecting the sensitivity and accuracy of the parameter .", "A high recovery is important for protein model discrimination , as typically there will be very few low RMSD models in the candidate set of predictions .", "The ContactScore metric also performs better than a simpler approach based on deviation of residue accessibilities in a model from their average values in a large dataset of proteins ( Bahadur and Chakrabarti , 2009 ) though an exhaustive comparison with other metrics for model discrimination has not been carried out .", "The efficiency of model discrimination using the ContactScore metric will likely be a function of quality of the decoy dataset as well as the accuracy of the contacts inferred from suppressor mutagenesis .", "In a dataset where very few low RMSD models are present or where not all contacts identified are true contacts , the model recovery will be lower than for the CcdB decoy dataset described here .", "Further , the small number of ( Parent inactive mutant , suppressor ) pairs identified here will not be sufficient for larger proteins , thereby affecting the model discrimination efficiency .", "However , most of these limitations can be overcome by identifying a higher number of ( Parent inactive mutant , suppressor ) pairs for the protein of interest and by combining residue contacts inferred from saturation mutagenesis with other experimental or computational constraints .", "The saturation suppressor mutagenesis approach was extended to the important case of membrane proteins .", "Many membrane proteins adopt multiple conformations ( Tokuriki and Tawfik , 2009 ) and membrane mimetics used to solubilize and stabilize membrane proteins can affect their conformations ( Cross et al . , 2013 ) .", "There are two reported structures of the integral membrane protein DgkA , one solved by X-ray crystallography ( Li et al . , 2013 ) and one by solution NMR ( Van Horn et al . , 2009 ) which differ significantly from each other .", "Using our suppressor methodology , we unambiguously identified six residue-residue contacts which were all present in the crystal structure but were spatially distant in the NMR structure .", "This suggests that the conformation of the protein in the lipidic ( monoacylglycerol ) cubic phase conditions ( used in X-ray crystallography [Li et al . , 2013] ) is the functional conformer in-vivo and is not an artifact resulting from the presence of thermostabilizing mutations and minor distortions that might result from crystal contacts .", "Our results are also consistent with a recent reanalysis of oriented sample solid state NMR data for the protein in liquid crystalline bilayers ( Murray et al . , 2014 ) which showed better overall agreement with the crystal than with the solution NMR structure .", "Broad application of suppressor methodology to systems where no structural information is available requires accurate discrimination of buried from exposed active-site residues and of distal from proximal suppressors .", "As discussed above , buried and active-site residues can be distinguished based on their mutational sensitivity patterns as well as from data on mutant protein levels , sensitivity to chaperone overexpression patterns ( Tokuriki and Tawfik , 2009 ) and residue conservation patterns ( Melamed et al . , 2015 ) .", "Distal suppressors are likely to be on the surface ( Bank et al . , 2015 ) .", "If the correlation of mutational sensitivity/RankScore with depth seen for CcdB holds for other globular proteins , this should allow distinction of local and global suppressors in such systems , as long as the majority of global suppressors lie on the surface .", "However , unambiguous distinction between global and local suppressors will likely not always be possible with a limited number of ( Parent inactive mutation , suppressor ) pairs .", "Also , in some cases , a single suppressor mutation may not be sufficient to restore the activity of a parent inactive mutant .", "In such cases , the network of correlating residues will be more complex and additional experimental data will be required to identify a set of mutations which will suppress a parent inactive mutant .", "Unlike globular proteins , little is known about sensitivity to mutation in membrane proteins or natively unfolded proteins .", "Until such data becomes available it will be challenging to apply this methodology to these systems .", "In the case of DgkA , we had the much simpler objective of distinguishing between two possible structures .", "While distinguishing between global and local suppressors maybe more challenging in membrane proteins , given sufficient double mutant data , it should be straightforward because each global suppressor should suppress a much larger number of parent inactive mutants than all local suppressors .", "The average relative frequency of obtaining proximal versus distal suppressors is currently unknown .", "If a single-site saturation mutagenesis library is enriched for inactive mutants ( Parent inactive mutants ) , subjected to random mutagenesis and screened for suppressors , the resulting population will be enriched for global suppressors ( Bershtein et al . , 2006; 2008 ) .", "This is because a global suppressor will suppress multiple parent inactive mutants .", "However , for a specific parent inactive mutant , it is not obvious that global suppressors will dominate .", "A recent study examined a library of the 75 amino acid RRM domain of the yeast poly-A binding protein ( Melamed et al . , 2013 ) .", "Functional scores for 1246 single and 39 , 912 double mutants were obtained .", "Epistatic interactions were enriched for residue pairs with short sequence spacing ( <5 ) and short distance ( 10–15Å ) .", "Another recent study reported exhaustive screening of single and double mutants of GB1 ( Olson et al . , 2014 ) .", "The majority of pairs displaying positive epistasis had Cβ-Cβ distances <8 Å .", "Both of the above studies indicate that local suppressors may occur at a higher frequency than global ones with respect to individual parent inactive mutants but more data is required to confirm this .", "Experimental approaches discussed previously to identify second-site suppressors used random mutagenesis and/or directed evolution to generate suppressor libraries .", "Although these libraries have high diversity and mutations at multiple residue positions , they typically do not have more than a single base substitution at any codon .", "It should be noted that single base changes can sample only 39% of all possible amino acid substitutions .", "Hence , they do not exhaustively sample all possible second-site suppressors for a given inactivating mutation .", "This is important , since for a given parent inactive mutant there appear to be only a few local suppressors , and these could well be absent in a library generated by conventional random mutagenesis .", "Computational approaches to identify spatially proximal residues require a large number of homologous sequences to be present ( typically greater than five times the length of the protein ) ( Kamisetty et al . , 2013 ) .", "These approaches do not work well for proteins like CcdB due to the limited evolutionary diversity in the multiple sequence alignment .", "Another constraint is that the range of substitutions sampled at a given position during natural evolution is limited by functional and stability constraints , that can be relaxed in a laboratory setting .", "For the protein DgkA , for which there are several sequences , the results of the five computational approaches to identify co-varying residues were more consistent with the crystal structure of DgkA than with the NMR structure .", "Several of the contacts identified by our suppressor approach were not identified by the sequence co-variation .", "Since our experimental strategy identifies only a few ( Parent inactive mutant , suppressor ) , a direct comparison of these results with predictions by computational methods is not possible .", "However , our approach , provides complementary information to these existing methods , and can be usefully combined with them to guide protein structure prediction .", "The approach described here uses saturation-suppressor mutagenesis to identify spatially proximal residues .", "The library generation design adopted here for CcdB , constructs the second-site saturation library in the background of individual inactive mutants chosen from a single-site saturation mutagenesis library comprising of ~1430 mutants of CcdB ( Adkar et al . , 2012 ) .", "Second-site suppressors can be either proximal or distal to the mutation site .", "Virtually all suppressors identified in the study ( for CcdB ) increased the thermal stability relative to the original parent inactive mutant .", "Proximal suppressors are likely to ameliorate packing defects and hence restore stability .", "These have previously been reported to restore activity ( Machingo et al . , 2001 ) , increase thermal stability and restore packing ( Pakula and Sauer , 1989 ) .", "Residues distant from the site have previously been found to function by either increasing global thermodynamic stability ( Araya et al . , 2012; Bershtein et al . , 2008; Pakula and Sauer , 1989 ) , increasing activity relative to the wild type protein without any substantial increase in stability ( Hecht and Sauer , 1985 ) or improving foldability without much effect on the thermodynamic stability ( Sideraki et al . , 2001 ) .", "In the present study , local suppressors could be obtained for each of the five parent inactive mutants in CcdB and five of six parent inactive mutants in DgkA , regardless of the location and nature of the parent inactive mutant .", "The distal suppressor R10G in CcdB increased the Tm by 8oC , relative to WT CcdB ( Supplementary file 2 ) which rescues the destabilized mutant L36A .", "Global suppressors have been shown to often comprise of consensus/ancestral mutations ( Bershtein et al . , 2008 ) .", "We analyzed the consensus/ancestral mutations for CcdB .", "The consensus sequence was obtained from a multiple sequence alignment of 350 homologs using MATLAB and the ancestral sequence was obtained using the FastML server ( Ashkenazy et al . , 2012 ) .", "The likely global suppressors we obtained in the present study are R10G and E11 ( R/K/P ) .", "At R10 the ancestral and consensus amino acids are P and R respectively and at E11 they are A and N respectively .", "Hence , at least for these two positions , the ancestral/consensus amino acids were different from the experimentally obtained suppressors .", "Further experiments are required to ascertain whether the ancestral/consensus amino acids will also act as global suppressors .", "Advances in the field of protein structure prediction integrate various computational approaches with distance restraints derived from cross-linking experiments and mass spectrometry ( Young et al . , 2000 ) , sparse NOE data ( Bowers et al . , 2000; Li et al . , 2003; Thompson , et al . , 2012 ) , residual dipolar coupling data ( Haliloglu et al . , 2003; Qu et al . , 2004 ) , chemical shift data ( Shen et al . , 2008 ) from NMR experiments , co-varying residues identified from statistical analysis of genomic data ( Hopf et al . , 2012; Jones et al . , 2012; Ovchinnikov et al . , 2014; Ovchinnikov et al . , 2015; Sulkowska , et al . , 2012 ) to determine structure .", "The combination of site-directed mutagenesis or doped oligonucleotide based synthetic library generation strategies with deep sequencing has expanded our understanding of sequence , structure , function relationships ( Tripathi and Varadarajan , 2014 ) .", "High resolution mutational analyses using single-site saturation mutagenesis have facilitated understanding the influence of each residue on a protein’s structure , stability , activity , specificity and fitness ( Adkar et al . , 2012; Fowler et al . , 2010; Roscoe et al . , 2013; Tripathi and Varadarajan , 2014 ) .", "Expanding this landscape by integrating spatial constraints isolated from paired mutational phenotypes can greatly advance our efforts to construct highly accurate structural models especially where evolutionary information is sparse , because of the paucity of homologous sequences .", "Unbiased examination of all pairwise mutant combinations is currently not feasible because the large library size cannot easily be sequenced at sufficient depth using deep sequencing ( 100C2 × 400 or 2 × 106 for a 100 residue protein [Tripathi and Varadarajan , 2014] ) .", "However , with continuous improvements in deep sequencing and mutant generation technologies this is likely to change soon .", "The methodology outlined here can be similarly applied to any protein or protein complex where mutation can be coupled to a phenotypic readout .", "In the two systems studied here , we identified intramolecular suppressors of core packing defects , and active-site mutants were excluded from the set of parent inactive mutants .", "However , a similar approach can be used to obtain structural information on biomolecular protein:protein complexes .", "In this case , single-site saturation mutagenesis libraries at active-site residues of one partner would be screened against parent inactive mutants located , at active-site residue of the other partner to identify inter-molecular suppressors .", "These in turn would provide constraints to build structural models of the complex .", "In these proof of principle studies , mutant identities were determined after single ( DgkA ) or multiple ( CcdB ) rounds of screening , using Sanger sequencing .", "In the case of CcdB , residue burial was accurately inferred exclusively from mutational data .", "Proximal and distal suppressors could be accurately distinguished based on their mutational sensitivity when present as single mutants and all proximal suppressors were in close contact with the corresponding parent inactive mutant .", "For other systems it remains to be seen how well this approach will work and how many suppressors will be required for accurate model discrimination .", "However , as with earlier studies using single mutant libraries ( Adkar et al . , 2012; Fowler et al . , 2010; Tripathi and Varadarajan , 2014 ) , enrichment of mutant pairs at each stage during selection or screening can be monitored using deep sequencing .", "Using this approach it will be possible to compare phenotypes of large numbers of partial loss-of function single mutants and corresponding double mutants .", "This data will help distinguish local from global suppressors and provide multiple constraints to guide macromolecular structure prediction and determination .", "These efforts may prove beneficial in resolving the gap between protein sequence and structure and also in the isolation of mutants with improved stability and foldability , relative to WT ." ], [ "The function ( Rs ) ( Bahadur and Chakrabarti , 2009 ) evaluates the deviation from the average packing properties of all residues in a given structural model .", "Rs is calculated using the following formula , where ASAxi and <ASAx> are the accessible surface area values of residue X at position i and the average ASA of residue X in a large dataset , respectively ( Bahadur and Chakrabarti , 2009 ) .", "Rs=∑i = 1whole chain|ASAxi−<ASAx>|<ASAx> The Rs parameter was calculated for all the 10 , 659 models in the CcdB decoy set .", "The ASA was calculated using NACCESS ( v2 . 1 . 1 ) ( Hubbard , 1992 ) ." ] ]
[ "Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction .", "Using Escherichia coli CcdB as the test case , we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information .", "In this methodology , for each of five inactive CcdB mutants , exhaustive screens for suppressors were performed .", "Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants .", "Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset .", "Suppressor methodology was also applied to the integral membrane protein , diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different .", "Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in vivo .", "The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists ." ]
[ "Common techniques to determine the three-dimensional structures of proteins can help researchers to understand these molecules’ activities , but are often time-consuming and do not work for all proteins .", "Proteins are made of chains of amino acids .", "When a protein chain folds , some of these amino acids interact with other amino acids and these contacts dictate the overall shape of the protein .", "This means that identifying the pairs of contacting amino acids could make it possible to predict the protein’s structure .", "Interactions between pairs of contacting amino acids tend to remain conserved throughout evolution , and if a mutation alters one of the amino acids in a pair then a 'compensatory' change often occurs to alter the second amino acid as well .", "Compensatory mutations can suggest that two amino acids are close to each other in the three-dimensional shape of a protein , but the computational methods used to identify such amino acid pairs can sometimes be inaccurate .", "In 2012 , researchers generated mutants of a bacterial protein called CcdB with changes to single amino acids that caused the protein to fail to fold correctly .", "Now , Sahoo et al . – who include two of the researchers involved in the 2012 work – have developed an experimental method to identify contacting amino acids and use the CcdB protein as a test case .", "The approach involved searching for additional mutations that could restore the activity of five of the original mutant proteins when the proteins were produced in yeast cells .", "The rationale was that any secondary mutations that restored the activity must have corrected the folding defect caused by the original mutation .", "Sahoo et al . then predicted how close the amino acids affected by the secondary mutations were to the amino acids altered by the original mutations .", "This information was used to select reliable three-dimensional models of CcdB from a large set of possible structures that had been generated previously using computer models .", "Next , the technique was applied to a protein called diacylglycerol kinase A . The structure of this protein had previously been inferred using techniques such as X-ray crystallography and nuclear magnetic resonance , but there was a mismatch between the two methods .", "Sahoo et al . found that the amino acid contacts derived from their experimental method matched those found in the crystal structure , suggesting that the functional protein structure in living cells is similar to the crystal structure .", "In the future , the experimental approach developed in this work could be combined with existing methods to reliably guide protein structure prediction ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
MicroRNA-mediated repression of nonsense mRNAs
elife-03032-v2
[ [ "Eukaryotic cells are constantly at risk for various types of mutations .", "Although many of the mutations are benign , a high number of mutations have detrimental consequences .", "Among these mutations , the nonsense mutation is a severe type that converts a coding codon into a stop codon , leading to the premature termination of translation and the expression of proteins truncated at the carboxyl terminus .", "These truncated protein products often have deleterious dominant-negative or gain-of-function effects that interfere with normal biological processes in cells .", "Indeed , many inherited genetic disorders , such as β-thalassemia ( Chang and Kan , 1979 ) and Duchenne muscular dystrophy ( Koenig et al . , 1987; Monaco et al . , 1988 ) , are caused by germline nonsense mutations .", "Moreover , nonsense mutations in critical tumor suppressor genes are associated with prevalent cancer types such as breast cancer ( Miki et al . , 1994 ) and colorectal cancer ( Powell et al . , 1992; Rowan et al . , 2000 ) .", "A recent large-scale genome-wide study revealed that even healthy individuals carry dozens of nonsense mutations ( MacArthur et al . , 2012 ) .", "In addition , transcriptional errors , mis-splicing , or even alternative splicing ( Danckwardt et al . , 2002; Lewis et al . , 2003; Wollerton et al . , 2004 ) also frequently lead to nonsense mutations .", "Accordingly , cells have evolved a surveillance system known as nonsense-mediated mRNA decay ( NMD ) to eliminate these aberrant transcripts .", "Great efforts have been made to uncover the mechanism by which cells detect and selectively degrade nonsense mRNAs .", "One well known mechanism for recognizing premature termination codon ( PTC ) -containing transcripts is exon–exon junction complex ( EJC ) -dependent NMD ( EJC-NMD ) .", "During splicing , a multi-protein EJC complex is deposited at the 5′ side of each exon–exon junction , which is subsequently displaced by the translating ribosome during the pioneer round of translation .", "An EJC will remain bound to the mRNA and trigger NMD efficiently if the ribosome stalls at a PTC located at least 50 nucleotides ( nt ) upstream of the final exon–exon junction ( Maquat , 2004 ) .", "However , nonsense transcripts that originate from naturally intronless genes are immune to EJC-NMD , as are transcripts with PTCs located within the last exon or less than 50 nt upstream of the last exon–exon junction .", "Other EJC-independent NMD mechanisms , such as long 3′ untranslated regions ( UTRs ) ( Buhler et al . , 2006; Eberle et al . , 2008; Singh et al . , 2008; Hogg and Goff , 2010; Zund et al . , 2013 ) and upstream open reading frames ( uORFs ) ( Matsui et al . , 2007 ) are also involved in the degradation of PTC-containing mRNAs .", "The diverse degradation pathways for nonsense mRNAs indicate the complexity of mRNA quality control mechanisms in living cells .", "Another mechanism for post-transcriptional gene regulation is mediated by microRNAs ( miRNAs ) , a class of small non-coding RNAs that are present in essentially every organ and tissue of the body .", "In animals , miRNAs are processed from hairpin precursors and assemble with Argonaute ( Ago ) family proteins into RNA-induced silencing complexes ( RISCs ) to regulate gene expression ( Bartel , 2004 ) .", "By imperfectly base-pairing with miRNA-responsive elements ( miREs ) in target mRNAs ( primarily through nucleotides 2–7 , the seed region of the miRNA ) , miRNAs exert their repressive effects by promoting RNA degradation through accelerated deadenylation and/or by inhibiting translation ( Wu and Belasco , 2008b ) .", "The richness and variety of cellular miRNAs , as well as the versatility of miRNA:miRE seed matches render miRNAs one of the most flexible molecules to govern transcriptome integrity .", "In this study , we demonstrate that miRNAs also selectively target and repress the expression of nonsense mRNAs by both expedited poly ( A ) tail removal and translational repression .", "We present evidence that naturally occurring cancer-causing nonsense mRNAs are repressed by miRNA-mediated surveillance .", "Furthermore , we show that miRNA-mediated surveillance and EJC-NMD function additively .", "We propose that miRNAs may serve as a novel component of the cellular mRNA quality control system that eliminates nonsense mRNAs ." ], [ "The vast majority of known functional and conserved miREs reside within the 3′ UTR of mRNAs ( Bartel , 2004 ) .", "By contrast , reports of miRNAs efficiently targeting ORFs are sparse ( Duursma et al . , 2008; Forman et al . , 2008; Tay et al . , 2008; Huang et al . , 2010; Schnall-Levin et al . , 2011 ) .", "Interestingly , recent studies describing the transcriptome-wide identification of miREs revealed prevalent RISC binding in the coding region ( Chi et al . , 2009; Hafner et al . , 2010; Helwak et al . , 2013 ) , raising a fascinating question about the biological significance of these ORF miREs .", "Previously , we have shown that miRNAs cause expedited removal of the poly ( A ) tails from their mRNA targets through the recognition of miREs in the 3′ UTR .", "By accelerating this initial and rate-limiting step of mRNA decay , miRNAs efficiently reduce the cellular concentration of their target mRNAs ( Wu et al . , 2006; Figure 1—figure supplement 2A ) .", "Theoretically , premature translation termination at a nonsense mutation should cause the ORF region downstream of the PTC to acquire a 3′ UTR identity .", "If any functional miRE is located within this redefined 3′ UTR region , this nonsense mRNA may become miRNA-sensitive and be subject to rapid miRNA-mediated deadenylation and then decay .", "In this manner , miRNAs could serve as a surveillance system against nonsense mRNAs .", "To test this hypothesis , we used a transiently inducible β-globin ( BG ) reporter system and a well-established transcriptional pulse-chase assay to analyze the effect of a PTC on mRNA deadenylation in human cells ( Shyu et al . , 1989 ) .", "Briefly , transcription was induced by removing tetracycline ( tet ) from the culture medium for a short period so as to obtain a nearly homogeneous population of BG mRNAs that subsequently underwent synchronous deadenylation and degradation .", "RNA samples collected at different time points after induction were subjected to site-specific cleavage by RNase H to produce 3′ BG mRNA fragments which facilitate accurate measurement of the poly ( A ) tail length via gel electrophoresis and Northern blotting .", "The natural sequence of BG mRNA does not harbor any miREs; therefore , we first modified the reporter by inserting a let-7a miRE sequence ( Figure 1F ) in-frame into the ORF of the last exon to create LastEx-L7 .", "We found that this insertion did not significantly accelerate poly ( A ) shortening in HeLa-tTA cells , where let-7a is naturally abundant ( Figure 1A , compare LastEx-L7 and TBG ) .", "This result indicates that ORF miREs are not functionally efficient .", "Interestingly , a point mutation ( AAA to TAA ) in the last exon that introduced a PTC 16 nt upstream of the let-7a miRE of LastEx-L7 ( LastEx-PTC-L7 ) significantly accelerated the shortening of the 3′ fragment ( Figure 1A , upper portion , compare LastEx-L7 and LastEx-PTC-L7 , time points 3 and 4 . 5 ) , but not the 5′ fragment , of BG mRNA ( Figure 1A , bottom portion ) , a finding indicative of expedited poly ( A ) removal .", "Furthermore , treatment of the RNA samples with oligo ( dT ) and RNase H caused the BG 3′ fragments that previously appeared as diffuse bands after electrophoresis to migrate uniformly to a position corresponding to fully deadenylated mRNA ( Figure 1B ) , which constitutes additional evidence that the decrease in the length of BG 3′ fragments was due to trimming of the poly ( A ) tails .", "The expedited deadenylation disappeared when the let-7a miRE was mutated ( LastEx-PTC-L7M ) , indicating that the rapid poly ( A ) removal was specifically induced by endogenous let-7a in the cells ( Figure 1A ) .", "As a consequence of accelerated deadenylation , the BG mRNA bearing both the let-7a miRE and the upstream PTC decayed much faster than counterparts that lacked the PTC or the let-7a miRE or bore the mutant let-7a miRE , with the half-life decreasing from >6 hr to <3 hr ( Figure 1—figure supplement 1A , B ) .", "LastEx-L7 decayed slightly faster than the control BG mRNAs ( TBG and LastEx-PTC ) , which suggests that the miRE located in the ORF may still have residual activity .", "Similarly , the deadenylation rate of another BG reporter with a PTC located 11 nt upstream of the last exon–exon junction was also accelerated in a miRNA-dependent manner ( Figure 1C , compare PTC102-L7 and PTC102-L7M , time points 1 . 5 , 3 , and 4 . 5 ) .", "Expedited poly ( A ) shortening was not observed for BG mRNA bearing the PTC in the last exon or 11 nt upstream of the last exon–exon junction but no downstream miRE ( Figure 1A , LastEx-PTC and Figure 1C , PTC102 ) , because the PTC alone is unable to trigger EJC-NMD when located in the last exon or less than 50 nt upstream of the last exon–exon junction .", "Similar results were obtained with BG reporters containing one miR-21 miRE ( Figure 1—figure supplement 2B , C and data not shown ) , providing evidence that miRNA-mediated deadenylation of nonsense mRNAs is not miRNA-type-specific . 10 . 7554/eLife . 03032 . 003Figure 1 . A PTC potentiates rapid miRNA-mediated deadenylation of nonsense mRNAs .", "( A ) The influence of a PTC on BG mRNAs with or without a downstream miRE .", "Cytoplasmic RNA was collected at the indicated times after transcriptional arrest by adding tetracycline ( tet ) .", "The RNA samples were then treated with RNase H and an oligodeoxynucleotide complementary to codons 74–81 of BG mRNA to generate 5′ and 3′ fragments , which were then separated by electrophoresis on a denaturing polyacrylamide gel and detected by Northern blotting .", "Left panel: BG mRNA deadenylation with or without a PTC in the last exon .", "TBG contains an intact BG ORF .", "A PTC was introduced into TBG at codon 121 within the last exon to generate LastEx-PTC .", "Both constructs harbor no miRE in their ORFs .", "Right panel: the influence of let-7a on the deadenylation rate of BG mRNAs harboring one let-7a miRE in its ORF with or without an upstream PTC mutation .", "LastEx-L7 contains one let-7a miRE in the last exon of the BG ORF .", "LastEx-PTC-L7 has a PTC at the same position as LastEx-PTC , which is 16 nt upstream of the let-7a miRE .", "Two nucleotides within LastEx-PTC-L7 let-7a miRE seed were changed to create a synonymous codon in LastEx-PTC-L7M .", "The positions of the ORF start site and original stop codon are indicated by arrows .", "The borders of the original ORF/3′ UTR and redefined ORF/3′ UTR upon PTC mutation are indicated by solid or dashed lines below the constructs .", "Markers A ( 0 ) and A ( 160 ) correspond in size to BG mRNA 3′ fragments bearing no poly ( A ) or a 160-nt poly ( A ) tail , respectively .", "( B ) Confirmation of poly ( A ) tail shortening by treatment with oligo ( dT ) and RNase H . The same LastEx-PTC-L7 RNA as in A were further treated with oligo ( dT ) and RNase H and analyzed by Northern blotting .", "( C ) Induction by let-7a of accelerated deadenylation of nonsense BG mRNAs that do not conform to the ‘50 nt boundary rule’ of EJC-NMD .", "PTC102-L7 contains a PTC mutation 11 nt upstream of the last exon–exon junction and a let-7a miRE in the last exon of the ORF .", "PTC102-L7M is identical to PTC102-L7 except for a mutated let-7a miRE .", "PTC102 contains the PTC only .", "( D ) The influence of let-7a on the deadenylation rate of BG mRNAs harboring one let-7a miRE in its ORF with a retained last intron .", "TBG-IR-L7 contains one let-7a miRE in the last exon of the BG ORF and a retained last intron that creates a PTC 505 nt upstream of the let-7a miRE .", "Two nucleotides within the TBG-IR-L7 let-7a miRE seed were mutated as in A . TBG-IR only has the retained intron but no miRE .", "( E ) The influence of let-7a on the deadenylation rate of BG mRNAs harboring one let-7a miRE in its ORF in the absence of translation .", "hp-LastEx-L7 contains a 40-nt inverted repeat in its 5′ UTR to block translation initiation .", "hp-LastEx-L7M is identical to hp-LastEx-L7 except for a mutated let-7a miRE .", "( F ) Duplexes expected for the let-7a miRE or its mutant counterpart base-paired with let-7a . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 00310 . 7554/eLife . 03032 . 004Figure 1—figure supplement 1 . A PTC potentiates rapid miRNA-mediated decay of nonsense mRNAs .", "( A ) Decay of β-globin ( BG ) mRNAs .", "Cytoplasmic RNA was collected at the indicated times after transcriptional arrest by adding tetracycline ( tet ) and analyzed by Northern blotting .", "To more accurately quantify the half-life of BG mRNA , RNA samples were collected at later times than when monitoring mRNA deadenylation in Figure 1 .", "( B ) Graphs of the concentration of each BG mRNA in A as a function of time .", "All values were normalized to EGFP mRNA , a co-transfected constitutively transcribed internal standard . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 00410 . 7554/eLife . 03032 . 005Figure 1—figure supplement 2 . A PTC potentiates rapid miR-21-mediated deadenylation of nonsense mRNAs .", "( A ) Left panel: the influence of let-7a on the deadenylation rate of BG mRNA harboring a let-7a miRE in the 3′ UTR .", "Right panel: the influence of miR-21 on the deadenylation rate of BG mRNA harboring a miR-21 miRE in the 3′ UTR .", "( B ) Left panel: deadenylation of BG mRNAs with or without a PTC in the last exon .", "TBG contains an intact BG ORF .", "A PTC was introduced into TBG at codon 116 within the last exon to generate LastEx-PTC .", "Neither construct harbors an miRE in its ORFs .", "Right panel: the influence of miR-21 on the deadenylation rate of BG mRNAs harboring one miR-21 miRE in its ORF with or without an upstream PTC mutation .", "LastEx-21 contains one miR-21 miRE in the last exon of the BG ORF .", "LastEx-PTC-21 has a PTC at the same position as LastEx-PTC , which is 22 nt upstream of the miR-21 miRE .", "Two nucleotides within the miR-21 miRE seed of LastEx-PTC-21 were mutated to create a synonymous codon in LastEx-PTC-21M .", "The positions of the ORF start site and the original stop codon are indicated by arrows .", "The borders of the original ORF/3′ UTR and redefined ORF/3′ UTR upon PTC mutation are indicated by solid or dashed lines below the constructs .", "( C ) miRNA-induced accelerated deadenylation of nonsense BG mRNA that does not conform to the ‘50 nt boundary rule’ of EJC-NMD .", "PTC102-21 contains a PTC 11 nt upstream of the last exon–exon junction and a miR-21 miRE in the last exon of the ORF .", "PTC102-21M is identical to PTC102-21 except for a mutated miR-21 miRE .", "( D ) The influence of miR-21 on the deadenylation rate of BG mRNA harboring a miR-21 miRE in its ORF in the absence of translation .", "hp-LastEx-21 contains a 40-nt inverted repeat in its 5′ UTR to block translation initiation .", "hp-LastEx-21M is identical to hp-LastEx-21 except for a mutated miR-21 miRE .", "( E ) Duplexes expected for the miR-21 miRE or its mutant counterpart base-paired with miR-21 . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 00510 . 7554/eLife . 03032 . 006Figure 1—figure supplement 3 . A large stem-loop structure in the 5′ UTR blocks translation of BG mRNA . A Renilla luciferase ( RL ) ORF was fused to the 5′ side of the BG ORF so as to facilitate precise quantification of protein production .", "Levels of the RL-BG protein and mRNA were determined by assaying luciferase activity and by qRT-PCR , respectively .", "Amounts of RL protein and mRNA were normalized to a co-transfected firefly luciferase control . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 006 Besides point mutations , alternative splicing also serves as an important source of PTCs .", "Recent studies suggest that intron retention , a splicing event that often creates PTCs , functions as a general mechanism that controls the expression of many genes in the cells ( Galante et al . , 2004; Yap et al . , 2012; Wong et al . , 2013 ) .", "We speculated that the PTC-containing transcripts generated by intron retention could also be targeted by miRNAs .", "To test this possibility , we generated a BG reporter ( TBG-IR-L7 ) by mutating the splicing sites in the last intron of LastEx-L7 , which abolished the splicing of the last intron and created a PTC 505 nt upstream of the let-7a miRE .", "As expected , we found that TBG-IR-L7 underwent rapid deadenylation in a miRNA-dependent manner; and that absence of the downstream miRE ( TBG-IR ) or mutations in the miRE seed region ( TBG-IR-L7M ) completely abolished this accelerated deadenylation ( Figure 1D ) .", "Interestingly , TBG-IR is deadenylated slightly faster than wild-type TBG , probably due to the presence of potential miREs in the longer region between the PTC and the native stop codon ( 570 nt ) that resulted from intron retention; these miREs may be recognized by the highly expressed endogenous miRNAs in HeLa-tTA cells , such as miR-10a/b and miR-17/20a ( data not shown ) .", "Collectively , these observations demonstrate that a PTC , introduced either by point mutation or alternative splicing , is able to potentiate miRNA-mediated rapid deadenylation of the mRNA by unmasking ORF miREs downstream of it .", "An essential feature that distinguishes the 3′ UTR from the ORF is the absence of translating ribosomes .", "The PTC may serve as a roadblock to stop ribosomes and mark the new boundary between the ORF and 3′ UTR .", "We speculated that blocking translation would cause the miRE within the ORF to behave as if it were in the 3′ UTR and efficiently trigger accelerated miRNA-mediated mRNA deadenylation , even in the absence of an upstream PTC .", "To test this hypothesis , we placed a large stem-loop structure at the 5′ UTR of LastEx-L7 ( hp-LastEx-L7 ) to block translation initiation ( Chen et al . , 1995; Wu et al . , 2006; Figure 1—figure supplement 3 ) and examined its deadenylation rate .", "As expected , blocking translation in this manner caused LastEx-L7 mRNA , which normally is deadenylated and decays slowly , to undergo rapid miRNA-mediated deadenylation and decay ( half-life of >6 hr vs <3 hr ) ( Figure 1E , Figure 1—figure supplement 1A , B ) , suggesting that translating ribosomes may indeed have interfered with miRNA-RISC binding to the miREs located in the ORF and thus masked their repressive function .", "Similar results were obtained with another set of BG reporters containing a miR-21 miRE ( Figure 1—figure supplement 2D ) .", "Together , these data suggest that miRNAs selectively accelerate the deadenylation of nonsense mRNAs by stably binding to miREs in the ORF downstream of the PTC .", "The immunity of PTC-free mRNA to miRNA-mediated repression may be due to masking of ORF miREs by the translating ribosomes , which is consistent with a previous study using constitutively transcribed luciferase reporters ( Gu et al . , 2009 ) .", "NMD exerts its repressive power primarily by promoting RNA degradation; however , miRNA-mediated repression usually involves both mRNA decay and translational repression .", "To determine whether translational repression is involved when miRNAs exert their repressive effects against nonsense mRNAs , we constructed a modified luciferase reporter that has a fragment containing two in-frame miR-125b miREs ( Figure 2A ) followed by an additional in-frame stop codon fused to the 3′ end of the luciferase ORF ( TAA-2E ) .", "In this construct , the original stop codon of the luciferase ORF served as a PTC .", "This PTC ( TAA ) was mutated to TCA to create a PTC-free counterpart ( TCA-2E ) ( Figure 2B ) , which produced a luciferase protein with an additional 46 amino acids at the carboxyl terminal .", "Measurement of the steady-state mRNA level by qRT-PCR showed specific and significant repression of TAA-2E in the presence of miR-125b ( Figure 2C ) , observations consistent with the results of the BG deadenylation assay shown in Figure 1A and Figure 1—figure supplement 2B .", "Moreover , the measurement of luciferase activity revealed an even greater reduction of the PTC-containing reporter at the protein level ( Figure 2D ) , indicating that translational repression plays a prominent role in the repression of nonsense messages by miRNAs ( Figure 2E ) .", "Since no intron is present downstream of the PTC , this reporter ( TAA-2E ) is immune to EJC-NMD and the repression is most likely contributed by miRNAs . 10 . 7554/eLife . 03032 . 007Figure 2 . A PTC potentiates miRNA-mediated translational repression of nonsense mRNAs .", "( A ) Duplex expected for the miR-125b miRE base-paired with miR-125b .", "( B ) The reporter constructs used in C to E . TAA-2E contains two miR-125b miREs 69 nt downstream of the original stop codon ( which now serves as a PTC ) of the firefly luciferase ( FL ) gene .", "This PTC ( TAA ) was mutated to codon TCA to generate TCA-2E .", "The borders of the ORF/3′ UTR and redefined ORF/3′ UTR upon PTC mutation are indicated by solid or dashed lines below the constructs .", "( C ) mRNA quantification for TAA-2E and TCA-2E in the presence and absence of miR-125b , as determined by qRT-PCR .", "The error bars represent the standard deviation of multiple measurements .", "( D ) Protein quantification by analyzing luciferase activity for TAA-2E and TCA-2E in the presence and absence of miR-125b .", "( E ) Contribution of translational repression by miRNA-mediated surveillance .", "The repression ratios for TAA-2E and TCA-2E were calculated from normalized levels of firefly luciferase protein ( black bars ) and mRNA ( gray bars ) in the absence versus the presence of miR-125b .", "By dividing the repression ratio for protein production and that for mRNA concentration , the repression ratio for translation efficiency ( protein yield per mRNA molecule , white bars ) was determined .", "A repression ratio for translation efficiency that is >1 indicates that part of the total repression observed at the protein level is attributable to inhibition of translation .", "( F ) Schematic illustration of the reporter designs used in G . The same 22-nt miR-125b miRE as in A was introduced in-frame into different positions before or after a PTC of a modified firefly luciferase reporter gene to obtain a series of miRE-containing plasmids .", "A graph that illustrates the different methods for calculating the distance between an upstream or a downstream miRE and the PTC is shown below the construct .", "( G ) Boundary rule for miRNA-mediated surveillance .", "Each firefly luciferase construct that contains one miR-125b miRE at a different position was co-transfected with a Renilla luciferase reporter into HEK293 cells in the presence or absence of miR-125b .", "The relative FL expression level was calculated from the normalized levels of firefly luciferase in the absence versus the presence of miR-125b . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 007 EJC-NMD generally complies with the ‘50 nt boundary rule’ .", "To determine whether any boundary rule for miRNA-mediated surveillance exists , we constructed a series of plasmids in which a miR-125b miRE was inserted in-frame at various locations before or after the PTC of a modified luciferase reporter mRNA ( Figure 2F ) .", "Measurement of luciferase activity revealed that an miRE has to be located at least 10 nt downstream of the PTC to trigger miRNA-mediated repression effectively ( Figure 2G ) .", "This observation defines a distinct boundary rule for miRNAs to successfully repress PTC-containing messages and also suggests that the size of the footprint of RISC is much smaller than that of the EJC , which makes miRNA-mediated surveillance more versatile in recognizing and repressing nonsense mRNAs .", "Altogether , these results demonstrate that a PTC can potentiate miRNA-mediated deadenylation and translational inhibition of nonsense mRNAs , via redefinition of ORF/3′ UTR identities and unmasking of downstream miREs .", "Next , we asked whether any naturally occurring nonsense mRNAs are subjected to regulation by miRNA-mediated surveillance .", "We found APC ( adenomatous polyposis coli ) , a tumor suppressor gene that is frequently mutated in colorectal cancer , to be of particular interest .", "Most of the mutations in the APC gene that have been identified in clinical studies are point mutations or frameshift indels that create a PTC and result in the expression of a truncated version of the APC protein .", "Interestingly , the majority of known APC mutations are clustered in a hotspot region ( designated as the MCR in Figure 3A ) within the last exon of the ORF ( Miyoshi et al . , 1992 ) , rendering these mutants immune to EJC-NMD .", "Meanwhile , bioinformatic analysis based on the seed match rule predicts numerous potential miREs between the MCR and the native stop codon in APC mRNA .", "All of these features make APC a nearly ideal paradigm for the study of miRNA-mediated surveillance . 10 . 7554/eLife . 03032 . 008Figure 3 . PTC-containing APC mRNAs are natural substrates repressed by miRNA-mediated surveillance .", "( A ) Schematic representation of APC mRNA .", "The ORF is shown in blue .", "The 5′ UTR and 3′ UTR are shown in gray .", "Exon–exon boundaries are indicated by vertical lines in the mRNA .", "The positions of representative miREs and PTC sites are indicated above or below the mRNA by solid lines and arrows , respectively .", "MCR refers to the mutation cluster region .", "( B ) Validation of miRE function by luciferase assays .", "A vector expressing both a firefly luciferase ( FL ) transcript harboring one potential miRE in its 3′ UTR and a control Renilla luciferase ( RL ) transcript was co-transfected with cognate miRNA mimics into HEK293 cells .", "The relative FL expression level represents the firefly/Renilla luciferase ratio for pRF-miRE relative to the no miRE control pRF-con .", "( C ) Validation of miRE function in an APC minigene .", "A vector expressing both an HA-tagged truncated APC ( APC-PTC1450 ) and a control HA-tagged EGFP was co-transfected with cognate miRNA mimics into HEK293 cells .", "The mutant counterpart of the APC minigene ( APC-PTC1450-mut ) contains two altered nucleotides that abolish miRE:miRNA complementarity without changing the identity of the encoded amino acid .", "( D ) Western blot analysis of endogenous truncated APC in SW480 cells and full-length APC in HEK293 cells by using an anti-APC antibody .", "( E ) Change in PTC-APC mRNA levels in SW480 cells upon miR-29a knockdown .", "Cytoplasmic RNA was extracted from the SW480 cell lines used in F , and the levels of APC and GAPDH mRNA were determined by qRT-PCR .", "The relative APC mRNA level was calculated by normalizing to GAPDH mRNA .", "( F ) Upregulation of endogenous truncated APC upon miR-29a knockdown .", "SW480 cells were transduced with lentiviruses encoding a miR-29a decoy ( TuD-29a ) or a control decoy ( TuD-NC ) .", "Endogenous truncated APC was probed with an anti-APC antibody .", "GAPDH served as a loading control .", "Changes in the levels of endogenous miR-29a and an untargeted control ( miR-26a ) were determined by Northern blotting .", "5S rRNA served as a loading control .", "( G ) Downregulation of endogenous truncated APC upon miR-29a overexpression .", "SW480 cells were transduced with lentiviruses encoding miR-29a or a control small RNA ( siEGFP ) .", "Western and Northern assays were performed as in F . ( H ) Invariant concentration of endogenous wild-type APC upon miR-29a overexpression .", "HEK293 cells were transduced with lentiviruses encoding miR-29a or a control small RNA ( siEGFP ) .", "Western and Northern assays were performed as in F except that Tubulin served as the loading control in the Western assay .", "( I ) Duplexes expected for the miR-29a miREs base-paired with miR-29a .", "( J ) Ribonucleoprotein immunoprecipitation ( RIP ) analysis of PTC-APC mRNA associated with Ago2 in SW480 cells upon miR-29a knockdown .", "SW480 cells used in F were transduced with a low amount of lentiviruses ( MOI <0 . 3 ) expressing FLAG-tagged Ago2 .", "Anti-FLAG RIP followed by qRT-PCR was performed to compare the binding of endogenous PTC-APC mRNAs to Ago2 .", "The amount of Ago2-associated PTC-APC mRNA was normalized to MYC , an endogenous target of let-7c .", "The relative Ago2-RIP efficiency was calculated from the normalized amount of PTC-APC mRNA in the presence of a miR-29a decoy ( TuD-29a ) versus a control decoy ( TuD-NC ) .", "( K ) RIP analysis of PTC-APC mRNA associated with Ago2 in SW480 cells upon miR-29a overexpression .", "SW480 cells used in G were transduced with a low amount of lentiviruses ( MOI <0 . 3 ) expressing FLAG-tagged Ago2 .", "RIP assays were performed as in J . The relative Ago2-RIP efficiency was calculated from the normalized amount of Ago2-associated PTC-APC mRNA in the presence of miR-29a versus a control small RNA ( siEGFP ) .", "( L ) The 3′ UTR of APC mRNA contains no miR-29a miRE .", "The sequence of a full length APC 3′ UTR was cloned to the 3′ UTR of a firefly luciferase reporter .", "This reporter plasmid ( pFL-APC-3′UTR ) or a control plasmid ( pFL ) was co-transfected with a miR-29a mimic or a control small RNA ( siNC ) into HEK293 cells .", "The relative FL expression level was calculated from the normalized levels of firefly luciferase activity for pFL-APC-3′UTR versus pFL in the presence of the miR-29a mimic or siNC .", "( M ) RIP analysis of ectopically expressed PTC-APC mRNA associated with Ago2 in HEK293 cells .", "A PTC-containing APC minigene plasmid with wild-type ( APC-PTC1450 ) or mutant miR-29a miREs ( APC-PTC1450-mut ) was co-transfected with the miR-29a mimic or a control small RNA ( siNC ) into HEK293 cells that stably expressed FLAG-tagged Ago2 .", "Anti-FLAG RIP followed by qRT-PCR was performed to compare the binding of the mRNAs to Ago2 .", "The amount of Ago2-associated PTC-APC mRNA or its miRE mutant counterpart was normalized to HOXD10 , an endogenous target of miR-10a .", "The relative Ago2-RIP efficiency was calculated from the normalized levels of Ago2-associated PTC-APC mRNA bearing wild-type ( APC-PTC1450 ) or mutant ( APC-PTC1450-mut ) miR-29a miREs in the presence of the miR-29a mimic versus siNC . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 00810 . 7554/eLife . 03032 . 009Figure 3—figure supplement 1 . Duplexes expected for functional miRNAs base-paired with cognate miREs .", "( A ) Reporter constructs and strategy for miRE screening of APC mRNA .", "The cDNA region between codon 1450 and the stop codon of APC mRNA was fused to the 3′ end of the firefly luciferase ( FL ) ORF such that the original stop codon of the luciferase gene functioned as a PTC .", "The PTC-STOP region of APC mRNA was examined for predicted miREs .", "To test each putative miRE , a cognate miRNA mimic and a control small RNA ( siEGFP ) were co-transfected with the reporter into HEK293 cells .", "As an internal control , a Renilla luciferase ( RL ) reporter was simultaneously transfected .", "miRNAs that caused strong repression were selected for further validation .", "( B ) Duplexes expected for miR-24 , miR-127-5p , and miR-146b base-paired with their cognate miREs . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 00910 . 7554/eLife . 03032 . 010Figure 3—figure supplement 2 . Repressive activity of miR-29a miREs within the PTC-STOP region of APC minigenes bearing the natural APC 3′ UTR . The constructs were the same as in Figure 3C except that they contained the full length 3′ UTR of APC mRNA .", "Western assays were performed as in Figure 3C . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 01010 . 7554/eLife . 03032 . 011Figure 3—figure supplement 3 . Full functionality of miREs in the PTC-STOP region in the presence of 3′ UTR-dependent repression by miRNAs . The PTC-STOP region and the entire 3′ UTR of APC mRNA were fused to the 3′ end of a firefly luciferase ( FL ) ORF so that the stop codon of luciferase served as a PTC .", "miREs for miR-29a ( ORF miRE ) and miR-135b ( 3′ UTR miRE ) were mutated individually or simultaneously .", "HEK293 cells were co-transfected with each reporter together with equal amounts of miR-29a and miR-135b or a control small RNA ( siNC ) .", "Relative FL expression represents the ratio of firefly/Renilla luciferase for each reporter relative to pFL-APC-29M+135M . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 01110 . 7554/eLife . 03032 . 012Figure 3—figure supplement 4 . Successful knockdown of endogenous miR-29a by TuD-29a .", "( A ) Schematic representation of the structure of the TuD decoy RNA for miR-29a .", "( B ) Reporter constructs used in C and D . pRF-29 contains the miR-29a miRE originating from APC , whereas pRF-29M contains a mutant form of the miR-29a miRE .", "Both reporters express Renilla luciferase ( RL ) from a second promoter in the same construct as a control .", "( C ) Successful knockdown of endogenous miR-29a in SW480 cells by TuD-29a .", "The relative expression level of pRF-29 was elevated in TuD-29a-expressing cells compared to TuD-NC-expressing control cells .", "The relative FL expression level was calculated from the normalized levels of firefly luciferase activity for pRF-29 versus pRF-29M .", "( D ) Tissue-specific effect of miRNA-mediated surveillance .", "pRF-29 is efficiently repressed by endogenous miR-29a in colon-derived SW480 cells but not in kidney-derived HEK293 cells .", "The relative FL expression level was calculated as in C . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 01210 . 7554/eLife . 03032 . 013Figure 3—figure supplement 5 . Confirmation of interaction between miR-29a and its miREs in APC mRNA . A mutant miR-29a mimic that restored seed complementarity with the mutant miR-29a miREs was co-transfected with APC-PTC1450 minigene plasmids bearing wild-type or mutant miR-29a miREs .", "Western assays were performed as in Figure 3C .", "The mutated nucleotides are underlined in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 013 To investigate whether PTC-containing APC mRNA is specifically targeted by certain miRNAs , we performed miRE screening using a reporter that has the region between a PTC at codon 1450 and the native stop codon ( the PTC-STOP region ) of APC mRNA fused to the 3′ end of the luciferase ORF ( Figure 3—figure supplement 1A ) .", "The top 45 miRNA candidates were chosen for the screening based on their general abundance in human tissues and the predicted thermal stability of the duplex they may form with an APC miRE .", "For each miRNA selected , we co-transfected HEK293 cells with the luciferase reporter plasmid and a synthetic miRNA mimic or a control small RNA , and examined protein production by measuring luciferase activity 36 hr after transfection .", "A decrease in luciferase activity when co-transfected with a miRNA mimic would indicate that the selected miRNA may have the potential to repress PTC-containing APC ( PTC-APC ) expression through miRE ( s ) in the PTC-STOP region .", "Using this method , we identified several miRNAs with a strong repressive effect ( Supplementary file 1 ) , although others that are naturally highly expressed in HEK293 cells even without transfection may have been missed .", "To verify that the repression is due to the direct interaction between the selected miRNA and an miRE in the PTC-APC mRNA , we mapped the corresponding miREs of the miRNAs via the 2–7 seed match alignment ( Figure 3A , I , Figure 3—figure supplement 1B ) , and then inserted the ∼30-nt-long sequence surrounding the predicted miREs into the 3′ UTR of a luciferase reporter .", "Multiple miREs exhibited specific responses to the cognate miRNAs ( Figure 3B ) , which verifies their functionality .", "To further confirm the repressive effect of these miRNAs on PTC-APC mRNA in its natural sequence context , we constructed minigene vectors that express both an HA-tagged PTC-APC bearing either a wild-type miRE or a mutant miRE with mismatches in the seed region and an HA-tagged EGFP that served as an internal control for more precise protein quantification .", "The minigene plasmids were co-transfected with cognate miRNA mimics into HEK293 cells .", "Western blotting revealed a marked increase in protein expression for the minigene constructs with mutant miREs ( Figure 3C ) .", "The unmasking of ORF miREs by PTCs may significantly augment repression by miREs in the 3′ UTR .", "To test this hypothesis , we constructed a pair of PTC-containing APC minigene plasmids that each contained the full-length APC 3′ UTR , one with wild-type miR-29a miREs ( APC-PTC1450-3’UTR ) and the other with mutant miREs ( APC-PTC1450-mut-3’UTR ) .", "Western blotting showed that , in the context of the natural APC 3′ UTR , the PTC was still able to potentiate repression by miR-29a miREs originally located in the ORF ( Figure 3—figure supplement 2 ) .", "In addition , to quantify the relationship between unmasked ORF miREs and pre-existing miREs in the 3′ UTR , we designed chimeric reporters in which the PTC-STOP region and the entire 3′ UTR of APC mRNA were fused to the 3′ end of a firefly luciferase ORF .", "The miR-29a miREs in the PTC-STOP region and a miR-135b miRE in the 3′ UTR of APC mRNA that had previously been reported to be functional ( Nagel et al . , 2008 ) were mutated , either individually or simultaneously .", "In the presence of both miRNAs , the wild-type chimeric reporter was repressed most efficiently , while mutating either the ORF miREs or the 3′ UTR miRE alleviated the repression ( Figure 3—figure supplement 3 ) .", "These observations indicate that ORF miREs unmasked by an upstream PTC are fully functional in the presence of repression by 3′ UTR miREs .", "We next sought to determine whether the expression of an endogenous PTC-APC mutant is downregulated by miRNAs .", "Our previous luciferase reporter- and minigene-based assays have identified that two miR-29a miREs ( Figure 3I ) are present in the PTC-STOP region of APC nonsense mRNA ( Figure 3A–C ) .", "Interestingly , the seed regions of the miR-29a miREs embedded in the APC ORF are highly conserved across several species .", "Therefore , miREs of miR-29a were selected for subsequent investigations .", "SW480 is a colorectal cell line that naturally expresses a truncated APC protein ( caused by a PTC mutation at codon 1338 ) that can be readily detected by Western blotting with a specific antibody ( Figure 3D ) , and high levels of endogenous miR-29a ( Figure 3F , G , Figure 3—figure supplement 4B , D ) , which render it a suitable cell line to investigate the repression mediated by miR-29a on the endogenous APC nonsense mutant .", "We transduced SW480 cells with lentiviruses encoding a TuD miRNA decoy ( Figure 3—figure supplement 4A ) , which has been proven a very effective and specific antagonizer of miRNAs ( Haraguchi et al . , 2009 ) , to inhibit endogenous miR-29a .", "TuD-expressing cells were cultured for 3 days before APC was examined by Western blotting .", "An approximate twofold increase in truncated APC expression was observed for SW480 cells in which miR-29a was knocked down ( Figure 3F , upper panel ) .", "We also measured the mRNA level of PTC-APC in SW480 cells and found that the knockdown of miR-29a caused a ∼1 . 6-fold increase of PTC-APC mRNA ( Figure 3E ) , which is consistent with the important role of translational repression by miRNAs .", "The successful knockdown of endogenous miR-29a expression was confirmed by Northern blotting ( Figure 3F , middle panel ) and a reporter assay ( Figure 3—figure supplement 4B , C ) ; by contrast , the abundance of an untargeted endogenous miRNA , miR-26a , remained unchanged ( Figure 3F , bottom panel ) , suggesting the upregulation observed for PTC-APC was specifically induced by the inhibition of miR-29a .", "To test if miR-29a-mediated repression is specific for PTC-APC but not wild-type APC ( WT-APC ) , we generated doxycycline ( dox ) -inducible miR-29a-overexpressing SW480 and HEK293 stable cell lines and examined the amount of endogenous APC protein after inducing miRNA expression for 3 days .", "HEK293 cells naturally express the full-length APC protein ( Figure 3D ) and low levels of miR-29a ( Figure 3H , Figure 3—figure supplement 4B , D ) .", "The amount of WT-APC protein remained unchanged after the induction of miR-29a expression to much higher levels in the cells ( Figure 3H ) because the miR-29a miREs are located within the PTC-STOP region but not the 3′ UTR of APC mRNA ( Figure 3A ) .", "In contrast , SW480 cells that overexpress miR-29a produced a lower amount of truncated APC compared to cells that overexpressed a non-functional small RNA that did not affect the already high expression levels of endogenous miR-29a ( Figure 3G ) .", "These results support that miR-29a specifically represses the PTC-APC and does not impair the expression of WT-APC .", "To further prove that the repressive effects of miR-29a on PTC-APC mRNA are direct , we performed ribonucleoprotein immunoprecipitation ( RIP ) assays to examine the association of PTC-APC mRNA with Ago2 , the component of the RISC complex that directly binds the miRNA and its target mRNA .", "The level of endogenous PTC-APC mRNA associated with Ago2 showed a mild but reproducible decrease when miR-29a was knocked down ( Figure 3J ) and a significant increase upon miR-29a overexpression in SW480 cells ( Figure 3K ) .", "The 3′ UTR of APC mRNA contains no miR-29a miREs , which was determined by comparing the expression of a luciferase reporter bearing a full-length APC 3′ UTR in the presence of miR-29a versus a negative control small RNA ( siNC ) ( Figure 3L ) .", "Therefore , the changes in the levels of Ago2-associated PTC-APC mRNA in SW480 cells are most likely due to a direct effect of miR-29a targeting its miREs within the PTC-STOP region .", "Moreover , the binding efficiency of APC-PTC1450 mRNA bearing wild-type or mutant miREs to Ago2 was compared by co-transfecting HEK293 cells with the minigene construct and a miR-29a mimic or a control small RNA .", "In the presence of miR-29a , the APC-PTC1450 mRNA that harbors wild-type miR-29a miREs has a much stronger association with Ago2 than does its counterpart that contains the mutant miREs ( Figure 3M ) .", "In addition , a mutant version of the miR-29a mimic that restored base-pairing of the 2–7 seed with the mutant miR-29a miRE also restored repression of APC mRNA ( Figure 3—figure supplement 5 ) , supporting the conclusion that repression of PTC-APC expression is achieved through the direct interaction of miR-29a with the miREs we mapped .", "Altogether , these observations represent novel evidence that the expression of a naturally occurring nonsense mRNA is selectively repressed by endogenous miRNAs in the cells .", "Although APC provides an excellent case for studying miRNA-mediated surveillance , most of the reported nonsense mRNAs that harbor PTCs located upstream of the last exon should be EJC-NMD sensitive .", "We therefore sought to determine whether these EJC-NMD-competent transcripts are simultaneously subjected to miRNA-mediated surveillance .", "BRCA1 , a tumor suppressor gene that is frequently mutated in breast cancer , was chosen for further investigation .", "Unlike APC , mutations that lead to the expression of a truncated version of BRCA1 are scattered along the ORF ( Castilla et al . , 1994 ) ; therefore , most BRCA1 nonsense mutants contain introns downstream of the PTC and are EJC-NMD-sensitive ( Perrin-Vidoz et al . , 2002 ) .", "We amplified the cDNA of the PTC-STOP region of one clinically identified BRCA1 mutant ( with a PTC at codon 526 ) ( Perrin-Vidoz et al . , 2002 ) and fused it to the 3′ end of a firefly luciferase coding region to create a chimera such that the original stop codon of the luciferase gene serves as a PTC in the fused reporter ( Figure 4A ) .", "This reporter ( pFL-BRCA1 ) has no downstream introns and harbors wild-type miREs; therefore , it is expected to be sensitive to miRNA only .", "pFL-BRCA1in is identical to pFL-BRCA1 except that two downstream introns were incorporated to render the transcript sensitive to EJC-NMD in addition to miRNA .", "The expression level of pFL-BRCA1in was significantly lower compared to pFL-BRCA1 , indicating that EJC-NMD was contributing to the repressive activity ( Figure 4C , left panel ) .", "When GW182 , a key component of RISC , was knocked down >70% by small interfering RNAs ( siRNAs ) in HeLa-tTA cells ( Figure 4C , right panel ) , the expression level of pFL-BRCA1 was significantly increased , which indicates that miRNAs were indeed involved in the repression .", "More importantly , knockdown of GW182 also caused a comparable extent of upregulation of pFL-BRCA1in , suggesting that miRNA-mediated repression of PTC-containing mRNAs is independent of the presence of downstream introns ( Figure 4C , left panel ) .", "Thus , miRNA-mediated surveillance and EJC-NMD are not mutually exclusive , and the repressive effects caused by both mechanisms are additive . 10 . 7554/eLife . 03032 . 014Figure 4 . The repressive effects of miRNA-mediated surveillance and EJC-NMD are additive .", "( A ) The reporter constructs used in C and D . pFL-BRCA1 and pFL-BRCA1-mut have no introns , whereas pFL-BRCA1in and pFL-BRCA1in-mut each harbor two introns downstream of PTC .", "The miREs for miR-137 and miR-544 were mutated to synonymous codons in pFL-BRCA1-mut and pFL-BRCA1in-mut .", "( B ) Identification of the surveillance mechanism pertinent to each reporter .", "( C ) Left panel: miRNA-mediated surveillance and EJC-NMD are not mutually exclusive .", "Knockdown of RISC core component GW182 alleviated the repression of BRCA1 reporters , regardless of the presence of the downstream introns .", "The relative FL expression level represents the firefly/Renilla luciferase ratio in the presence of a control siRNA ( siNC ) versus an siRNA targeting GW182 ( siGW182 ) .", "Right panel: expression level of GW182 protein in HeLa-tTA cells treated with siNC or siGW182 .", "Tubulin served as a loading control .", "( D ) Additive effects of miRNA-mediated surveillance and EJC-NMD .", "Each reporter in A was co-transfected with miR-137 , miR-544 , or a control small RNA ( siNC ) , together with a Renilla luciferase reporter into HEK293 cells .", "The relative FL expression level represents the firefly/Renilla luciferase ratio of each EJC-NMD- and/or miRNA-responsive reporter relative to pFL-BRCA1-mut .", "( E ) Reporter constructs for miRE function validation of candidates identified from HCT-116 exome and RNA sequencing data .", "The PTC-STOP region of each candidate was fused to a firefly luciferase ( FL ) ORF in the same manner as for APC and BRCA1 .", "A control Renilla luciferase ( RL ) reporter was expressed from a second promoter in the same construct ( pRF-candidate ) .", "Each potential miRE identified from the screening was mutated to obtain a series of miRE mutant reporters ( pRF-candidate-miREmut ) .", "( F–I )", "Experimental verification of functional miREs in the PTC-STOP region of selected candidates .", "The wild-type or miRE mutant version of each candidate reporter was co-transfected into HEK293 cells with a cognate miRNA mimic .", "The activity of each miRE ( fold increase ) was calculated from the normalized levels of firefly luciferase activity for pRF-candidate versus pRF-candidate-miREmut in the presence of the cognate miRNA mimic . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 01410 . 7554/eLife . 03032 . 015Figure 4—figure supplement 1 . Experimental verification of functional miREs in the PTC-STOP region of additional nonsense mutant mRNAs . Six additional candidates identified from HCT-116 cells were subjected to experimental verification of predicted miREs in their PTC-STOP regions .", "The wild-type or miRE mutant version of the reporter for each candidate was co-transfected into HEK293 cells with a cognate miRNA mimic .", "The activity of each miRE was calculated as in Figure 4F–I . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 015 Next , we identified several potential miREs between the PTC and the natural stop codon of BRCA1 nonsense mutant mRNA by using the similar screening method for APC in HEK293 cells ( data not shown ) .", "miREs targeted by miR-137 and miR-544 were mutated to create pFL-BRCA1-mut , which is not responsive to either EJC-NMD or miR-137 and miR-544 , and pFL-BRCA1in-mut , which is insensitive to miR-137 and miR-544 but responsive to EJC-NMD ( Figure 4A , B ) .", "As expected , the expression level of EJC-NMD-sensitive reporter pFL-BRCA1in-mut was significantly lower compared to pFL-BRCA1-mut ( Figure 4D , compare black and light gray bars ) .", "When co-transfected with miR-137 or miR-544 in HEK293 cells ( which do not naturally express these two miRNAs ) , the expression of the miRNA-specific reporter pFL-BRCA1 was significantly suppressed compared to pFL-BRCA1-mut , confirming the role of miRNA-mediated surveillance in repressing its target ( Figure 4D , middle and right , compare black and dark gray bars ) .", "Importantly , a markedly stronger repression was observed for the reporter expected to be sensitive to both EJC-NMD and miRNA ( pFL-BRCA1in ) when co-transfected with miR-137 or miR-544 but not when co-transfected with a control small RNA ( Figure 4D , compare black and white bars ) .", "This observation further validates that miRNA-mediated surveillance and EJC-NMD can act additively .", "To determine if miRNA-mediated surveillance is a general mechanism that helps to eliminate nonsense transcripts , we sought to identify more potentially functional ORF miREs in other naturally occurring nonsense mRNAs .", "We performed exome sequencing of HCT-116 cells and identified 188 heterozygous nonsense mutations .", "Analysis of transcriptome data ( Djebali et al . , 2012 ) indicates that 47 of them were actively expressed in HCT-116 cells ( Supplementary file 2 ) .", "We chose 16 candidates that contain PTC-STOP regions longer than 400 nt for further experimental validation of miREs by using a similar strategy for APC and BRCA1 .", "We found that 11 of the 16 candidates contained at least one functional miRE in the PTC-STOP regions ( Figure 4E–I , Figure 4—figure supplement 1 ) .", "Several of these candidates , such as ITGA6 , USP1 , and PPM1J , harbored multiple functional miREs that were responsive to different miRNAs ( Figure 4F–H ) .", "Other candidates , such as KTN1 and RAD54L2 , contained at least one strong miRE ( Figure 4I ) .", "As only a limited number of miRNAs have been screened by our luciferase assays , additional miREs may be found by increasing the screened miRNAs .", "Considering the well-known facts that multiple miRNAs can repress the same mRNA simultaneously and the effects of miREs are additive ( Doench and Sharp , 2004 ) , the presence of multiple active miREs within these PTC-STOP regions could result in a pronounced repressive effect .", "Together , our results strongly support the view that miRNA-mediated surveillance may serve as a general mechanism that downregulates various nonsense mRNAs in human cells ( Figure 5 ) . 10 . 7554/eLife . 03032 . 016Figure 5 . Model of miRNA-mediated surveillance system . The coding region of an mRNA may contain multiple potential miREs .", "Usually , miRNAs cannot stably bind to their cognate miREs that are embedded within the ORF of a normal transcript under active translation .", "However , upon nonsense mutation , the translating ribosome stalls at the PTC so that miREs downstream of the PTC are unmasked , triggering miRNA-mediated deadenylation and translational repression . DOI: http://dx . doi . org/10 . 7554/eLife . 03032 . 016" ], [ "In this study , we have described evidence that a PTC is sufficient to induce miRNA-mediated downregulation of nonsense mRNAs by unmasking miREs located between the PTC and the natural stop codon of the mRNA .", "We have also experimentally verified that nonsense mutants of APC , BRCA1 , and a few other genes are subjected to miRNA-mediated surveillance in human cells .", "Our findings indicate that besides their established roles in regulating gene expression , miRNAs may serve as a novel surveillance system to reduce aberrant mRNAs bearing PTCs and their potentially harmful truncated protein products , thus functioning as an important supplement to other mRNA surveillance systems in mammalian cells .", "Unlike classic NMD , miRNA-mediated surveillance is EJC-independent and recognizes its targets only if an miRE is embedded in the PTC-STOP region ( Figure 1A ) at a position as close as 10 nt downstream of the PTC ( Figure 2F , G ) .", "The repressive influence of miRNA is enhanced by combining accelerated mRNA degradation and translational inhibition ( Figure 2C–E ) .", "Furthermore , miRNA-mediated surveillance can work together with other surveillance mechanisms , such as EJC-NMD , in an additive manner ( Figure 4C , D ) , which strengthens the repressive activity and expands the substrate spectrum of the cellular mRNA quality control system .", "An estimated one-third of genetic diseases are associated with truncated proteins produced from nonsense mRNAs ( Kuzmiak and Maquat , 2006 ) .", "Indeed , truncated BRCA1 has been shown to antagonize wild-type BRCA1 function in a dominant-negative manner ( Fan et al . , 2001; Sylvain et al . , 2002 ) , and several lines of evidence support the view that truncated APC contributes to colorectal cancer by causing spindle misalignment that eventually leads to chromosomal instability ( Fodde et al . , 2001; Tighe et al . , 2004; Quyn et al . , 2010 ) .", "As predicted by a 2–7 seed match algorithm , the nonsense mutants of APC and BRCA1 contain many potential conventional miREs in their PTC-STOP regions and several of them are experimentally validated in the cell lines , suggesting that these disease-causing mutants may be targets of miRNA-mediated surveillance .", "Recent studies have revealed that many unconventional miREs exist in animals ( Shin et al . , 2010; Helwak et al . , 2013 ) , raising the possibility that many more functional miREs may be present than would be predicted by a search algorithm based on the 2–7 seed match rule .", "In addition , targets for only a limited number of miRNAs were sought , which could also lead us to underestimate the frequency with which miREs are present in the PTC-STOP regions .", "The abundance of some endogenous miRNAs is already very high in HEK293 cells , which could lead them to falsely be scored as negative in our mimic-based screening .", "Such miRNAs could be identified by performing the screening in multiple cell lines that have different miRNA profiles .", "Moreover , the contribution of miRNAs to the overall level of repression may be even greater if we count the inhibitory effect of miRNAs on translation , which would not be evident in the RNA sequencing data ( MacArthur et al . , 2012 ) .", "Interestingly , we found that a luciferase reporter harboring the miR-29a miREs from the PTC-STOP region of APC nonsense mutant is efficiently repressed only in colon-derived SW480 cells but is not in kidney-derived HEK293 cells ( Figure 3—figure supplement 4B , D ) , which correlates well with the relatively high abundance of miR-29a in SW480 cells and its undetectable level of expression in HEK293 cells ( Figure 3F–H ) .", "This finding implies that a nonsense mRNA may have a very different fate in distinct tissues depending on which miRNAs are highly expressed .", "It is well established that the expression of many miRNAs are tissue- or cell type-specific and are dynamically regulated during cell differentiation or under different physiological conditions ( Houbaviy et al . , 2003; Liu et al . , 2004; Lu et al . , 2005; Marsit et al . , 2006; Landgraf et al . , 2007; Liang et al . , 2007 ) .", "Some nonsense mRNAs may escape miRNA-mediated surveillance due to the lack of expression of certain miRNAs in some tissues or under some stress conditions , thereby increasing the risk of tissue-specific diseases .", "In addition to many identified nonsense messages resulting from genomic mutations , evidence suggests that ∼35% of alternatively spliced gene products contain PTCs ( Lewis et al . , 2003; Wollerton et al . , 2004 ) .", "Transcripts with retained introns , for example , are efficiently degraded by miRNAs , as shown by our BG reporter assays ( Figure 1D ) .", "Considering that introns tend to be very long and rich in repeats , we believe many more transcripts with retained introns may be targeted by miRNAs .", "Consistent with this idea , more than 10% of Ago-CLIP reads map to introns ( Chi et al . , 2009; Hafner et al . , 2010 ) .", "Our computational analyses of transcriptome and published Ago-CLIP sequencing data from two human cell lines revealed that hundreds of PTC-containing transcripts generated by intron retention are indeed bound by Ago in the PTC-STOP regions ( Supplementary file 3 ) , which makes them attractive candidates of miRNA-mediated surveillance .", "Taken together , we have uncovered a new role for miRNAs in mRNA quality control .", "This miRNA-mediated surveillance system acts in parallel with other systems , such as EJC-NMD , to provide extra protection for the cells against nonsense mutations .", "We have also demonstrated that APC and BRCA1 nonsense mutant mRNAs are downregulated by multiple miRNAs that specifically target the PTC-STOP regions , thereby providing a feasible means to eliminate such deleterious nonsense transcripts without impairing their wild-type counterparts ." ], [ "The rabbit β-globin coding region was amplified by PCR from the pBBB plasmid ( Shyu et al . , 1989 ) and placed downstream of a tet-off promoter to generate the plasmid TBG .", "LastEx-PTC was constructed by mutating one nucleotide at codon 121 to introduce a PTC in TBG .", "The LastEx-L7 and LastEx-PTC-L7 plasmids were constructed by inserting one let-7a miRE originating from human LIN-28 ( GCACAGCCTATTGAACTACCTCA ) in-frame into TBG and LastEx-PTC .", "hp-LastEx-L7 is identical to LastEx-L7 except for the presence of a stable hairpin ( GGGGCGCGTGGTGGCGGCTGCAGCCGCCACCACGCGCCCC ) in the β-globin 5′ UTR 29 nt upstream of the initiation codon .", "A Renilla luciferase ORF was fused to the 5′ end of BG ORF to create a RL-BG chimeric reporter , and then the stable hairpin was placed in the 5′ UTR at the same position as hp-LastEx-L7 to generate hp-RL-BG .", "Two nucleotides within the let-7a miRE seed region were mutated without changing the amino acids to generate LastEx-PTC-L7M and hp-LastEx-L7M .", "Two nucleotides of the 5′ splicing site and three nucleotides of the 3′ splicing site within the final intron of TBG or LastEx-L7 were mutated to create TBG-IR or TBG-IR-L7 .", "Two nucleotides within the let-7a miRE seed region were mutated without changing the amino acids to generate TBG-IR-L7M .", "For the second set of TBG plasmids containing a miR-21 miRE , all the other designs are identical to the TBG plasmids with a let-7a miRE except for two differences: ( 1 ) the PTC site was introduced at codon 116 , and ( 2 ) an artificial miR-21 miRE complementary to miR-21 with three centered mismatches ( TCAACATCAGAGAGATAAGCTA ) was inserted in-frame into TBG .", "The plasmid 3′UTR-L7 has been described ( Wu et al . , 2006 ) .", "The let-7a miRE between the NheI and XbaI sites was replaced by a miR-21 miRE to generate 3′UTR-21 .", "PTC102 , PTC102-L7 , and PTC102-L7M are identical to LastEx-PTC , LastEx-PTC-L7 , and LastEx-PTC-L7M , respectively , except that the PTC site was introduced at codon 102 .", "The let-7a miRE or its mutant in PTC102-L7 or PTC102-L7M was replaced with a miR-21 miRE or its mutant to generate PTC102-21 and PTC102-21M .", "Two tandem miR-125b miREs ( GGTATCACAAGTTACAATCTCAGGGATAGCCAAGGTATCACAAGTTACAATCTCAGGGATAGCCAATTCTTAAT ) were inserted between the EcoRI and XbaI sites 59 nt downstream of the firefly luciferase ORF with modified 3′ UTR sequences containing an additional stop codon to generate TAA-2E .", "The original stop codon of the luciferase gene was disrupted by a point mutation to obtain TCA-2E .", "One miR-125b miRE was inserted in-frame into a modified firefly luciferase plasmid at different positions before or after the PTC to generate a series of miRE-containing luciferase plasmids .", "The APC minigene plasmid was constructed by placing an HA-tagged APC ORF with or without its natural 3′ UTR downstream of the CMV promoter .", "An HA-tagged EGFP ORF was driven by an SV40 promoter from the same plasmid backbone .", "PTCs and the corresponding miRE mutations were generated by site-directed mutagenesis .", "miRE screening plasmids for APC , BRCA1 , and all of the selected candidates from HCT-116 cells were constructed by fusing corresponding PTC-STOP regions to the 3′ end of a firefly luciferase ORF .", "miRE validation plasmids were constructed either by inserting ∼30 nt sequences that surround predicted miRE seeds into the 3′ UTR of a firefly luciferase reporter gene or by fusing the whole PTC-STOP region of each candidate with the mutated miRE seed to the 3′ end of the firefly luciferase ORF .", "The last 738 nt of APC coding region ( PTC-STOP region ) plus the entire 3′ UTR were fused to the 3′ end of a firefly luciferase ORF to generate pFL-APC-WT .", "The miR-29a miREs and miR-135b miRE were disrupted individually or together by site-directed mutagenesis to generate pFL-APC-29M , pFL-APC-135M , and pFL-APC-29M+125M .", "The sequence of the APC natural 3′ UTR was cloned downstream of a firefly luciferase gene to generate pFL-APC-3′UTR .", "Mature miR-29a or a negative control small RNA sequence siEGFP ( AACTTCAGGGTCAGCTTGCCG ) was cloned into the inducible TRIPZ lentiviral shRNA vector to generate miRNA-overexpression lentiviruses .", "A miRNA decoy sequence ( TuD-NC or TuD-29a ) was cloned into the GIPZ constitutive lentiviral vector to generate miRNA-knockdown lentiviruses .", "pFL-BRCA1 is identical to the BRCA1 miRE screening plasmid .", "pFL-BRCA1in harbors two introns downstream of the luciferase stop codon .", "Functional miR-137 and miR-544 miREs within the BRCA1 PTC-STOP region were subjected to a synonymous mutation to generate pFL-BRCA1-mut and pFL-BRCA1in-mut .", "HEK293 , 293T , HCT-116 , and SW480 cells were purchased from ATCC ( Manassas , Virgina , USA ) .", "HeLa-tTA cells were purchased from Clontech ( Mountain View , California , USA ) .", "All cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum ( Invitrogen , Carlsbad , California , USA ) .", "To produce the lentiviruses , 293T cells were transfected with a virus vector encoding the expression cassette as well as the VSVG and ΔR8 . 91 plasmids .", "Viruses were harvested at 48 and 72 hr post-transfection .", "HEK293 , HCT-116 , and SW480 stable cell lines were generated by transduction with lentiviruses in the presence of 8 μg/ml of polybrene overnight , followed by a 1-week puromycin and/or hygromycin selection .", "Endogenous GW182 of HeLa-tTA cells was depleted using protocols described elsewhere ( Piao et al . , 2010 ) .", "siRNAs were synthesized by GenePharma ( Shanghai , China ) and the mature sequences are as follows: siNC: UUCUCCGAACGUGUCACGUUU; siGW182: UUGAGCACGGAGAUUAGGCUG .", "HeLa-tTA cells were plated on 35-mm plates 1 day before transfection in DMEM containing 20 ng/ml tet .", "In total , 350 ng of the BG reporter plasmid was used for transfection .", "The transcription of BG mRNA was induced by removing tet 12 hr after transfection .", "After 3 hr of induction , tet was added to a final concentration of 1 μg/ml to block the transcription of BG .", "Cytoplasmic RNA was then isolated at various time intervals .", "Equal amounts of RNA were treated with RNase H in the presence of an oligodeoxynucleotide ( GGTTGTCCAGGTGACTCAGACCCTC ) complementary to codons 74–81 within the BG coding region .", "For BG reporters with a retained last intron , RNA samples were treated with RNase H in the presence of an oligodeoxynucleotide ( CAGTGTATATCATTGTAACCATAAA ) complementary to a region within the last intron which is 81 nt upstream of the final exon .", "The digested RNA samples were then analyzed by electrophoresis ( 5 . 5% PAGE with 8 M urea ) and Northern blotting as previously described ( Wu et al . , 2006; Wu and Belasco , 2008a ) .", "For measuring BG mRNA half-life , a constitutively transcribed EGFP mRNA was co-transfected with BG plasmids .", "In the miR-125b repression assays , HEK293 cells cultured in a 12-well plate were transfected with a firefly luciferase reporter ( TAA-2E , TCA-2E , or a series of firefly luciferase reporters with one miR-125b miRE embedded before or after the PTC , 10 ng ) , pRL ( 10 ng ) , and a plasmid encoding or not encoding miR-125b ( pMIR125b or pMIR125bΔ , respectively; 480 ng ) .", "In the miRE screening assays of APC , BRCA1 , and computationally identified candidates from HCT-116 cells , HEK293 cells cultured in a 24-well plate were transfected with a firefly luciferase reporter containing the corresponding PTC-STOP region of the selected candidate ( 20 ng ) , pRL ( 10 ng ) , and 10 pmol of a synthetic miRNA mimic .", "In the miRE validation assays , HEK293 cells cultured in a 24-well plate were transfected with a vector encoding both firefly and Renilla luciferase ( 10 ng ) and 10 pmol of the cognate miRNA mimics .", "In the PTC-STOP and 3′ UTR miRE additive effect assay , HEK293 cells cultured in a 24-well plate were transfected with a firefly luciferase reporter having a partial ORF and the entire 3′ UTR of APC fused to the 3′ end of the luciferase ORF ( pFL-APC-WT or its miRE mutant counterparts , 10 ng ) , pRL ( 10 ng ) , and 10 pmol of siNC or miR-29a and miR-135b mimic mixture .", "To examine potential miR-29a miREs in the natural APC 3′ UTR , HEK293 cells cultured in a 24-well plate were transfected with a normal firefly luciferase reporter ( pFL , 10 ng ) or a reporter bearing a full length APC 3′ UTR sequence in the luciferase 3′ UTR ( pFL-APC-3′UTR , 10 ng ) , pRL ( 10 ng ) , and 10 pmol of siNC or miR-29a mimics .", "In the miRNA-mediated surveillance and EJC-NMD additive effect assay , HEK293 cells cultured in a 24-well plate were transfected with a firefly luciferase reporter harboring the BRCA1 PTC-STOP region with or without introns ( pFL-BRCA1 or its miRE mutant version pFL-BRCA1-mut , 20 ng; pFL-BRCA1in or its miRE mutant version pFL-BRCA1in-mut , 33 . 3 ng ) , pRL ( 10 ng ) , and 10 pmol of siNC or miRNA mimics .", "In the GW182 knockdown assay , siRNA-treated HeLa-tTA cells cultured in a 24-well plate were transfected with pFL-BRCA1 ( 20 ng ) or pFL-BRCA1in ( 33 . 3 ng ) , together with 10 ng pRL .", "In the tissue-specific repression of miRNA-mediated surveillance and miR-29a knockdown assays , HEK293 cells , SW480 cells , and TuD-NC- or TuD-29a-overexpressing SW480 cells cultured in a 24-well plate were transfected with a vector encoding both a firefly and a Renilla luciferase reporter ( 20 ng ) with the APC miR-29a miREs or the mutant form in the 3′ UTR of the firefly luciferase gene .", "In all luciferase assays , values represent means±SD from at least three independent experiments .", "In total , 2 μg cytoplasmic RNA isolated from HEK293 cells was treated with 1 U DNase I ( Fermentas , Burlington , Ontario , Canada ) and was then reverse transcribed using M-MLV ( TAKARA , Otsu , Shiga , Japan ) , according to the manufacturer's instructions .", "Real-time PCR was performed on a StepOnePlus real-time PCR system ( Applied Biosystems , Foster City , California , USA ) with Power SYBR Green PCR Master Mix ( Applied Biosystems , Foster City , California , USA ) .", "The PCR mixtures were heated to 95°C for 10 min and then subjected to 40 amplification cycles ( 15 s at 95°C , 1 min at 60°C ) .", "For protein expression from the APC minigene construct , 1 μg of the minigene vector encoding both HA-APC and HA-EGFP and 100 pmol of the synthetic miRNA mimics were transfected into HEK293 cells cultured in 6-well plates using Lipofectamine 2000 ( Invitrogen , Carlsbad , California , USA ) .", "After 36 hr , protein extracts of transfected cells were separated on 7 . 5% polyacrylamide-SDS gels by electrophoresis .", "For blotting full length APC , 6% gels were used .", "After electrophoresis , the gels were cut according to the pre-stained protein size marker .", "The portion that contained the internal control ( HA-EGFP , GAPDH , or Tubulin ) was transferred to a nitrocellulose membrane using standard transfer buffer ( 25 mM Tris , 192 mM glycine , 10% methanol ) at 200 mA for 2 hr .", "The portion that contained the APC protein was transferred with a different transfer buffer ( 50 mM Tris , 380 mM glycine , 0 . 2% SDS , 5% methanol ) at 25 V for 20 hr .", "After blocking with 5% non-fat milk in phosphate-buffered saline ( PBS ) containing 0 . 05% Tween-20 ( PBST ) for 30 min at room temperature , the blot was probed for 1 hr at room temperature with anti-HA antibodies ( 1:3000; ImB , Shanghai , China ) , anti-APC antibodies ( 1:200; Millipore , Billerica , Massachusetts , USA ) , anti-GAPDH antibodies ( 1:3000; Bioworld , St . Louis Park , Minnesota , USA ) , or anti-α-Tubulin antibodies ( 1:5000; Sigma-Aldrich , St . Louis , Missouri , USA ) diluted in PBST buffer with 1% BSA , then incubated with a secondary antibody conjugated to horseradish peroxidase ( 1:10 , 000; Jackson ImmunoResearch Laboratories , West Grove , Pennsylvania , USA ) in 5% non-fat milk , and detected with an Immun-Star HRP chemiluminescence kit ( Bio-Rad , Hercules , California , USA ) .", "For validation of GW182 knockdown in HeLa-tTA cells , an anti-GW182 antibody ( 1:1000; MBL , Nagoya , Japan ) was used .", "For RIP assays of ectopically expressed PTC-APC mRNAs , HEK293 cells that stably express FLAG-tagged Ago2 were plated in 60-mm plates 1 day before transfection .", "A total of 2 μg of APC-PTC1450 or APC-PTC1450-mut plasmid with 150 pmol of siNC or miR-29a mimic was transfected into the cells .", "Then 36 hr after transfection , cells were trypsinized , collected , and washed with 10 ml PBS twice .", "The pelleted cells were lysed in 200 μl PLB buffer ( 100 mM KCl , 5 mM MgCl2 , 10 mM HEPES , 0 . 5% NP-40 , 1 mM DTT , 100 U/ml RNase inhibitor ) .", "The cleared lysate was incubated with anti-FLAG affinity gel ( Sigma-Aldrich , St . Louis , Missouri , USA ) for 1 hr .", "Beads were washed with 1 ml NT-2 buffer ( 50 mM Tris , 150 mM NaCl , 1 mM MgCl2 , 0 . 05% NP-40 ) five times .", "Washed beads were re-suspended in 250 μl NT-2 buffer and RNA was isolated using Trizol LS Reagent ( Ambion , Austin , Texas , USA ) and analyzed by qRT-PCR .", "For RIP assays of endogenous PTC-APC mRNAs , SW480 cells that stably express FLAG-tagged Ago2 were cultured in one 100-mm plate and were used for RIP assay after they reached 80% confluency ." ] ]
[ "Numerous studies have established important roles for microRNAs ( miRNAs ) in regulating gene expression .", "Here , we report that miRNAs also serve as a surveillance system to repress the expression of nonsense mRNAs that may produce harmful truncated proteins .", "Upon recognition of the premature termination codon by the translating ribosome , the downstream portion of the coding region of an mRNA is redefined as part of the 3′ untranslated region; as a result , the miRNA-responsive elements embedded in this region can be detected by miRNAs , triggering accelerated mRNA deadenylation and translational inhibition .", "We demonstrate that naturally occurring cancer-causing APC ( adenomatous polyposis coli ) nonsense mutants which escape nonsense-mediated mRNA decay ( NMD ) are repressed by miRNA-mediated surveillance .", "In addition , we show that miRNA-mediated surveillance and exon–exon junction complex-mediated NMD are not mutually exclusive and act additively to enhance the repressive activity .", "Therefore , we have uncovered a new role for miRNAs in repressing nonsense mutant mRNAs ." ]
[ "To produce a protein from a gene , the sequence of the gene must be transcribed to produce a molecule of messenger RNA ( mRNA ) .", "The sequence of the mRNA is then read in groups of three letters at a time ( called codons ) , and each codon instructs for a particular amino acid to be added into the protein .", "Some codons , however , do not code for an amino acid and instead these ‘stop codons’ mark the end of a protein .", "If a DNA letter is added , lost , or changed , this mutation can sometimes produce a stop codon too early in the mRNA sequence .", "This is called a nonsense mutation , and produces truncated proteins that either work incorrectly or do not work at all , which can harm the organism .", "For example , people with a nonsense mutation in the human tumor suppressor gene called APC—which normally stops uncontrolled cell growth and division—are more likely to develop colon cancer than people without this mutation .", "Cells in the body employ several different surveillance mechanisms to detect nonsense mutations .", "The best-known mechanism involves a large protein group called the exon–exon junction complex ( EJC ) , which binds to sites within the mRNA .", "The cellular translation machinery removes all the EJCs bound to a normal mRNA during the production of proteins .", "If the translation machinery reaches a stop codon too early , so that EJCs located downstream of it are not removed , the mRNA molecule is destroyed .", "However , this mechanism does not work for all genes—including APC .", "Very short sections of RNA called microRNAs regulate protein production by causing mRNAs to degrade and by inhibiting their translation , and Zhao et al . have now found that microRNAs also act as a defense against nonsense mutations in the APC gene .", "A premature stop codon exposes sites further along the mRNA molecule that microRNA molecules bind to , which triggers the breaking down of the mRNA and inhibits its translation .", "The microRNA surveillance system works independently of the system involving the EJC .", "However , both mechanisms can work in parallel alongside each other , which provides extra protection against nonsense mutations .", "Zhao et al . also found that microRNAs can protect against nonsense mutations in several other types of gene found in human cells .", "Therefore , microRNA surveillance is likely to be a common method employed by cells to restrict the production of potentially harmful truncated proteins ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
A family of photoswitchable NMDA receptors
elife-12040-v2
[ [ "NMDA receptors are ligand-gated ion channels at excitatory synapses throughout the nervous system .", "They trigger long-term potentiation ( LTP ) and long-term depression ( LTD ) of synaptic strength and are implicated in memory formation , synapse development , circuit refinement , neuropsychiatric disorders , excitotoxicity and neurodegeneration ( Lau and Zukin , 2007; Nabavi et al . , 2014 ) .", "NMDA receptors are heterotetramers , consisting of two glycine-binding GluN1 subunits paired with two glutamate-binding GluN2 and/or glycine-binding GluN3 subunits , yielding a combinatorial diversity of channel composition and function ( Paoletti et al . , 2013; Traynelis et al . , 2010 ) .", "The glutamate-binding GluN2 subunits ( GluN2A-D ) are encoded by four genes , which appear at specific developmental stages , bind distinct regulatory proteins , and are situated at diverse cellular locations ( Hardingham and Bading , 2010 ) .", "The complexity of subunit stoichiometry and receptor localization has made it difficult to unravel the roles of NMDA receptor signaling in circuit function and behavior .", "Much has been learned about the function of NMDA receptors from pharmacological and genetic manipulations that target specific receptor subtypes ( Foster et al . , 2010; Tang et al . , 1999; Traynelis et al . , 2010 ) .", "However , pharmacological agents need to be used at low concentrations to maintain selectivity , resulting in slow onset , and are slow to wash out due to high affinity , are difficult to confine spatially , and generally cannot be targeted to a specific cell .", "Genetic manipulations , like gene-knockout or RNA-interference , provide subunit-specificity , but are either for the life of the organism or , when conditional , slow to turn on and cannot typically be turned off .", "These chronic effects can lead to circuit changes and compensation ( Nakazawa et al . , 2004; Rossi et al . , 2015 ) .", "Moreover , the ability to confine these manipulations to specific brain regions and cell types is limited .", "The problems of spatial and temporal control have been addressed by the advent of caged versions of glutamate , NMDA and the pore blocker MK-801 , which can be photo-uncaged in very small volumes at precise times ( Huang et al . , 2005; Kohl et al . , 2011; Matsuzaki et al . , 2001; Palma-Cerda et al . , 2012; Rodríguez-Moreno et al . , 2011 ) .", "MK-801 can work from inside the cell and , therefore , can be loaded via the patch pipet , whereas glutamate and NMDA cannot and so cannot be targeted precisely to a specific cell; furthermore , none of these compounds are selective for receptor subtype .", "A recent development has been the subunit-specific control of an ion channel with a photo-reactive unnatural amino acid that enables photo-inactivation .", "So far , this methodology has been applied to a potassium channel ( Kang et al . , 2013 ) , AMPA receptor ( Klippenstein et al . , 2014 ) and GluN2B-containing NMDA receptors ( Zhu et al . , 2014 ) .", "However , photo-inactivation requires intense and prolonged irradiation with UV light and , importantly , is irreversible .", "To overcome the above obstacles , we set out to endow individual GluN subunits with fast and reversible light-switching via the site-directed , on-cell attachment of a Photoswitched Tethered Ligand ( PTL ) .", "We employed PTLs from the 'MAG' family ( Figure 1a ) , which consist of Maleimide ( for covalent attachment to a cysteine residue substituted onto the water exposed surface of the ligand binding domain of the GluN subunit ) , a photo-isomerizable Azobenzene linked to a Glutamate ligand ( for synthesis see [Volgraf et al . , 2006] ) .", "Illumination with near UV light ( 360–405 nm; violet light ) isomerizes MAG into the bent cis-configuration , whereas illumination with blue-green light ( 460–560 nm; green light ) isomerizes MAG to the trans-configuration ( Figure 1a ) ( Gorostiza and Isacoff , 2008 ) .", "By choosing geometrically favorable positions for introduction of the cysteine attachment site ( Figure 1b and Figure 1—figure supplement 1 ) and altering the length of the MAG molecule by varying the linker via additional glycine ( s ) ( Figure 1a , dashed brackets; n ) , the glutamate moiety of MAG can be designed to either engage or obstruct the ligand binding pocket in the cis configuration , and withdraw from the ligand binding pocket in the trans configuration , yielding light-dependent gating ( Figure 1c and Figure 2a , but see also [Numano et al . , 2009] ) .", "PTLs , including MAGs , have been employed to generate light-gated ionotropic kainate receptors ( Janovjak et al . , 2010; Reiner et al . , 2015; Szobota et al . , 2007; Volgraf et al . , 2006 ) , metabotropic glutamate receptors ( Levitz et al . , 2013 ) , nicotinic acetylcholine receptors ( Tochitsky et al . , 2012 ) , P2X receptors ( Lemoine et al . , 2013 ) and GABAA receptors ( Lin et al . , 2014 ) . 10 . 7554/eLife . 12040 . 003Figure 1 . Photo-agonism of NMDA receptors in HEK293 cells and hippocampal neurons .", "( a ) MAG photoswitches showing chemical structure and cartoon depiction .", "MAG0 and MAG1 differ in length ( brackets , n = 0 , 1 ) ( Gorostiza et al . , 2007 ) , whereas L-MAG and D-MAG ( Levitz et al . , 2013 ) differ in stereochemistry ( red asterisk ) .", "Illumination with 360–405 nm light photoisomerizes MAG from the elongated trans- azobenzene ( red ) configuration to the bent cis configuration; illumination at ~460–560 nm returns MAG to the trans isomer , as does slow thermal relaxation in the dark .", "The maleimide group ( cyan ) allows attachment to an engineered cysteine .", "( b ) Space filled , crystal structure of GluN2A’s Ligand Binding Domain ( LBD , PDB-2A5S ( Furukawa et al . , 2005 ) ) showing the site of glutamate binding ( blue sticks ) as well as the nearby V713 position ( green spheres ) , which was mutated to a cysteine to which maleimide tethers and yields photo-agonism .", "( c ) Cartoon depiction of a photo-agonized NMDA receptor showing , for simplicity , only one of the two LiGluN1a-wt subunits ( purple ) co-assembling with one of the two engineered LiGluN2A subunits ( pink ) .", "The MAG photoswitch ( color-coded sticks; as shown in", "a ) is covalently attached to the LBD ( cyan dot ) to endow the channel with light-sensitivity .", "The cis configuration allows docking of the glutamate headgroup ( orange triangle ) into the binding pocket ( middle cartoon ) , inducing LBD closure and channel opening ( rightward cartoon ) .", "For simplicity the ligand of the GluN1 subunit ( glycine ) is not shown .", "Light- and glutamate/NMDA-induced currents in", "( d ) HEK293 cells or", "( e ) hippocampal neurons ( from wt rats ) expressing GluN1a-wt and GluN2A ( V713C ) labeled with L-MAG1 .", "Photo-current is elicited by 380 nm light ( ~3 mW/mm2 ) ( violet bar ) and turned off by 500 nm light ( ~3 mW/mm2 ) ( green bar ) .", "Full activation is induced by application of 1 mM glutamate or NMDA ( black bar ) .", "( f ) Representative trace ( averaged from 4 consecutives sweeps ) for fast MAG photoisomerization by an intense 375 nm light pulse ( 2 ms at ~20 W/mm2 ) leading to rapid activation and opening of GluN2A ( V713C ) ( red trace is a monoexponential fit , τ indicated ) .", "( g-h )", "Representative traces ( 2 cells ) for tuning the off kinetics of the photo-current by applying high intensity ( ~12 mW/mm2 , average of 8 consecutive sweeps ) for fast deactivation", "( g ) or low green light intensity ( ~1 . 5 mW/mm2 , average trace from 4 consecutives sweeps ) for slow deactivation", "( h ) .", "( i ) Representative image ( left , scale bar 10 µm ) and trace ( right ) showing the bistability of the photoswitch in a cultured hippocampal neurons ( from wt rats ) transfected with GluN2A ( V713C ) -only .", "The cis state of MAG is photo-stable , so that following illumination ( targeted line-scanning ) by a brief 405 nm laser pulse ( 2 s ) over a large region of the dendritic tree ( dashed violet box ) induces an inward current ( black trace , ON ) that persists in the dark without visible decay for tens of seconds ( see also Figure 1—figure supplement 3 ) .", "Likewise , following closure of the channel ( black trace- OFF ) with brief green light ( 488 nm ) illumination ( 2 sec ) , the channel remains shut and no current is observed unless triggered anew by violet light .", "Spontaneous EPSCs are observed during the opening and closing of the channel . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 00310 . 7554/eLife . 12040 . 004Figure 1—figure supplement 1 . Screen of GluN2A cysteine positions and MAG variants .", "( a ) Amino acids near the glutamate binding site of GluN2A were individually replaced with cysteine by site-directed mutagenesis , co-transfected with wild-type GluN1a into HEK293 cells , and tested with several MAG variants: L-MAG0 , L-MAG1 , L-MAG2 and D-MAG1 , previously used ( Levitz et al . , 2013; Volgraf et al . , 2006 ) .", "Photo-responses were detected by calcium-imaging with Fura2-AM and characterized as agonist or antagonist by photo-switching in the absence and presence of 1 mM glutamate , respectively .", "Most photo-responses , albeit small , were detected during illumination with 380 nm light ( violet ) , one during illumination at 500 nm ( green ) , and several exhibited no photo-response ( grey ) .", "The colors in the first column correspond to the sites group-colored in the structure of GluN2A’s LBD on the left ( PDB-2A5S [Furukawa et al . , 2005] ) .", "The largest agonistic and antagonistic photo-responses yielded by violet light illumination ( emphasized by solid boxes and colored fonts ) were GluN2A ( V713C ) conjugated to L-MAG1 and GluN2A ( G712C ) conjugated to L-MAG0 , respectively , and were subsequently scrutinized using patch clamping techniques ( examples shown in b , c and see main figures ) .", "( b-c )", "Repeatable manipulation of photo-switching speed .", "Near-UV and green light illumination was switched at varying frequencies to mimic different modes of transmissions and or experimental paradigms .", "Representative traces from cultured hippocampal neurons ( from wt rats ) showing the versatility of the photo-switching technique and responses in neurons .", "Illumination at 405 nm ( violet bars ) is used to open the channels ( downward inflection of the current ) , whereas 488 nm ( green bars , upward inflection of the current ) to close them .", "This could be done repeatedly at low", "( b ) or high switching frequency", "( c ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 00410 . 7554/eLife . 12040 . 005Figure 1—figure supplement 2 . Pharmacological characterization of GluN2A ( V713C ) , GluN2A ( G712C ) , GluN2B ( V714C ) and GluN1a ( E406C ) .", "Wild-type ( wt ) and engineered GluNRs were expressed in Xenopus oocytes and ligand-induced currents were recorded by two-electrode voltage-clamp ( Vm = −60 mV , 0 Mg2+ ) .", "( a ) GluN1a-wt and GluN2A ( wt , V713C or 712C ) currents ( normalized ) in response to increasing glutamate concentrations alone ( black , red , blue , respectively ) , summarized in", "( b ) .", "( c ) GluN1a-wt and GluN2B ( wt or V714C ) currents ( normalized ) in response to increasing glutamate concentrations alone ( black , green respectively ) , summarized in", "( d ) .", "( e ) GluN1a ( wt or E406C ) and GluN2B-wt currents ( normalized ) in response to increasing glycine concentrations alone ( black , purple , respectively ) , summarized in", "( f ) .", "( g-h )", "Activation kinetics of GluNRs containing the LiGluNx subunits .", "Activation kinetics were assessed by fast uncaging of 500 µM MNI-glutamate over the entire HEK293 cells ( pink bar- 1 ms pulse , 375 nm , ~50 W/mm2 ) of ( Matsuzaki et al . , 2001 ) during whole cell voltage-clamp recording .", "The uncaging elicited rapid activation of wt and engineered GluN2A ( V713C or G712C ) subunits", "( g ) or wt and engineered GluN2B ( V714C ) subunits", "( h ) co-expressed with GluN1a-wt in HEK293 cells .", "In g , bar graphs show mean ± SD , n . s . , not significant , one way ANOVA , Dunnet post hoc test or h; two-tailed , unpaired t-test , n shown within bars .", "Non parametric Mann-Whitney Rank Sum Test . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 00510 . 7554/eLife . 12040 . 006Figure 1—figure supplement 3 . Lack of perturbation of neurons by MAG or LiGluN2A ( G712C ) .", "( a ) MAG-exposure does not cause cell death or damage membrane integrity of cultured hippocampal neurons .", "Representative images of neurons ( 15 DIV ) from wt animals following incubation for 45 min with either L-MAG1 , 0 . 3% DMSO ( in NMDG buffer ) or NMDG buffer alone ( see Materials and methods ) showed no reduction in cell viability ( green labeling ) or membrane integrity ( red labeling , neurites ) compared to untreated cells ( no treatment prior labeling ) and Summary ( right ) ( N coverslips= no treatment ( no treatm . ) - 2 , +NMDG- 2 , +DMSO- 5 , +L-MAG1- 9 ) .", "Values are mean ± SEM ( n . s . , not significant , one way ANOVA , post hoc Tukey test , all pairwise comparison ) .", "( b ) Expression of GluN2A ( G712C ) combined with exposure to L-MAG0 does not perturb electrophysiological properties of neurons ( from wt rats ) in comparison to non-transfected neurons that were also exposed to MAG .", "No change in resting potential ( left ) or resting membrane resistance ( right ) was observed between non-transfected ( black bars ) and GFP-NR2A ( G712V ) -transfected ( cyan bars ) neurons .", "( c-d )", "No effect of MAG on native receptors or channels .", "( Left )", "Representative trace showing a non-transfected hippocampal neuron , though treated with L-MAG1 , that does not display any light-responses to either 380 nm light ( violet bar ) or 500 nm light ( green bar ) ; before or during application ( and washout ) of 1 mM NMDA .", "Summary of the effect of light on the current in individual non-transfected neurons ( exposed to L-MAG1 , middle panel ) and box plot representation with median and outliers ( filled circles , right panel ) displayed .", "( d ) Transfection of GFP-GluN2A ( G712C ) ( orange bar ) did alter GluNR-expression compared to GFP only -transfected neurons ( green bar ) , as no change in total current amplitude induced by 1 mM NMDA was observed .", "To note , these observations are in line with earlier reports demonstrating that MAG ( and related PTLs ) do not have non-specific action or adverse effect on neuronal physiology ( Caporale et al . , 2011; Lemoine et al . , 2013; Levitz et al . , 2013; Li et al . , 2012; Szobota et al . , 2007; Tochitsky et al . , 2012; Volgraf et al . , 2006 ) .", "( e ) Representative trace showing the bistability of the photoswitch .", "The cis state of MAG is photo-stable , so that following activation by a brief 380 nm light pulse the current persists in the dark without visible decay for 60 s ( see also Figure 1i and [Reiner and Isacoff , 2014] ) .", "Statistics in panels", "( b ) and", "( d ) are shown as mean ± SEM , tested with two-tailed , unpaired t-test; n . s . - not-significant , n , shown within bars . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 00610 . 7554/eLife . 12040 . 007Figure 2 . Rapid development of light-agonized LiGluN2B subunit , based on LiGluN2A .", "( a ) Partial sequence alignment ( left ) and overlaid crystal structures ( ribbon ) of the LBDs of GluN2A ( blue , PDB-2A5S ( Furukawa et al . , 2005 ) ) and GluN2B ( grey , PDB- 4PE5 [Karakas and Furukawa , 2014] ) showing the high degree of similarity between the two LBDs and the corresponding mutation in GluN2B ( V714 ) to that of LiGluN2A ( V713 ) ( dashed circle , Valine; color-coded spheres ) .", "( b ) Representative trace of photo-agonism of LiGluN2B ( V714C ) .", "A HEK293 cell transfected with GluN1a and LiGluN2B ( V714C ) , and labeled with L-MAG1 , when illuminated with 380 nm light ( violet bars ) produces an inward photo-current that can be turned off by 510 nm light ( green bars ) .", "Note the small increase in current during glutamate perfusion , suggesting that L-MAG1 may act as a stronger agonist than glutamate .", "( c ) LiGluN2B ( V714C ) photocurrents are of similar size , but slower to turn off than those of LiGluN2A ( V713C ) .", "Hippocampal neurons ( from wt rats ) , transfected with either LiGluN2A ( top black trace ) or -2B ( bottom grey trace ) exhibit similar inward photo-currents , during which barrages of spontaneous EPSCs emerge ( violet bars ) , demonstrating that photo-activation of LiGluNs receptors causes action potential firing of presynaptic neurons ( see also Figure 4 and Figure 4 supplements ) .", "LiGluN2B photocurrents deactivate ~3 times slower than those of LiGluN2A ( insets ) , as well as display consistently longer enduring observable EPSCs , summarized in d-f .", "( d ) Box plot representation of median , outliers ( filled circles ) and individual data points ( filled squares ) are displayed .", "Red trace in", "( f ) is a monoexponential fit , τ indicated .", "Statistics in panels are shown as Box plots of the data ( not normally distributed , see Figure 2—figure supplement 2 ) , showing median , outliers ( filled circles ) and individual data points ( filled squares in", "d ) .", "Significance was tested using a nonparametric Mann-Whitney Rank Sum Test ( see Materials and methods ) , ***p<0 . 001 , n . s . - not-significant , n shown next to plots . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 00710 . 7554/eLife . 12040 . 008Figure 2—figure supplement 1 . Photo-activation of LiGluN2A and -2B drives action potential firing in hippocampal neurons .", "( a-b )", "Representative traces ( current clamp ) of neurons transfected with LiGluN2A ( V713C ) ( a , black trace ) or LiGluN2B ( V714C ) ( b , grey ) ; labeled with L-MAG1 and illuminated with 380 nm ( violet bars ) or 510 nm ( green bars ) light .", "Neurons ( from wt rats ) , held at -45 to -50 mV , during green light illumination display very little activity ( i . e . action potentials , AP ) .", "When switching to violet light and photo-activation of LiGluN2A or -2B , neurons display a significant increase in firing frequency ( FAP , frequency plots shown below traces ) , summarized for LiGluN2A ( V713C ) or -2B ( V714C ) in panels", "( c ) and", "( d ) , respectively .", "The lag from the appearance of violet light to initiation of an action potential ( time to excitation , top insets ) and lag to cessation of firing after toggling back to green light ( time to de-excitation , bottom insets ) are shown and summarized in panels", "( f ) and", "( g ) respectively .", "( e ) No difference in firing frequency was observed between neurons transfected with LiGluN2A ( V713C ) or -2B ( V714C ) .", "Statistics in panels are shown as mean ± SEM , tested with two-tailed , unpaired t-test; *p<0 . 05 , ***p<0 . 001 , n . s . - not-significant , n shown within bars . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 00810 . 7554/eLife . 12040 . 009Figure 2—figure supplement 2 . Summary of nonparametric statistics for Figure 2 . Results from Normality and nonparametric Mann-Whitney Rank Sum Tests are displayed for results shown in Figure 2d–f . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 009 We now report a novel family of four Light-gated GluN subunits , or LiGluNs: 1 ) a light-activated GluN2A , 2 ) a light-activated GluN2B , 3 ) a light-antagonized GluN2A and 4 ) a light-antagonized GluN1 , isoform 1a , ( GluN1a ) .", "The first three LiGluN subunits enable selective manipulation of GluN2A- or GluN2B-containing receptors , whereas the fourth operates as a general controller of all plasma membrane NMDA receptors , owing to the obligatory occurrence of GluN1 in all NMDA receptors .", "We show that LiGluN-containing NMDA receptors function normally , incorporate into synapses , and that their expression does not alter NMDA receptor expression levels .", "Photoswitching can be sculpted to generate NMDA receptor currents that mimic the fast ( GluN2A-like ) or slow ( GluN2B-like ) deactivation kinetics of native excitatory postsynaptic currents ( EPSCs ) .", "Widefield illumination and photo-activation of LiGluN2A or LiGluN2B-containing receptors in primary hippocampal neurons robustly drives activity , whereas photo-antagonism of LiGluN2A and LiGluN1a reversibly block excitatory synaptic currents .", "Spatially-targeted photo-activation of LiGluN2A-containing receptors on single dendritic spines can be used to trigger a spine-specific increase in calcium and the spine expansion that is associated with LTP .", "Complementarily to this , photo-antagonism of LiGluN2A-containing receptors can block LTP-induction via Schaffer collateral stimulation or prevent spine expansion .", "LiGluNs fulfill a major promise of chemical optogenetic photo-pharmacology by providing the kind of spatio-temporally precise control that is obtained with heterologous microbial opsins , that over-ride normal cellular signals , but in this case to control the native neuronal signaling proteins of the synapse that play central roles in synaptic plasticity , learning and memory .", "Together , these tools may open the door to advanced studies of receptor biophysics , and their function in synapses and neural circuits ." ], [ "We , and others , have previously generated light-controlled receptors and channels to manipulate cellular excitability ( e . g . see reviews [Kramer et al . , 2013; Szobota and Isacoff , 2010; Tye and Deisseroth , 2012] ) by photo-regulating the flux of ions across the cell membrane .", "Here , we asked whether light-activated NMDA receptors could be engineered , which would traffic to synapses and function normally and thereby engage the cellular mechanisms of neuronal plasticity .", "Our previous success with light-gated glutamate receptors ( Levitz et al . , 2013; Szobota et al . , 2007 ) along with other reports demonstrating that exogenously expressed GluN2 subunits efficiently co-assemble with endogenous GluN1 subunits and traffic properly to synapses ( Barria and Malinow , 2002 ) , suggested to us that this could be achieved by designing GluN subunits that contain a single cysteine attachment site for anchoring the MAG PTL .", "We therefore systematically introduced single cysteine point mutations at different locations on the GluN2A ligand-binding domain ( LBD ) with proximity and accessibility to the glutamate binding site ( Figure 1—figure supplement 1a ) , and following conjugation to different MAG variants ( Figure 1a ) , assessed the effects of photoswitching in HEK293 cells , first using calcium-imaging and then following with voltage-clamp recordings of the most promising candidates , as shown below .", "We found several attachment positions that yielded light-responses , however focused on variants that when conjugated to L-MAG0 or L-MAG1 , yielded the largest light-responses .", "We initially focused on photo-agonism of the light-gated GluN2A subunits with the cysteine attachment site introduced at residue 713 ( LiGluN2A ( V713C ) ) .", "When LiGluN2A ( V713C ) was co-expressed with wildtype ( wt ) GluN1a , photoisomerization of L-MAG1 to its cis configuration by illumination with 380 nm light ( Figure 1a , c ) generated an inward current ( Figure 1d , e- violet bars ) in both HEK293 cells ( 37 . 3 ± 2 . 2% of the total current induced by 1 mM glutamate , n = 5 , Figure 1d ) and in primary cultured hippocampal neurons ( 30 . 8 ± 5 . 9% of the total current induced by 1 mM NMDA , n = 6 , Figure 1e ) .", "This photocurrent could then be completely turned off by photoisomerization of L-MAG1 to trans by illumination at 488 nm ( Figure 1d , e- green bars ) .", "Since the isomerization of the azobenzene is fast ( Figure 1f ) ( Reiner and Isacoff , 2014 ) and fully reversible , opening and closing of the channels could be accomplished by toggling between near-UV and green-light illumination , at various frequencies and repeatedly , to mimic various physiological patterns ( Figure 1—figure supplement 1b , c ) .", "No inhibition of the current was observed during photo-agonism in the presence of saturating glutamate or NMDA , suggesting that when MAG in its cis form competes with free glutamate in LiGluN2A , and is as good an agonist as glutamate .", "Dose-response profiling of NMDA receptors containing LiGluN2A ( V713C ) ( as well as other variants , see below ) shows preservation of the glutamate potency ( EC50 ) as seen in wt receptors that contain the GluN2A-wt subunit ( Figure 1—figure supplement 2a , b ) ( Anson et al . , 1998; Chen et al . , 2005; Hedegaard et al . , 2012; Paoletti et al . , 2013 ) .", "Moreover , LiGluN2A ( V713C ) -containing receptors have wt activation kinetics , as assessed by fast MNI-glutamate uncaging that was designed to mimic the brief rise of glutamate that occurs in synapses due to vesicle release ( Figure 1—figure supplement 2g ) .", "Importantly , MAG labeling had no effect on the health , resting membrane potential or membrane resistance of cultured hippocampal neurons and yielded no photo-responses in neurons that did not express the cysteine-modified GluN2 subunit ( Figure 1—figure supplement 3a–c ) , indicating a lack of action on native glutamate receptors or other channels .", "Moreover , expression of the LiGluN2A subunit did not significantly change the amplitude of NMDA-induced current in hippocampal neurons ( Figure 1—figure supplement 3d ) , suggesting that it competes with native LiGluN2 subunits for assembly with the native GluN1 , so that the total number of synaptic NMDA receptors is preserved , but now subject to photo-control .", "Together , these results show that NMDA receptors containing the LiGluN subunits should support normal synaptic transmission and plasticity , while providing specific photo-control over these processes .", "MAG photoswitching provides two advantageous properties for the remote control of NMDA receptors .", "First , since the speed of MAG photoswitching depends on light intensity so that switching can be achieved either with short high intensity pulses or longer low intensity pulses ( Gorostiza et al . , 2007; Reiner and Isacoff , 2014 ) , it should be possible to sculpt the photocurrent to resemble NMDA receptor excitatory postsynaptic currents ( EPSCNMDA ) .", "Using LiGluN2A ( V713C ) conjugated to L-MAG1 , a 2 ms pulse of 375 nm light could be used to trigger substantial receptor activation ( Figure 1f ) , with the fast rise kinetics comparable to those of EPSCNMDA kinetics observed for GluN2A-containing synaptic NMDA receptors ( Bidoret et al . , 2009; Erreger and Traynelis , 2005; Vicini et al . , 2001; Yuan et al . , 2009 ) .", "Then , off-photoswiching could be triggered over tens of milliseconds ( Figure 1g ) , as typical of GluN2A , or more slowly ( Figure 1h ) to mimic the slower GluN2B deactivation kinetics ( Erreger and Traynelis , 2005; Vicini et al . , 1998 ) .", "Triggering these EPSCNMDA waveforms by only regulating light intensity avoids the need for piezo-driven fast perfusion systems and a cocktail of inhibitors typically used to isolate EPSCsNMDA .", "Since variations in the off-kinetics leads to variation in the intracellular Ca2+-concentration , this kind of control could be employed to study the role of Ca2+ and specific NMDA receptor subtypes in the induction of LTP and LTD , as it is still debated how the magnitude , temporal pattern and NMDA receptor subunit type contribute to plasticity changes ( Cummings et al . , 1996; Köhr et al . , 2003; Pawlak et al . , 2005; Shipton and Paulsen , 2014; Zhou et al . , 2005 ) .", "A second advantageous property is the bistability of MAG photoswitches ( Gorostiza et al . , 2007 ) .", "After switching with a brief 380 nm light pulse , photo-agonism ( or photo-antagonism , as described below ) is sustained in the dark for extended periods of time , until it is reversed by a brief 500 nm light pulse , as illustrated in both hippocampal neurons , ( Figure 1i ) and HEK293 cells ( Figure 1—figure supplement 3e ) .", "Our scan to identify effective cysteine attachment sites for MAGs in LiGluN2A provided a guide for the development of a LiGluN2B based on the homology of its LBD with that of GluN2A .", "Hence , we introduced a cysteine at position 714 of GluN2B , corresponding to the 713 position that yielded the photo-agonism in GluN2A ( Figure 2a ) .", "Indeed , conjugation of L-MAG1 to LiGluN2B ( V714C ) that was coexpressed with GluN1a in HEK293 cells yielded GluN2B-containing NMDA receptors which were activated by light ( Figure 2b ) .", "Unlike with LiGluN2A ( V713C ) , photo-activation of LiGluN2B ( V714C ) in the presence of saturating glutamate yielded a small increase in current ( Figure 2b ) , suggesting that L-MAG1 is a more potent agonist than glutamate .", "Importantly , LiGluN2B ( V714C ) -containing receptors displayed the same glutamate affinity and activation kinetics as wt GluN2B receptors ( Figure 1—figure supplement 2c , d and h ) , indicating that , as with LiGluN2A ( V713C ) , the orthogonal light control is obtained over receptors that otherwise function normally .", "Light-agonistic LiGluN2A and LiGluN2B subunits were examined side-by-side in hippocampal neurons .", "We expressed either LiGluN2A or LiGluN2B alone , relying on them to co-assemble with the endogenous pool of GluN1a subunits ( Barria and Malinow , 2002 ) .", "Following labeling with L-MAG1 , each of these variants gave rise to photocurrents in neurons ( Figure 2c ) .", "The LiGluN2A and LiGluN2B photocurrents were similar in amplitude and activation kinetics ( Figure 2d and e and Figure 2—figure supplement 2 ) , but the LiGluN2B photocurrent turned off ~3 times more slowly under identical light conditions ( Figure 2c , inset and f and Figure 2—figure supplement 2 ) , reminiscent of the slower deactivation kinetics of GluN2B- than GluN2A-containing receptors ( Bidoret et al . , 2009; Vicini et al . , 1998 ) .", "Widefield photoactivation at 380 nm of both LiGluN2A and LiGluN2B triggered an increase in EPSC frequency and this was reversed—more slowly in LiGluN2B—by illumination at 510 nm ( Figure 2c ) , suggesting that photoactivation of NMDA receptors containing either LiGluN2A or LiGluN2B triggered action potential firing in presynaptic neurons .", "Indeed , in current clamp recording , photoactivation ( violet bars ) and photodeactivation ( green bars ) reliably and reproducibly elicited bouts of firing ( Figure 2—figure supplement 1a–e ) .", "Photoactivation of LiGluN2A elicited a faster rise of excitation ( Figure 2—figure supplement 1a–b , top insets , and", "f ) and photodeactivation of LiGluN2A elicited a faster ( ~three fold ) de-excitation ( Figure 2—figure supplement 1a–b , bottom insets , and", "g ) than neurons expressing LiGluN2B .", "Our initial screen also identified several LBD anchoring positions where light blocked the glutamate-induced current ( Figure 1—figure supplement 1a ) , suggesting that , instead of docking correctly in the glutamate binding pocket , the ligand end of MAG could obstruct access of free glutamate to the binding pocket ( Figure 3a ) .", "The largest photo-antagonism was generated by L-MAG0 anchored at G712C of the GluN2A subunit when expressed in hippocampal neurons ( 56 . 4 ± 2 . 9% inhibition of the total current induced by 1 mM NMDA , n = 7 ) ( Figure 3b , c and", "f ) , similar to the block observed in HEK293 cells ( 65 . 8 ± 4 . 3% inhibition of the total current induced by 1 mM glutamate in HEK cells , n = 9 ) .", "This photo-antagonism is on par with the antagonism of GluN2A by NVP-AAM077 , when applied at concentrations that maintain subunit selectivity ( Monaghan et al . , 2012; Neyton and Paoletti , 2006; Paoletti and Neyton , 2007 ) , and so , although incomplete ( likely due to labeling efficiency , see below and Figure 3—figure supplement 1 ) , would be predicted to have utility .", "Whereas the specific photo-antagonism of GluN2A enables fast , reversible inhibition of GluN2A-containing NMDA receptors only , we also sought to develop a general tool for photo-antagonizing all NMDA receptor-subtypes within a given neuron , by creating a photo-antagonized GluN1 .", "The GluN1 subunit is activated by glycine or D-serine , but not glutamate ( Figure 3b ) .", "In fact , GluN1 antagonists resemble MAG in that they consist of an α-amino acid attached to a larger structure that prevents closure of the LBD ( Inanobe et al . , 2005 ) .", "We therefore reasoned that MAG tethered near the GluN1 ligand binding pocket would prevent normal agonist binding when photo-docked and would thereby function as a photo-antagonist .", "Indeed , a screen of cysteine mutants around the glycine-binding pocket identified a labeling site , E406C , where L-MAG0 functioned as photo-antagonist ( 30 . 7 ± 1 . 7% inhibition of the total current neurons , n = 6 ) ( Figure 3b , d and", "f ) , without affecting the glycine potency of the subunit ( Figure 1—figure supplement 2e , f ) .", "When the photo-antagonizable GluN1a ( E406C ) and GluN2A ( G712C ) subunits were co-expressed in dissociated hippocampal neurons and incubated with L-MAG0 , their combined effect resulted in 73 . 8 ± 5 . 2% ( n = 4 ) photo-inhibition of the total NMDA-induced current in neurons ( Figure 3e , f ) . 10 . 7554/eLife . 12040 . 010Figure 3 . Photo-antagonism of NMDA receptors in hippocampal neurons .", "( a ) Photo-antagonism with L-MAG0 attached to LiGluN2A ( G712C ) , where the cis-configuration is thought to place the glutamate end of MAG near the binding pocket , where it impedes LBD closure or entry of free glutamate ( blue triangles , rightmost cartoon ) , leaving the channel closed .", "( b ) Space filled , crystal structures of GluN1a ( light grey ) and GluN2A ( dark grey ) LBDs ( PDB-2A5S , 2A5T ( Furukawa et al . , 2005 ) , respectively ) showing the sites of glycine and glutamate binding ( green and blue sticks , respectively ) as well as the nearby E406 and G712 positions ( red spheres ) which , when tethered to L-MAG0 , yield photo-antagonism .", "( c-e )", "Representative traces of photo-antagonism by 380 nm light ( violet bars ) of NMDA currents induced by 1 mM NMDA ( black bars ) in neurons ( from wt rats ) labeled with L-MAG0 that express either: b- GluN1a-wt and GluN2A ( G712C ) to photo-block the glutamate binding pocket of the GluN2A subunit; c- GluN1a ( E406C ) to photo-block the glycine binding site of GluN1a subunit; d- GluN1a ( E406C ) and GluN2A ( G712C ) to photo-block both classes of binding sites .", "( f ) Summary of the average ( ± SEM ) photo-antagonism ( red bars ) and photo-agonism ( green bars ) observed in neurons .", "n shown within bars . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 01010 . 7554/eLife . 12040 . 011Figure 3—figure supplement 1 . Moderate correlation between total current size and photo-current .", "( a ) Representative traces of photo-antagonism ( left ) and photo-agonism ( right ) , displaying the relative sizes of the total NMDA-induced current and the photo-responses ( inhibition/activation ) , plotted in b .", "( b ) A modest correlation ( Spearman , R = 0 . 709 , R2 = 0 . 502 ) , albeit significant ( p<0 . 001 ) , was obtained for the relationship between both types of currents , suggesting that the variation in the photo-current size cannot be fully ( ~50% ) explained by a simple increase in the content of NMDA receptors at the plasma membrane ( assuming consistent MAG-labeling efficiency ) .", "Variability in photo-current size is likely due to variability in labeling efficiency . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 011 Cultured hippocampal neurons from wt rats ( C57BL ) transfected with the photo-antagonizing GluN2A ( G712C ) subunit in combination with photo-antagonizing GluN1a ( E406C ) , labeled with L-MAG0 and under green light illumination , exhibited typical action potential activity under current clamp recordings .", "Photo-antagonism with 370 nm light hyperpolarized cells and suppressed firing ( Figure 4a , b ) , as seen with conventional NMDA receptor blockers .", "No such photo-effect was seen in non-transfected cells ( Figure 4c , d and Figure 4—figure supplement 2 ) .", "Reversal of photo-antagonism by 510 nm light elicited a gradual repolarization , reminiscent of physiological recovery ( Figure 4b ) .", "In voltage clamp , neurons transfected with the photo-antagonizing GluN2A ( G712C ) subunit alone , or in combination with the photo-antagonizing GluN1a ( E406C ) , displayed typical barrages of spontaneous EPSCNMDAevents ( Groc et al . , 2002; Ivenshitz and Segal , 2010; Kirson and Yaari , 1996 ) ( Figure 4—figure supplement 1a ) .", "During green light illumination , action currents were detected in some cells ( Figure 4—figure supplement 1a , arrow and inset ) and illumination with violet light decreased the inward current , the frequency of action currents ( Figure 4—figure supplement 1a , inset ) , and of spontaneous EPSCNMDA ( Figure 4—figure supplement 1a , and summary in", "d ) , with no effect of violet light on non-transfected neurons ( Figure 4—figure supplement 1b , right panel , summary in c and Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 12040 . 012Figure 4 . Photo-inhibition of neuronal activity with LiGluN1a and LiGluN2A .", "( a ) Representative trace showing a long recording ( current clamp ) of a neuron ( from wt rats ) transfected with LiGluN1a ( E406C ) and LiGluN2A ( G712C ) , labeled with L-MAG0 , and illuminated with 380 nm ( violet bars ) or 510 nm ( green bars ) light ( held at −40 mV , Mg2+-free , 20 µM CNQX ) .", "During green light illumination , the neuron displayed strong action potential activity that could be faithfully inhibited by near-UV light ( i . e . block is ON ) , as the photo-antagonism hyperpolarized the cell to suppress action potential firing ,", "( b ) , as typically seen with soluble GluNRs blockers .", "( c-d )", "Summary of photo-antagonism on membrane potential on individual transfected- and non-transfected neurons ( from wt rats )", "( c ) , and summary of the effect on membrane potential ( ΔVm ) is shown in", "( d ) .", "( e ) Summary for the reduction in firing frequency following violet light illumination of individual cells ( circles ) and averages ± SEM ( color-coded filled squares ) .", "Statistics in panel d are shown as Box plots of the data ( not normally distributed , see Figure 4—figure supplement 2 ) , showing median and outliers ( filled circles ) followed by a nonparametric Mann-Whitney Rank Sum Test ( see methods ) , ***p<0 . 001 , **p<0 . 01 , n . s . - not-significant , n shown next to plots .", "Statistics in panel e are shown as mean ± SEM , tested with two-tailed , paired t-test; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 01210 . 7554/eLife . 12040 . 013Figure 4—figure supplement 1 . Photo-antagonism of NMDA receptors in hippocampal neurons inhibits the development of EPSCs .", "( a ) Representative trace ( voltage clamp , −60 mV ) of a hippocampal neuron ( from wt rats ) transfected with LiGluN1a ( E406C ) and LiGluN2A ( G712C ) , labeled with L-MAG0 .", "During green light illumination ( i . e . no block ) , spontaneous EPSC can be seen ( FEPSC- frequency shown in plot below trace ) , as well as occasional current potentials ( arrow and asterisks in inset ) .", "During violet light ( i . e . during receptor block ) , the baseline current is reduced as well as the frequency of EPSCs ( shown in plot below trace ) and current potentials completely disappear .", "Note that the current potentials were cut in trace to ease the view of the much smaller EPSCs ( inset ) .", "( b ) Light-induced photo-antagonism reduces the current in individual transfected- , but not in non-transfected ( non-trans . ) , neurons ( from wt rats ) .", "( c ) Average ( ± SEM ) of the extent of current reduction in transfected and non-transfected neurons .", "( d ) Reduction in the frequency of EPSCs .", "During 370 nm illumination ( violet bar ) individual neurons display a consistent decrease in the frequency of spontaneous EPSCs ( filled squares , mean ± SEM ) .", "Statistics in panel c are shown as Box plots of the data ( not normally distributed , see Figure 4—figure supplement 2 ) , showing median and outliers ( filled circles ) followed by a nonparametric Mann-Whitney Rank Sum Test ( see methods ) , ***p<0 . 001 .", "Statistics in panel d are shown as mean ± SEM ( filled color-coded squares ) and individual cells are displayed ( circles ) .", "n , when displayed , appears next to bars .", "Hippocampal neurons obtained from wt rats . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 01310 . 7554/eLife . 12040 . 014Figure 4—figure supplement 2 . Summary of nonparametric statistics for Figure 4 . Results from Normality and nonparametric Mann-Whitney Rank Sum Tests are displayed for results shown in Figure 4c–e and for Figure 4—figure supplement 1b–d . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 014 The fundamental power of the highly selective photo-pharmacology that is utilized in chemical optogenetics is the ability to orthogonally and spatially control native signaling proteins in their physiological location in the cell- in the synapse for NMDA receptors .", "To test whether LiGluN2 subunits are incorporated into synapses , as shown earlier for wt GluN2A or GluN2B subunits ( Cummings et al . , 1996; Prybylowski et al . , 2002; Thomas et al . , 2006 ) , we turned to autaptic connections , in which hippocampal neurons from wt rats are grown at low density ( see Materials and methods ) that neurons synapse onto themselves ( Figure 5a ) , thereby serving as both the presynaptic cell which can be stimulated electrically as well as the postsynaptic cell , from which we can record synaptic currents ( Bekkers and Stevens , 1991 ) . 10 . 7554/eLife . 12040 . 015Figure 5 . Photo-block of synaptic transmission by LiGluNs in hippocampal autapses .", "( a ) ( left ) Representative images of hippocampal neurons ( from wt rats ) grown in low density forming autapses .", "( right )", "Schematics of hippocampal autapse used to measure the effect on the NMDA receptors EPSC ( blue trace , evoked NMDA EPSC- eEPSCNMDA ) of turning ON ( violet bar ) and OFF ( green bar ) the photo-antagonsim of LiGluN .", "( b ) No significant difference in eEPSCNMDA amplitude ( left ) or deactivation times ( bi-exponential weighted τdeact ) ( right ) in autaptic neurons expressing GluN2A ( G712C ) alone ( cyan ) or GluN2A ( G712C ) and GluN1a ( E406C ) ( red ) compared to non-transfected control neurons ( black ) ( from wt rats ) .", "Inset shows superimposed eEPSCNMDA for the three conditions .", "( c ) Autaptic NMDA receptor’s EPSCs in neurons transfected with GluN2A ( G712C ) and GluN1a ( E406C ) before photo-antagonism ( 1 , under 510 nm light ) .", "Individual EPSCNMDA ( grey ) are superimposed with average of 5 consecutive EPSCs ( green for 510 nm light; violet for 370 nm light ) .", "Photo-antagonism 370 nm light reversibly reduces the amplitude of the eEPSCNMDA .", "( d ) Time series of NMDA receptors EPSC amplitudes for cell shown in", "( c ) reveals repeated reversibility of photo-block of eEPSCNMDA , despite the typical rundown of the responses ( see [Goda and Stevens , 1998] ) .", "( e ) Non-transfected cell exposed to L-MAG0 does not exhibit light-dependent modulation of eEPSCNMDA amplitude .", "( f ) Summary of photo-block ( as % inhibition ) of autaptic NMDA receptors EPSCs in transfected and non-transfected neurons ( color scheme as in", "b ) .", "4 cells were excluded ( see Figure 5—figure supplement 1 ) .", "Statistics in panels b and c are shown as Box plots displaying medians , outliers ( filled circles ) and individual data points ( filled , color-coded squares ) , followed by a nonparametric ANOVA on ranks ( see Figure 5—figure supplement 1 ) , n . s . , not significant .", "Statistics for panel f are shown as mean ± SEM , tested with one way ANOVA , ***p<0 . 001 , **p<0 . 01 , all pairwise Tukey post hoc test , n shown in parentheses atop bars . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 01510 . 7554/eLife . 12040 . 016Figure 5—figure supplement 1 . Summary of nonparametric and parametric statistics for Figure 5 .", "( a ) Results from Normality tests and nonparametric Kruskal-Wallis One Way Analysis of Variance ( ANOVA on ranks ) are displayed for panels shown in Figure 5b and One Way ANOVA for Figure 5f .", "( b ) No correlation ( Spearman , R = 0 . 543 , R2 = 0 . 295 , p = 0 . 297 ) was obtained for the relationship between the effect of light ( i . e . photo-block of the EPSCNMDA;% inhibition ) and the total size of the synaptic NMDA-current ( EPSCNMDA ) pooled for both LiGluN2 and LiGluN1+2 groups .", "This suggests that the variation in the effect of light ( i . e . relative size of the block ) has no relationship with the total size of the synaptic current , in line with the observations displayed in Figure 3—figure supplement 1 .", "Dashed region- 4 non responding cells transfected with either LiGluN2A-only or with LiGluN2A and LiGluN1a are shown for: 1- comparison with responsive cells and 2- showing variability in labeling efficiency .", "Non-responding cells ( i . e . transfected neurons and exposed to MAG , but that exhibited less than 5% inhibition ) were excluded from the average shown in Figure 5f .", "This criterion was defined to differentiate between real photo-inhibition from variability in transmission rundown ( Goda and Stevens , 1998 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 016 Transfected neurons that formed autapses displayed eEPSCNMDA events that were similar in amplitude and kinetics to those seen in non-transfected cells ( Figure 5b and Figure 5—figure supplement 1a ) , indicating that LiGluN expression makes synaptic NMDA receptors sensitive to light without significantly changing their number , consistent with evidence presented above ( Figure 1—figure supplement 3d ) and with earlier reports which expressed wildtype subunits ( Prybylowski et al . , 2002 ) .", "In neurons that were transfected with the photo-antagonizing LiGluN2A ( G712C ) and labeled with L-MAG0 , the amplitude of the eEPSCNMDA was inhibited by illumination at 370 nm and this photo-antagonism was relieved by illumination at 510 nm ( Figure 5c , d and", "f ) .", "The photo-antagonism could be toggled on and off repeatedly ( Figure 5c , d ) , whereas non-transfected neurons showed no change in their eEPSCNMDA's amplitude as the wavelength of illumination was switched between 510 nm and 370 nm ( Figure 5e , f ) .", "The magnitude of the light-dependent inhibition of the synaptic eEPSCNMDA was not correlated with eEPSCNMDA amplitude , suggesting that the degree of inhibition varies primarily with labeling efficiency ( Figure 5—figure supplement 1b; % inhibition ranges from ~20-50% ) , but also see Figure 3—figure supplement 1 ) .", "Thus , LiGluN subunits incorporate into synaptic NMDA receptors and function normally in response to synaptically released glutamate , while providing for optical control over the synaptic transmission that is mediated by that specific receptor subtype .", "An attractive application of optical control over the function of specific NMDA receptors is to probe their function in synaptic plasticity in specific neural circuits .", "To determine whether such control is possible , we initially turned to the prototypical form of NMDA receptor-dependent plasticity , where tetanic stimulation of hippocampal CA3 Schaffer collateral axons evokes LTP at CA1 pyramidal cells ( Bliss and Collingridge , 1993; Lüscher and Malenka , 2012 ) ( Figure 6a ) .", "Organotypic slices from GluN2A-knockout neonate mice ( Sakimura et al . , 1995 ) were biolistically transfected with GluN1a ( E406C ) , GluN2A ( G712C ) and tdTomato ( typically 1–3 transfected CA1 neurons per slice , Figure 6b ) .", "Following incubation with L-MAG0 , neurons were illuminated with green light ( 497 nm for 2 s ) to place the majority of L-MAG0 in its inactive trans state .", "Then , Schaffer collaterals were stimulated at 0 . 03 Hz and evoked EPSCs were recorded from CA1 neurons for a 8-minute baseline period .", "Illumination was then applied for 2 s at either 390 nm ( photo-antagonism ON; Figure 6c , violet ) or 497 nm ( photo-antagonism OFF; Figure 6c , green ) .", "Because of the bistability of MAG ( Figure 1i and Figure 1—figure supplement 3e ) , LiGluNs remain blocked ( following violet light ) or unblocked ( following green light ) in the dark for many minutes .", "Following the brief illumination protocol , we immediately applied a tetanic stimulation with a pair of 1 s long 100 Hz bursts in the dark .", "Stimulation was then returned to 0 . 03 Hz for an extended period to measure changes in synaptic strength ( Figure 6c ) .", "Slices , in which L-MAG0 was maintained in trans ( photo-antagonism off ) by green light , exhibited robust LTP ( Figure 6c , black circles; EPSC amplitude increased to 185 + 31% ( n = 10 ) of baseline , averaged over 20–30 min . post-tetanus; summary in Figure 6d ) , whereas slices in which L-MAG0 was switched to cis ( photo-antagonism on ) by violet light did not ( Figure 6c , open circles; EPSC amplitude dropped to 67 + 28% ( n = 10 ) of baseline , averaged over 20–30 min . post-tetanus; summary in Figure 6d ) .", "Slices to which L-MAG0 was not added , but which were illuminated at 390 nm , exhibited the same level of LTP as those labeled with L-MAG0 and illuminated with 497 nm light ( 174 + 31 . 74% , n = 4 ) , demonstrating that the light protocol did not interfere with the generation of LTP and that the block of LTP-induction is a specific outcome of photo-antagonism .", "Thus , photo-antagonism of post-synaptic NMDA receptors can be used to gate the induction of LTP that is induced by presynaptic bursts of action potentials . 10 . 7554/eLife . 12040 . 017Figure 6 . LTP induction blocked by photo-antagonism of LiGluN1a ( E406C ) and LiGluN2A ( G712C ) in organotypic hippocampal slice from GluN2A-knockout neonate mice .", "( a ) Schematic of the hippocampus with stimulating electrode on Schaffer collaterals CA3 pyramidal axons that innervate pyramidal neurons of CA1 and recording pipette on a transfected CA1 neuron from Glu2A-KO mice .", "( b ) Transfected neuron in an organotypic hippocampal slice identified by tdTomato fluorescence after biolistic co-transfection of LiGluN1a ( E406C ) , LiGluN2A ( G712C ) and tdTomato .", "( c ) Following exposure of the slices to L-MAG0 , Schaffer Collaterals were electrically stimulated once every 30 s to obtain a baseline EPSC amplitude ( ranging from −8 to 0 min ) , followed by two high frequency trains ( tetanic stimulation: 1-s long trains at 100 Hz , separated by 20 s ) , followed by a return for 30 min to a one stimulus every 30 s .", "Normalized mean ± SEM of evoked EPSC amplitudes once every 30 s are shown ( ranging from 0 to 35 min ) .", "When the tetanic stimulation was preceded by illumination at 497 nm ( photo-antagonism OFF; green bar , filled symbols , n = 10 ) EPSC amplitude approximately doubled , as common for CA3-CA1 LTP .", "However , illumination at 390 nm ( photo-antagonism ON; violet bar , open symbols , n = 10 ) prevented the generation of LTP .", "Inset shows representative average EPSCs before ( black traces ) and after ( at t= 20–30 min ) the tetanic stimulation ( green and violet traces ) .", "( d ) Summary of the results shown in", "( c ) , averaged between t = 20–30 min .", "Statistics in panels are shown as mean ± SEM , tested with two-tailed , unpaired t-test , p as indicated , n shown within bars . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 017 Having shown that precisely timed light pulses can be used to control the activity of synaptic LiGluN containing NMDA receptors , we turned to a second major attraction of light , the ability to focus in small regions of interest , and attempted to control NMDA receptor function at single synapses .", "Dendritic spine expansion is a structural correlate of LTP , which can be induced at the level of single dendritic spines by the local photo-uncaging of glutamate ( Matsuzaki et al . , 2001; Matsuzaki et al . , 2004 ) .", "We asked whether highly local , subunit-specific photo-agonism of GluN2A can induce such structural changes at single spines .", "As with the experiments , above , which demonstrated the ability to block LTP induction by photo-antagonism of LiGluN2A-containing NMDA receptors , these experiments were performed in organotypic slices obtained from GluN2A-knockout neonate mice ( see Materials and methods ) .", "Since calcium entry through NMDA receptors is critical for synaptic plasticity , first we examined the ability of photo-agonized GluN2A ( V713C ) labeled with L-MAG1 to elicit spine-specific rises in Ca2+ in slices co-transfected with the soluble calcium reporter R-GECO1 . 0 ( Zhao et al . , 2011 ) .", "Illumination of a single spine head at 405 nm ( 100 µW for 100 µs/pixel ) elicited a rise in R-GECO fluorescence in the illuminated spine , with little or no response in nearby spines or in the dendritic shaft , consistent with spine head Ca2+-signaling compartmentalization ( Figure 7b , c ) ( Yuste , 2013 ) .", "We next asked whether photo-activation of GluN2A ( V713C ) can trigger spine expansion .", "Slices expressing GluN2A ( V713C ) , along with tdTomato as a reporter of spine size ( see Materials and methods and [Holtmaat et al . , 2005] ) , were labeled with L-MAG1 and imaged in a Mg2+-free , high Ca2+ solution containing TTX .", "Spine head illumination with 405 nm ( 100 µW/µm2 for 600 μs per pixel , ~200 ms per spine ) triggered an increase in spine head volume that peaked in ~5 min ( Ft/Fi = 1 . 67 ± 0 . 15 of baseline , n = 26 , p<0 . 001 , two-tailed , paired t-test ) and then declined to an elevated plateau that persisted for at least 45 min ( Ft/Fi = 1 . 31 ± 0 . 06 of baseline , n = 26 , p<0 . 001 , two-tailed , paired t-test ) ( Figure 7d and e , red symbols ) .", "Non-illuminated nearby spines did not change volume ( Figure 7e , black symbols ) , nor did illuminated spines in slices that were not labeled with L-MAG1 ( Figure 7d and e , blue symbols ) .", "These experiments show that:", "i ) LiGluN2A ( V713C ) traffics to synapses , as shown above for LiGluN2A ( G712C ) ,", "ii ) photo-agonism of GluN2A ( V713C ) can be used to induce a spine-specific elevation in internal calcium , and", "iii ) activation of post-synaptic GluN2A-containing receptors is sufficient to induce the morphological correlate of LTP . 10 . 7554/eLife . 12040 . 018Figure 7 . GluN2A ( V713C ) photo-agonism triggers calcium increase and expansion in single dendritic spines of organotypic hippocampal slice from GluN2A-knockout neonate mice .", "( a ) Schematic of photo-agonism with L-MAG1 conjugated to the LiGluN2A ( V713C ) subunit .", "( b ,", "c ) Single-spine calcium rise induced by photo-agonism .", "( b ) CA1 neurons co-expressing LiGluN2A ( V713C ) fused to R-GECO1 . 0 [LiGluN2A ( V713C ) -R-GECO1 . 0] and soluble R-GECO1 . 0 ( red ) as well as GFP ( green ) in merged low power image showing cluster of transfected neurons ( top ) and high-magnification image of dendritic shaft with several spines , showing the green , red and merged channels ( bottom ) .", "Photo-agonism with 405 nm illumination focuses on spine #1 ( red dashed circle ) , while R-GECO1 . 0 imaging measures calcium in spines # 1–4 and the dendritic shaft ( dashed green rectangle ) .", "( c ) Illumination of spine #1 in", "( b ) at 405 nm ( violet bar , with additional excitations at 488 and 561 nm to image eGFP ( green bar ) and R-GECO1 . 0 ( orange bar ) , respectively , elicits an increase in R-GECO1 . 0 fluorescence within the head of spine #1 ( red trace ) , while other spines ( black traces ) and the dendritic shaft ( green trace ) display little or no increase in fluorescence .", "( d ) Single spine expansion by photo-agonism of LiGluN2A .", "Spines from CA1 pyramidal neuron co-expressing GluN2A ( V713C ) and tdTomato and treated with L-MAG1 were stimulated with repetitive 405 nm laser scanning at the tip of the spine head , at ~1 Hz for a total amount of 1–2 min . ( Top ) Time lapse of a representative spine undergoing expansion after on-spine stimulation with 405 nm light ( violet bar on top of inset , dashed red circle ) .", "Non-illuminated , nearby spines ( white arrowheads ) do not exhibit any change in volume .", "( Bottom )", "Representative spine from a slice without L-MAG1 treatment showing no response to 405 nm light ( blue dashed circle ) .", "Numbers above images indicate time in min relative to photo-stimulation at t = 0 .", "( e ) Summary of results .", "Statistics in panel", "( e ) are shown as mean ± SEM , ***p<0 . 001 , two-tailed , paired t-test compared to baseline , Δt= -10–0 mi .", "n , from series of experiments as in", "( d ) , is shown in parentheses . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 018 We next asked whether photo-antagonism of GluN2A and GluN1a could be used to prevent glutamate-induced spine expansion at single dendritic spines in organotypic slices obtained from GluN2A-knockout neonate mice .", "Caged-glutamate ( MNI-glu ) was added to a Mg2+-free bathing solution for 1–2 min followed by photo-uncaging with 405 nm light at a spot of the same lateral size as the spine head , but located 0 . 5 µm away ( Figure 8—figure supplement 1a ) .", "The 405 nm illumination was well-confined ( Figure 8—figure supplement 1a ) and reliably induced spine expansion ( Figure 8—figure supplement 1b ) when LiGluN2A was transfected , as shown earlier for GluN2A-wt ( Lee et al . , 2009; Matsuzaki et al . , 2001; Matsuzaki et al . , 2004 ) , but stable expansion did not occur in slices that were not transfected with LiGluN2A ( Figure 8—figure supplement 1c ) .", "To determine if the spine expansion that is triggered by glutamate uncaging could be prevented by photo-antagonism of LiGluN ( Figure 8a ) , we co-expressed LiGluN2A ( G712C ) and LiGluN1a ( 406C ) with tdTomato and labeled the slices with L-MAG0 .", "Individual spines received two successive bouts of near-spine glutamate photo-uncaging , the first following induction of LiGluN photo-antagonism by 405 nm on the spine head ( 100 µW/µm2 for 600 µs per pixel ) , and the second following reversal of the photo-antagonism by 488 nm light ( 100 µW/µm2 for 150 µs per pixel ) ( Figure 8b , c ) .", "In this experimental design , the second bout of uncaging was crucial to determining that a spine that did not respond to the first bout of uncaging was nevertheless competent to expand , asmany spines are not ( Lynch et al . , 2013 ) .", "The first uncaging bout , during GluN1a/GluN2A photo-antagonism , induced only a small , transient increase in spine head volume , which peaked in 1 min ( Ft/Fi = 1 . 18 ± 0 . 07 of baseline , n = 12 , p>0 . 1 , two tailed , paired t-test ) and shrank back to near baseline ( Ft/Fi F = 1 . 05 ± 0 . 05 of baseline , n = 12 , p>0 . 1 , two tailed , paired t-test ) within 5 min ( Figure 8c , red symbols ) , indicating a lack of the long-lasting expansion that is associated with LTP ( Yang et al . , 2008 ) .", "After turning off the GluN1a/GluN2A photo-antagonism with green light , the second bout of near-spine glutamate uncaging induced a large persistent expansion ( Ft/Fi = 1 . 47 ± 0 . 13 compared to new moderately elevated baseline between t = 3 min and 15 min , n = 12 , p<0 . 01 , two tailed , paired t-test ) ( Figure 8c , red symbols ) .", "Nearby , non-illuminated spines did not respond ( Figure 8b and c , black symbols ) .", "These experiments show that photo-antagonism of GluN1a/GluN2A can block the induction of spine expansion and that this block is reversible , enabling a morphological correlate of LTP to be gated temporally at single synapses . 10 . 7554/eLife . 12040 . 019Figure 8 . Photo-antagonism prevents glutamate-induced expansion in single dendritic spines of organotypic hippocampal slice from GluN2A-knockout neonate mice .", "( a ) Schematic of a photo-antagonized NMDA receptors containing LiGluN1a ( E406C ) and LiGluN2A ( G712C ) conjugated to L-MAG0 .", "( b ) CA1 pyramidal neuron co-expressing LiGluN1a ( E406C ) , LiGluN2A ( G712C ) and tdTomato following treatment with L-MAG0 .", "( Top )", "Single spine receiving two bouts of near-spine glutamate-uncaging at 405 nm ( magenta spots , insets ) , the first after photo-antagonism was induced by 405 nm on-spine illumination ( violet dashed circle , left inset ) , the second following removal of the photo-antagonism by 488 nm on-spine illumination ( green dashed circle , right inset ) .", "Photo-block ( and relief of block ) of the receptors was done by stimulating the tip of the spine head with repetitive 405 nm ( or 488 ) laser scanning , at ~1 Hz for a total amount of 1 min .", "MNI-glutamate uncaging was performed slightly away from the spine head ( ~2 µm ) to avoid further stimulation of the receptors found on the spine head .", "The spine undergoes a small expansion in response to the first glutamate-uncaging ( p=0 . 06 , compared to baseline at t= -8 – 0 min ) , and a large expansion following removal of the photo-antagonism .", "( Bottom )", "A nearby non-illuminated spine ( arrowhead ) does not change size .", "Numbers above images indicate time in min relative to photo-stimulation at t = 0 .", "( c ) Summary of results from a series of experiments as in", "( b ) ( mean ± SEM , **p< 0 . 01 , two-tailed , paired t-test compared to baseline at t= 9–15 min , n shown in parentheses ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 01910 . 7554/eLife . 12040 . 020Figure 8—figure supplement 1 . Single spine spatial precision of photocontrol in organotypic hippocampal slice from GluN2A-knockout neonate mice .", "( a ) Photo-bleaching indicates spatial precision of \"on-spine\" photo-stimulation .", "High intensity illumination at 405 nm ( magenta arrow ) rastered over a single 1 µm diameter spine for 1 s ( white dashed circle in images , black symbols in graph ) leads to >70% confined bleaching of GFP in that spine ( white arrowhead in right image taken 3 . 5 s following the bleaching ) and only marginal bleaching ( <10% ) of the neighboring dendritic shaft ( red dashed circle in images , red symbols in graph ) or nearby spine ( green dashed circle in images , green symbols in graph ) .", "Numbers above images indicate time in seconds ( 0 before bleaching , 3 . 5 s after bleaching ) .", "( b ) ( Top ) Uncaging of MNI-glutamate ( 2 . 5 mM in nominally Mg2+-free solution , see Materials and methods ) near a single spine ( magenta spot in left image at time 0 or magenta arrow shown in plot ) of a CA1 pyramidal neuron from a cultured GluN2A-KO mice organotypic slice , biolistically-transfected with GluN1a ( E406C ) , GluN2A ( G712C ) and tdTomato , but not treated with L-MAG0 , triggers expansion of targeted spine ( magenta dashed circle ) , but not of nearby spines ( white arrowheads ) .", "Numbers above images indicate time in minutes ( 0 before uncaging , 10 and 20 min after uncaging ) .", "( Bottom )", "Summary of results from series of experiments as in ( Top ) .", "Slices , biolistically-transfected with GluN2A ( G712C ) but unexposed to MAG , following 1–2 min incubation with 2 . 5 mM MNI-glutamate exhibit long-lasting expansion , demonstrating the sufficiency of this time window to deliver the compound to spines and rescue of long lasting expansion .", "( c ) Spines , of CA1 neurons from a cultured GluN2A-KO mice organotypic slice , biolistically transfected with td-tomato only and unexposed to MAG , do not maintain increased volume following MNI-glu uncaging ( magenta arrow ) , reminiscent of earlier findings showing reduced hippocampal LTP ( Sakimura et al . , 1995 ) .", "Statistics in panels are shown as mean ± SEM , tested with two-tailed , paired t-test; ***p<0 . 001 , n displayed in plots . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 020 Having seen that LiGluNs provide optical control over NMDA receptor dependent transmission and plasticity in dissociated neurons and in brain slice , we turned to test their utility in vivo in larval zebrafish , Danio rerio .", "As a model of NMDA receptor-dependent plasticity , we examined the synaptic pruning that takes place in the developing visual system .", "NMDA receptors play an important role in the formation of sensory topographic maps ( Bliss and Collingridge , 1993; Lüscher and Malenka , 2012 ) .", "In many vertebrates , including zebrafish , exposure to soluble NMDA receptor antagonists during a critical period in development of retinotectal projections can disrupt the convergence of retinal ganglion cell axons , thus disrupting retinotopy ( Cline and Constantine-Paton , 1989; Ruthazer et al . , 2003; Schmidt , 1991; Zhang et al . , 1998 ) .", "In zebrafish larvae , one such critical period occurs between 5 and 7 days post fertilization ( dpf ) , coincident with the onset of behaviors that require visual acuity , such as prey capture .", "Larvae exposed to soluble NMDA receptor antagonists during this critical period exhibit enlarged RGC axonal arbors ( Schmidt et al . , 2000 ) .", "We asked whether photo-antagonism of LiGluNR2A ( G712C ) during the 5–7 dpf critical period would affect retinal ganglion cell axon growth .", "We generated a transgenic line of zebrafish expressing GluN2A ( G712C ) under the control the gal4-UAS expression system ( Scott et al . , 2007 ) .", "Double transgenic Tg[s1101t-gal4;UAS-GluN2A ( G712C ) ] fish were incrossed to produce embryos with GluN2A ( G712C ) expressed throughout the nervous system ( Figure 9b ) , including the retina and optic tectum .", "To visualize individual retinal ganglion cells , embryos were injected with DNA for GFP expression ( see methods ) to sparsely mark a subset of retinal ganglion cells projecting to the optic tectum ( Figure 9b , right panels ) ( Xiao and Baier , 2007 ) .", "At 5 dpf , healthy larvae expressing LiGluN2A ( G712C ) pan-neuronally , with sparse expression of GFP in retinal ganglion cells , were divided into three treatment groups: ( 1 ) L-MAG0 , ( 2 ) vehicle ( DMSO only ) , or ( 3 ) MK-801 .", "All larvae were mounted in agarose to image baseline axon arbor morphology , and then freed and transferred to a 48-well plate imaging chamber ( Levitz et al . , 2013 ) .", "Between 5 and 7 dpf , freely swimming larvae were exposed to a 10 s flash of 405 nm light once every 30 min ( Figure 9a ) .", "Because MAG is bistable ( see Figure 1 ) , we reasoned that brief bouts of isomerization into the photo-antagonizing state would be sufficient to provide long-term antagonism , while allowing the larvae to develop under predominantly natural visual stimuli .", "At 7 dpf , larvae were imaged a second time to assess the growth of each axon arbor . 10 . 7554/eLife . 12040 . 021Figure 9 . GluN2A ( G712C ) photo-antagonism disrupts refinement of retinal ganglion cell axon arbors in larval zebrafish in vivo .", "( a ) Cartoons depicting GluN2A ( G712C ) photo-antagonism ( top left ) , development of retinal ganglion cell projection ( top , right ) and timeline of the photo-antagonism assay ( bottom ) .", "( b ) ( Left ) Dorsal view of 5 dpf larva showing pan-neuronal expression pattern driven by s1101t-gal4 visualized by expression of UAS-GCaMP3 .", "Transverse ( right , top ) and dorsal ( right , bottom ) tectal projection of a retinal ganglion cell axon arbor labeled by mosaic expression of pou4f3:mGFP at 7 dpf .", "( c ) Axon arbors were traced and arbor radius measured by a 3-dimensional Sholl analysis counting the number of intersections encountered by concentric spheres centered on the first branch point .", "Arrows indicate arbor radius ( R ) at 5 dpf and 7 dpf .", "( d ) Prior to antagonism , Tg[s1101t-gal4; UAS:GluN2A ( G712C ) ] animals have a comparable distribution of arbor radii at 5 dpf as non-expressing animals ( without UAS , but mix of s1101t +/- ) ( n . s . , p >0 . 6 , Mann-Whitney Rank Sum Test , n = 19 , GluN2A ( G712C ) -expressing axons and 10 non-expressing axons ) .", "Box plot whiskers indicate 5% and 95% percentiles .", "Dots above whiskers represent outliers .", "( e ) Representative selection of retinal ganglion cell axon arbors in transgenic animals at 5 dpf ( green ) and 7 dpf ( magenta ) .", "Larvae were treated at 5 dpf with either 150 µM L-MAG0 in 0 . 3% DMSO , 0 . 3% DMSO alone , or 25 µM MK-801 , and subjected to 10 s flashes of 405 nm light at 30 min intervals from 128–168 hpf .", "( f ) In animals where GluN2A ( G712C ) was photo-antagonized by MAG , retinal ganglion cell axon arbors grew significantly more compared to DMSO-treated control groups ( left-to-right: ΔR = 6 . 9% , n = 10 axons , ΔR = 1 . 2% , n = 9 axons; ΔR = 16 . 4% , n = 12 axons ) .", "This overgrowth was comparable to the change in arbor radius observed in animals treated with MK-801 ( ΔR = 13 . 8% , n = 10 axons ) .", "For statistical analysis of the relative change in arbor radius ( ΔR = ( R7dpf-R5dpf ) /R5dfp ) , we initially compared untreated –wt animals and untreated transgenic animals ( two left bars ) and observed no statistical difference ( mean ± SEM , n . s . not significant , two-tailed , unpaired t-test ) .", "This enabled the comparison between the animals from the same transgenic background ( second to fourth bar ) for the effect of photo-inhibition on radius ( mean ± SEM , n . s . not significant , **p<0 . 01 , *p<0 . 05 , tested with one way ANOVA , all pairwise Tukey post hoc test ( see Figure 9—figure supplement 1c ) .", "Individual data points are shown for f ( open circles ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 02110 . 7554/eLife . 12040 . 022Figure 9—figure supplement 1 . MAG treatment of zebrafish larvae in the absence of LiGluN2A expression does not affect behavior . Wildtype larvae were treated at 5 dpf with either 150 µM MAG0 in 0 . 3% DMSO ( n = 48 ) or 0 . 3% DMSO alone ( n = 48 ) for 40 min , rinsed thoroughly in fresh fish water , and allowed to develop as two separate groups without further treatment in large common dishes of E3 .", "( a ) MAG treatment in the absence of LiGluN2A expression does not affect innate escape reflex or the habituation of this reflex .", "Zebrafish at 7dpf were subjected to an acousto-vibrational stimulus protocol ( top ) designed to assay", "( i ) sensitivity ,", "( ii ) responsiveness ,", "( iii ) habituation in response to repeated stimuli , and", "( iv ) spontaneous dishabitutation ( n = 48 animals ) .", "Movies were scored for response within 60 ms of the stimulus .", "Escape probabilities were calculated as the mean number of responders per group ( mean ± SEM ) .", "No significant difference was seen between the MAG-treated ( blue ) and DMSO-treated groups ( green ) in any phase of the assay .", "( b ) At 7 dpf , individual fish were transferred to single wells of 48-well plates and spontaneous swimming was filmed at 10 fps .", "Fish were tracked in each well ( blue = MAG-treated; green = DMSO-treated ) by detecting changes in pixel intensity between movie frames using Matlab ( see Materials and methods ) .", "Empty wells represent fish that were excluded from statistical analysis if the automated tracking algorithm failed for more than 15% of the 10 min imaging session ( manual inspection revealed that the automated tracking failed when fish spent extended time near a partially obscured well edge; in total , 10% were excluded ) .", "( bottom )", "Summary shows that MAG and DMSO treated animals showed no significant difference in cumulative distance traveled during spontaneous swim bouts , swim bout frequency or duration , or average speed , or time spent resting .", "( n=41 for MAG0 , n=43 for DMSO-treated animals , n . s . , not significant , two-tailed , unpaired t-test ) .", "Values mean ± SEM .", "( c ) Statistical results of a one way ANOVA ( all pairwise Tukey post hoc test ) , performed for Figure 9f . DOI: http://dx . doi . org/10 . 7554/eLife . 12040 . 022 The size of a retinal ganglion cell axon arbor was determined by a 3-dimensional Sholl analysis ( Figure 9c ) , which compared z-stacks obtained at 5 dpf and 7 dpf .", "At 5 . 25 dpf ( 126 ± 2 hr post fertilization ) , the size distribution of retinal ganglion cell axon arbors from Tg[s1101t-gal4;UAS-GluN2A ( G712C ) ] larvae was indistinguishable that of siblings lacking the UAS-GluN2A ( G712C ) transgene ( Figure 9d ) ( p>0 . 7 , two- two-tailed , unpaired t-test , n = 19 Tg[s1101t-gal4;UAS-GluN2A ( G712C ) ] and n = 10 control axons ) , indicating that gal4-driven expression of UAS-GluN2A ( G712C ) does not alter the initial development of the retinotectal projection .", "However , by 7 dpf , axon arbors in Tg[s1101t-gal4;UAS-GluN2A ( G712C ) ] larvae that were illuminated episodically to induce photo-antagonism showed a significant increase in arbor radius ( Figure 9f , ΔR = 16 . 4 ± 1 . 7% , n = 12 axons ) , compared to illuminated vehicle-treated ( ΔR = 1 . 2 ± 3 . 4% , n = 9 axons ) or wild-type siblings ( ΔR = 6 . 9% ± 2 . 6% , n = 10 axons ) ( Figure 9—figure supplement 1a ) .", "Tg[s1101t-gal4;UAS-GluN2A ( G712C ) ] larvae that were not treated with MAG , but were instead reared in 25 µM MK-801 , under the same conditions from 5–7 dpf , showed a comparable increase in arbor radius ( ΔR = 13 . 8 ± 3 . 7% , n = 10 axons ) , indicating that the efficacy of photo-antagonism was on par with a relatively strong dose of a soluble NMDA receptor blocker .", "MAG treatment alone had no effect on development compared to vehicle-treated siblings , based on an assessment of spontaneous swim behavior , escape behavior and habituation of escape behavior ( Figure 9—figure supplement 1b and Materials and methods ) .", "These results demonstrate that extended photo-antagonism of LiGluN2A ( G712C ) mimics the effect of in vivo chronic systemic exposure to a soluble NMDAR antagonist , producing a block of activity-dependent remodeling of retinotectal projections ." ], [ "We have used chemical optogenetics to engineer light-controlled NMDA receptors that can be used to study the mechanisms of NMDA receptor function and of synaptic transmission and plasticity .", "We generated a family of four light-gated NMDA receptor-subunits that provide reversible spatiotemporal control of receptor activity and synaptic plasticity via a photochemical switch that either activates or antagonizes specific 'LiGluN' subunits: LiGluN1a , LiGluN2A and LiGluN2B .", "The photo-agonized subunits can be photoactivated to yield persistent currents or be sculpted in time to mimic synaptic activation of the receptors ( Figure 1 ) .", "Photo-antagonism blocks the effects of perfused ligands ( glutamate/NMDA ) and of synaptically released glutamate , thus controlling NMDA receptor mediated EPSCs and LTP .", "Spatial confinement of photo-antagonism to a single dendritic spine blocks the spine expansion that is associated with LTP ( Figures 2–6 and 8 ) .", "As a complement to this , single-spine photo-agonism evokes spine-specific Ca2+-transients and spine expansion ( Figure 7 ) .", "We also report a transgenic zebrafish line that expresses the light-antagonized version of GluN2A , which enables chronic antagonism over days of larval development after a single application of MAG ( Figure 9 ) .", "We take advantage of the bistability of MAG ( Figure 1i and Figure 1—figure supplement 1e ) to produce chronic photo-antagonism with short pulses of light at long intervals over a period of 40 hr in freely swimming fish , thereby minimizing visual stimulation and avoiding photo-toxicity .", "We find that the photo-antagonism is as potent as MK-801 in interfering with the normal NMDA receptor dependent pruning process , producing an overgrowth of retinal ganglion cell axons in the optic tectum .", "Conveniently , in the zebrafish system , MAG can simply be added to the water to be taken up systemically , as shown earlier ( Janovjak et al . , 2010; Levitz et al . , 2013; Szobota et al . , 2007; Wyart et al . , 2009 ) .", "For photo-controlled NMDA receptors to work under physiological expression conditions , one would want LiGluN subunits to replace native subunits , rather than increasing the pool of NMDA receptors .", "Fortunately , in some preparations , GluN2A overexpression does not appear to increase synaptic content of NMDA receptors ( Barria and Malinow , 2002; Prybylowski et al . , 2002 ) , most likely because of the limiting supply of the native obligatory partner GluN1 subunit .", "We find that expression of LiGluN2A makes NMDA receptors sensitive to light without altering whole cell NMDA-induced current or NMDA receptor EPSCs evoked by action potential-driven glutamate release at autaptic synapses .", "The degree of photo-block of NMDA receptor EPSCs in autapses from neurons from wt rats ( Figure", "5 ) is small on average ( mean 25–35% , range ~20–50% without inclusion of non-responding cells , see Figure 5—figure supplement 1 ) , but shows that functional light-controlled receptors can be obtained in a wild-type background where the LiGluN subunits compete with wt subunits to assemble into receptors that are properly trafficked to the plasma membrane of the synapses .", "It should be noted that , the expression of the photoswitchable subunits in a wild-type background where the native subunit is expressed yielded variable effects , ranging from very strong block to no block at all ( Figure 5—figure supplement 1b ) .", "This may result from a combination of factors , including variation in expression level that results in variable incorporation into GluN2-containing receptors .", "Nevertheless , in responding cells , regardless of the magnitude of fractional optical control , the control is reproducible ( Figure 3—figure supplement 1 and Figure 5—figure supplement 1b ) .", "The degree of photo-block in other preparations , such as organotypic slices from the GluN2A-KO mice , where we demonstrated block of LTP ( Figure", "6 ) and either induction or block of spine expansion ( Figures 7–8 ) , is likely greater because of absence of the native GluN2A subunit .", "The chemical optogenetic approach is selective for the subunit that bears the cysteine anchoring site for the MAG photoswitch .", "Thus , our toolset allows for the direct and specific optical manipulation of GluN2A or GluN2B receptors and should make it possible to address the roles of these subunits in synaptic plasticity , circuit function and memory ( Foster et al . , 2010; Kohl et al . , 2011; Liu et al . , 2004; Massey et al . , 2004; Sakimura et al . , 1995; Weitlauf et al . , 2005 ) .", "In addition , owing to the ability to localize and shape light , it should also be possible to determine how synaptic transmission and circuit function are shaped by receptor type at various subcellular locations and within single dendritic spines .", "Chemical labeling and azobenzene isomerization have limitations that need to be considered .", "While neurons in culture and slice are easily incubated with MAG , and MAG can be readily delivered systemically in zebrafish when added to the swimming media ( Figure 9 ) ( Janovjak et al . , 2010; Levitz et al . , 2013; Szobota et al . , 2007; Wyart et al . , 2009 ) , we do not know the efficiency of this process and incomplete conjugation ( i . e . partial occupancy ) may contribute to the incomplete photo-agonism and photo-antagonism that we observe .", "In addition , a small fraction of MAG remains in trans during 380 nm illumination ( Gorostiza et al . , 2007 ) , and there may be differences in occupancy in the cis state of MAG molecules that are bound to the cysteine at the two possible stereochemistries of attachment to the maleimide ( Figure 1a ) ( Numano et al . , 2009 ) .", "We have recently solved two of these challenges for photoswitching a metabotropic glutamate receptor by replacing the maleimide-cysteine conjugation with the bio-orthogonal and very efficient conjugation of a benzylguanine photoswitch to a SNAP tag fused to the N-terminal of the LBD ( Broichhagen et al . , 2015 ) .", "Because the cysteine-reactive maleimide of MAG hydrolyzes , receptor conjugation will occur in the first hours after addition of MAG .", "Only those receptors that are on the plasma membrane during this time window will be labeled and become photo-controllable .", "As a result , the time frame of an experiment is limited by receptor-turnover , and for in vivo experiments extending several days , MAG may need to be reapplied .", "It also means that for studies of development , cells born after MAG application will not be under light control .", "This will mean that the tools do not work for some applications , or require an extra step ( i . e . MAG reapplication ) .", "In some cases it could actually provide an advantage to selectively antagonize or agonize based on receptor 'birthdate' .", "Recent advances in the design of azobenzene-based photo-switches have yielded new classes of MAG ( Kienzler et al . , 2013 ) that exhibit favorable two-photon absorption ( Carroll et al . , 2015; Gascón-Moya et al . , 2015; Izquierdo-Serra et al . , 2014 ) .", "This is particularly advantageous for light confinement ( Emiliani et al . , 2015 ) , especially for small subcellular compartments , and for deeper tissue penetration , for in vivo applications .", "Fortunately , in preparations such as C . elegans , Drosophila and zebrafish , where precise genetic targeting can be readily achieved ( i . e . of the photoswitch-ready subunit ) and where the nervous system is accessible to light and less scattering , it is already possible with conventional 1-photon illumination to accomplish MAG photo-control of glutamate receptors , as previously shown in zebrafish sensory neurons ( Szobota et al . , 2007 ) , the central pattern generator circuit for swimming ( Janovjak et al . , 2010; Wyart et al . , 2009 ) , pan-neuronally ( Levitz et al . , 2013 ) , and as shown here in transgenic zebrafish expressing the LiGluN2A ( G712C ) subunit ( Figure 9 ) .", "In conclusion , we have engineered a palette of light-controlled NMDA receptor subunits , which enable the fast and reversible remote control of specific receptor subtypes , in specific cells and thereby with presynaptic versus postsynaptic selectivity .", "These properties should enable a new level of study of the molecular mechanism of function of NMDA receptors and how specific NMDA receptors participate in synaptic transmission , integration and plasticity ." ], [ "Cysteine point mutations were introduced near , but outside of the glutamate-binding site of GluN2A , GluN2B and GluN1a using site-directed mutagenesis and verified by full sequencing .", "HEK293 or HEK293T cells were maintained in DMEM with 5% FBS on poly-L-lysine-coated glass coverslips at ~3 x 106 cells per milliliter and transiently co-transfected with GluN1a and GluN2A plasmids at a DNA ratio of 1:2 using Lipofectamine 2000 ( Invitrogen/Life technologies , Carlsbad , CA ) .", "Calcium imaging or patch clamping was performed 12–48 hr after transfection .", "Dissociated postnatal hippocampal neurons ( P0-P5 ) were prepared from Sprague Dawley rats ( Charles River ) at high density ( 80 K cells/coverslip ) and transfected as described previously ( Berlin et al . , 2015; Szobota et al . , 2007 ) , using 1 µg of cDNA encoding the NMDA receptor subunit ( s ) and 0 . 2 µg of GFP .", "For autaptic recordings , cells were plated at a lower density ( 20 K cell/coverslip ) with the extracellular medium enriched with 15 mM KCl , to promote the formation of autaptic connections .", "As an additional precaution , we transfected these neurons at very low efficiency so that very few neurons in a dish expressed LiGluN .", "We took this additional provision so that the illumination would only affect the population of LiGluN receptors located on the recorded cell , without affecting the activity of other cells in the vicinity ( of which the majority are non-transfected even under normal transfection methods nonetheless ) , as is the case of other soluble blockers .", "For Xenopus oocyte mRNA injection , we have cloned GluNRs and LiGluNRs into pGEM-HJ , synthesized mRNA in vitro and injected into defolliculated oocytes; as previously described ( Berlin et al . , 2010 ) .", "All experiments on hippocampal slices ( physiological experiments on LTP induction and single spine expansion experiments ) were in cultured ( organotypic ) slices from GluN2A-knockout mice ( Sakimura et al . , 1995 ) .", "Briefly , slices ( 400 µm thick ) were prepared at postnatal days 6–8 as previously described ( Fuller and Dailey , 2007 ) .", "Slices were cut in ice-cold , oxygenated dissection solution ( 95% O2/5% CO2 ) containing ( in mM ) 1 CaCl2 , 10 dextrose , 4 KCl , 5 MgCl2 , 26 NaHCO3 , 233 sucrose , 0 . 0005% phenol red , and grown on cell culture inserts ( Millipore , EMD ) in culture medium consisting of neurobasal medium ( no L-glutamine , GIBCO ) , horse serum ( Hyclone ) , insulin ( Sigma ) , ascorbic acid ( Sigma ) , Glutamax ( GIBCO ) , penicillin-streptomycin ( GIBCO ) and HEPES ( pH 7 . 4 ) at 34°C .", "Medium was supplemented with 4 µM cytosine β-D-arabinofuranoside hydrochloride ( AraC , Sigma ) the day after .", "Slices were biolistically transfected with gold particles ( 1 µm , Bio-Rad ) covered with the appropriate DNA combination ( O'Brien and Lummis , 2006 ) .", "AraC was withdrawn from the media prior to transfection and 10 µM MK-801 ( Tocris ) was added at that time .", "The MK-801 was removed on the day of experiment .", "HEK293 cells were loaded in recording solution with 5 μM Fura2-AM ( Molecular Probes ) for 30 min at 37°C , 5% CO2 .", "The recording solution contained ( in mM ) : 150 NaCl , 5 KCl , 0 . 2 CaCl2 , 10 D-glucose , 10 D-sucrose , 10 HEPES , 0 . 01 EDTA , 0 . 05 glycine , pH 7 . 4 .", "Cells were labeled with 50–100 µM MAG in glycine-free recording solution .", "Changes in intracellular [Ca2+] in individual cells were measured from Fura2-AM fluorescence intensity by brief ( <1 s , ~1 . 5 µW/mm2 ) excitation at 350 nm and 380 nm at 5 s intervals and by detecting emission at 510 nm .", "Patch clamp recordings used an Axopatch 200 A amplifier in the whole cell mode .", "Recordings were carried out 12 to 48 hr after transfection in HEK cells and after 15 DIV for hippocampal neurons .", "Cells were pre-treated with 1 mM DTT for 5 min , rinsed for 10 min , and incubated with 50–100 µM MAG ( and for GluN2A ( G712C ) , 500 µM AP5 ) for 30 min at 37°C , 5% CO2 .", "The labeling solution contained ( in mM ) : 150 NMDG-HCl , 3 KCl , 0 . 5 CaCl2 , 5 MgCl2 , 10 HEPES , 5 D- glucose , pH 7 . 4 .", "Cells were voltage-clamped at -60 mV .", "Pipettes had resistances of 2–8 MΩ and were filled with a solution containing , for HEK cells ( in mM ) : 110 D-gluconic acid , 30 CsCl , 4 NaCl , 5 HEPES , 5 BAPTA , 0 . 5 CaCl2 , 2 MgCl2 , pH 7 . 3 , and for neurons ( in mM ) : 136 . 5 K-gluconate , 17 . 5 KCl , 9 NaCl , 1 MgCl2 , 10 HEPES , 0 . 2 EGTA , pH 7 . 3 .", "The extracellular recording solution for HEK cells was as described above for calcium-imaging experiments .", "For dissociated rat hippocampal neurons , when assessing the relative photo-current ( or block ) compared to the total NMDA- induced current the extracellular recording solution , nominally Mg2+-free and with high external Ca2+ , contained ( in mM ) : 138 NaCl , 1 . 5 KCl , 10 D-glucose , 3 . 7 CaCl2 , 5 HEPES , pH 7 . 4 and 0 . 05 glycine .", "All other experiments with cultured neurons were performed with 2 . 5 mM external Ca2+ .", "Illumination was applied using a Polychrome monochromator ( TILL Photonics ) ( also see below ) coupled to the back port of an Olympus IX70 inverted microscope .", "Light intensity measured at the 40x objective was ~3 mW/mm2 .", "Recording of autapses was performed using a standard protocol as described in ( Levitz et al . , 2013 ) .", "Briefly , cells we held at −70 and depolarized to +20 or 40 mV for 3–5 ms , then returned to −70 mV .", "For recording NMDA-dependent spontaneous or evoked EPSC ( sEPSCNMDA and eEPSCNMDA , respectively ) , cells were incubated with 20 µM CNQX , in nominally Mg2+-free extracellular recording solution and with 2 . 5 mM CaCl2 .", "To note , during autaptic recordings we consistently observed the gradual reduction in eEPSCNMDA amplitude ( transfected and non-transfected neurons ) , consistent with previous reports ( Goda and Stevens , 1998 ) .", "Hippocampal slices were obtained from GluN2A-knockout neonate mice and electrophysiological recordings to measure LTP induction were done on at 6–8 d in vitro .", "Just before recording , slices were incubated at room temperature ( ~25°C ) with 100 µM TCEP for 1 min , rinsed for 2 min , and incubated for 45 min with 250 μM MAG and 500 µM AP5 diluted in the NMDG-labeling solution ( see above ) .", "Slices were rinsed twice in labeling solution before recording .", "Whole-cell patch-clamp recordings were performed on an upright Zeiss AxioExaminer using an Axopatch 200B amplifier ( Molecular Devices ) .", "A bipolar stimulating electrode was placed along the Schaffer collateral pathway and post-synaptic currents were recorded in whole-cell mode from transfected CA1 pyramidal neurons ( Figure 6 ) .", "The internal solution contained ( in mM ) : 142 CsCl , 2 MgCl2 , 1 EGTA , 10 HEPES , 0 . 4 Na3GTP , 4 . 4Na2ATP , 5 QX314 , pH 7 . 4 .", "Slices were perfused with a medium containing ( in mM ) : 118 . 9 NaCl , 2 . 5 KCl , 2 NaH2PO4 , 26 . 2 NaHCO3 , 11 D-Glucose , 2 . 5 CaCl2 , 1 . 3 MgCl2 and 0 . 01 glycine , pH 7 . 4 when saturated with 95% O2 / 5% CO2 .", "The light used for photoswitching was from a DG-4 ( Sutter Instruments ) coupled to the microscope and projected onto the sample through a 40× objective .", "Light intensity measured at the sample was approximately 43 mW/mm2 at 390 nm and 51 mW/mm2 at 497 nm .", "EPSCs were recorded in voltage-clamp mode and the tetanus ( consisting of two 1 s trains of 100 Hz separated by 20 s ) was delivered in current-clamp mode .", "Two-electrode voltage clamp experiments were performed in Xenopus laevis oocytes injected with 50 nl mRNA of the different GluN subunits at 1–1 . 5 ng/oocyte .", "GluN2A ( wt , G712C , V713C ) and GluN2B ( wt , V714C ) were injected with GluN1a-wt at a ratio of 1:1 .", "GluN1a ( E406C ) was injected with GluN2B-wt .", "Cells were then incubated in ND-96 ( 96 mM NaCl , 2 mM KCl , 1 . 8 mM CaCl2 , 1 mM MgCl2 , 50 mg/ml gentamicin , 2 . 5 mM Na-pyruvate and 5 mM HEPES , pH 7 . 6 ) at 18°C for 24 hr .", "For measuring glutamate efficacy , cells were clamped at −60 mV and perfused with Mg2+-free extracellular solution containing ( mM ) : 100 NaCl , 0 . 3 BaCl2 , 5 HEPES ( adjusted with KOH to 7 . 3 ) , 100 µM Glycine and 10 µM DTPA ( zinc chelator- added before the experiment ) .", "Glutamate concentrations ranged from 0 . 1 to 100 µM .", "For Glycine efficacy of GluN1a ( wt , E406C ) , extracellular solution contained 10 µM glutamate and glycine concentrations ranged from 0 . 1 to 10 µM .", "Recordings were performed with the use of a Dagan CA-1 amplifier ( Dagan Corporation ) , controlled by the Digidata-1440 board and pClamp10 software package ( Axon Instruments ) .", "Most electrophysiological experiments were performed with illumination that was applied to the entire field of view using a Polychrome V monochromator ( TILL photonics ) through 20 or 40x objectives .", "For organotypic slice recordings ( Figure", "6 ) illumination was applied using a Lambda DG4 high speed wavelength switcher ( Sutter instruments ) , with a 380 nm and a 500 nm filters through a 20x objective .", "Fast , millisecond photoswitching ( Figure 1f–h ) and fast photouncaging ( Figure 1—figure supplement 2g , h ) was achieved with a laser spot illumination system ( Reiner and Isacoff , 2014 ) .", "In brief , the output of a 375/488 nm dual diode laser module ( Omicron LDM: 375 nm 200 mW multi-mode , 488 nm 80 mW single-mode ) was coupled into a UV/VIS multi-mode fiber ( OZ Optics , 10 μm , NA 0 . 1 ) , the divergence of the exiting light reduced by threefold magnification of the fiber end , and the collimated beam directed to the objective .", "The laser output was controlled with TTL pulses and analog power modulation .", "Single spine illumination was performed on a confocal microscope ( Zeiss 780-upright confocal ) equipped with a 405 nm laser ( 100 μW at the objective ) .", "To note , we consistently used near-UV illumination ( 365–405 nm ) for photoactivation , as nucleotides , DNA and proteins do not efficiently absorb in this region ( Forné et al . , 2012; Schmid , 2001 ) , making these wavelengths and technique undamaging and compatible with biological preparations .", "Targeted illumination with simultaneous electrophysiological recordings were performed on a Zeiss 780-upright confocal microscope ( e . g . Figure 1i , dashed box and Figure 1—figure supplement 1b , c ) .", "For sculpting the photo-activation and deactivation profile of light-agonized GluN2A ( V713C ) the 488 nm light intensity was modulated .", "Typically 4–8 traces were averaged and the apparent activation and photo-deactivation kinetics were fitted with single exponential functions .", "For photouncaging experiments , 4-methoxy-7-nitroindolinyl-caged-L-glutamate ( MNI-glutamate , Tocris ) was added to the bath ( 0 . 5 mM ) and uncaged with a short 375 nm laser pulse centered at the cell ( ~50 W/mm2 , ~ 15 μm spot diameters ) .", "Uncaging pulses of 0 . 5 ms , 1 ms and 2 ms yielded identical activation kinetics , demonstrating that these reflected the intrinsic receptor activation kinetics rather than concentration-dependent second order binding .", "Deactivation due to diffusion of glutamate out of the uncaging site occurred on the timescale of seconds and became slower , as more glutamate was uncaged with longer pulse lengths .", "Rise times ( 10–90% ) were used to describe the speed of activation and were compared to wt-receptors using two-tailed , unpaired t-tests .", "Hippocampal cells ( 15 DIV ) were assayed for cellular and membrane viability following different treatments:", "1 ) incubation with 300 µM L-MAG1 in NMDG-labeling solution ( see above ) ,", "2 ) with 0 . 3% DMSO ,", "3 ) with NMDG-labeling solution or", "4 ) untreated .", "Cells were labeled using the Neurite outgrowth kit ( Molecular Probes ) and red and green fluorescence was acquired using a Zeiss 780-upright confocal microscope .", "Assessment was done by comparing multiple coverslips ( N= 2–9 ) at same cellular density .", "Confocal images of red ( neurite content ) and green ( cell viability ) fluorescence were taken under identical imaging settings and identical region sizes ( as described by the manufacturer ) .", "Data is presented as mean ± SEM and compared using one-way analysis of variance ( ANOVA ) with a post hoc Tukey test , all-pairwise analysis ( n . s . ; not significant ) .", "Single spine imaging experiments were performed in organotypic slices obtained from GluN2A-knockout neonate mice at room temperature ( ~25°C ) in Mg2+-free and high-Ca2+ACSF containing ( in mM ) : 118 . 9 NaCl , 2 . 5 KCl , 1 NaH2PO4 , 26 . 2 NaHCO3 , 11 glucose , 4–5 CaCl2 , 0 . 001 TTX and 2 . 5 MNI-glutamate , oxygenated with 95% O2 and 5% CO2 , as typically described elsewhere ( Matsuzaki et al . , 2004; Okamoto et al . , 2004; Harvey and Svoboda , 2007; Harvey et al . , 2008; Makino and Malinow , 2009 ) .", "TTX and glycine were added and MK-801 removed at least 25 min before start of the imaging experiments .", "Images were taken using a laser scanning microscope; Zeiss 780-upright confocal microscope .", "Photo-uncaging of MNI-glutamate and photo-activation of MAG ( 300 µM ) for either photo-agonism or photo-antagonism was triggered by illumination with a 405 nm laser at ~1 Hz for a total of 1–2 min , as previously described for MNI-uncaging ( Matsuzaki et al . , 2004; Nishiyama and Yasuda , 2015 ) .", "To test the ability of photo-agonism to induce a rise in calcium in single spines , we expressed a fusion of the calcium indicator R-GECO1 . 0 ( Zhao et al . , 2011 ) to the C-terminus of GluN2A ( V713C ) ( GluN2A ( V713C ) -R-GECO ) and co-transfected with additional soluble R-GECO , to improve the calcium signal detection within the entire spine head , under excitation at 561 nm , and eGFP , to enable simultaneous imaging of spine size under excitation at 488 nm .", "In the photo-agonized ( 100 µW for 100 µs/pixel ) spine , the R-GECO signal decayed over tens of seconds , even though the photo-agonized GluN2A containing NMDAR remains open without decay for tens of seconds , due to bleach induced by 3 different illuminating lasers ( 405 , 488 and 561 nm ) .", "We applied this unique combination of laser-illumination , to bypass the known artifacts of R-GECO , which undergoes unique reversible bleaching ( Shaner et al . , 2008 ) .", "Spine morphological changes were assessed by time-lapse Z-stack images of single spines collected using a 20x/NA=1 . 0 water immersion objective ( digital zoom: 10–13 ) by imaging space-filling cytosolic tdTomato with a 561 nm laser .", "3-dimensional projection images ( of 512x512 or 1024x1024 pixels ) were exported ( Zen 2011 software , Zeiss ) for analysis with a custom MATLAB program ( Peled and Isacoff , 2011 ) .", "We measured the fluorescence of the spine ( as shown in [Zhang et al . , 2008] ) , which is proportional to spine-head volume ( Holtmaat et al . , 2005 ) , and normalized it to the fluorescence of the shaft to correct for any changes that may have occurred to fluorescence; resulting from bleach or movement ( Nimchinsky et al . , 2004 ) .", "Nearby spines typically consisted of spines located on the same dendrite ( <10 µm from photo-stimulated spine ) found in the same field of view as the photostimulated spine or atop dendrites that ran across the field of view , nearby the photostimulated region .", "To determine whether the ability to induce spine expansion was restored to CA1 neuronal spines , in slices prepared from GluN2A-knockout mice , neurons were transfected with the GluN2A ( V713C ) subunit and single spines were assessed for expansion by uncaging MNI-glutamate .", "MNI-glutamate ( 2 . 5 mM final concentration ) was allowed to diffuse into the slice during 1–2 min of superfusion with Mg2+-free bathing solution , in the presence of 2 µM TTX , before photo-uncaging .", "Photo-uncaging with 405 nm light ( 100 µW for 100 µs/pixel ) at a spot of the same lateral size as the spine head , but located 0 . 5 µm away ( Figure 8—figure supplement 1b ) .", "This illumination reliably induced expansion of the spine below the spot of illumination ( Figure 8—figure supplement 1b , dashed circle ) ( Hardingham and Bading , 2010; Lee et al . , 2009 ) , indicating rescue of structural plasticity by expression of the cysteine-bearing subunits .", "To generate a stable transgenic line of LiGluN2A zebrafish , we inserted the rat-GluN2A ( G712C ) gene with a N-terminal GFP tag under control of a 10X-UAS sequence into a pT2KXIG-delta-IN vector containing Tol2 transposon recognition sites ( Suster et al . , 2009 ) using EcoRI and NotI restriction sites .", "The resulting pT2-LiGluN2A ( G712C ) construct was diluted to 50 ng/μL with 0 . 25 ng Tol2 transposase and 0 . 05% Phenol Red ( Sigma ) .", "Injected embryos were raised to sexual maturity and outcrossed to identify founder ( F0 ) fish .", "F1 fish were genotyped for GluN2A ( G712C ) using primers 5’-TGCA GAG AAT CGG ACC CAC T-3’ and 5’-TCG ATC ACT GCC CTC ACT GT-3’ .", "F1 Tg[UAS:GluN2A ( G712C ) ] fish were outcrossed to a pan-neuronal transgenic Gal4 line , Tg ( s1101t-gal4 ) .", "Double transgenic Tg[s1101t- gal4;UAS-GluN2A ( G712C ) ]; fish grew to sexual maturity with no obvious phenotypes .", "To determine whether exposure to MAG affected the health or development of zebrafish larvae , we assayed basic spontaneous and evoked behaviors of freely swimming animals for up to 2 days post-treatment .", "5 dpf wildtype larvae were bathed in either with 150 µM MAG or vehicle only under the same conditions as used in the LiGluN2A experiments .", "Fish were transferred to 48-well plates ( 1 fish per well with 800 µL fresh E3 ) and placed in an imaging arena ( Levitz et al . , 2013 ) .", "After a 10 min acclimation period , spontaneous behavior was filmed for 10 min .", "Movies were analyzed , using custom made Matlab programs , to determine population statistics for metrics representative of spontaneous motor behaviors: distance traveled , time spent resting , place preference , and the frequency and duration of spontaneous swim bouts .", "MAG-treated animals exhibited no significant difference in any measure compared with DMSO-treated controls .", "For visualization of individual retinal ganglion cells , embryos from incrosses of Tg[s1101t-gal4;; UAS-GluN2A ( G712C ) ] fish were injected with pou4f3-gal4:UAS-mGFP DNA ( mGFP; monomeric membrane-bound due to palmitoylation sequence from gap43 ) .", "Injected embryos were reared in E3 containing 0 . 005% 1-phenyl 2-thiourea ( PTU ) and screened based on GFP expression at 48 hpf .", "Typically , injected embryos had 1–3 non-overlapping labeled axons .", "At 120 hpf , pou4f3- gal4:UAS-mGFP positive fish were assessed for spontaneous swimming and inflated swim bladder as markers for normal development .", "Healthy animals were divided into two groups to receive treatment in either 150 µM MAG-0 in E3 with 0 . 3% DMSO or in E3 with 0 . 3% DMSO for 40 min at 28 . 6°C .", "All fish then were rinsed twice in fresh E3 and allowed to recover for 2 hr at 28 . 6°C .", "Fish were then embedded in 1 . 4% low-melting agarose in E3 for an initial imaging session to measure baseline morphology of the RGC projections .", "Following the baseline imaging session , each animal was freed from agarose and transferred to fresh E3 containing 0 . 005% PTU to recover from anesthesia .", "Fish typically recovered spontaneous swimming within 1 hr .", "Between 128 hpf and 168 hpf , fish were subjected to a photo-antagonism protocol described below .", "Each fish was then remounted in agarose for a second imaging session to assess RGC growth .", "Following the second imaging session , fish were euthanized in 0 . 4% tricaine .", "Genomic DNA was then extracted from individual whole animals in 60 µl volume ( Meeker et al . , 2007 ) and genotyped for the UAS-GluN2A ( G712C ) transgene using the PCR primers described above .", "At 128 hpf , each fish was transferred to a 48 well plate in 800 µl E3 with 0 . 005% PTU and placed in a custom imaging chamber equipped with an array of 405 nm LEDs centered under each well .", "The LED array was controlled by a DAQ ( National Instruments ) programmed to produce 10 s flashes every 30 min .", "The imaging chamber maintained ambient lighting on a 14 hr light/10 hr dark cycle with an ambient temperature of ~26°C .", "Twenty minutes prior to each imaging session , each fish was mounted dorsal-side up in a glass-bottom dish in 1 . 4% low-melting-point agarose dissolved in E3 , and anesthetized with 0 . 02% tricaine .", "Z-stacks with 0 . 35–0 . 5 µm steps were obtained using a 2-photon Zeiss LSM710 microscope equipped with a 3 W Ti-Sapphire laser ( Coherent , Chameleon Ultra ) tuned to 920 nm .", "Minimal laser intensity was used ( <20 mW measured at the front of the objective ) .", "A long working distance water-dipping 40X/1 . 0 NA objective was used to acquire all images .", "In each 3- dimensional image , RGC axon arbors were traced using the Simple Neurite Tracer plug-in for ImageJ ( Longair et al . , 2011 ) .", "Sholl analysis was performed on each isolated 3-dimension arbor , where spheres were centered on the first branch point remaining at 7 dpf ( Figure 9c , inset ) .", "MAG photo-switches are now available commercially from Aspira​ Scientific: http://www . aspirasci . com/neuroscience-probes All animal experiments were done under oversight by the University of California institutional review board ( Animal Care and Use committee ) .", "This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health .", "All of the animals were handled according to approved institutional animal care and use committee ( IACUC ) protocols ( AUP-2015-04-7437 ) of the University of California .", "Every effort was made to minimize suffering .", "Results are shown as mean ± SEM .", "Data sets of >3 points of data were tested for Normality ( normal distribution ) , followed by a multiple group comparison , using one-way analysis of variance ( ANOVA ) with a post hoc Tukey test .", "Data sets which failed the normality test were followed by ANOVA on ranks ( Mann-Whitney Rank Sum Test ) .", "All-pairwise analysis and two group comparisons were done using two-tailed , T-test or paired T-test , when applicable ( SigmaPlotTM , 2011 , Systat Software Inc . , San Jose , CA ) .", "Rank Sum test was applied whenever normality failed for two group comparison .", "Asterisks indicate statistically significant differences as follows: *p<0 . 05; **p<0 . 01; ***p<0 . 001 , n . s . , not significant .", "Box plots display medians , 10th and 90th percentiles and outliers ( filled circles ) .", "Individual data points , when displayed , are found to the right of the box plot as filled squares ." ] ]
[ "NMDA receptors , which regulate synaptic strength and are implicated in learning and memory , consist of several subtypes with distinct subunit compositions and functional properties .", "To enable spatiotemporally defined , rapid and reproducible manipulation of function of specific subtypes , we engineered a set of photoswitchable GluN subunits ( 'LiGluNs' ) .", "Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation .", "Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion , the morphological correlate of LTP .", "Photo-antagonism of GluN2A alone , or in combination with photo-antagonism of GluN1a , reversibly blocks excitatory synaptic currents , prevents the induction of long-term potentiation and prevents spine expansion .", "In addition , photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish .", "By providing precise and rapidly reversible optical control of NMDA receptor subtypes , LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission , integration and plasticity ." ]
[ "Within the nervous system , neurons are organized into extensive networks via connections called synapses .", "To signal across a synapse , one neuron releases chemical messengers that bind to “receptors” on the surface of the neighboring cell .", "The ease with which neurons can communicate can change depending on how often a synapse is used .", "This adaptability is known as synaptic plasticity , and is central to the formation of memories .", "NMDA receptors are one group of receptors that play an important role in synaptic plasticity .", "There are several types of NMDA receptor , which are made up of different combinations of protein subunits and have different properties .", "This means that each type contributes to synaptic plasticity in a slightly different way .", "Other receptors found in neurons have been studied using a technique called chemical optogenetics , which allows the activity of modified proteins to be turned on and off by light .", "Now , Berlin , Szobota et al . have designed a toolbox that enables the activity of four of the NMDA receptor subunits to be controlled with light , which activates or blocks the NMDA receptors that they form ( which includes several of the main receptor types ) .", "Thus , how these types of NMDA receptor contribute to synaptic plasticity can be investigated .", "The toolbox can be used to control synaptic plasticity under a wide range of conditions .", "Plasticity can be induced or prevented in either single synaptic connections or large regions containing many thousands of synapses .", "The approach works in individual neurons grown artificially in the laboratory , in brain slices and in the living brain .", "Furthermore , synaptic plasticity can be controlled precisely to affect single synaptic events ( which occur in milliseconds ) or it can be controlled over several days to study whether this affects how neurons develop .", "The next steps will be to expand the toolset so that the activity of all the NMDA receptor subtypes can be controlled using light .", "Further studies could then incorporate the receptors into the brains of mammals to study how the receptors’ activity affects a range of processes including memory formation and disease ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "tools and resources", "genetics and genomics" ]
Systematic imaging reveals features and changing localization of mRNAs in Drosophila development
elife-05003-v1
[ [ "Cell differentiation is accompanied by polarization and segregation of membranes , cytoplasm , and organelles .", "A powerful mechanism to generate subcellular asymmetries used by eukaryotes and even prokaryotes is mRNA localization in combination with controlled protein translation ( reviewed in Medioni et al . , 2012 ) .", "Long-range mRNA transport in most metazoans relies on the polarized cytoskeleton and the microtubule minus- and plus-end motor complexes .", "mRNA enrichment at microtubule minus-ends is aberrant in mutants that affect the dynein motor complex , while plus-end directed transport requires kinesin molecules ( reviewed in Bullock , 2011; Medioni et al . , 2012 ) Mechanistic dissection of several canonical localization examples showed that , mRNAs localize through cis-regulatory sequences , zipcodes , which are often present in the 3′UTR of the transcript ( reviewed in Jambhekar and Derisi , 2007 ) and zipcode-binding proteins that initiate the formation of transport competent ribonucleoproteins ( RNPs ) ( Dienstbier et al . , 2009; Bullock et al . , 2010; Chao et al . , 2010; Dix et al . , 2013 ) .", "mRNAs can also harbour two antagonizing localization signals that act consecutively in cells and direct mRNAs sequentially to opposing microtubule ends ( Ghosh et al . , 2012; Jambor et al . , 2014 ) , suggesting that transport RNPs could be regulated .", "It has further been shown that some mRNA localization elements are active in several cell types suggesting that the mRNA transport machinery is widely expressed and mRNA localization elements function in a cell-type independent manner ( Kislauskis et al . , 1994; Bullock and Ish-Horowicz , 2001; Snee et al . , 2005; Jambor et al . , 2014 ) .", "In addition to microtubule-based transport , some mRNAs can enrich by trapping to a localized anchoring activity ( Forrest and Gavis , 2003; Sinsimer et al . , 2011 ) or by hitch-hiking along with a localization-competent mRNA ( Jambor et al . , 2011 ) .", "Recent live-imaging studies revealed that the same mRNA can , depending on the cell type , use both diffusion and active transport mechanisms ( Park et al . , 2014 ) .", "Furthermore , in vitro data showed that mRNA transport along microtubules can occur both uni- and bi-directionally , suggesting mRNAs can switch between processive and diffusive transport modes ( Soundararajan and Bullock , 2014 ) .", "mRNA localization is perhaps best characterized in the oocyte of Drosophila melanogaster ( D . melanogaster ) where localization of oskar , bicoid , and gurken is instrumental for setting up the embryonic axes ( Berleth et al . , 1988; St Johnston et al . , 1989; Ephrussi et al . , 1991; Neuman-Silberberg and Schüpbach , 1993 ) .", "However , more recent work suggests that mRNA localization is not occurring only for few singular mRNAs but instead is a widespread cellular feature that affects a large proportion of expressed mRNAs ( Shepard et al . , 2003; Blower et al . , 2007; Lecuyer et al . , 2007; Zivraj et al . , 2010; Cajigas et al . , 2012 ) .", "How a cell distinguishes localized from ubiquitous transcripts and orchestrates transport of many mRNAs remains enigmatic .", "It is conceivable that each localized mRNA carries its own zipcode sequence that directs it to a specific subcellular location .", "However , despite wealth of data on co-localized transcripts , computational methods thus far fail to detect such signals in a reliable manner .", "Alternatively co-packaging of several mRNA species , only one of which carries specific localization signal , has been shown in at least two cases ( Lange et al . , 2008; Jambor et al . , 2011 ) .", "It is also unclear to what extent the mRNA localization status is subject to tissue specific regulation .", "Here , we describe a genome-wide image-based resource that unravels the global landscape of mRNA localization in the Drosophila ovary by combining stage-specific mRNA sequencing with systematic fluorescent in situ hybridizations ( FISH ) and imaging .", "The localized transcripts show characteristic gene level features , such as longer and highly conserved 3′UTRs , which clearly distinguish subcellular enriched from ubiquitous mRNAs .", "Comparing mRNA localizations across the sampled time-points showed that the localization status of the majority of mRNAs changes in the oocyte as oogenesis progresses .", "These changing localizations are not due to alternative gene expression since the germline cells of the Drosophila ovary show only little transcriptional change .", "Integrative analysis of ovary localization data together with similar data from embryos ( Lecuyer et al . , 2007 ) also revealed that mRNA localizations differ across cell types .", "Therefore , mRNA localization is widespread in cells and is highly regulated ." ], [ "To globally investigate post-transcriptional regulation through mRNA localization , we systematically probed and imaged the expression and subcellular distributions of mRNAs in egg-chambers mass isolated from Drosophila ovaries .", "We combined stage-specific mRNA sequencing ( 3Pseq and RNAseq ) with genome-wide fluorescent in situ hybridization ( FISH ) .", "RNA sequencing data , expression pattern annotations ( using a hierarchical controlled vocabulary-http://tomancak-srv1 . mpi-cbg . de/cgi-bin-public/ovary_annotation_hierarchy . pl ) and images ( representative 2D images and all original z-stacks ) are collected in a publicly accessible database , the Dresden Ovary Table , DOT ( http://tomancak-srv1 . mpi-cbg . de/DOT/main ) ( Figure 1—figure supplement 1A , B ) .", "This genome-wide resource also integrates data on tissue-specific gene expression ( Tomancak et al . , 2002 , 2007 ) and subcellular mRNA localization ( Lecuyer et al . , 2007 ) in Drosophila embryos .", "Based on our in situ hybridization screen , we identified 3475 mRNAs as being expressed and most of these mRNAs were also detectable by RNA sequencing .", "Both sequencing techniques were in good agreement with each other ( Figure 1A , Figure 2—figure supplement 1A , Figure 5—figure supplement 1A ) .", "Of the expressed genes , 64% showed ubiquitous mRNA distribution in ovary cells throughout oogenesis ( ubiquitous ) , but we also observed mRNA expressions restricted to subsets of cells ( cellular ) and mRNAs that asymmetrically localized in the cytoplasm ( subcellular ) or to the nuclei of cells ( nuclear ) . 10 . 7554/eLife . 05003 . 003Figure 1 . Summary of the fluorescent in situ hybridization ( FISH ) screen in ovaries .", "( A ) Summary of key numbers of the screen .", "For each of the 6091 FISH experiments , we annotated the signal as no signal , ubiquitous , or specific .", "Specific and some ubiquitous signals were imaged .", "( B ) Schematic of exemplary subcellular expression patterns .", "( B′ )", "Exemplary subcellular expression patterns .", "In the syncytial early egg-chamber , 591 mRNAs are transported from the site of transcription in the nurse cells into the developing oocyte: mRNAs are either restricted to a cortical domain ( fwe ) or detectable in the entire ooplasm ( Imp ) .", "mRNAs also simultaneously enriched in the oocyte portion of the syncytial egg-chamber and at the apical membrane of the somatic epithelial cells ( Shroom ) .", "Five mRNAs were specifically excluded from the oocyte portion and enriched in the nurse cells ( Nacalpha ) .", "Few mRNAs were enriched anterior in stage 2–7 oocytes ( mus209 ) .", "mRNAs showed ubiquitous granules in the cytoplasm ( CG17494 ) or rarely ring-like staining patterns ( RpS6 , inset [10 × 10 μm] showing only the RNA channel ) .", "mRNAs enriched around the nucleus of the oocyte and/or the nurse cells ( msk ) varying from a ring around the entire nucleus to restricted localization in sub-areas of the perinuclear space ( spoon ) .", "Apical enrichment ( CG43693 ) or basal localization ( CG12171 ) was detected in late epithelial somatic cells .", "Anterior and posterior RNA localization varied between diffuse ( fs ( 1 ) N , yemalpha ) and tight cortical enrichments ( Lcp65Ac , mus210 ) .", "( C ) Distribution of subcellular localized mRNAs in subcategories .", "Note: mRNAs can appear in more than one subgroup .", "( D ) GO-term enrichment analysis of ubiquitous , cellular , nuclear , and subcellular gene sets . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 00310 . 7554/eLife . 05003 . 004Figure 1—figure supplement 1 . Experimental outline and database features .", "( A ) Overview of the experimental procedure for transcriptome and genome-wide in situ hybridization experiments and evaluation .", "( B ) Screenshot of the publicly available Dresden ovary table , DOT , and key search and download functions . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 00410 . 7554/eLife . 05003 . 005Figure 1—figure supplement 2 . GO-term enrichment analysis for gene sets . GO-terms associated with ubiquitous , subcellular , cellular , nuclear , oocyte enriched , anterior and posterior gene sets .", "Shown is also analysis of all ‘localization competent’ mRNAs .", "Bar plots show the p-values for each GO-term calculated by the modified Fisher Exact test , which results in the EASE score p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 005 Subcellular mRNA localization affected 790 mRNAs ( 22% ) but was limited to small number of subcellular domains ( Figure 1B–C ) .", "The largest group was 591 mRNAs that were enriched in the oocyte portion of the syncytial egg-chamber during early oogenesis ( fwe , Imp , Shroom ) .", "At this stage , the microtubule minus ends of the polarized microtubule cytoskeleton are also concentrated in the oocyte ( reviewed in Steinhauer and Kalderon , 2006 ) .", "At mid-oogenesis the oocyte establishes its own polarized microtubule cytoskeleton ( Steinhauer and Kalderon , 2006 ) and at this stage , we observed 106 mRNAs enriched towards the anterior and 119 mRNAs enriched at the posterior pole .", "The quality of these localizations ranged from tight ( mus210 , Lcp65Ac ) to diffuse association ( yemalpha , fs ( 1 ) N ) at the anterior-dorsal , the entire anterior or the posterior cortex .", "mRNAs were also detected in subcellular domains of the nurse ( msk , spoon ) and somatic epithelial cells ( CG43693 , CG12171 ) .", "For few mRNAs , we observed previously unknown ovary accumulations , for example mRNAs in cytoplasmic granules ( CG17494 ) , depleted from the oocyte ( Nacalpha ) , showing cortical enrichment ( Actn ) , or forming ring-like structures ( CG14639 , Figure 1B' , Figure 2—figure supplement 1E ) .", "The 309 mRNAs ( 13% ) of the cellular category were predominantly expressed in the somatic epithelium ( follicle cells ) and often restricted to a subset of epithelial cells at specific oogenesis stages ( Figure 2A , B ) .", "191 RNAs were detectable specifically in ovarian nuclei , mostly of the endocycling , polyploid nurse cells , but also in epithelial cells and in 29 cases in the oocyte nucleus ( Figure 2C , D ) .", "The RNAs in ovarian nuclei were visible from stage 9 of oogenesis onwards and their localization changed appearance from stage 9 to 10 ( Figure 2—figure supplement 1B ) .", "Nuclear patterns varied from ring-like signal to dispersed foci or widespread distribution in the nucleoplasm and were not linked to the chromosomal position of the genes ( Figure 2—figure supplement 1C ) .", "Precursors of micro RNAs and long non-coding RNAs also showed varying degrees of nuclear enrichments ( Figure 2—figure supplement 1D ) . 10 . 7554/eLife . 05003 . 006Figure 2 . Summary of cellular and nuclear expression patterns .", "( A , C )", "Exemplary FISH experiments for the cellular ( A ) and nuclear ( C ) expression sets .", "RNA is shown in green and the DNA ( labelled with DAPI ) is shown in magenta .", "Scale bars: 30 μm .", "( A ) tutl is expressed in cap cells at the tip of the germarium , while Ect3 mRNA is detectable in the somatic epithelial cells of the germarium .", "Several mRNAs are expressed in mosaic pattern , indicating cell cycle control in somatic epithelial cells ( His3 . 3A , Obp99a ) and in nurse cells ( His3 . 3A ) .", "Expression in the anterior and posterior follicle cells is often seen simultaneously ( CG11275 , CG11147 , Nep2 ) .", "Some mRNAs were expressed only in anterior follicle cells that become migratory border cells ( Men-b ) or in posterior follicle cells ( CG9336 ) .", "CG8303 is expressed in the somatic cells destined to become columnar epithelium .", "aop is exclusively seen in follicle cells that will give rise to the squamous epithelium and several mRNAs are specifically expressed here at later stages ( ImpL2 , CG7997 ) .", "mRNAs are also expressed in cells forming the border of columnar and squamous epithelial cells ( inx2 ) .", "( C ) Nuclei enrichments of RNAs in nurse cells varies from a ring-like expression ( CG11076 ) to foci in a discrete area ( Dbp80 ) , widespread foci ( CG10962 ) , or nucleoplasm signal ( Rm62 ) .", "RNAs are also detectable in epithelial cell nuclei ( pip ) and for 28 RNAs also in the oocyte nucleus ( e . g . , CG5819 ) .", "Greyscale image shows the respective RNA staining only in a zoomed-in view .", "( B ) The cellular gene set was subcategorized according to the specific cellular expression pattern .", "Individual mRNAs can fall into several of these subgroups .", "( D ) Instances of nuclear RNA enrichments in nurse cells , epithelial cells , and the oocyte . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 00610 . 7554/eLife . 05003 . 007Figure 2—figure supplement 1 . FISH screen results and controls .", "( A ) Estimate of false-positive/negative rate of the in situ screen using comparison with the independent transcriptomics data .", "A gene was classified as falsely positive if it was annotated as ubiquitous or specific by FISH but was not detectable by either 3Pseq or RNAseq at any time-point of oogenesis .", "In 20% of the experiments we failed to detect in situ signal ( ‘no signal’ ) although the transcript was detected at least at one time point by at least one deep sequencing method .", "These may represent false negative results , possibly due to non-functional RNA probes , however , we nevertheless included them in the downstream analysis in the no signal category .", "( B ) mRNA enrichments in the somatic epithelial cells overlaying the oocyte ( CG14639 ) and at the cortex of nurse cells ( Actn ) .", "RNA signal shown in green .", "DNA , labelled with DAPI , is shown in magenta .", "Scale bar 30 μm .", "( C ) CG9609 and Doa mRNAs detected in the oocyte nucleus showing the enrichment over time at stages 9 , 10A and 10B .", "At stage 9 only few small mRNA foci are visible , at stage 10 the mRNAs were enriched in proximity of the DNA in two large foci ( see arrows ) .", "( D ) Karyogram showing the chromosomal position of genes for nuclear , anterior , and posterior RNA localization classes .", "Neither nuclear RNA genes , which often appear in foci-like enrichments nor anterior or posterior class genes appear clustered on the chromosome .", "( E ) Examples of FISH experiment detecting distributions of non-coding RNA ( in green ) .", "While pri-miRNA-318 is enriched in somatic epithelial cell nuclei , pri-miRNA-303 , pri-miRNA-31-b and the long non-coding RNA CR42862 are restricted to nuclei of the germline nurse cells .", "Scale bar 30 μm , DNA in magenta . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 007 In summary , our screen revealed countless new instances of tissue-specific gene expression and mRNA localization in the ovary .", "The relatively low number of different subcellular localization sites allowed us to group mRNAs into subcellular localization gene-sets containing tens to hundreds of co-regulated genes .", "The division of RNAs into gene sets enabled us to address whether genes within each class are functionally related ( Figure 1D , Figure 1—figure supplement 2 ) .", "Gene Ontology ( GO ) analysis showed that the subcellular gene set is distinct from the cellular and the nuclear gene sets .", "Consistent with their respective expression , cellular genes are enriched for epithelial development , lipid trafficking and cuticle formation , nuclear genes for RNA regulatory processes and the subcellular gene set for reproductive processes , cytoskeleton organization , and cell cycle regulation .", "Anterior and posterior gene sets differed: anterior genes were enriched for microtubule terms and , being localized in proximity to the meiotic oocyte nucleus , are additionally associated with chromosome and cell cycle regulation terms .", "The posterior mRNAs associated strongly with signalling , cell fate commitment , and membrane organization terms .", "The GO analysis suggests that mRNAs that co-localize in the cytoplasm are functionally related .", "We next asked whether the proteins encoded by the mRNAs show physical interactions .", "To this end , we analysed the protein interaction data ( mentha interactome database [Calderone et al . , 2013] ) , which revealed that proteins of the posterior gene set participate in significantly more protein–protein interactions than of the anterior gene set ( Figure 3B ) .", "This suggests that the close proximity of their transcripts in the cell could be of functional importance .", "The gene sets defined by our ovary screen also maintained distinct expression patterns during embryogenesis .", "Genes of the subcellular sets are enriched among genes expressed in the central nervous system and epithelia , suggesting an interesting relatedness of these polarized tissues ( Figure 3A , Figure 3—figure supplement 1 ) .", "Thus , gene sets defined by ovary expression are co-regulated also beyond oogenesis . 10 . 7554/eLife . 05003 . 008Figure 3 . Localized mRNAs show gene set specific features .", "( A ) Linear hierarchy plot ( Tomancak et al . , 2007 ) showing stage- and tissue-specific re-expression of the ovary gene sets in embryogenesis .", "( B ) Protein interaction analysis per gene set revealed that posterior genes , but not anterior genes , share significantly more protein–protein interactions than would be expected by chance .", "( C ) Boxplot showing the median mRNA expression level is significantly higher in the posterior gene set compared to anterior mRNAs ( C′: Kolmogorov–Smirnov p-value: 3 . 9e-06 ) .", "Shown are 3Pseq quantifications from late ovary mRNA ( for early , full ovaries and early embryogenesis see Figure 3—figure supplement 2A ) .", "For description of gene sets see Supplementary file 1 .", "( D–E )", "Distributions of median 3′UTR length ( D ) and conservation of the 3′UTR sequence ( E , across 24 Drosophila species ) for gene sets .", "( D′–E′ )", "Results of a non-parametric randomization test to show that ( D′ ) ubiquitous and subcellular genes ( p-value = 0 ) and anterior and posterior genes ( p-value = 0 . 0018 ) have significantly different median 3′UTR lengths ( i . e . , no or little overlap of densities ) and ( E′ ) that ubiquitous genes are significantly less conserved in their 3′UTRs than subcellular genes ( p-value: 0 ) and posterior genes show higher conservation than anterior genes ( p-value: 0 . 0032 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 00810 . 7554/eLife . 05003 . 009Figure 3—figure supplement 1 . Ovary gene sets have specific expression patterns during embryogenesis . Linear hierarchy ( Tomancak et al . , 2007 ) plot showing at which embryonic stage and in which tissue the oogenesis gene sets are re-expressed during embryogenesis .", "Each colour-coded bar represents organ systems of the embryo from its stage-specific anlagen to primordia to final differentiated structures .", "The width of the bar is proportional to the frequency with which this annotation term was used in the embryo data set; the height corresponds to a z-score of over- ( above axis ) or under-representation ( below axis ) of the term in the set of genes defined by ovary annotation .", "The following oogenesis gene sets are shown: ubiquitous , nuclear , cellular , subcellular , no signal , nurse cells perinuclear , oocyte-enriched , oocyte anterior and oocyte posterior and apical in epithelial cells .", "Genes expressed ubiquitously in the ovary mostly remained ubiquitous in the embryo and were additionally enriched in meso- and endoderm; genes of the cellular gene set are enriched in ectoderm/epidermis cells of the late embryo; subcellular genes were highly expressed in the ectoderm and nervous system of the embryo .", "Most ‘no signal’ genes are also underrepresented in almost all stages and tissues of embryogenesis , apart from the PNS and ectodermal derivatives in the late stages of embryogenesis .", "Perinuclear enriched genes are highly expressed in meso- and endoderm tissues .", "Oocyte enriched , oocyte anterior , and oocyte posterior genes are overall very similarly expressed during embryogenesis , being high in the polarized CNS and ectoderm tissues . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 00910 . 7554/eLife . 05003 . 010Figure 3—figure supplement 2 . Gene features of subcellular enriched mRNAs .", "( A ) Boxplots showing the median mRNA expression measured by 3Pseq per gene set in early and full ovaries and in 0–2 hr embryos .", "At the onset of embryogenesis , the cellular mRNAs were almost as low as the ‘no signal’ class , confirming their predominant expression in somatic cells that at this time-point have undergone apoptosis .", "Accompanying the boxplot is the matrix of statistical significance tests ( Kolmogorov–Smirnov ) of the null hypothesis that the distributions of median expression values across the subcellular gene sets are the same .", "Statistically significant differences ( p < 0 . 01 ) are shown in dark grey , while gene sets that did not differ significantly are shown in light grey ( p > 0 . 01 ) .", "( B–I )", "Boxplots showing the median gene length ( B ) , exon length ( C ) , intron length ( D ) , 5′UTR length ( E ) , intron number ( F ) , exon number ( G ) , intron proportion ( H ) , and exon proportion ( I ) for each gene set and the corresponding significance level calculated by the Kolmogorov–Smirnov ( KS ) test .", "Statistically significant differences ( p < 0 . 01 ) are shown in dark grey , while gene sets that did not differ significantly are shown in light grey ( p > 0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 01010 . 7554/eLife . 05003 . 011Figure 3—figure supplement 3 . Embryo localized mRNAs also have long , conserved 3′UTRs . Distributions of median 3′UTR length ( A ) and conservation of the 3′UTR sequence ( B , across 24 Drosophila species ) for embryo gene sets .", "Shown are genes that are ubiquitously expressed during embryogenesis , genes whose RNAs were enriched at the apical or basal membrane in blastoderm embryos and RNAs at the posterior pole . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 01110 . 7554/eLife . 05003 . 012Figure 3—figure supplement 4 . Cytoplasmic but not nuclear mRNA localization requires the cytoskeleton .", "( A ) Localization of anterior and posterior mRNAs is lost upon microtubule depolymerization by colchicine .", "Shown are the anterior mRNAs fs ( 1 ) K10 and milt ( examples for diffuse-anterior and tight-anterior localization ) and the and posterior mRNA vkg and zpg ( examples for diffuse-posterior and tight-posterior ) .", "The appearance of the ubiquitous mRNA msl-2 is unchanged in colchicine treated egg-chambers .", "For details see Supplementary file", "8 . ( B ) Summary of the quality of all anterior and posterior mRNA distributions tested in colchicine treated egg-chambers ( round aggregates , tiny aggregates , dispersed and diffuse aggregates ) .", "Diffuse aggregates were observed for those mRNAs that in wild type egg-chambers showed a diffuse posterior enrichment ( e . g . , Figure 1B’: fs ( 1 ) N ) .", "( C ) mRNA localization in proximity to the nucleus is lost in colchicine treated egg-chambers ( Scp2 ) , RNAs localized partially nuclear and partially perinuclear loose the cytoplasmic localization ( CG11076 ) while strictly nuclear RNAs are unaffected by microtubule depolymerization ( rhi ) .", "( A , C )", "FISH experiments showing the RNA in green; DNA ( labelled with DAPI ) is shown in magenta ( A ) or blue ( C ) , the nuclear membrane is stained with WGA dye shown in red ( C ) .", "Scale bar 30 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 01210 . 7554/eLife . 05003 . 013Figure 3—figure supplement 5 . Posterior mRNA localization is impaired in posterior localization pathway mutants .", "( A ) Localization of the novel posterior candidate mRNAs vkg , TwdlG , PI3K21B , and zpg is lost in egg-chambers that prematurely depolymerize the microtubules ( flies homozygous for SpireRP ) , are mutant for the RNA binding protein Staufen ( flies homozygous for StauD3 ) or mutant for the EJC protein Barentz ( flies homozygous for Btz1 ) .", "The candidate mRNAs are mis-localized in a manner similar to oskar mRNA .", "In Btz1 egg-chambers a weak enrichment of vkg mRNA remained that in rare instances is also observed for oskar mRNA .", "( B ) In egg-chambers either lacking functional Oskar protein or posterior oskar mRNA , the localization of the candidate posterior mRNAs was lost: In Oskar protein mutant egg-chambers ( osk84/Df ( 3R ) pXT103 ) , oskar mRNA is initially localized at stage 9 but successively detaches from the posterior pole resulting in reduced oskar mRNA at the posterior pole from stage 10 onwards .", "A similar reduction from stage 9 to 10 was seen for vkg and TwdlG mRNAs , while PI3K21B ( already in wild type being localized at low levels ) and zpg mRNA ( in wild type localized after stage 9 ) never showed localization .", "In egg-chambers that do not express posterior oskar mRNA ( oskar 3′UTR/+; oskA87/Df ( 3R ) pXT103; Jenny et al . , 2006 ) none of the candidate posterior mRNAs localized .", "( Expression of the non-localizing oskar 3′UTR is necessary to rescue the early oogenesis arrest . ) ( A–B ) FISH experiments showing the RNA in green and DNA ( labelled with DAPI ) in magenta .", "Scale bars 30 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 013 We next investigated whether there are further global features that could set localized mRNAs apart from ubiquitous ones .", "Ovarian expressed mRNAs differed in their expression levels over several orders of magnitude .", "Using our stage specific 3Pseq data , we analysed the expression levels for each gene set .", "Ubiquitous and subcellular mRNA expression levels were overall comparable however , the posterior class was expressed significantly higher than all other localization classes , including the related anterior mRNAs ( Figure 3C–C' , Figure 3—figure supplement 2A ) .", "Considering how seemingly inefficient posterior transport is ( Zimyanin et al . , 2008 ) , higher expression levels could be an additional measure to ensure that enough mRNAs will eventually localize .", "In particular , the late phase accumulation of posterior localized mRNAs in the enlarged oocyte ( Forrest and Gavis , 2003; Sinsimer et al . , 2011 ) could benefit from high expression levels .", "Yet , expression level alone cannot account for subcellular localization .", "We therefore compared the gene-level variables of each localization class and revealed that subcellular mRNAs had significantly longer 3′UTR sequences and this was more pronounced for the posterior localization class ( Figure 3D , D' ) .", "The posterior gene set further showed longer gene structures , longer 5′UTRs , longer exons and introns , a higher number of exons and introns , and a higher intron proportion compared to ubiquitous and anterior mRNAs ( Figure 3—figure supplement 2B–H ) .", "Consistent with the observation that localized mRNAs are enriched in non-coding portions , the exon proportion was the highest in the ubiquitous gene set ( Figure 3—figure supplement 2I ) .", "The high intron proportion of posterior genes is particularly interesting in light of the recent finding that the stable deposition of the exon junction complex , required for posterior oskar mRNA localization , is correlated with long intron-containing genes ( Ashton-Beaucage et al . , 2010; Ghosh et al . , 2012 ) .", "Localized genes not only had longer 3′UTRs , but also showed higher 3′UTR sequence conservation than ubiquitous genes , and again this was significantly more pronounced in the posterior gene set ( Figure 3E , E' ) .", "We also observed longer and more conserved 3′UTRs in the embryo localized mRNAs ( apical , posterior ) compared to the embryo ubiquitous mRNAs ( Figure 3—figure supplement 3A , B based on data from [Lecuyer et al . , 2007] ) , indicating that these features are not specific to oocyte-localized mRNAs .", "The posterior gene set shows clearly distinct functional and gene architectural features compared to all the other categories .", "We therefore decided to investigate whether the cytoplasmic localization of the novel candidate mRNAs depends on the known components of RNA localization machinery in the oocyte .", "First , we probed the dependency of mRNA localization on the microtubule cytoskeleton .", "Transport of known mRNAs towards the anterior and the posterior pole of the oocyte requires an intact microtubule cytoskeleton ( reviewed in Steinhauer and Kalderon , 2006 ) .", "We observed that the localization of all new anterior and posterior candidate mRNAs is lost in colchicine-treated egg-chambers , while ubiquitously distributed mRNAs or RNA foci in the nucleoplasm , that lacks a microtubule cytoskeleton , were unaffected by the colchicine treatment ( Figure 3—figure supplement 4A–C , Supplementary file 8 ) .", "However , mRNA localization requires more than an intact microtubule cytoskeleton .", "We therefore next investigated the localization of candidate posterior mRNAs in mutant egg-chambers that affect the localization of the known posterior mRNA , oskar .", "Posterior transport of oskar mRNA requires components of the exon junction complex , the RNA binding protein Staufen and an intact microtubule cytoskeleton ( van Eeden et al . , 2001; St Johnston et al . , 1989; Ephrussi et al . , 1991; Hachet and Ephrussi , 2001 , 2004; Micklem et al . , 2000 ) .", "The posterior enrichment of the selected candidate mRNAs was severely reduced in egg-chambers mutant for an exon junction complex component ( Btz1 ) , that has a disrupted cytoskeleton ( SpireRP ) or that lack Staufen ( StauD3 ) protein .", "The localization of all candidate posterior mRNAs resembled the mis-localized oskar mRNA in these mutant conditions ( Figure 3—figure supplement 5A ) .", "Oskar protein is a known to be required for the assembly of functional pole plasm and the subsequent localization of mRNAs such as nanos ( Ephrussi et al . , 1991; Ephrussi and Lehmann , 1992 ) .", "Therefore , we next investigated whether the novel candidate mRNAs also require Oskar protein for their posterior localization .", "We used genetic combinations that result in lack of Oskar protein ( osk84/Df ( 3R ) pXT103 ) .", "In these Oskar protein deficient egg-chambers oskar mRNA is initially localized at the posterior pole at stage", "9 . However , the mRNA becomes successively detached from stage 10 onwards due to the lack of Oskar protein-mediated RNA anchoring ( Ephrussi et al . , 1991; Vanzo and Ephrussi , 2002 ) .", "The novel candidates initially localized in the absence of Oskar protein at stage 9 but their posterior localization was reduced from stage 10 onwards ( Figure 3—figure supplement 5B ) .", "Based on these experiments , we propose that the initial posterior localization of the candidate mRNAs , unlike the localization of nanos mRNA , is independent from Oskar protein .", "Interestingly , if we completely remove posterior oskar RNA from the egg-chambers ( oskA87/Df ( 3R ) pXT103 ) ( Jenny et al . , 2006 ) , we do not observe posterior signal for any of the novel candidate mRNAs , both at stage 9 and stage 10 ( Figure 3—figure supplement 5B ) .", "We propose that the novel candidate mRNAs require oskar mRNA to initially reach the posterior pole and Oskar protein to remain stably anchored at the posterior pole beyond stage", "9 . The notable exception is zpg mRNA , that adopts posterior localization only at late stage 9/early stage 10 egg-chambers .", "Based on our experiments , we cannot determine whether zpg mRNA requires oskar mRNA or protein for its localization .", "Our findings revealed that co-localized mRNAs share global features and have similar cytoplasmic requirements for their localization .", "However , as seen with zpg , mRNAs within gene sets differ in the precise timing and consequently regulation of their localization .", "We therefore investigated the time-course of mRNA localizations in detail .", "In the oocyte , mRNAs can be oocyte-enriched , anterior or posterior localized ( Figure 4A ) .", "By comparing exemplary mRNAs across oogenesis time points ( Figure 4A' ) , we observed that after being oocyte-enriched mRNAs could enrich at either anterior ( Dok ) or posterior pole ( ZnT35C ) , but also de-localize and show ubiquitous distribution ( exu ) .", "Conversely , mRNAs that showed ubiquitous distribution during early oogenesis could adopt posterior localization at later stages ( aret ) .", "These examples show that multiple combinations of mRNA distributions from early to late oogenesis are possible and mRNAs that belong to the same gene set early are not necessarily grouped together at other time points . 10 . 7554/eLife . 05003 . 014Figure 4 . mRNA localizations change across time-points .", "( A ) Schematic of changing mRNA distributions in germline cells ( nurse cells , oocyte ) in stage 4–7 and stage 9–10 egg-chambers .", "( A′ )", "Exemplary mRNAs that show diverging combinations of mRNA localizations over the course of oogenesis: After initially being oocyte enriched at stage 2–7 , Dok mRNA becomes detectable at the anterior pole , ZnT35C mRNA at the posterior pole and exu mRNA becomes ubiquitously distributed at stage 9/10 .", "aret mRNA being ubiquitously distributed at stage 2–7 becomes weakly detectable at the posterior pole .", "( B ) Schematic of mRNA distributions in ovary and embryonic cell types .", "( B′ ) mRNA expressions in ovarian and embryonic cells .", "All embryo data are from http://fly-fish . ccbr . utoronto . ca/ .", "Sdc mRNA is localized where microtubules minus ends are enriched ( Callaini and Anselmi , 1988; Clark et al . , 1997; Delanoue and Davis , 2005 ) in the syncytial egg-chamber , in epithelial cells of the ovary and of the stage 4–5 embryo .", "Bsg25D mRNA is oocyte enriched , then localizes at the anterior pole in the oocyte but enriches at the posterior pole in the early embryo .", "Similarly , ssp2 mRNA enriches in the oocyte during oogenesis and localizes towards the posterior pole in early embryos but during late oogenesis undergoes a ubiquitous phase .", "CG14814 mRNA is initially ubiquitous , then shows perinuclear localization and in early embryos is enriched at the posterior pole .", "( A′–B′ ) : FISH showing the RNA in green and DNA ( labelled with DAPI ) in magenta .", "Scale bar 30 μm .", "Embryo data are from http://fly-fish . ccbr . utoronto . ca/ ( Lecuyer et al . , 2007 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 014 The same dynamics was also apparent when we compared localizations beyond the oocyte ( Figure 4B ) .", "Maternal mRNAs that eventually enriched at the posterior pole during early embryogenesis showed any combination of mRNA distributions during oogenesis ( Figure 4B' ) .", "For example , the anterior , ubiquitous and perinuclear mRNAs Bsg25D , ssp2 and CG14814 are all eventually enriched at the posterior pole in early embryos ( Lecuyer et al . , 2007; fly-fish . ccbr . utoronto . ca ) .", "The changes in mRNA localization status within the oocyte over time prompted us to ask whether this could be explained by transcriptional regulation during oogenesis .", "Are we observing different transcript variants that differ in their cytoplasmic distribution ?", "Alternative splicing was previously shown to differentially regulate mRNA localization by producing localized and non-localized isoforms of the same gene ( Whittaker et al . , 1999; Horne-Badovinac and Bilder , 2008 ) .", "We therefore probed our stage-specific transcriptomic data for changes in gene and isoform expression .", "For the exemplary mRNAs shown in Figure 4 , we could not detect significant changes in the expressed isoform ( measured by RNAseq ) or the 3′UTR end ( measured by 3Pseq; Figure 5A ) . 10 . 7554/eLife . 05003 . 015Figure 5 . mRNA expression is stable during oogenesis .", "( A ) Changing localization of ZnT35C , exu , aret , Dok , Bsg25D and ssp mRNAs across time-points ( see Figure", "4 ) does not coincide with a change in transcript expression: the expressed 3′UTRs ( sampled by 3′prime sequencing , red ) and transcript isoforms ( sampled by RNAseq , green ) do not change from early oogenesis to late oogenesis/early embryogenesis .", "( B ) Pair-wise correlation of early/late 3Pseq data revealed that the stage-specific transcriptomes were highly similar ( Pearson Correlation: 0 . 79 ) ; only few genes , highlighted in black , were significantly up- or down-regulated ( p-value adjusted for multiple testing <0 . 1 ) .", "( C ) Correlation analysis of expressed transcript isoform ( deduced from RNAseq data ) revealed that from early to late ovaries almost no transcript-isoforms significantly changed in their expression level .", "Transcripts with significant changes are shown in black .", "( D ) Only ∼300 genes ( early-full: 298; late-full: 308; full-embryo: 346 ) changed their mean-weighted 3′UTR length that is indicative of an alternative polyadenylation .", "Alternative UTR form usage across oogenesis was found for 1 ( early-late oogenesis ) /4 ( late-full oogenesis ) anterior mRNAs ( red ) and for 4 ( early to late oogenesis ) /5 ( late to full oogenesis ) posterior mRNAs ( blue ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 01510 . 7554/eLife . 05003 . 016Figure 5—figure supplement 1 . The transcriptome shows little variation over the course of oogenesis .", "( A ) Scatterplot showing high correlation ( Pearson Correlation 0 . 71 ) between RNAseq and 3Pseq sequencing results .", "( B ) Stage-specific sequencing reveals that ∼5500 genes ( grey ) were detected by both mRNA sequencing ( RNAseq ) and 3′prime end sequencing ( 3Pseq ) .", "Additional 1000–2000 mRNAs were captures with either RNAseq ( green ) or 3Pseq ( red ) only , suggesting that at each time point about half of the D . melanogaster genome was expressed .", "( C ) Venn diagram showing that most genes , 85% , are continuously expressed as they are detectable at each time point of oogenesis ( 3Pseq: red; RNAseq: green ) .", "( D ) Across oogenesis RNA levels were highly correlated , suggesting minimal changes in the stage-specific transcriptomes ( Pair-wise correlation , Pearson Correlation: 0 . 77; significantly up/down regulated genes shown in black: p-value adjusted for multiple testing <0 . 1 , see also Figure 5B ) .", "( E ) GO-term analysis of genes significantly up- ( arrow up ) or down- ( arrow down ) regulated during oogenesis/early embryogenesis .", "Particularly genes encoding components of the extra-embryonic layers ( vitelline membrane , ECM , cuticle ) changed expression levels .", "( F ) nanos mRNA significantly changes gene expression from early to full ovaries measured by RNAseq ( green ) and 3Pseq ( red ) .", "Below: gene model .", "( G ) Boxplot showing that the vast majority of genes expressed only one 3′UTR form during oogenesis , suggesting low prevalence of alternative polyadenylation .", "( H ) Correlation of expressed transcripts ( measured by RNAseq ) revealed that only few genes significantly ( shown in black ) changed the expressed isoform during oogenesis ( see also Figure 5C ) .", "( I ) Only few subcellular localized mRNAs showed significant changes in transcript expression ( see H ) .", "( J ) The number of transcripts expressed per gene did not change during oogenesis did not differ between ubiquitous and subcellular gene sets ( highlighted: anterior [red] and posterior [blue] genes ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 016 We next asked whether global transcriptional changes occur that could explain differential mRNA localization during oogenesis .", "In agreement with results from gene expression analyses of whole ovaries measured by microarray ( Chintapalli et al . , 2007 ) and RNAseq ( Graveley et al . , 2011 ) , we find that about half of the D . melanogaster genes were expressed at each sampled time point and the vast majority of these expressed transcripts , 85% , were detectable at every time point from early oogenesis until embryogenesis ( Figure 5—figure supplement 1B , C ) .", "Also the expression levels across time points were highly correlated ( Figure 5B , Figure 5—figure supplement 1D ) , suggesting that the transcriptome remained constant throughout oogenesis .", "Significant up- or down regulation of gene expression levels was only observed for 626 transcripts and among them are only rare examples of germline-specific transcripts ( padj < 0 . 1 , Figure 5B: black data points , Supplementary files 2–4 , Figure 5—figure supplement 1E–F ) .", "Instead , GO-term analysis associated genes under differential expression with extracellular matrix , vitelline membrane , and cuticle formation , consistent with their expression in the somatic epithelial cells ( Figure 5—figure supplement 1E ) .", "Across the entire oogenesis , we also could not detect shortening or lengthening of the 3′UTRs , changes in the number of transcript ends and while 55% of genes were expressed in alternative isoforms , the vast majority ( >99% ) of genes showed no change in isoform expression ( Figure 5C–D , Figure 5—figure supplement 1G–J , Supplementary files 5–7 ) .", "Furthermore , the ubiquitous gene set showed similar transcript diversity as subcellular genes .", "Therefore , changing expression levels , isoform expression , and alternative polyadenylation cannot explain the changing localization of the majority of mRNAs .", "The stability of the transcriptome from egg chamber formation until the onset of zygotic transcription also suggests that oogenesis is not dependent on transcriptional changes but rather on post-transcriptional regulation of the expressed transcripts , in particular through mRNA localization .", "A substantial portion of expressed mRNAs is localized during oogenesis .", "Given that the transcriptome is rather stable yet individual mRNAs show changing localizations across oogenesis , we next analysed mRNA localizations during this period globally .", "Within one cell , the oocyte , only few mRNAs are localized at all time points , while the majority of localizations is temporary with intermittent ubiquitous phases ( Figure 6A ) .", "The oocyte has a highly polarized microtubule cytoskeleton that undergoes dramatic re-polarizations across oogenesis ( reviewed in Steinhauer and Kalderon , 2006 ) .", "All mRNA localizations we observed were at sites that are known to enrich for microtubule plus or minus ends .", "Microtubule orientation is a hallmark of cell polarity .", "In order to compare localizations across oogenesis stages , we categorized the localized mRNAs as being in proximity to microtubule minus- or plus- ends ( plus and minus category Figure 6B , inset ) .", "We do not show direct association of all localised mRNAs with microtubules .", "However , microtubule cytoskeleton is required for RNA localization ( Steinhauer and Kalderon , 2006 ) and the oocyte enriched , anterior and posterior localizations categories correspond to where the microtubule minus and plus ends are enriched ( Theurkauf et al . , 1992; Januschke et al . , 2006 ) . 10 . 7554/eLife . 05003 . 017Figure 6 . mRNA localizations are changing across cell types and within cells over time .", "( A ) In the oocyte , most mRNAs of the subcellular category also have phases with ubiquitous mRNA distribution .", "( B ) The number of localized mRNAs in the oocyte varies over oogenesis time points .", "mRNAs are grouped according to their relative localization with respect to the polarised microtubule cytoskeleton ( Steinhauer and Kalderon , 2006 ) .", "Red = mRNAs that localize where microtubule minus ends are enriched ( minus category ) , blue = mRNAs in proximity of microtubule plus ends ( plus category ) .", "( C ) Time course of clustered single mRNA localizations .", "Each line represents an mRNA , indicated below are the oogenesis time-points .", "Localizations to the poles of the oocyte are colour-coded in red ( minus category ) or blue ( plus category ) ; ubiquitous phases of the mRNA are shown in grey .", "A summary of the trend of mRNA localizations in each cluster and the number of entries is shown to the right .", "( D ) Overlap of mRNAs localized in either germline ( oocyte , nurse cells ) , epithelial ( follicle cells ) and embryonic cell types shown as a Venn-diagram: Only 5 ( <1% ) mRNAs localized in all sampled cell types , 89 ( 14% ) mRNAs localized in at least two cell types .", "The largest group in each cell type was mRNAs localized in proximity to sites known to be enriched for microtubule minus-ends ( Callaini and Anselmi , 1988; Clark et al . , 1997; Delanoue and Davis , 2005 ) ; in the early egg-chamber: oocyte-enriched; in somatic epithelial follicle cells: apical; in embryonic epithelial cells: apical .", "Only 3 mRNAs ( <1% ) showed this localization in all cell types , 29 mRNAs ( 9% ) in two cell types . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 01710 . 7554/eLife . 05003 . 018Figure 6—figure supplement 1 . Changing localization of mRNAs in ovaries and embryos .", "( A ) Expanded dendrogram from Figure 6C including the data for the first two time-points of embryogenesis ( Lecuyer et al . , 2007 ) .", "mRNAs at the anterior ( minus category ) and posterior ( plus category ) poles further decrease from oogenesis into early embryogenesis; more posterior ( plus category ) mRNAs remain localized in the embryo than anterior ( minus category ) mRNAs .", "A rise in localization of the oocyte-localized mRNAs was only observed at stage 4–5 of embryogenesis; the rise was more pronounced in the minus category ( i . e . , apical localization in embryo cells ) .", "( B ) Minus and plus categories expanded to the embryo localization data .", "( C ) Comparison of the in situ hybridization screens in ovaries ( our data ) and Drosophila embryos ( Lecuyer et al . , 2007 ) .", "In total 1674 mRNAs are subcellularly localized during oogenesis and/or embryogenesis and therefore are ‘localization competent’ ( see also Figure 1—figure supplement 2 ) .", "( D ) Venn diagram of the mRNAs showing nuclear enrichment in either oogenesis or embryogenesis .", "Only three mRNAs are nuclear at both developmental time-points . DOI: http://dx . doi . org/10 . 7554/eLife . 05003 . 018 At the different time-points of oogenesis the number of localized mRNAs varied; it was the highest at stage 2–7 and dropped to around 100 mRNAs at stage 8 and increased only slightly again towards stage 10 of oogenesis ( Figure 6B ) .", "Also , the number of mRNAs in the minus and plus categories changed yet at different rates: during early oogenesis , the majority of mRNAs are in the minus category but the number of genes in this category rapidly dropped at stage 8 and further decreased throughout oogenesis .", "In contrast , the mRNAs in the plus category were increasing towards the end of oogenesis ( Figure 6B ) .", "To understand these trends in more detail , we plotted individual mRNAs over the course of oogenesis and clustered them by the localization category .", "Using this ‘localization-dendrogram’ , we revealed that the changing localization of single mRNAs ( Figure", "4 ) is a global feature of localized mRNAs and occurred at all stages of oogenesis ( Figure 6C ) .", "Using the localization dendrogram , we observed several groups: mRNAs that remained in the minus category at all time points , mRNAs that switched from minus category to ubiquitous distribution ( this was by far the biggest category ) , mRNAs that switch from minus to plus category ( with and without intermittent ubiquitous distribution ) and ubiquitous mRNAs that become localized and affiliated with the plus category .", "It is noteworthy that such de novo localization of an initially ubiquitous transcript was not observed for the minus category .", "The dendrogram also revealed that such changes in localization occurred at all time points of oogenesis: mRNAs could switch from minus category to ubiquitous distribution at stage 8 , 9 or 10 or enrich in the plus category , that is , adopt posterior localization at stage 9 or 10 of oogenesis .", "These localization time-courses recapitulate the localization pattern of the well-characterized , singular mRNAs such as oskar , gurken , nanos , and bicoid; however , our data show that each of them occurs for multiple co-regulated mRNAs .", "It has been shown that mRNA localization to the oocyte portion of the syncytial egg-chamber , to the apical side of somatic epithelial cells of the ovary and of embryonic epithelial cells in the embryo ( stage 4–5 ) is functionally equivalent ( Bullock and Ish-Horowicz , 2001; Jambor et al . , 2014 ) .", "Indeed , we observed mRNAs that were oocyte enriched and apical in epithelial cells ( Figure 4B' Sdc ) .", "How general is this phenomenon ?", "Does the majority of mRNAs localize to equivalent sites in different cell types or is it a property of singular mRNAs ?", "To address these questions , we use again the microtubule polarity as a universal proxy of cell polarity that enables comparison of equivalent localization sites across tissues .", "This allows us to extend the minus and plus categories in ovaries to include data from embryos ( Lecuyer et al . , 2007 ) .", "Minus category includes additionally apical sets from embryo and plus category includes pole plasm and basal embryonic enrichment categories ( Figure 6—figure supplement 1B ) .", "We do not include pole cell annotations in the plus category since this is not a subcellular localization but rather a cell-specific expression pattern .", "The posterior pole plasm and anterior embryo categories reflect the polarity of embryonic body axis and do not imply any microtubule-related localization mechanism .", "It is for instance known that some RNAs become restricted to the posterior pole by selective degradation protection mechanism ( reviewed in Lipshitz and Smibert , 2000 ) .", "This grouping enables us to compare localization of mRNAs between life cycle stages ( ovaries and embryos ) and cell types ( germline and epithelial cells ) .", "To address whether oogenesis localized transcripts remain localized into early embryogenesis , we extended the localization dendrogram to stage 1–3 ( maternally loaded transcripts ) and stage 4–5 ( after the onset of zygotic transcription ) of embryogenesis ( Figure 6—figure supplement 1A , B ) .", "This revealed that only very few of the minus and plus category mRNAs remained localized in embryogenesis: only three of the minus category mRNAs and a few more in the plus category .", "The plus category increased slightly at stage 1–3 of embryogenesis as a few ubiquitous oogenesis transcripts became localized .", "A rise of mRNAs in the minus category was only detectable at stage 4–5 of embryogenesis ( apical localization ) when the initiation of zygotic transcription occurs .", "We conclude that mRNAs are differentially localized in different developmental contexts .", "Since many oocyte localized genes are also expressed in the somatic epithelium of the ovary and again during embryogenesis , we next wondered whether localization is preserved in different cell types .", "We took advantage of the wealth of FISH data now available for Drosophila and combined our data for the somatic epithelial cells and the germline ( nurse cells , oocyte ) cells of the ovary with the FISH screen performed on embryonic cells ( Lecuyer et al . , 2007 ) .", "These screens in combination covered 9114 genes of which 1674 mRNAs showed subcellular localization at least at one time point either during oogenesis or embryogenesis and thus are ‘localization competent’ ( Figure 6—figure supplement 1C ) .", "Filtering of the data sets for mRNAs that were probed by FISH in all three cell types resulted in 720 mRNAs of which only five mRNAs were localized in all three , and 89 mRNAs were localized in two cell types ( Figure 6D ) .", "Strikingly , the data also show that with respect to microtubule polarity , only three mRNAs were in each cell type localized to the side where also microtubule minus ends are enriched ( Dok , Sdc , CG12006; Figure 6D ) .", "Other types of localization , for example , nuclear RNA enrichment , had similarly minimal overlap across cell types ( Figure 6—figure supplement 1D ) .", "The relative lack of mRNA localization to equivalent subcellular destinations indicates that while many mRNAs are localization competent , their localization appears to be cell type specific and developmentally regulated ." ], [ "We generated a comprehensive resource , the Dresden Ovary Table ( DOT , http://tomancak-srv1 . mpi-cbg . de/DOT/main . html ) that includes stage-specific transcriptomic and image-based RNA expression and subcellular localization data for the entire oogenesis from cystoblast division to the beginning of embryogenesis .", "Our resource consists of 52 , 000 carefully selected , annotated , stage-specific images of ovarian gene expression , and localization patterns that can be searched online or downloaded for in-depth computational analysis .", "The curated images are linked to 32 , 000 raw 3D image stacks available for interactive browsing that will facilitate further discovery .", "The ovary data set is integrated with similar data on gene expression and RNA localization patterns in Drosophila embryos ( Tomancak et al . , 2007 ) enabling comparisons between tissues on a gene-by-gene basis .", "All visual expression patterns are described with controlled vocabularies facilitating searches and grouping of co-regulated genes .", "Together , this resource represents one of the most comprehensive databases of spatio-temporal gene expression patterns for two intensively studied developmental systems .", "The vast majority of the patterns shown in DOT are novel , often providing the very first data for computationally predicted genes .", "This makes the resource an excellent starting point for in-depth mechanistic studies and for enrichment analysis of gene sets generated in other genomics studies .", "The global analysis of the annotation data allowed us to define gene sets of co-localized mRNAs and show that localized , particularly posterior mRNAs , have a more complex gene structure , longer and higher conserved non-coding features and higher expression levels than ubiquitous mRNAs .", "These properties of localised mRNAs are significant for both the oogenesis and embryogenesis data sets .", "Although they are not by themselves predictive of the localization status of individual mRNAs , it will be interesting to examine these properties in other cellular and developmental contexts .", "Our analysis , for example , predicts that the neuronal transcripts that must reach the distant synaptic compartments , would encode long , highly expressed transcripts analogous to the posterior localization gene set in the oocyte .", "Similarly to embryonic cells , ovarian cells also show prevalent subcellular mRNA localizations .", "In contrast to the embryo system ( Lecuyer et al . , 2007 ) , ovarian cells displayed more homogenous subcellular enrichments .", "With few exceptions , the candidate mRNAs localized at sites known to be enriched for either microtubule minus or plus ends .", "Curiously , we observed that the ovarian localized mRNAs themselves are strongly enriched for genes with cytoskeletal functions , as were localized mRNAs in the embryo ( Lecuyer et al . , 2007 ) .", "This is particularly interesting in light of a recent model suggesting a self-organizing principle for the polarized cytoskeleton in mouse neurites through localized mRNAs and localized translation ( Preitner et al . , 2014 ) .", "A local source of cytoskeletal proteins , for example , in the early oocyte could be beneficial to allow the rapid re-organization and growth of the cytoskeleton at the transition from early to mid-oogenesis .", "Next to cytoskeletal regulating factors , anterior mRNAs that localize in proximity to the meiotic oocyte nucleus were enriched for terms assigning a cell cycle regulating function .", "It will be interesting to investigate whether anterior mRNA localization affects meiosis , a process shown to be regulated through translational control ( Tadros et al . , 2007; Benoit et al . , 2008; Cui et al . , 2013; Kronja et al . , 2014 ) .", "Curiously at stage 9 and 10 , we also identified mRNAs enriched in the nuclei of the oocyte .", "These mRNAs could either be nurse cell transcripts imported into the meiotic oocyte nucleus or else the controversial instances of transcription from the meiotic nucleus ( Saunders and Cohen , 1999; Cáceres and Nilson , 2005 ) .", "Cross-tissue and time-course analyses revealed the changing mRNA localization profile during development and that the well-described , canonical examples of mRNA localization in the ovary ( Berleth et al . , 1988; St Johnston et al . , 1989; Ephrussi et al . , 1991; Neuman-Silberberg and Schüpbach , 1993 ) represent classes of co-regulated mRNAs .", "Considering that the transcriptome appears stable , we find it surprising that the same mRNA isoforms and thus the same primary sequences show such differential localizations during oogenesis .", "Such pervasive changes in localization status of mRNAs contradict the model that mRNAs localize through sequence encoded mRNA zipcodes ( reviewed in Medioni et al . , 2012 ) and that the general localization machinery is active in all cell types analysed ( Bullock and Ish-Horowicz , 2001; Jambor et al . , 2014 ) .", "It will therefore be interesting to investigate whether specificity of mRNA localization is based on selective , cell-type-specific mRNA regulation machinery or a zipcode signal that is under specific temporal control .", "mRNAs can , for example , harbour two consecutively acting localization signals that direct mRNAs sequentially to opposing microtubule ends ( Ghosh et al . , 2012; Jambor et al . , 2014 ) .", "However , how one signal is de-activated and the other activated is yet unknown .", "Alternatively , only few mRNAs could have a zipcode for their localization and the vast majority would be co-transported with these regulated mRNAs in large transport granules .", "Finally , it is also conceivable that subcellular mRNAs could be locally trapped by unidentified physical properties of subcellular cytoplasmic domains or that similarly to the early embryo , some localization patterns result from protection from general cytoplasmic degradation ( reviewed in Lipshitz and Smibert , 2000 ) .", "All these mechanisms could be active consecutively or in combination during development , which could result in the observed diversity in mRNA localization .", "Regardless of the specific mechanisms of mRNA transport , our genome-wide analysis shows that mRNA localization is a phenomenon contingent on the cellular context and is most likely highly regulated during development .", "It also highlights that oocytes do not rely on transcriptional but on post-transcriptional mechanisms to regulate gene expression , in particular ( but likely not limited to ) mRNA localization .", "Our resource enables the transition from deep mechanistic dissection of singular mRNA localization events towards systemic examination of how mRNAs transcribed in the nucleus distribute in cells and how this affects cellular architecture and cell behaviour in development ." ], [ "Flies were grown under standard laboratory conditions , fed for 2 days with fresh yeast at 21 and 25°C .", "For isolation of egg-chambers , we developed a mass isolation protocol ( see below ) that allows us to enrich separated egg-chambers of all stages .", "We isolated total mRNA using TRIreagent ( Sigma Aldrich , Germany ) from stage 1 to 7 egg-chambers , including the germline stem cells , from stage 8 to 10 egg-chambers and from total ovaries containing mainly stage 11 and older egg-chambers .", "Additionally , RNA from 0 to 2 hr embryos was isolated .", "We used two complementary mRNA sequencing approaches; standard whole mRNA sequencing ( RNAseq ) and a sequencing method , 3Pseq , that captures specifically the sequence adjacent to the poly ( A ) tail .", "Our 3Pseq protocol is similar to the SAPAS method described previously ( Fu et al . , 2011 ) .", "In contrast to SAPAS , total mRNA was fragmented chemically , resulting in 200 nucleotide long molecules .", "cDNA was generated and amplified using a polyT primer terminating with a dinucleotide made of non-T followed by a random base and a 5′ template switch primer; both primers containing Illumina adaptors .", "This allowed us to capture each expressed polyadenylated mRNA once and thereby precisely quantify expression level ( Vineeth Surendranath and Andreas Dahl , personal communication ) .", "Of the ∼50 million ( 3Pseq ) and 100 million ( RNAseq ) Illumina reads , we mapped 70% ( 3Pseq ) and 90% ( RNAseq ) to the D . melanogaster release 5 . 52 genome with Bowtie .", "Quantification was done using HTSeq ( Anders and Huber , 2010 ) .", "Normalization and differential expression was done using DESeq ( Anders and Huber , 2010 ) .", "Noise thresholds of 70 and 50 counts , for RNAseq and 3Pseq respectively , were derived from observing the distributions of normalized counts .", "3′UTR forms were assigned by overlaying annotated Flybase UTR forms with 3Pseq reads lying within 200 nucleotides of the annotated 3′UTR end .", "Alternate Polyadenylation events were called by calculating the mean-weighted UTR length ( Ulitsky et al . , 2012 ) , a difference of 200 nucleotides in the mean-weighted lengths corresponding to two biological stages resulted in the gene being considered as undergoing Alternate Polyadenylation .", "We used an established protocol for in situ hybridization in 96-well plates ( Tomancak et al . , 2007 ) with minor adaptations ( see below ) : we added an over-night wash step after hybridization , incubate the anti-DIG antibody over night and used fluorescent tyramides for probe detection .", "Each experiment was evaluated and imaged using a wide-field microscope ( Zeiss Axioplan Imaging , Zeiss , Germany ) equipped with an optical sectioning device ( DSD1 , Andor Technology , UK ) to generate confocal-like z-stacks .", "We developed a controlled vocabulary to describe the cell types and relevant subcellular structures for oogenesis for germline and somatic cells ( http://tomancak-srv1 . mpi-cbg . de/cgi-bin-public/ovary_annotation_hierarchy . pl ) .", "Experiments showing no detectable FISH signal were classified as ‘no signal at all stages’ , while experiments resulting in a homogeneous signal throughout oogenesis were classified as ‘ubiquitous signal at all stages’ .", "Gene expression patterns were imaged up to stage 10B of oogenesis after which cuticle deposition prevents probe penetration .", "Each pattern that did not fall in the above-mentioned classes was imaged at all stages of oogenesis in several individual egg-chambers per time point .", "We collected 3D images and used custom scripts in FIJI ( Schindelin et al . , 2012 ) to manually select and orient representative 2D images that were uploaded to the Dresden Ovarian-expression Table ( DOT ) ( http://tomancak-srv1 . mpi-cbg . de/DOT/main ) .", "The 2D images remain linked to the original image stacks and all the raw stacks that were used to create an exemplary 2D image are available for interactive inspection using a simple image browsing cgi script .", "Thus , the record of each in situ experiment for a given gene consists of a set of 2D images assigned to a specific oogenesis stage and described using annotation terms selected from the controlled vocabulary .", "For definition of broad classifications , subclass grouping and embryo annotation class definition , see Supplementary file 1 .", "The binary matrix summarizes the data of our screen in tabular form , which facilitates access to the multidimensional image annotation data and integrates them with the RNAseq and 3Pseq data .", "The binary matrix is a freeze from September 2013 , based on which our analyses were done .", "The binary matrix is provided as a flat file for independent bioinformatics investigation of the data set ( http://tomancak-srv1 . mpi-cbg . de/cgi-bin-public/dump_binary_matrix_ovary . pl ? db=insitu_ovaries ) .", "The matrix contains the following information for each annotated gene: the FlyBase ID; the expression levels as raw as well as normalized counts from RNAseq and 3Pseq experiments for early- , late- and full ovaries and 0–2 hr embryos; the pair-wise comparison of expression over the time course analysed , raw and normalized; the mean-weighted length indicating alternative 3′UTR expression .", "The binary matrix additionally contains the annotation of FISH expression patterns .", "The expression terms are from the controlled vocabulary ( CV ) .", "If the CV term is true its value is equal to one , otherwise it is zero .", "If a gene is annotated twice during the screen , the CV values are summed up and thus result in values >1 .", "We also provide information which clone was used to prepare the FISH probe; the classification into broad annotation classes ( ‘no signal’; ‘ubiquitous’; ‘specific’ , see ‘Results’ . All reliable genes in these categories were used for the analysis and the table in Figure 1A ) ; classification of specific expression patterns into subclasses ( ‘cellular’ , ‘subcellular’ , ‘nuclear’ ) ; reliability status: ‘reliable’ and ‘non-reliable’ ( genes probed with more than one RNA probe that resulted in conflicting annotations [n = 247] , were labelled as ‘not reliable’ . 185 ‘unreliable’ cases resulted from a ‘no signal’ vs ‘ubiquitous’ or ‘no signal’ vs ‘specific’ annotations , here we assume one of the probes to be non-functional . 57 ‘unreliable’ annotations were due to different probes giving a ‘ubiquitous’ and ‘specific’ signal , respectively . One possibility is that probes were specific to different isoforms of the gene ) ; pn-status: comparison of sequencing and FISH results ( TN = true negative: genes expressed below cut-off in either RNAseq or 3Pseq and giving a ‘no signal’ in FISH experiments .", "FN = false negatives: genes expressed below cut-off in either RNAseq or 3Pseq and giving a ‘ubiquitous’ or ‘specific’ in FISH signal .", "TP = true positives: genes expressed above cut-off in either RNAseq or 3Pseq and giving a ‘ubiquitous’ or ‘specific’ in FISH signal .", "FP = false positives: genes expressed above cut-off in either RNAseq or 3Pseq and resulting in a ‘no signal’ FISH annotation ( see Figure 1—figure supplement 2A ) ) .", "For GO-term enrichment of gene sets we used the DAVID web server ( Huang da et al . , 2009 ) .", "Terms or features enriched at a false discovery rate ( FDR ) of ≤10% and/or a Benjamini p-value of <0 . 1 were considered significant .", "Two stringencies were applied: the standard FDR cut-off ( ≤10% ) or the more stringent ‘Benjamini’ p-value ( ≤0 . 1 ) .", "Flies were fed for 15 hr at 25°C with fresh yeast paste supplemented with 50 μg/ml colchicine ( Cha et al . , 2002 ) .", "The effect of colchicine on individual egg-chambers was determined by scoring the detachment of the oocyte nucleus from the anterior cortex and its migration towards the centre of the oocyte .", "To test posterior localization in mutants that affect oskar mRNA localization we used ovaries from homozygous SpireRP ( Manseau and Schupbach , 1989 ) , StauD3 ( St Johnston et al . , 1991 ) , and Btz1 ( van Eeden et al . , 2001 ) flies .", "Further , we analysed egg-chambers from osk84/Df ( 3R ) pXT103 flies lacking functional Oskar protein ( Lehmann and Nüsslein-Volhard , 1986 ) and from oskar3′UTR/+;oskA87/Df ( 3R ) pXT103 flies that entirely lack endogenous oskar mRNA but develop past the early oogenesis arrest characteristic for oskar RNA null flies due to a transgenic source of oskar 3′UTR ( Jenny et al . , 2006 ) that is incapable posterior localization .", "For analysis of the annotated gene features , we used the flybase gff data ( D . melanogaster release 5 . 52 ) .", "For each gene , we defined the most used UTR form as the form that was most highly expressed ( relative to any other forms expressed from the same gene ) and which had UTR ends that overlapped by ± 200 bp with a FlyBase annotated UTR end .", "From this data , we extracted unique 3′UTR lengths for each gene .", "Sequence conservation of 3′UTRs was measured as median phyloP scores ( Pollard et al . , 2010 ) across all bases in the most used UTR form for 3′UTR sequence alignments across 24 Drosophila species ( using the D . melanogaster UTR co-ordinates ) .", "PhyloP scores were calculated using the R package Rphast ( Hubisz et al . , 2011 ) .", "Median UTR lengths and conservation scores were bootstrapped by re-sampling genes with replacement from selected annotation sets 100 , 000 times and calculating median values for each re-sample .", "p-values were calculated as the number of re-samples in which the annotation group with a lower median value was greater than or equal to the re-sampled median of the annotation group to which it was being compared , divided by 100 , 000 .", "A manually curated D . melanogaster protein–protein interaction network was downloaded from the mentha interactome database ( Calderone et al . , 2013 ) .", "To test whether genes belonging to certain annotation groups participated in more protein–protein interactions within the annotation group than expected by chance , we adopted the following randomization-based approach .", "A random sample , the size of the number of genes in an annotation group that participate in at least one interaction in the total protein interactome , was taken from the total set of genes belonging to the protein interactome , and the number of protein interactions within this random sample was scored , minus loops .", "This was repeated 100 , 000 times to generate a distribution of the number of interactions obtained by randomly sampling the number of genes belonging to the annotation group from the total interactome .", "The p-value was calculated as the number of randomly sampled networks that had as many or more interactions as the real annotation group divided by 100 , 000 .", "Flies were fed with fresh yeast and kept for 1–2 days at 25°C . Mixed sex flies were narcotized with CO2 for a maximum of 5 min before proceeding to step 3 . Narcotized flies were immediately immersed in 4% Formaldehyde in PBS ( for FISH experiments ) or in PBS supplemented with 0 . 1% Tween-10 ( for ovarian extract or total RNA isolation ) .", "Flies were rapidly processed twice through a grinding mill adaptor at a fine setting ( grade step ‘3’ ) on a standard food processor ( Kitchen Aid ) .", "The ground flies were size-separated using 850 , 450 , and 212 μm sieves successively , resulting in a flow-through highly enriched for separated egg-chambers of all stages . Collection of mass-isolated material:a .", "For FISH experiments , the co-isolation of testis and gut materials did not disturb the subsequent analysis and the material was allowed to settle by gravity and to be fixed for additional 15 min in 4% Formaldehyde , resulting in an overall fixation time of 20 min .", "The supernatant was then removed , the material washed twice in 1×PBS and then transferred stepwise into 100% methanol for storage at −20°C . b . For isolation of total RNA , we manually selected egg-chambers at early stages ( germarium to stage 7 , previtellogenesis ) , late stages ( stage 9–10 , postvitellogenesis ) , and full ovaries highly enriched for stage 11+ egg-chambers using a stereomicroscope .", "For each stage we collected at least 10 μl of total material that was frozen immediately .", "Mass isolated egg-chambers were transferred stepwise ( MeOH/PBT 3:1; MeOH/PBS 1:1; MeOH/PBS 1:3 ) into PBT0 . 1% ( each wash few minutes ) .", "Egg-chambers were then washed 6× in PBT0 . 1% , 5 min each . Egg-chambers were briefly washed in PBT0 . 1%/Hyb 1:1 . Pre-hybridization of egg-chambers was done in 200 μl hybridization buffer at 55°C for 1 hr . Egg-chambers were then added to a 96-well plate and hybridized over-night at 55°C in 200 μl hybridization buffer with Dextran Sulfate supplemented with 2 μl of probe . 100 μl of warm Wash Buffer was added to each well and immediately removed together with probe-solution . Egg-chambers were rinsed once with 150 μl of Wash Buffer and then washed four times for one hour at 55°C in Wash Buffer . Egg-chambers were then washed five times for 1 hr at 55°C in 150 μl PBT0 . 1% , the last wash was done over-night at 55°C . Egg-chambers were washed twice for 1 hr at room temperature in 150 μl PBT0 . 1% .", "The primary antibody ( Anti-Digoxigenin-POD Fab Fragments [Roche , Germany] ) was diluted 1:200 in PBT0 . 1% and egg-chambers were incubated in 200 μl antibody solution overnight . Egg-chambers were rinsed with 150 μl of PBT0 . 1% and then washed ten times for 30 min at RT in 150 μl of PBT0 . 1% .", "For detection egg-chambers were incubated with Cy3-Tyramides ( Perkin–Elmer , Boston Mass . ) 1:70 diluted in amplification buffer for 30 min . Egg-chambers were then washed ten times for 30 min at room temperature in 150 μl of PBT0 . 1% .", "DAPI , diluted 1:1000 , was included in one wash step . All PBT0 . 1% was removed and ∼50 μl mounting medium was added ." ] ]
[ "mRNA localization is critical for eukaryotic cells and affects numerous transcripts , yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown .", "We combined transcriptomics and systematic imaging to determine the tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis .", "mRNA localization is widespread in the ovary and detectable in all of its cell types—the somatic epithelial , the nurse cells , and the oocyte .", "Genes defined by a common RNA localization share distinct gene features and differ in expression level , 3′UTR length and sequence conservation from unlocalized mRNAs .", "Comparison of mRNA localizations in different contexts revealed that localization of individual mRNAs changes over time in the oocyte and between ovarian and embryonic cell types .", "This genome scale image-based resource ( Dresden Ovary Table , DOT , http://tomancak-srv1 . mpi-cbg . de/DOT/main . html ) enables the transition from mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells ." ]
[ "To make a protein , the DNA sequence that encodes it must first be ‘transcribed’ to build a molecule of messenger RNA ( called mRNA for short ) .", "Although many mRNA molecules are found throughout a cell , some are ‘localized’ to certain areas; and recent evidence suggests that this mRNA localization may be more common than previously thought .", "Not much is known about how cells identify which mRNAs need to be localized , or how these molecules are then transported to their destination .", "The localization process has been studied in most detail in the developing egg cell—also known as an oocyte—of the fruit fly species Drosophila melanogaster .", "These studies have identified few mRNA molecules that , if they are not carefully localized within the cell , cause the different parts of the fly embryo to fail to develop correctly when the oocyte is fertilized .", "Jambor et al . created an open-access online resource called the ‘Dresden Ovary Table’ that shows how 5862 mRNA molecules are distributed in several cell types involved in oocyte production in the ovary of female D . melanogaster flies .", "This resource consists of a combination of three-dimensional fluorescent images and measurements of mRNA amounts recorded at different stages in the development of the oocyte .", "Using the resource , Jambor et al . demonstrate that all of the cell types that make up the ovary localize many different mRNA molecules to several distinct destinations within the cells .", "The localized mRNAs share certain features , with mRNAs localized in the same part of the cell showing the most similarities .", "For example , localized mRNAs have longer so-called 3′ untranslated regions ( 3′UTR ) that carry regulatory information and these sequences are also more evolutionarily conserved .", "Further , when the mRNA molecules in the oocyte were examined at different times during its development and compared with the embryo , the majority of these mRNAs were found to change where they are localized as the organism develops .", "The resource can be used to gain insight into specific genetic features that control the distribution of mRNAs .", "This information will be instrumental for cracking the ‘RNA localization code’ and understanding how it affects the activity of proteins in cells ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "computational and systems biology", "neuroscience" ]
Training deep neural density estimators to identify mechanistic models of neural dynamics
elife-56261-v3
[ [ "New experimental technologies allow us to observe neurons , networks , brain regions , and entire systems at unprecedented scale and resolution , but using these data to understand how behavior arises from neural processes remains a challenge .", "To test our understanding of a phenomenon , we often take to rebuilding it in the form of a computational model that incorporates the mechanisms we believe to be at play , based on scientific knowledge , intuition , and hypotheses about the components of a system and the laws governing their relationships .", "The goal of such mechanistic models is to investigate whether a proposed mechanism can explain experimental data , uncover details that may have been missed , inspire new experiments , and eventually provide insights into the inner workings of an observed neural or behavioral phenomenon ( Herz et al . , 2006; Gerstner et al . , 2012; O'Leary et al . , 2015; Baker et al . , 2018 ) .", "Examples for such a symbiotic relationship between model and experiments range from the now classical work of Hodgkin and Huxley , 1952 , to population models investigating rules of connectivity , plasticity and network dynamics ( van Vreeswijk and Sompolinsky , 1996; Prinz et al . , 2004; Vogels et al . , 2005; Potjans and Diesmann , 2014; Litwin-Kumar and Doiron , 2012 ) , network models of inter-area interactions ( Sporns , 2014; Bassett et al . , 2018 ) , and models of decision making ( Gold and Shadlen , 2007; Wang , 2008 ) .", "A crucial step in building a model is adjusting its free parameters to be consistent with experimental observations .", "This is essential both for investigating whether the model agrees with reality and for gaining insight into processes which cannot be measured experimentally .", "For some models in neuroscience , it is possible to identify the relevant parameter regimes from careful mathematical analysis of the model equations .", "But as the complexity of both neural data and neural models increases , it becomes very difficult to find well-fitting parameters by inspection , and automated identification of data-consistent parameters is required .", "Furthermore , to understand how a model quantitatively explains data , it is necessary to find not only the best , but all parameter settings consistent with experimental observations .", "This is especially important when modeling neural data , where highly variable observations can lead to broad ranges of data-consistent parameters .", "Moreover , many models in biology are inherently robust to some perturbations of parameters , but highly sensitive to others ( Gutenkunst et al . , 2007; O'Leary et al . , 2015 ) , for example because of processes such as homeostastic regulation .", "For these systems , identifying the full range of data-consistent parameters can reveal how multiple distinct parameter settings give rise to the same model behavior ( Foster et al . , 1993; Prinz et al . , 2004; Achard and De Schutter , 2006; Alonso and Marder , 2019 ) .", "Yet , despite the clear benefits of mechanistic models in providing scientific insight , identifying their parameters given data remains a challenging open problem that demands new algorithmic strategies .", "The gold standard for automated parameter identification is statistical inference , which uses the likelihood p⁢ ( 𝐱|𝜽 ) to quantify the match between parameters 𝜽 and data 𝐱 .", "Likelihoods can be efficiently computed for purely statistical models commonly used in neuroscience ( Truccolo et al . , 2005; Schneidman et al . , 2006; Pillow et al . , 2008; Yu et al . , 2009; Macke et al . , 2011; Cunningham and Yu , 2014; Pandarinath et al . , 2018 ) , but are computationally intractable for most mechanistic models .", "Mechanistic models are designed to reflect knowledge about biological mechanisms , and not necessarily to be amenable to efficient inference: many mechanistic models are defined implicitly through stochastic computer simulations ( e . g . a simulation of a network of spiking neurons ) , and likelihood calculation would require the ability to integrate over all potential paths through the simulator code .", "Similarly , a common goal of mechanistic modeling is to capture selected summary features of the data ( e . g . a certain firing rate , bursting behavior , etc… ) , not the full dataset in all its details .", "The same feature ( such as a particular average firing rate ) can be produced by infinitely many realizations of the simulated process ( such as a time-series of membrane potential ) .", "This makes it impractical to compute likelihoods , as one would have to average over all possible realizations which produce the same output .", "Since the toolkit of ( likelihood-based ) statistical inference is inaccessible for mechanistic models , parameters are typically tuned ad-hoc ( often through laborious , and subjective , trial-and-error ) , or by computationally expensive parameter search: a large set of models is generated , and grid search ( Prinz et al . , 2003; Tomm et al . , 2011; Stringer et al . , 2016 ) or a genetic algorithm ( Druckmann et al . , 2007; Hay et al . , 2011; Rossant et al . , 2011; Van Geit et al . , 2016 ) is used to filter out simulations which do not match the data .", "However , these approaches require the user to define a heuristic rejection criterion on which simulations to keep ( which can be challenging when observations have many dimensions or multiple units of measurement ) , and typically end up discarding most simulations .", "Furthermore , they lack the advantages of statistical inference , which provides principled approaches for handling variability , quantifying uncertainty , incorporating prior knowledge and integrating multiple data sources .", "Approximate Bayesian Computation ( ABC ) ( Beaumont et al . , 2002; Marjoram et al . , 2003; Sisson et al . , 2007 ) is a parameter-search technique which aims to perform statistical inference , but still requires definition of a rejection criterion and struggles in high-dimensional problems .", "Thus , computational neuroscientists face a dilemma: either create carefully designed , highly interpretable mechanistic models ( but rely on ad-hoc parameter tuning ) , or resort to purely statistical models offering sophisticated parameter inference but limited mechanistic insight .", "Here , we propose a new approach using machine learning to combine the advantages of mechanistic and statistical modeling .", "We present SNPE ( Sequential Neural Posterior Estimation ) , a tool that makes it possible to perform Bayesian inference on mechanistic models in neuroscience without requiring access to likelihoods .", "SNPE identifies all mechanistic model parameters consistent with observed experimental data ( or summary features ) .", "It builds on recent advances in simulation-based Bayesian inference ( Papamakarios and Murray , 2016; Lueckmann et al . , 2017; Greenberg et al . , 2019; Cranmer et al . , 2020 ) : given observed experimental data ( or summary features ) 𝐱o , and a mechanistic model with parameters 𝜽 , it expresses both prior knowledge and the range of data-compatible parameters through probability distributions .", "SNPE returns a posterior distribution p⁢ ( 𝜽|𝐱o ) which is high for parameters 𝜽 consistent with both the data 𝐱o and prior knowledge , but approaches zero for 𝜽 inconsistent with either ( Figure 1 ) .", "Similar to parameter search methods , SNPE uses simulations instead of likelihood calculations , but instead of filtering out simulations , it uses all simulations to train a multilayer artificial neural network to identify admissible parameters ( Figure 1 ) .", "By incorporating modern deep neural networks for conditional density estimation ( Rezende and Mohamed , 2015; Papamakarios et al . , 2017 ) , it can capture the full distribution of parameters consistent with the data , even when this distribution has multiple peaks or lies on curved manifolds .", "Critically , SNPE decouples the design of the model and design of the inference approach , giving the investigator maximal flexibility to design and modify mechanistic models .", "Our method makes minimal assumptions about the model or its implementation , and can for example also be applied to non-differentiable models , such as networks of spiking neurons .", "Its only requirement is that one can run model simulations for different parameters , and collect the resulting synthetic data or summary features of interest .", "While the theoretical foundations of SNPE were originally developed and tested using simple inference problems on small models ( Papamakarios and Murray , 2016; Lueckmann et al . , 2017; Greenberg et al . , 2019 ) , here we show that SNPE can scale to complex mechanistic models in neuroscience , provide an accessible and powerful implementation , and develop validation and visualization techniques for exploring the derived posteriors .", "We illustrate SNPE using mechanistic models expressing key neuroscientific concepts: beginning with a simple neural encoding problem with a known solution , we progress to more complex data types , large datasets and many-parameter models inaccessible to previous methods .", "We estimate visual receptive fields using many data features , demonstrate rapid inference of ion channel properties from high-throughput voltage-clamp protocols , and show how Hodgkin–Huxley models are more tightly constrained by increasing numbers of data features .", "Finally , we showcase the power of SNPE by using it to identify the parameters of a network model which can explain an experimentally observed pyloric rhythm in the stomatogastric ganglion ( Prinz et al . , 2004 ) –in contrast to previous approaches , SNPE allows us to search over the full space of both single-neuron and synaptic parameters , allowing us to study the geometry of the parameter space , as well as to provide new hypotheses for which compensation mechanisms might be at play ." ], [ "SNPE performs Bayesian inference on mechanistic models using only model-simulations , without requiring likelihood evaluations .", "It requires three inputs: a model ( i . e . computer code to simulate data from parameters ) , prior knowledge or constraints on parameters , and data ( outputs from the model or the real system it describes , Figure 1 ) .", "SNPE runs simulations for a range of parameter values , and trains an artificial neural network to map any simulation result onto a range of possible parameters .", "Importantly , a network trained to maximize log-probability ( of parameters given simulation results ) will learn to approximate the posterior distribution as given by Bayes rule ( Papamakarios and Murray , 2016 ) ( see Materials and methods for details , Figure 1 ) .", "After training on simulated data with known model parameters , SNPE can perform Bayesian inference of unknown parameters for empirical data .", "This approach to Bayesian inference never requires evaluating likelihoods .", "SNPE’s efficiency can be further improved by using the running estimate of the posterior distribution to guide further simulations toward data-compatible regions of the parameter space ( Papamakarios and Murray , 2016; Lueckmann et al . , 2017; Greenberg et al . , 2019 ) .", "Below , we apply SNPE to a range of stochastic models in neuroscience .", "We first illustrate SNPE on linear-nonlinear ( LN ) encoding models , a special case of generalized linear models ( GLMs ) .", "These are simple , commonly used phenomenological models for which likelihood-based parameter estimation is feasible ( Brown et al . , 1998; Paninski , 2004; Pillow , 2007; Gerwinn et al . , 2010; Polson et al . , 2013; Pillow and Scott , 2012 ) , and which can be used to validate the accuracy of our approach , before applying SNPE to more complex models for which the likelihood is unavailable .", "We will show that SNPE returns the correct posterior distribution over parameters , that it can cope with high-dimensional observation data , that it can recover multiple solutions to parameter inference problems , and that it is substantially more simulation efficient than conventional rejection-based ABC methods .", "An LN model describes how a neuron’s firing rate is modulated by a sensory stimulus through a linear filter 𝜽 , often referred to as the receptive field ( Pillow et al . , 2005; Chichilnisky , 2001 ) .", "We first considered a model of a retinal ganglion cell ( RGC ) driven by full-field flicker ( Figure 2a ) .", "A statistic that is often used to characterize such a neuron is the spike-triggered average ( STA ) ( Figure 2a , right ) .", "We therefore used the STA , as well as the firing rate of the neuron , as input 𝐱o to SNPE .", "( Note that , in the limit of infinite data , and for white noise stimuli , the STA will converge to the receptive field [Paninski , 2004]–for finite , and non-white data , the two will in general be different . )", "Starting with random receptive fields 𝜽 , we generated synthetic spike trains and calculated STAs from them ( Figure 2b ) .", "We then trained a neural conditional density estimator to recover the receptive fields from the STAs and firing rates ( Figure 2c ) .", "This allowed us to estimate the posterior distribution over receptive fields , that is to estimate which receptive fields are consistent with the data ( and prior ) ( Figure 2c ) .", "For LN models , likelihood-based inference is possible , allowing us to validate the SNPE posterior by comparing it to a reference posterior obtained via Markov Chain Monte Carlo ( MCMC ) sampling ( Polson et al . , 2013; Pillow and Scott , 2012 ) .", "We found that SNPE accurately estimates the posterior distribution ( Appendix 1—figure 1 and Appendix 1—figure 2 ) , and substantially outperforms Sequential Monte Carlo ( SMC ) ABC methods ( Sisson et al . , 2007; Beaumont et al . , 2009; Figure 2d ) .", "If SNPE works correctly , its posterior mean filter will match that of the reference posterior – however , it is not to be expected that either of them precisely matches the ground-truth filter ( Figure 2c and Appendix 1—figure 1 ) : In the presence of finite sampling and stochasticity , multiple different filters could have plausibly given rise to the observed data .", "A properly inferred posterior will reflect this uncertainty , and include the true filters as one of many plausible explanations of the data ( but not necessarily as the ‘mean’ of all plausible explanations ) ( Appendix 1—figure 2 ) .", "Increasing the number of Bernoulli samples in the observed data leads to progressively tighter posteriors , with posterior samples closer to the true filter ( Appendix 1—figure 3 ) .", "Furthermore , SNPE closely agrees with the MCMC reference solution in all these cases , further emphasizing the correctness of the posteriors inferred with SNPE .", "As a more challenging problem , we inferred the receptive field of a neuron in primary visual cortex ( V1 ) ( Niell and Stryker , 2008; Dyballa et al . , 2018 ) .", "Using a model composed of a bias ( related to the spontaneous firing rate ) and a Gabor function with eight parameters ( Jones and Palmer , 1987 ) describing the receptive field’s location , shape and strength , we simulated responses to 5 min random noise movies of 41 × 41 pixels , such that the STA is high-dimensional , with a total of 1681 dimensions ( Figure 2e ) .", "This problem admits multiple solutions ( as e . g . rotating the receptive field by 180° ) .", "As a result , the posterior distribution has multiple peaks ( ‘modes’ ) .", "Starting from a simulation result 𝐱o with known parameters , we used SNPE to estimate the posterior distribution p⁢ ( 𝜽|𝐱o ) .", "To deal with the high-dimensional data 𝐱o in this problem , we used a convolutional neural network ( CNN ) , as this architecture excels at learning relevant features from image data ( Krizhevsky et al . , 2012; Simonyan and Zisserman , 2015 ) .", "To deal with the multiple peaks in the posterior , we fed the CNN’s output into a mixture density network ( MDN ) ( Bishop , 1994 ) , which can learn to assign probability distributions with multiple peaks as a function of its inputs ( details in Materials and methods ) .", "Using this strategy , SNPE was able to infer a posterior distribution that tightly enclosed the ground truth simulation parameters which generated the original simulated data 𝐱o , and matched a reference MCMC posterior ( Figure 2f , posterior over all parameters in Appendix 1—figure 4 ) .", "For this challenging estimation problem with high-dimensional summary features , an SMC-ABC algorithm with the same simulation-budget failed to identify the correct receptive fields ( Figure 2g ) and posterior distributions ( Appendix 1—figure 5 ) .", "We also applied this approach to electrophysiological data from a V1 cell ( Dyballa et al . , 2018 ) , identifying a sine-shaped Gabor receptive field consistent with the original spike-triggered average ( Figure 2i; posterior distribution in Appendix 1—figure 6 ) .", "We next show how SNPE can be efficiently applied to estimation problems in which we want to identify a large number of models for different observations in a database .", "We considered a flexible model of ion channels ( Destexhe and Huguenard , 2000 ) , which we here refer to as the Omnimodel .", "This model uses eight parameters to describe how the dynamics of currents through non-inactivating potassium channels depend on membrane voltage ( Figure 3a ) .", "For various choices of its parameters 𝜽 , it can capture 350 specific models in publications describing this channel type , cataloged in the IonChannelGenealogy ( ICG ) database ( Podlaski et al . , 2017 ) .", "We aimed to identify these ion channel parameters 𝜽 for each ICG model , based on 11 features of the model’s response to a sequence of five noisy voltage clamp protocols , resulting in a total of 55 different characteristic features per model ( Figure 3b , see Materials and methods for details ) .", "Because this model’s output is a typical format for functional characterization of ion channels both in simulations ( Podlaski et al . , 2017 ) and in high-throughput electrophysiological experiments ( Dunlop et al . , 2008; Suk et al . , 2019; Ranjan et al . , 2019 ) , the ability to rapidly infer different parameters for many separate experiments is advantageous .", "Existing fitting approaches based on numerical optimization ( Destexhe and Huguenard , 2000; Ranjan et al . , 2019 ) must repeat all computations anew for a new experiment or data point ( Figure 3c ) .", "However , for SNPE the only heavy computational tasks are carrying out simulations to generate training data , and training the neural network .", "We therefore reasoned that by training a network once using a large number of simulations , we could subsequently carry out rapid ‘amortized’ parameter inference on new data using a single pass through the network ( Figure 3d; Speiser et al . , 2017; Webb et al . , 2018 ) .", "To test this idea , we used SNPE to train a neural network to infer the posterior from any data 𝐱 .", "To generate training data , we carried out 1 million Omnimodel simulations , with parameters randomly chosen across ranges large enough to capture the models in the ICG database ( Podlaski et al . , 2017 ) .", "SNPE was run using a single round , that is , it learned to perform inference for all data from the prior ( rather than a specific observed datum ) .", "Generating these simulations took around 1000 CPU-hours and training the network 150 CPU-hours , but afterwards a full posterior distribution could be inferred for new data in less than 10 ms . As a first test , SNPE was run on simulation data , generated by a previously published model of a non-inactivating potassium channel ( McTavish et al . , 2012; Figure 3b ) .", "Simulations of the Omnimodel using parameter sets sampled from the obtained posterior distribution ( Figure 3e ) closely resembled the input data on which the SNPE-based inference had been carried out , while simulations using ‘outlier’ parameter sets with low probability under the posterior generated current responses that were markedly different from the data 𝐱o ( Figure 3f ) .", "Taking advantage of SNPE’s capability for rapid amortized inference , we further evaluated its performance on all 350 non-inactivating potassium channel models in ICG .", "In each case , we carried out a simulation to generate initial data from the original ICG model , used SNPE to calculate the posterior given the Omnimodel , and then generated a new simulation 𝐱 using parameters sampled from the posterior ( Figure 3f ) .", "This resulted in high correlation between the original ICG model response and the Omnimodel response , in every case ( >0 . 98 for more than 90% of models , see Appendix 1—figure 7 ) .", "However , this approach was not able to capture all traces perfectly , as for example it failed to capture the shape of the onset of the bottom right model in Figure 3g .", "Additional analysis of this example revealed that this example is not a failure of SNPE , but rather a limitation of the Omnimodel: in particular , directly fitting the steady-state activation and time-constant curves on this specific example yielded no further quantitative or qualitative improvement , suggesting that the limitation is in the model , not the fit .", "Thus , SNPE can be used to reveal limitations of candidate models and aid the development of more verisimilar mechanistic models .", "Calculating the posterior for all 350 ICG models took only a few seconds , and was fully automated , that is , did not require user interactions .", "These results show how SNPE allows fast and accurate identification of biophysical model parameters on new data , and how SNPE can be deployed for applications requiring rapid automated inference , such as high-throughput screening-assays , closed-loop paradigms ( e . g . for adaptive experimental manipulations or stimulus-selection [Kleinegesse and Gutmann , 2019] ) , or interactive software tools .", "The Hodgkin–Huxley ( HH ) model ( Hodgkin and Huxley , 1952 ) of action potential generation through ion channel dynamics is a highly influential mechanistic model in neuroscience .", "A number of algorithms have been proposed for fitting HH models to electrophysiological data ( Prinz et al . , 2003; Huys et al . , 2006; Pospischil et al . , 2008; Rossant et al . , 2011; Meliza et al . , 2014; Van Geit et al . , 2016; Ben-Shalom et al . , 2019 ) , but ( with the exception of Daly et al . , 2015 ) these approaches do not attempt to estimate the full posterior .", "Given the central importance of the HH model in neuroscience , we sought to test how SNPE would cope with this challenging non-linear model .", "As previous approaches for HH models concentrated on reproducing specified features ( e . g . the number of spikes , [Pospischil et al . , 2008] ) , we also sought to determine how various features provide different constraints .", "We considered the problem of inferring eight biophysical parameters in a HH single-compartment model , describing voltage-dependent sodium and potassium conductances and other intrinsic membrane properties , including neural noise , making the model stochastic by nature ( Figure 4a , left ) .", "We simulated the neuron’s voltage response to the injection of a square wave of depolarizing current , and defined the model output 𝐱 used for inference as the number of evoked action potentials along with six additional features of the voltage response ( Figure 4a , right , details in Materials and methods ) .", "We first applied SNPE to observed data 𝐱o created by simulation from the model , calculating the posterior distribution using all seven features in the observed data ( Figure 4b ) .", "The posterior contained the ground truth parameters in a high probability-region , as in previous applications , indicating the consistency of parameter identification .", "The variance of the posterior was narrower for some parameters than for others , indicating that the seven data features constrain some parameters strongly ( such as the potassium conductance ) , but others only weakly ( such as the adaptation time constant ) .", "Additional simulations with parameters sampled from the posterior closely resembled the observed data 𝐱o , in terms of both the raw membrane voltage over time and the seven data features ( Figure 4c , purple and green ) .", "Parameters with low posterior probability ( outliers ) generated simulations that markedly differed from 𝐱o ( Figure 4c , magenta ) .", "Genetic algorithms are commonly used to fit parameters of deterministic biophysical models ( Druckmann et al . , 2007; Hay et al . , 2011; Van Geit et al . , 2016; Gouwens et al . , 2018 ) .", "While genetic algorithms can also return multiple data-compatible parameters , they do not perform inference ( i . e . find the posterior distribution ) , and their outputs depend strongly on user-defined goodness-of-fit criteria .", "When comparing a state-of-the-art genetic algorithm ( Indicator Based Evolutionary Algorithm , IBEA , [Bleuler et al . , 2003; Zitzler and Künzli , 2004; Van Geit et al . , 2016] ) to SNPE , we found that the parameter-settings favored by IBEA produced simulations whose summary features were as similar to the observed data as those obtained by SNPE high-probability samples ( Appendix 1—figure 10 ) .", "However , high-scoring IBEA parameters were concentrated in small regions of the posterior , that is , IBEA did not identify the full space of data-compatible models .", "To investigate how individual data features constrain parameters , we compared SNPE-estimated posteriors based ( 1 ) solely on the spike count , ( 2 ) on the spike count and three voltage-features , or ( 3 ) on all 7 features of 𝐱o .", "As more features were taken into account , the posterior became narrower and centered more closely on the ground truth parameters ( Figure 4d , Appendix 1—figure 8 ) .", "Posterior simulations matched the observed data only in those features that had been used for inference ( e . g . applying SNPE to spike counts alone identified parameters that generated the correct number of spikes , but for which spike timing and subthreshold voltage time course were off , Figure 4e ) .", "For some parameters , such as the potassium conductance , providing more data features brought the peak of the posterior ( the posterior mode ) closer to the ground truth and also decreased uncertainty .", "For other parameters , such as VT , a parameter adjusting the spike threshold ( Pospischil et al . , 2008 ) , the peak of the posterior was already close to the correct value with spike counts alone , but adding additional features reduced uncertainty .", "While SNPE can be used to study the effect of additional data features in reducing parameter uncertainty , this would not be the case for methods that only return a single best-guess estimate of parameters .", "These results show that SNPE can reveal how information from multiple data features imposes collective constraints on channel and membrane properties in the HH model .", "We also inferred HH parameters for eight in vitro recordings from the Allen Cell Types database using the same current-clamp stimulation protocol as in our model ( Allen Institute for Brain Science , 2016; Teeter et al . , 2018; Figure 4f , Appendix 1—figure 9 ) .", "In each case , simulations based on the SNPE-inferred posterior closely resembled the original data ( Figure 4f ) .", "We note that while inferred parameters differed across recordings , some parameters ( the spike threshold , the density of sodium channels , the membrane reversal potential and the density of potassium channels ) were consistently more strongly constrained than others ( the intrinsic neural noise , the adaptation time constant , the density of slow voltage-dependent channels and the leak conductance ) ( Appendix 1—figure 9 ) .", "Overall , these results suggest that the electrophysiological responses measured by this current-clamp protocol can be approximated by a single-compartment HH model , and that SNPE can identify the admissible parameters .", "We next aimed to demonstrate how the full posterior distribution obtained with SNPE can lead to novel scientific insights .", "To do so , we used the pyloric network of the stomatogastric ganglion ( STG ) of the crab Cancer borealis , a well-characterized neural circuit producing rhythmic activity .", "In this circuit , similar network activity can arise from vastly different sets of membrane and synaptic conductances ( Prinz et al . , 2004 ) .", "We first investigated whether data-consistent sets of membrane and synaptic conductances are connected in parameter space , as has been demonstrated for single neurons ( Taylor et al . , 2006 ) , and , second , which compensation mechanisms between parameters of this circuit allow the neural system to maintain its activity despite parameter variations .", "While this model has been studied extensively , answering these questions requires characterizing higher dimensional parameter spaces than those accessed previously .", "We demonstrate how SNPE can be used to identify the posterior distribution over both membrane and synaptic conductances of the STG ( 31 parameters total ) and how the full posterior distribution can be used to study the above questions at the circuit level .", "For some biological systems , multiple parameter sets give rise to the same system behavior ( Prinz et al . , 2004; Marder and Goaillard , 2006; Gutierrez et al . , 2013; Fisher et al . , 2013; Marder et al . , 2015; Alonso and Marder , 2019 ) .", "In particular , neural systems can be robust to specific perturbations of parameters ( O'Leary et al . , 2014; Marder et al . , 2015; O'Leary and Marder , 2016 ) , yet highly sensitive to others , properties referred to as sloppiness and stiffness ( Goldman et al . , 2001; Gutenkunst et al . , 2007; Machta et al . , 2013; O'Leary et al . , 2015 ) .", "We studied how perturbations affect model output using a model ( Prinz et al . , 2004 ) and data ( Haddad and Marder , 2018 ) of the pyloric rhythm in the crustacean stomatogastric ganglion ( STG ) .", "This model describes a triphasic motor pattern generated by a well-characterized circuit ( Figure 5a ) .", "The circuit consists of two electrically coupled pacemaker neurons ( anterior burster and pyloric dilator , AB/PD ) , modeled as a single neuron , as well as two types of follower neurons ( lateral pyloric ( LP ) and pyloric ( PY ) ) , all connected through inhibitory synapses ( details in Materials and methods ) .", "Eight membrane conductances are included for each modeled neuron , along with seven synaptic conductances , for a total of 31 parameters .", "This model has been used to demonstrate that virtually indistinguishable activity can arise from vastly different membrane and synaptic conductances in the STG ( Prinz et al . , 2004; Alonso and Marder , 2019 ) .", "Here , we build on these studies and extend the model to include intrinsic neural noise on each neuron ( see Materials and methods ) .", "We applied SNPE to an extracellular recording from the STG of the crab Cancer borealis ( Haddad and Marder , 2018 ) which exhibited pyloric activity ( Figure 5b ) , and inferred the posterior distribution over all 31 parameters based on 18 salient features of the voltage traces , including cycle period , phase delays , phase gaps , and burst durations ( features in Figure 5B , posterior in Figure 5c , posterior over all parameters in Appendix 1—figure 11 , details in Materials and methods ) .", "Consistent with previous reports , the posterior distribution has high probability over extended value ranges for many membrane and synaptic conductances .", "To verify that parameter settings across these extended ranges are indeed capable of generating the experimentally observed network activity , we sampled two sets of membrane and synaptic conductances from the posterior distribution .", "These two samples have widely disparate parameters from each other ( Figure 5c , purple dots , details in Materials and methods ) , but both exhibit activity highly similar to the experimental observation ( Figure 5d , top left and top right ) .", "We then investigated the geometry of the parameter space producing these rhythms ( Achard and De Schutter , 2006; Alonso and Marder , 2019 ) .", "First , we wanted to identify directions of sloppiness , and we were interested in whether parameter settings producing pyloric rhythms form a single connected region , as has been shown for single neurons ( Taylor et al . , 2006 ) , or whether they lie on separate ‘islands’ .", "Starting from the two parameter settings showing similar activity above , we examined whether they were connected by searching for a path through parameter space along which pyloric activity was maintained .", "To do this , we algorithmically identified a path lying only in regions of high posterior probability ( Figure 5c , white , details in Materials and methods ) .", "Along the path , network output was tightly preserved , despite a substantial variation of the parameters ( voltage trace 1 in Figure 5d , Appendix 1—figure 12 ) .", "Second , we inspected directions of stiffness by perturbing parameters off the path .", "We applied perturbations that yield maximal drops in posterior probability ( see Materials and methods for details ) , and found that the network quickly produced non-pyloric activity ( voltage trace 2 , Figure 5d; Goldman et al . , 2001 ) .", "Note that , while parameter set 2 seems to lie in regions of high probability when inspecting pairwise marginals , it in fact has low probability under the full posterior distribution ( Appendix 1—figure 13 ) .", "In identifying these paths and perturbations , we exploited the fact that SNPE provides a differentiable estimate of the posterior , as opposed to parameter search methods which provide only discrete samples .", "Overall , these results show that the pyloric network can be robust to specific perturbations in parameter space , but sensitive to others , and that one can interpolate between disparate solutions while preserving network activity .", "This analysis demonstrates the flexibility of SNPE in capturing complex posterior distributions , and shows how the differentiable posterior can be used to study directions of sloppiness and stiffness .", "Experimental and computational studies have shown that stable neural activity can be maintained despite variable circuit parameters ( Prinz et al . , 2004; Marder and Taylor , 2011; O’Leary , 2018 ) .", "This behavior can emerge from two sources ( Marder and Taylor , 2011 ) : either , the variation of a certain parameter barely influences network activity at all , or alternatively , variations of several parameters influence network activity , but their effects compensate for one another .", "Here , we investigated these possibilities by using the posterior distribution over membrane and synaptic conductances of the STG .", "We began by drawing samples from the posterior and inspecting their pairwise histograms ( i . e . the pairwise marginals , Figure 6a , posterior over all parameters in Appendix 1—figure 11 ) .", "Consistent with previously reported results ( Taylor et al . , 2009 ) , many parameters seem only weakly constrained and only weakly correlated ( Figure 6b ) .", "However , this observation does not imply that the parameters of the network do not have to be finely tuned: pairwise marginals are averages over many network configurations , where all other parameters may take on diverse values , which could disguise that each individual configuration is finely tuned .", "Indeed , when we sampled parameters independently from their posterior histograms , the resulting circuit configurations rarely produced pyloric activity , indicating that parameters have to be tuned relative to each other ( Appendix 1—figure 14 ) .", "This analysis also illustrates that the ( common ) approach of independently setting parameters can be problematic: although each parameter individually is in a realistic range , the network as a whole is not ( Golowasch et al . , 2002 ) .", "Finally , it shows the importance of identifying the full posterior distribution , which is far more informative than just finding individual parameters and assigning error bars .", "In order to investigate the need for tuning between pairs of parameters , we held all but two parameters constant at a given consistent circuit configuration ( sampled from the posterior ) , and observed the network activity across different values of the remaining pair of parameters .", "We can do so by calculating the conditional posterior distribution ( details in Materials and methods ) , and do not have to generate additional simulations ( as would be required by parameter search methods ) .", "Doing so has a simple interpretation: when all but two parameters are fixed , what values of the remaining two parameters can then lead to the desired network activity ?", "We found that the desired pattern of pyloric activity can emerge only from narrowly tuned and often highly correlated combinations of the remaining two parameters , showing how these parameters can compensate for one another ( Figure 6c ) .", "When repeating this analysis across multiple network configurations , we found that these ‘conditional correlations’ are often preserved ( Figure 6c , left and right ) .", "This demonstrates that pairs of parameters can compensate for each other in a similar way , independently of the values taken by other parameters .", "This observation about compensation could be interpreted as an instance of modularity , a widespread underlying principle of biological robustness ( Kitano , 2004 ) .", "We calculated conditional correlations for each parameter pair using 500 different circuit configurations sampled from the posterior ( Figure 6d ) .", "Compared to correlations based on the pairwise marginals ( Figure 6b ) , these conditional correlations were substantially stronger .", "They were particularly strong across membrane conductances of the same neuron , but primarily weak across different neurons ( black boxes in Figure 6d ) .", "Finally , we tested whether the conditional correlations were in line with experimental observations .", "For the PD and the LP neuron , it has been reported that overexpression of the fast transient potassium current ( IA ) leads to a compensating increase of the hyperpolarization current ( IH ) , suggesting a positive correlation between these two currents ( MacLean et al . , 2003; MacLean et al . , 2005 ) .", "These results are qualitatively consistent with the positive conditional correlations between the maximal conductances of IA and IH for all three model neurons ( Figure 6e top ) .", "In addition , using the dynamic clamp , it has been shown that diverse combinations of the synaptic input strength and the maximal conductance of IH lead to similar activity in the LP and the PD neuron ( Grashow et al . , 2010; Marder , 2011 ) .", "Consistent with these findings , the non-zero conditional correlations reveal that there can indeed be compensation mechanisms between the synaptic strength and the maximal conductance of IH of the postsynaptic neuron ( Figure 6e bottom ) .", "Overall , we showed how SNPE can be used to study parameter dependencies , and how the posterior distribution can be used to efficiently explore potential compensation mechanisms .", "We found that our method can predict compensation mechanisms which are qualitatively consistent with experimental studies .", "We emphasize that these findings would not have been possible with a direct grid-search over all parameters: defining a grid in a 31-dimensional parameter space would require more than 231 > 2 billion simulations , even if one were to use the coarsest-possible grid with only two values per dimension ." ], [ "SNPE builds on recent advances in machine learning and in particular in density-estimation approaches to likelihood-free inference ( Papamakarios and Murray , 2016; Le et al . , 2017a; Lueckmann et al . , 2017; Chan et al . , 2018; Greenberg et al . , 2019 , reviewed in Cranmer et al . , 2020 ) .", "We here scaled these approaches to canonical mechanistic models of neural dynamics and provided methods and software-tools for inference , visualization , and analysis of the resulting posteriors ( e . g . the high-probability paths and conditional correlations presented here ) .", "The idea of learning inference networks on simulated data can be traced back to regression-adjustment methods in ABC ( Beaumont et al . , 2002; Blum and François , 2010 ) .", "Papamakarios and Murray , 2016 first proposed to use expressive conditional density estimators in the form of deep neural networks ( Bishop , 1994; Papamakarios et al . , 2017 ) , and to optimize them sequentially over multiple rounds with cost-functions derived from Bayesian inference principles .", "Compared to commonly used rejection-based ABC methods ( Rubin , 1984; Pritchard et al . , 1999 ) , such as MCMC-ABC ( Marjoram et al . , 2003 ) , SMC-ABC ( Sisson et al . , 2007; Liepe et al . , 2014 ) , Bayesian-Optimization ABC ( Gutmann and Corander , 2016 ) , or ensemble methods ( Britton et al . , 2013; Lawson et al . , 2018 ) , SNPE approaches do not require one to define a distance function in data space .", "In addition , by leveraging the ability of neural networks to learn informative features , they enable scaling to problems with high-dimensional observations , as are common in neuroscience and other fields in biology .", "We have illustrated this capability in the context of receptive field estimation , where a convolutional neural network extracts summary features from a 1681 dimensional spike-triggered average .", "Alternative likelihood-free approaches include synthetic likelihood methods ( Wood , 2010; Costa et al . , 2013; Wilkinson , 2014; Meeds and Welling , 2014; Papamakarios et al . , 2019a; Lueckmann et al . , 2019; Durkan et al . , 2018 ) , moment-based approximations of the posterior ( Barthelmé and Chopin , 2014; Schröder et al . , 2019 ) , inference compilation ( Le et al . , 2017b; Casado et al . , 2017 ) , and density-ratio estimation ( Hermans et al . , 2020 ) .", "For some mechanistic models in neuroscience ( e . g . for integrate-and-fire neurons ) , likelihoods can be computed via stochastic numerical approximations ( Chen , 2003; Huys and Paninski , 2009; Meliza et al . , 2014 ) or model-specific analytical approaches ( Huys et al . , 2006; Hertäg et al . , 2012; Pozzorini et al . , 2015; Ladenbauer et al . , 2018; René et al . , 2020 ) .", "How big is the advance brought by SNPE relative to ‘conventional’ brute-force approaches that aim to exhaustively explore parameter space ?", "A fundamental difference from grid search approaches that have been applied to neuroscientific models ( Prinz et al . , 2003; Caplan et al . , 2014; Stringer et al . , 2016 ) is that SNPE can perform Bayesian inference for stochastic models , whereas previous approaches identified parameters whose deterministic model-outputs were heuristically ‘close’ to empirical data .", "Depending on the goal of the analysis , either approach might be preferable .", "SNPE , and Bayesian inference more generally , is derived for stochastic models .", "SNPE can , in principle , also be applied to deterministic models , but a rigorous mathematical interpretation or empirical evaluation in this regime is beyond the scope of this study .", "SNPE also differs conceptually and quantitatively from rejection-ABC , in which random parameters are accepted or rejected based on a distance-criterion .", "SNPE uses all simulations during training instead of rejecting some , learns to identify data features informative about model parameters rather than relying on the user to choose the correct data features and distance metric , and performs considerably better than rejection-ABC , in particular for problems with high-dimensional observations ( Figure 2 ) .", "Another advantage over grid search and rejection-ABC is that SNPE can ‘amortize’ inference of parameter posteriors , so that one can quickly perform inference on new data , or explore compensation mechanisms , without having to carry out new simulations , or repeatedly search a simulation database .", "We should still note that SNPE can require the generation of large sets of simulations , which can be viewed as a brute-force step , emphasising that one of the main strengths of SNPE over conventional brute-force approaches relies on the processing of these simulations via deep neural density estimators .", "Our approach is already finding its first applications in neuroscience–for example , Oesterle et al . , 2020 have used a variant of SNPE to constrain biophysical models of retinal neurons , with the goal of optimizing stimulation approaches for neuroprosthetics .", "Concurrently with our work , Bittner et al . , 2019 developed an alternative approach to parameter identification for mechanistic models and showed how it can be used to characterize neural population models which exhibit specific emergent computational properties .", "Both studies differ in their methodology and domain of applicability ( see descriptions of underlying algorithms in our prior work [Lueckmann et al . , 2017; Greenberg et al . , 2019] and theirs [Loaiza-Ganem et al . , 2017] ) , as well in the focus of their neuroscientific contributions .", "Both approaches share the overall goal of using deep probabilistic inference tools to build more interpretable models of neural data .", "These complementary and concurrent advances will expedite the cycle of building , adjusting and selecting mechanistic models in neuroscience .", "Finally , a complementary approach to mechanistic modeling is to pursue purely phenomenological models , which are designed to have favorable statistical and computational properties: these data-driven models can be efficiently fit to neural data ( Brown et al . , 1998; Truccolo et al . , 2005; Pillow , 2007; Pillow et al . , 2008; Schneidman et al . , 2006; Macke et al . , 2011; Yu et al . , 2009; Pandarinath et al . , 2018; Cunningham and Yu , 2014 ) or to implement desired computations ( Sussillo and Abbott , 2009 ) .", "Although tremendously useful for a quantitative characterization of neural dynamics , these models typically have a large number of parameters , which rarely correspond to physically measurable or mechanistically interpretable quantities , and thus it can be challenging to derive mechanistic insights or causal hypotheses from them ( but see e . g . Mante et al . , 2013; Sussillo and Barak , 2013; Maheswaranathan et al . , 2019 ) .", "When fitting mechanistic models to data , it is common to target summary features to isolate specific behaviors , rather than the full data .", "For example , the spike shape is known to constrain sodium and potassium conductances ( Druckmann et al . , 2007; Pospischil et al . , 2008; Hay et al . , 2011 ) .", "When modeling population dynamics , it is often desirable to achieve realistic firing rates , rate-correlations and response nonlinearities ( Rubin et al . , 2015; Bittner et al . , 2019 ) , or specified oscillations ( Prinz et al . , 2004 ) .", "In models of decision making , one is often interested in reproducing psychometric functions or reaction-time distributions ( Ratcliff and McKoon , 2008 ) .", "Choice of summary features might also be guided by known limitations of either the model or the measurement approach , or necessitated by the fact that published data are only available in summarized form .", "Several methods have been proposed to automatically construct informative summary features ( Blum et al . , 2013; Jiang et al . , 2017; Izbicki et al . , 2019 ) .", "SNPE can be applied to , and might benefit from the use of summary features , but it also makes use of the ability of neural networks to automatically learn informative features in high-dimensional data .", "Thus , SNPE can also be applied directly to raw data ( e . g . using recurrent neural networks [Lueckmann et al . , 2017] ) , or to high-dimensional summary features which are challenging for ABC approaches ( Figure 2 ) .", "In all cases , care is needed when interpreting models fit to summary features , as choice of features can influence the results ( Blum et al . , 2013; Jiang et al . , 2017; Izbicki et al . , 2019 ) .", "A key advantage of SNPE is its general applicability: it can be applied whenever one has a simulator that allows to stochastically generate model outputs from specific parameters .", "Furthermore , it can be applied in a fully ‘black-box manner’ , that is , does not require access to the internal workings of the simulator , its model equations , likelihoods or gradients .", "It does not impose any other limitations on the model or the summary features , and in particular does not require them to be differentiable .", "However , it also has limitations which we enumerate below .", "First , current implementations of SNPE scale well to high-dimensional observations ( ∼1000s of dimensions , also see Greenberg et al . , 2019 ) , but scaling SNPE to even higher-dimensional parameter spaces ( above 30 ) is challenging ( note that previous approaches were generally limited to less than 10 dimensions ) .", "Given that the difficulty of estimating full posteriors scales exponentially with dimensionality , this is an inherent challenge for all approaches that aim at full inference ( in contrast to just identifying a single , or a few heuristically chosen parameter fits ) .", "Second , while it is a long-term goal for these approaches to be made fully automatic , our current implementation still requires choices by the user: as described in Materials and methods , one needs to choose the type of the density estimation network , and specify settings related to network-optimization , and the number of simulations and inference rounds .", "These settings depend on the complexity of the relation between summary features and model parameters , and the number of simulations that can be afforded .", "In the documentation accompanying our code-package , we provide examples and guidance .", "For small-scale problems , we have found SNPE to be robust to these settings .", "However , for challenging , high-dimensional applications , SNPE might currently require substantial user interaction .", "Third , the power of SNPE crucially rests on the ability of deep neural networks to perform density estimation .", "While deep nets have had ample empirical success , we still have an incomplete understanding of their limitations , in particular in cases where the mapping between data and parameters might not be smooth ( e . g . near phase transitions ) .", "Fourth , when applying SNPE ( or any other model-identification approach ) , validation of the results is of crucial importance , both to assess the accuracy of the inference procedure , as well as to identify possible limitations of the mechanistic model itself .", "In the example applications , we used several procedures for assessing the quality of the inferred posteriors .", "One common ingredient of these approaches is to sample from the inferred model , and search for systematic differences between observed and simulated data , e . g . to perform posterior predictive checks ( Cook et al . , 2006; Talts et al . , 2018; Liepe et al . , 2014; Lueckmann et al . , 2017; Greenberg et al . , 2019; Figure 2g , Figure 3f , g , Figure 4c , and Figure 5d ) .", "These approaches allow one to detect ‘failures’ of SNPE , that is , cases in which samples from the posterior do not reproduce the data .", "However , when diagnosing any Bayesian inference approach , it is challenging to rigorously rule out the possibility that additional parameter-settings ( e . g . in an isolated ‘island’ ) would also explain the data .", "Thus , it is good practice to use multiple initializations of SNPE , and/or a large number of simulations in the initial round .", "There are challenges and opportunities ahead in further scaling and automating simulation-based inference approaches .", "However , in its current form , SNPE will be a powerful tool for quantitatively evaluating mechanistic hypotheses on neural data , and for designing better models of neural dynamics ." ], [ "Code implementing SNPE based on Theano , is available at http://www . mackelab . org/delfi/ .", "An extended toolbox based on PyTorch is available at http://www . mackelab . org/sbi/ ( Tejero-Cantero et al . , 2020 ) .", "To perform Bayesian parameter identification with SNPE , three types of input need to be specified: For each problem , our goal was to estimate the posterior distribution p⁢ ( 𝜽|𝐱o ) .", "To do this , we used SNPE ( Papamakarios and Murray , 2016; Lueckmann et al . , 2017; Greenberg et al . , 2019 ) .", "Setting up the inference procedure required three design choices: We emphasize that SNPE is highly modular , that is , that the the inputs ( data , the prior over parameter , the mechanistic model ) , and algorithmic components ( network architecture , probability density , optimization approach ) can all be modified and chosen independently .", "This allows neuroscientists to work with models which are designed with mechanistic principles—and not convenience of inference—in mind .", "Furthermore , it allows SNPE to benefit from advances in more flexible density estimators , more powerful network architectures , or optimization strategies .", "With the problem and inference settings specified , SNPE adjusts the network weights ϕ based on simulation results , so that p⁢ ( 𝜽|𝐱 ) ≈qF⁢ ( 𝐱 , ϕ ) ⁢ ( 𝜽 ) for any 𝐱 .", "In the first round of SNPE , simulation parameters are drawn from the prior p⁢ ( 𝜽 ) .", "If a single round of inference is not sufficient , SNPE can be run in multiple rounds , in which samples are drawn from the version of qF⁢ ( 𝐱o , ϕ ) ⁢ ( 𝜽 ) at the beginning of the round .", "After the last round , qF⁢ ( 𝐱o , ϕ ) is returned as the inferred posterior on parameters 𝜽 given observed data 𝐱o .", "If SNPE is only run for a single round , then the generated samples only depend on the prior , but not on 𝐱o: in this case , the inference network is applicable to any data ( covered by the prior ranges ) , and can be used for rapid amortized inference .", "SNPE learns the correct network weights ϕ by minimizing the objective function ∑jℒ⁢ ( 𝜽j , 𝐱j ) where the simulation with parameters 𝜽j produced result 𝐱j .", "For the first round of SNPE ℒ⁢ ( 𝜽j , 𝐱j ) =-log⁡qF⁢ ( 𝐱j , ϕ ) , while in subsequent rounds a different loss function accounts for the fact that simulation parameters were not sampled from the prior .", "Different choices of the loss function for later rounds result in SNPE-A ( Papamakarios and Murray , 2016 ) , SNPE-B ( Lueckmann et al . , 2017 ) or SNPE-C algorithm ( Greenberg et al . , 2019 ) .", "To optimize the networks , we used ADAM with default settings ( Kingma and Ba , 2014 ) .", "The details of the algorithm are below: Algorithm 1: SNPE Input: simulator with ( implicit ) density p⁢ ( 𝐱|𝜽 ) , observed data 𝐱o , prior p⁢ ( 𝜽 ) , density family qψ , neural network F⁢ ( 𝐱 , ϕ ) , number of rounds , simulation count for each round Nr randomly initialize ϕ p~1⁢ ( 𝜽 ) :=p⁢ ( 𝜽 ) N:=0 for r=1 to R do for i=1⁢…⁢Nr do sample 𝜽N+i∼p~r⁢ ( 𝜽 ) simulate 𝐱N+i∼p⁢ ( 𝐱|𝜽N+i ) N←N+Nr train ϕ←arg⁢minϕ⁢∑j=1Nℒ⁢ ( 𝜽j , 𝐱j ) p~r⁢ ( 𝜽 ) :=qF⁢ ( 𝐱o , ϕ ) ⁢ ( 𝜽 ) return qF⁢ ( 𝐱o , ϕ ) ⁢ ( 𝜽 ) In Papamakarios and Murray , 2016 , it was shown that the procedure described above ( i . e . sample from the prior , train a flexible density estimator by minimizing the log-loss ℒ⁢ ( 𝜽j , 𝐱j ) =-∑jlog⁡qF⁢ ( 𝐱j , ϕ ) ⁢ ( 𝜽j ) ) can be used to perform Bayesian inference without likelihood evaluations .", "For the multi-round case , in which samples are no longer drawn from the prior , but adaptively generated from a ( generally more focused ) proposal distribution , the loss function needs to be modified .", "Different variants of SNPE differ in how exactly this is done: As described above , SNPE approximates the posterior distribution with flexible neural density estimators: either a mixture density network ( MDN ) or a masked autoregressive flow ( MAF ) .", "Below , we provide a few more details about these density estimators , how we chose their respective architectures , and when to choose one or the other .", "The MDN outputs the parameters of a mixture of Gaussians ( i . e . mixture weights , and for each component of the mixture , the mean vector and covariance entries ) .", "Thus , for an MDN composed of K components , we chose an architecture with at least as many units per layer as K ( 1+Nθ+Nθ ( Nθ+1 ) /2 ) −1 , where Nθ is the number of parameters to infer , to ensure enough flexibility to approximate well the parameters of the mixture of Gaussians .", "For example , when inferring the parameters of the Hodgkin-Huxley model given in vitro recordings from mouse cortex ( Allen Cell Types Database , https://celltypes . brain-map . org/data ) , we infer the posterior over eight parameters with a mixture of two Gaussians , and the MDN needs at least 89 units per layer .", "Across applications , we found two layers to be sufficient to appropriately approximate the posterior distribution .", "MAF is a specific type of normalizing flow , which is a highly flexible density estimator ( Rezende and Mohamed , 2015; Papamakarios et al . , 2017; Papamakarios et al . , 2019b ) .", "Normalizing flows consist of a stack of bijections which transform a simple distribution ( usually a multivariate Gaussian distribution ) into the target distribution .", "Each bijection is parameterized by a specific type of neural network ( for MAF: a Masked Autoencoder for Distribution Estimation , or MADE ) .", "In our experiments , five stacked bijections are enough to approximate even complex posterior distributions .", "Depending on the size of the parameter and data space , each neural network had between [50 , 50] and [100 , 100 , 100] hidden units .", "When using SNPE in a single-round , we generally found superior performance for MAFs as compared to MDNs .", "When running inference across multiple rounds , training MAFs leads to additional challenges which might impede the quality of inference ( Greenberg et al . , 2019; Durkan et al . , 2020 ) .", "We used a Linear-Nonlinear ( LN ) encoding model ( a special case of a generalized linear model , GLM , [Brown et al . , 1998; Paninski , 2004; Truccolo et al . , 2005; Pillow , 2007; Pillow et al . , 2008; Gerwinn et al . , 2010] ) to simulate the activity of a neuron in response to a univariate time-varying stimulus .", "Neural activity zi was subdivided in T=100 bins and , within each bin i , spikes were generated according to a Bernoulli observation model , zi∼Bern⁢ ( η⁢ ( 𝐯i⊤⁢𝒇+β ) ) , where 𝐯i is a vector of white noise inputs between time bins i-8 and i , 𝒇 a length-9 linear filter , β is the bias , and η⁢ ( ⋅ ) =exp⁡ ( ⋅ ) / ( 1+exp⁡ ( ⋅ ) ) is the canonical inverse link function for a Bernoulli GLM .", "As summary features , we used the total number of spikes N and the spike-triggered average 1N⁢𝐕𝐳 , where 𝐕=[v1 , v2 , … , vT] is the so-called design matrix of size 9×T .", "We note that the spike-triggered sum 𝐕𝐳 constitutes sufficient statistics for this GLM , that is that selecting the STA and N together as summary features does not lead to loss of model relevant information over the full input-output dataset {𝐕 , 𝐳} .", "We used a Gaussian prior with zero mean and covariance matrix Σ=σ2 ( F⊤F ) −1 , where 𝐅 encourages smoothness by penalizing the second-order differences in the vector of parameters ( De Nicolao et al . , 1997 ) .", "For inference , we used a single round of 10 , 000 simulations , and the posterior was approximated with a Gaussian distribution ( 𝜽∈ℝ10 , 𝐱∈ℝ10 ) .", "We used a feedforward neural network with two hidden layers of 50 units each .", "We used a Polya Gamma Markov Chain Monte Carlo sampling scheme ( Polson et al . , 2013 ) to estimate a reference posterior .", "In Figure 2d , we compare the performance of SNPE with two classical ABC algorithms , rejection ABC and Sequential Monte Carlo ABC as a function of the number of simulations .", "We report the relative error in Kullback-Leibler divergence , which is defined as: ( 1 ) DKL ( pM⁢C⁢M⁢C ( 𝜽|𝐱 ) ||p^ ( 𝜽|𝐱 ) ) DKL ( pM⁢C⁢M⁢C ( 𝜽|𝐱 ) ||p ( 𝜽 ) ) , and which ranges between 0 ( perfect recovery of the posterior ) and 1 ( estimated posterior no better than the prior ) .", "Here , pM⁢C⁢M⁢C⁢ ( 𝜽|𝐱 ) is the ground-truth posterior estimated via Markov Chain Monte Carlo sampling , p^⁢ ( 𝜽|𝐱 ) is the estimated posterior via SNPE , rejection ABC or Sequential Monte Carlo ABC , and p⁢ ( 𝜽 ) is the prior .", "For the spatial receptive field model of a cell in primary visual cortex , we simulated the activity of a neuron depending on an image-valued stimulus .", "Neural activity was subdivided in bins of length Δ⁢t=0 . 025⁢s and within each bin i , spikes were generated according to a Poisson observation model , zi∼Poiss⁢ ( η⁢ ( 𝐯i⊤⁢𝒉+β ) ) , where 𝐯i is the vectorized white noise stimulus at time bin i , 𝒉 a 41 × 41 linear filter , β is the bias , and η⁢ ( ⋅ ) =exp⁡ ( ⋅ ) is the canonical inverse link function for a Poisson GLM .", "The receptive field 𝒉 is constrained to be a Gabor filter:h ( gx , gy ) =gexp⁡ ( −x′2+r2y′22σ2 ) cos⁡ ( 2πfx′−ϕ ) x′= ( gx−x ) cos⁡ψ− ( gy−y ) sin⁡ψy′= ( gx−x ) sin⁡ψ+ ( gy−y ) cos⁡ψσ=2log⁡22πf2w+12w−1 , where ( gx , gy ) is a regular grid of 41 × 41 positions spanning the 2D image-valued stimulus .", "The parameters of the Gabor are gain g , spatial frequency f , aspect-ratio r , width w , phase ϕ ( between 0 and π ) , angle ψ ( between 0 and 2⁢π ) and location x , y ( assumed within the stimulated area , scaled to be between −1 and 1 ) .", "Bounded parameters were transformed with a log- , or logit-transform , to yield unconstrained parameters .", "After applying SNPE , we back-transformed both the parameters and the estimated posteriors in closed form , as shown in Figure 2 .", "We did not transform the bias β .", "We used a factorizing Gaussian prior for the vector of transformed Gabor parameters[log⁡g , log⁡f , log⁡r , log⁡w , l0 , π⁢ ( ϕ ) , l0 , 2⁢π⁢ ( ψ ) , l-1 , 1⁢", "( x ) , l-1 , 1⁢", "( y ) ] , where transforms l0 , π⁢ ( X ) =log⁡ ( X/ ( 2⁢π-X ) ) , l0 , 2⁢π⁢ ( X ) =log⁡ ( X/ ( π-X ) ) , l-1 , 1⁢ ( X ) =log⁡ ( ( X+1 ) / ( 1-X ) ) ensured the assumed ranges for the Gabor parameters ϕ , ψ , x , y .", "Our Gaussian prior had zero mean and standard deviations [0 . 5 , 0 . 5 , 0 . 5 , 0 . 5 , 1 . 9 , 1 . 78 , 1 . 78 , 1 . 78] .", "We note that a Gaussian prior on a logit-transformed random variable logit⁢X with zero mean and standard deviation around 1 . 78 is close to a uniform prior over the original variable X . For the bias β , we used a Gaussian prior with mean −0 . 57 and variance 1 . 63 , which approximately corresponds to an exponential prior e⁢x⁢p⁢ ( β ) ∼E⁢x⁢p⁢ ( λ ) with rate λ=1 on the baseline firing rate exp⁡ ( β ) in absence of any stimulus .", "The ground-truth parameters for the demonstration in Figure 2 were chosen to give an asymptotic firing rate of 1 Hz for 5 min stimulation , resulting in 299 spikes , and a signal-to-noise ratio of −12dB .", "As summary features , we used the total number of spikes N and the spike-triggered average 1N⁢𝐕𝐳 , where 𝐕=[v1 , v2 , … , vT] is the stimulation video of length T=300/Δ⁢t=12000 .", "As for the GLM with a temporal filter , the spike-triggered sum 𝐕𝐳 constitutes sufficient statistics for this GLM .", "For inference , we applied SNPE-A with in total two rounds: an initial round serves to first roughly identify the relevant region of parameter space .", "Here we used a Gaussian distribution to approximate the posterior from 100 , 000 simulations .", "A second round then used a mixture of eight Gaussian components to estimate the exact shape of the posterior from another 100 , 000 simulations ( 𝜽∈ℝ9 , 𝐱∈ℝ1682 ) .", "We used a convolutional network with five convolutional layers with 16 to 32 convolutional filters followed by two fully connected layers with 50 units each .", "The total number of spikes N within a simulated experiment was passed as an additional input directly to the fully-connected layers of the network .", "Similar to the previous GLM , this model has a tractable likelihood , so we use MCMC to obtain a reference posterior .", "We applied this approach to extracelullar recordings from primary visual cortex of alert mice obtained using silicon microelectrodes in response to colored-noise visual stimulation .", "Experimental methods are described in Dyballa et al . , 2018 .", "In order to illustrate the competitive performance of SNPE , we obtained a posterior estimate with a classical ABC method , Sequential Monte Carlo ( SMC ) ABC ( Sisson et al . , 2007; Beaumont et al . , 2009 ) .", "Likelihood-free inference methods from the ABC family require a distance function d⁢ ( 𝐱o , 𝐱 ) between observed data 𝐱o and possible simulation outputs 𝐱 to characterize dissimilarity between simulations and data .", "A common choice is the ( scaled ) Euclidean distance d⁢ ( 𝐱o , 𝐱 ) =||𝐱-𝐱o||2 .", "The Euclidean distance here was computed over 1681 summary features given by the spike-triggered average ( one per pixel ) and a single summary feature given by the ‘spike count’ .", "To ensure that the distance measure was sensitive to differences in both STA and spike count , we scaled the summary feature ‘spike count’ to account for about 20% of the average total distance ( other values did not yield better results ) .", "The other 80% were computed from the remaining 1681 summary features given by spike-triggered averages .", "To showcase how this situation is challenging for ABC approaches , we generated 10 , 000 input-output pairs ( 𝜽i , 𝐱i ) ∼p⁢ ( 𝐱|𝜽 ) ⁢p⁢ ( 𝜽 ) with the prior and simulator used above , and illustrate the 10 STAs and spike counts with closest d⁢ ( 𝐱o , 𝐱i ) in Appendix 1—figure 5a .", "Spike counts were comparable to the observed data ( 299 spikes ) , but STAs were noise-dominated and the 10 ‘closest’ underlying receptive fields ( orange contours ) showed substantial variability in location and shape of the receptive field .", "If even the ‘closest’ samples do not show any visible receptive field , then there is little hope that even an appropriately chosen acceptance threshold will yield a good approximation to the posterior .", "These findings were also reflected in the results from SMC-ABC with a total simulation budget of 106 simulations ( Appendix 1—figure 5b ) .", "The estimated posterior marginals for ‘bias’ and ‘gain’ parameters show that the parameters related to the firing rate were constrained by the data 𝐱o , but marginals of parameters related to shape and location of the receptive field did not differ from the prior , highlighting that SMC-ABC was not able to identify the posterior distribution .", "The low correlations between the ground-truth receptive field and receptive fields sampled from SMC-ABC posterior further highlight the failure of SMC-ABC to infer the ground-truth posterior ( Appendix 1—figure 5c ) .", "Further comparisons of neural-density estimation approaches with ABC-methods can be found in the studies describing the underlying machine-learning methodologies ( Papamakarios and Murray , 2016; Lueckmann et al . , 2019; Greenberg et al . , 2019 ) .", "We simulated non-inactivating potassium channel currents subject to voltage-clamp protocols as:IK=g¯Km ( V−EK ) , where V is the membrane potential , g¯K is the density of potassium channels , EK is the reversal potential of potassium , and m is the gating variable for potassium channel activation .", "m is modeled according to the first-order kinetic equationdmdt=m∞ ( V ) −mτm ( V ) , where m∞⁢ ( V ) is the steady-state activation , and τm⁢ ( V ) the respective time constant .", "We used a general formulation of m∞⁢ ( V ) and τm⁢ ( V ) ( Destexhe and Huguenard , 2000 ) , where the steady-state activation curve has two parameters ( slope and offset ) and the time constant curve has six parameters , amounting to a total of 8 parameters ( θ1 to θ8 ) : m∞ ( V ) =11+e−θ1V+θ2τm ( V ) =θ4e−[θ5 ( V−θ3 ) +θ6 ( V−θ3 ) 2]+e[θ7 ( V−θ3 ) +θ8 ( V−θ3 ) 2] .", "Since this model can be used to describe the dynamics of a wide variety of channel models , we refer to it as Omnimodel .", "We modeled responses of the Omnimodel to a set of five noisy voltage-clamp protocols ( Podlaski et al . , 2017 ) : as described in Podlaski et al . , 2017 , the original voltage-clamp protocols correspond to standard protocols of activation , inactivation , deactivation , ramp and action potential , to which we added Gaussian noise with zero mean and standard deviation 0 . 5 mV .", "Current responses were reduced to 55 summary features ( 11 per protocol ) .", "Summary features were coefficients to basis functions derived via Principal Components Analysis ( PCA ) ( 10 per protocol ) plus a linear offset ( one per protocol ) found via least-squares fitting .", "PCA basis functions were found by simulating responses of the non-inactivating potassium channel models to the five voltage-clamp protocols and reducing responses to each protocol to 10 dimensions ( explaining 99 . 9% of the variance ) .", "To amortize inference on the model , we specified a wide uniform prior over the parameters: θ1∈𝒰 ( 0 , 1 ) , θ2∈𝒰 ( −10 . , 10 . ) , θ3∈𝒰 ( −120 . , 120 . ) , θ4∈𝒰 ( 0 . , 2000 ) , θ5∈𝒰 ( 0 . , 0 . 5 ) , θ6∈𝒰 ( 0 , 0 . 05 ) , θ7∈𝒰 ( 0 . , 0 . 5 ) , θ8∈𝒰 ( 0 , 0 . 05 ) .", "For inference , we trained a shared inference network in a single round of 106 simulations generated by sampling from the prior ( 𝜽∈ℝ8 , 𝐱∈ℝ55 ) .", "The density estimator was a masked autoregressive flow ( MAF ) ( Papamakarios et al . , 2017 ) with five MADES with [250 , 250] hidden units each .", "We evaluated performance on 350 non-inactivating potassium ion channels selected from IonChannelGenealogy ( ICG ) by calculating the correlation coefficient between traces generated by the original model and traces from the Omnimodel using the posterior mode ( Appendix 1—figure 7 ) .", "We simulated a single-compartment Hodgkin–Huxley type neuron with channel kinetics as in Pospischil et al . , 2008 , CmdVdt=gl ( El−V ) +g¯Nam3h ( ENa−V ) +g¯Kn4 ( EK−V ) +g¯Mp ( EK−V ) +Iinj+ση ( t ) dqdt=q∞ ( V ) −qτq ( V ) , q∈{m , h , n , p} , where V is the membrane potential , Cm is the membrane capacitance , gl is the leak conductance , El is the membrane reversal potential , g¯c is the density of channels of type c ( Na+ , K+ , M ) , Ec is the reversal potential of c , ( m , h , n , p ) are the respective channel gating kinetic variables , and σ⁢η⁢ ( t ) is the intrinsic neural Gaussian noise .", "The right hand side of the voltage dynamics is composed of a leak current , a voltage-dependent Na+ current , a delayed-rectifier K+ current , a slow voltage-dependent K+ current responsible for spike-frequency adaptation , and an injected current Iinj .", "Channel gating variables q have dynamics fully characterized by the neuron membrane potential V , given the respective steady-state q∞⁢ ( V ) and time constant τq⁢ ( V ) ( details in Pospischil et al . , 2008 ) .", "Two additional parameters are implicit in the functions q∞⁢ ( V ) and τq ( V ) : VT adjusts the spike threshold through m∞ , h∞ , n∞ , τm , τh and τn; τmax scales the time constant of adaptation through τp⁢ ( V ) ( details in Pospischil et al . , 2008 ) .", "We set ENa=53 mV and EK=-107 mV , similar to the values used for simulations in Allen Cell Types Database ( http://help . brain-map . org/download/attachments/8323525/BiophysModelPeri . pdf ) .", "We applied SNPE to infer the posterior over eight parameters ( g¯Na , g¯K , gl , g¯M , τmax , VT , σ , El ) , given seven voltage features ( number of spikes , mean resting potential , standard deviation of the resting potential , and the first four voltage moments , mean , standard deviation , skewness and kurtosis ) .", "The prior distribution over the parameters was uniform , 𝜽∼𝒰⁢ ( plow , phigh ) , where plow=[0 . 5 , 10-4 , 10-4 , 10-4 , 50 , 40 , 10-4 , 35] and phigh=[80 , 15 , 0 . 6 , 0 . 6 , 3000 , 90 , 0 . 15 , 100] .", "These ranges are similar to the ones obtained in Pospischil et al . , 2008 , when fitting the above model to a set of electrophysiological recordings .", "For inference in simulated data , we used a single round of 100 , 000 simulations ( θ∈R8 , x∈R7 ) .", "The density estimator was a masked autoregressive flow ( MAF ) ( Papamakarios et al . , 2017 ) with five MADES with [50 , 50] hidden units each .", "For the inference on in vitro recordings from mouse cortex ( Allen Cell Types Database , https://celltypes . brain-map . org/data ) , we selected eight recordings corresponding to spiny neurons with at least 10 spikes during the current-clamp stimulation .", "The respective cell identities and sweeps are: ( 518290966 , 57 ) , ( 509881736 , 39 ) , ( 566517779 , 46 ) , ( 567399060 , 38 ) , ( 569469018 , 44 ) , ( 532571720 , 42 ) , ( 555060623 , 34 ) , ( 534524026 , 29 ) .", "For each recording , SNPE-B was run for two rounds with 125 , 000 Hodgkin–Huxley simulations each , and the posterior was approximated by a mixture of two Gaussians .", "In this case , the density estimator was composed of two fully connected layers of 100 units each .", "We compared SNPE posterior with a state-of-the-art genetic algorithm ( Indicator Based Evolutionary Algorithm IBEA , [Bleuler et al . , 2003; Zitzler and Künzli , 2004] from the BluePyOpt package [Van Geit et al . , 2016] ) , in the context of the Hodgkin-Huxley model with 8 parameters and seven features ( Appendix 1—figure 10 ) .", "For each Hodgkin-Huxley model simulation i and summary feature j , we used the following objective score:ϵi⁢j=|xi⁢j-xo⁢jσj| , j=1 , … , 7 , where xi⁢j is the value of summary feature j for simulation i , xo⁢j is the observed summary feature j , and σj is the standard deviation of the summary feature j computed across 1000 previously simulated datasets .", "IBEA outputs the hall-of-fame , which corresponds to the 10 parameter sets with the lowest sum of objectives ∑j7ϵi⁢j .", "We ran IBEA with 100 generations and an offspring size of 1000 individuals , corresponding to a total of 100 , 000 simulations .", "We used extracellular nerve recordings made from the stomatogastric motor neurons that principally comprise the triphasic pyloric rhythm in the crab Cancer borealis ( Haddad and Marder , 2018 ) .", "The preparations were decentralized , that is , the axons of the descending modulatory inputs were severed .", "The data was recorded at a temperature of 11°C .", "See Haddad and Marder , 2018 for full experimental details .", "We simulated the circuit model of the crustacean stomatogastric ganglion by adapting a model described in Prinz et al . , 2004 .", "The model is composed of three single-compartment neurons , AB/PD , LP , and PD , where the electrically coupled AB and PD neurons are modeled as a single neuron .", "Each of the model neurons contains eight currents , a Na+ current INa , a fast and a slow transient Ca2+ current ICaT and ICaS , a transient K+ current IA , a Ca2+-dependent K+ current IKCa , a delayed rectifier K+ current IKd , a hyperpolarization-activated inward current IH , and a leak current Ileak .", "In addition , the model contains seven synapses .", "As in Prinz et al . , 2004 , these synapses were simulated using a standard model of synaptic dynamics ( Abbott and Marder , 1998 ) .", "The synaptic input current into the neurons is given by Is=gss ( Vpost−Es ) , where gs is the maximal synapse conductance , Vpost the membrane potential of the postsynaptic neuron , and Es the reversal potential of the synapse .", "The evolution of the activation variable s is given byd⁢sd⁢t=s¯⁢ ( Vpre ) -sτswiths¯⁢ ( Vpre ) =11+exp⁡ ( ( Vth-Vpre ) /δ ) ⁢and⁢τs=1-s¯⁢ ( Vpre ) k- .", "Here , Vpre is the membrane potential of the presynaptic neuron , Vth is the half-activation voltage of the synapse , δ sets the slope of the activation curve , and k- is the rate constant for transmitter-receptor dissociation rate .", "As in Prinz et al . , 2004 , two types of synapses were modeled since AB , LP , and PY are glutamatergic neurons whereas PD is cholinergic .", "We set Es=-70 mV and k-=1/40 ms for all glutamatergic synapses and Es=-80 mV and k-=1/100 ms for all cholinergic synapses .", "For both synapse types , we set Vth=-35 mV and δ=5 mV .", "For each set of membrane and synaptic conductances , we numerically simulated the rhythm for 10 s with a step size of 0 . 025 ms . At each time step , each neuron received Gaussian noise with mean zero and standard deviation 0 . 001 mV . ms−0 . 5 .", "We applied SNPE to infer the posterior over 24 membrane parameters and 7 synaptic parameters , that is , 31 parameters in total .", "The seven synaptic parameters were the maximal conductances gs of all synapses in the circuit , each of which was varied uniformly in logarithmic domain from 0 . 01⁢ nS to 1000⁢ nS , with the exception of the synapse from AB to LP , which was varied uniformly in logarithmic domain from 0 . 01⁢ nS to 10000⁢ nS .", "The membrane parameters were the maximal membrane conductances for each of the neurons .", "The membrane conductances were varied over an extended range of previously reported values ( Prinz et al . , 2004 ) , which led us to the uniform prior bounds plow=[0 , 0 , 0 , 0 , 0 , 25 , 0 , 0]⁢ mS cm-2 and phigh=[500 , 7 . 5 , 8 , 60 , 15 , 150 , 0 . 2 , 0 . 01]⁢ mS cm-2 for the maximal membrane conductances of the AB neuron , plow=[0 , 0 , 2 , 10 , 0 , 0 , 0 , 0 . 01]⁢ mS cm-2 and phigh=[200 , 2 . 5 , 12 , 60 , 10 , 125 , 0 . 06 , 0 . 04]⁢ mS cm-2 for the maximal membrane conductances of the LP neuron , and plow=[0 , 0 , 0 , 30 , 0 , 50 , 0 , 0]⁢ mS cm-2 and phigh=[600 , 12 . 5 , 4 , 60 , 5 , 150 , 0 . 06 , 0 . 04]⁢ mS cm-2 for the maximal membrane conductances of the PY neuron .", "The order of the membrane currents was: [Na , CaT , CaS , A , KCa , Kd , H , leak] .", "We used the 15 summary features proposed by Prinz et al . , 2004 , and extended them by three additional features .", "The features proposed by Prinz et al . , 2004 are 15 salient features of the pyloric rhythm , namely: cycle period T", "( s ) , AB/PD burst duration dABb", "( s ) , LP burst duration dLPb", "( s ) , PY burst duration dPYb", "( s ) , gap AB/PD end to LP start Δ⁢tAB-LPes", "( s ) , gap LP end to PY start Δ⁢tLP-PYes", "( s ) , delay AB/PD start to LP start Δ⁢tAB-LPss", "( s ) , delay LP start to PY start Δ⁢tLP-PYss", "( s ) , AB/PD duty cycle dAB , LP duty cycle dLP , PY duty cycle dPY , phase gap AB/PD end to LP start Δ⁢ϕAB-LP , phase gap LP end to PY start Δ⁢ϕLP-PY , LP start phase ϕLP , and PY start phase ϕPY .", "Note that several of these values are only defined if each neuron produces rhythmic bursting behavior .", "In addition , for each of the three neurons , we used one feature that describes the maximal duration of its voltage being above −30 mV .", "We did this as we observed plateaus at around −10 mV during the onset of bursts , and wanted to distinguish such traces from others .", "If the maximal duration was below 5 ms , we set this feature to 5 ms . To extract the summary features from the observed experimental data , we first found spikes by searching for local maxima above a hand-picked voltage threshold , and then extracted the 15 above described features .", "We set the additional 3 features to 5 ms . We used SNPE to infer the posterior distribution over the 18 summary features from experimental data .", "For inference , we used a single round with 18 . 5 million samples , out of which 174 , 000 samples contain bursts in all neurons .", "We therefore used these 174 , 000 samples with well defined summary features for training the inference network ( 𝜽∈ℝ31 , 𝐱∈ℝ18 ) .", "The density estimator was a masked autoregressive flow ( MAF ) ( Papamakarios et al . , 2017 ) with five MADES with [100 , 100 , 100] hidden units each .", "The synaptic conductances were transformed into logarithmic space before training and for the entire analysis .", "Previous approaches for fitting the STG circuit ( Prinz et al . , 2004 ) first fit individual neuron features and reduce the number of possible neuron models ( Prinz et al . , 2003 ) , and then fit the whole circuit model .", "While powerful , this approach both requires the availability of single-neuron data , and cannot give access to potential compensation mechanisms between single-neuron and synaptic parameters .", "Unlike Prinz et al . , 2004 , we apply SNPE to directly identify the full 31 dimensional parameter space without requiring experimental measurements of each individual neuron in the circuit .", "Despite the high-dimensional parameter space , SNPE can identify the posterior distribution using 18 million samples , whereas a direct application of a full-grid method would require 4 . 65⋅1021 samples to fill the 31 dimensional parameter space on a grid with five values per dimension .", "In order to find directions of robust network output , we searched for a path of high posterior probability .", "First , as in Prinz et al . , 2004 , we aimed to find two similar model outputs with disparate parameters .", "To do so , we sampled from the posterior and searched for two parameter sets whose summary features were within 0 . 1 standard deviations of all 174 , 000 samples from the observed experimental data , but that had strongly disparate parameters from each other .", "In the following , we denote the obtained parameter sets by 𝜽s and 𝜽g .", "Second , in order to identify whether network output can be maintained along a continuous path between these two samples , we searched for a connection in parameter space lying in regions of high posterior probability .", "To do so , we considered the connection between the samples as a path and minimized the following path integral: ( 2 ) ℒ ( γ ) =∫01−log⁡ ( pθ|x ( γ", "( s ) |xo ) ) ‖γ˙", "( s ) ‖ds .", "To minimize this term , we parameterized the path γ⁢", "( s ) using sinusoidal basis-functions with coefficients αn , k:γ", "( s ) =[∑k=1Kα1 , k⋅sin⁡ ( πks ) ⋮∑k=1KαN , k⋅sin⁡ ( πks ) ]+[∑k=K+12Kα1 , k⋅sin2⁡ ( πks ) ⋮∑k=K+12KαN , k⋅sin2⁡ ( πks ) ]+ ( 1−s ) ⋅θs+sθg These basis functions are defined such that , for any coefficients αn , k , the start and end points of the path are exactly the two parameter sets defined above:γ⁢ ( 0 ) =𝜽s γ⁢ ( 1 ) =𝜽g With this formulation , we have framed the problem of finding the path as an unconstrained optimization problem over the parameters αn , k .", "We can therefore minimize the path integral ℒ using gradient descent over αn , k .", "For numerical simulations , we approximated the integral in Equation 2 as a sum over 80 points along the path and use two basis functions for each of the 31 dimensions , that is , K=2 .", "In order to demonstrate the sensitivity of the pyloric network , we aimed to find a path along which the circuit output quickly breaks down .", "For this , we picked a starting point along the high-probability path and then minimized the posterior probability .", "In addition , we enforced that the orthogonal path lies within an orthogonal disk to the high-probability path , leading to the following constrained optimization problem:min𝜽⁡log⁡ ( p⁢ ( 𝜽|𝐱 ) ) s . t . nT⁢Δ⁢𝜽=0where n is the tangent vector along the path of high probability .", "This optimization problem can be solved using the gradient projection method ( Rosen , 1960 ) :Δ⁢𝜽=-P⁢ ( ∇⁡log⁡ ( p⁢ ( 𝜽|𝐱 ) ) ) ( ∇⁡log⁡ ( p⁢ ( 𝜽|𝐱 ) ) ) T⁢P⁢ ( ∇⁡log⁡ ( p⁢ ( 𝜽|𝐱 ) ) ) with projection matrix P=𝟙-1nT⁢n⁢n⁢nT and 𝟙 indicating the identity matrix .", "Each gradient update is a step along the orthogonal path .", "We let the optimization run until the distance along the path is 1/27 of the distance along the high-probability path .", "In order to investigate compensation mechanisms in the STG , we compared marginal and conditional correlations .", "For the marginal correlation matrix in Figure 6b , we calculated the Pearson correlation coefficient based on 1 . 26 million samples from the posterior distribution p⁢ ( 𝜽|𝐱 ) .", "To find the two-dimensional conditional distribution for any pair of parameters , we fixed all other parameters to values taken from an arbitrary posterior sample , and varied the remaining two on an evenly spaced grid with 50 points along each dimension , covering the entire prior space .", "We evaluated the posterior distribution at every value on this grid .", "We then calculated the conditional correlation as the Pearson correlation coefficient over this distribution .", "For the 1-dimensional conditional distribution , we varied only one parameter and kept all others fixed .", "Lastly , in Figure 6d , we sampled 500 parameter sets from the posterior , computed the respective conditional posteriors and conditional correlation matrices , and took the average over the conditional correlation matrices ." ] ]
[ "Mechanistic modeling in neuroscience aims to explain observed phenomena in terms of underlying causes .", "However , determining which model parameters agree with complex and stochastic neural data presents a significant challenge .", "We address this challenge with a machine learning tool which uses deep neural density estimators—trained using model simulations—to carry out Bayesian inference and retrieve the full space of parameters compatible with raw data or selected data features .", "Our method is scalable in parameters and data features and can rapidly analyze new data after initial training .", "We demonstrate the power and flexibility of our approach on receptive fields , ion channels , and Hodgkin–Huxley models .", "We also characterize the space of circuit configurations giving rise to rhythmic activity in the crustacean stomatogastric ganglion , and use these results to derive hypotheses for underlying compensation mechanisms .", "Our approach will help close the gap between data-driven and theory-driven models of neural dynamics ." ]
[ "Computational neuroscientists use mathematical models built on observational data to investigate what’s happening in the brain .", "Models can simulate brain activity from the behavior of a single neuron right through to the patterns of collective activity in whole neural networks .", "Collecting the experimental data is the first step , then the challenge becomes deciding which computer models best represent the data and can explain the underlying causes of how the brain behaves .", "Researchers usually find the right model for their data through trial and error .", "This involves tweaking a model’s parameters until the model can reproduce the data of interest .", "But this process is laborious and not systematic .", "Moreover , with the ever-increasing complexity of both data and computer models in neuroscience , the old-school approach of building models is starting to show its limitations .", "Now , Gonçalves , Lueckmann , Deistler et al . have designed an algorithm that makes it easier for researchers to fit mathematical models to experimental data .", "First , the algorithm trains an artificial neural network to predict which models are compatible with simulated data .", "After initial training , the method can rapidly be applied to either raw experimental data or selected data features .", "The algorithm then returns the models that generate the best match .", "This newly developed machine learning tool was able to automatically identify models which can replicate the observed data from a diverse set of neuroscience problems .", "Importantly , further experiments showed that this new approach can be scaled up to complex mechanisms , such as how a neural network in crabs maintains its rhythm of activity .", "This tool could be applied to a wide range of computational investigations in neuroscience and other fields of biology , which may help bridge the gap between ‘data-driven’ and ‘theory-driven’ approaches ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "medicine", "neuroscience" ]
Reporting and misreporting of sex differences in the biological sciences
elife-70817-v1
[ [ "Historically , biomedical research has not considered sex as a biological variable ( SABV ) .", "Including only one sex in preclinical studies—or not reporting sex at all—is a widespread issue ( Sugimoto et al . , 2019 ) .", "In a cross-disciplinary , quantitative assessment of the 2009 biomedical literature , Beery and Zucker , 2011 , found a concerning bias toward the use of males only .", "As awareness of this issue increased , in 2016 the National Institutes of Health ( NIH ) implemented a policy requiring consideration of SABV in the design , analysis , and reporting of all NIH-funded preclinical research ( NIH , 2015; Clayton , 2018 ) .", "By addressing the long-standing over-representation of male non-human animals and cells , the policy was intended not only to ameliorate health inequities but to improve rigor and reproducibility in biomedical research ( Clayton and Collins , 2014 ) .", "Since 2016 , NIH has made available a number of resources , including training modules , administrative funding supplements , and a center program focused on sex differences ( Arnegard et al . , 2020 ) .", "These efforts have resulted in the discovery of new sex differences across a wide spectrum of research fields ( Arnegard et al . , 2020 ) .", "Although the NIH policy does not explicitly require that males and females be compared directly with each other , the fact that more NIH-funded researchers must now study both sexes should lead to an increase in the frequency of such comparisons ( Maney , 2016 ) .", "For example , there should be more testing for sex-specific responses to experimental treatments .", "However , in a follow-up to Beery and Zucker , 2011 , study , Woitowich et al . , 2020 , showed evidence to the contrary .", "Their analysis revealed that between 2011 and 2019 , although the proportion of articles that included both sexes significantly increased ( see also Will et al . , 2017 ) , the proportion that treated sex as a variable did not .", "This finding contrasts sharply with expectations , given not only the NIH mandate but also numerous calls over the past decade to disaggregate all preclinical data by sex and to test for sex differences ( e . g . , Becker et al . , 2016; Potluri et al . , 2017; Shansky and Murphy , 2021; Tannenbaum et al . , 2019; Woitowich and Woodruff , 2019 ) .", "One potential barrier to SABV implementation is a lack of relevant resources; for example , not all researchers have received training in experimental design and data analysis that would allow them to test for sex differences using appropriate statistical approaches .", "This barrier is quite important not only because it prevents rigorous consideration of sex in the first place , but also because any less-than-rigorous test for sex differences creates risk for misinterpretation of results and dissemination of misinformation to other scientists and to the public ( Maney , 2016 ) .", "In other words , simply calling for the sexes to be compared is not enough if researchers are not trained to do so; if SABV is implemented haphazardly , it has the potential to decrease , rather than increase , rigor and reproducibility .", "In this study , our goal was to analyze recently published articles to determine how often sex differences are being reported and what statistical evidence is most often used to support findings of difference .", "To conduct this assessment , we leveraged the collection of articles originally curated by Woitowich et al . , 2020 , for their analysis of the extent to which SABV is being implemented .", "Their original list , which was itself generated using criteria developed by Beery and Zucker , 2011 , included 720 articles published in 2019 in 34 scholarly journals within nine biological disciplines .", "Of those , Woitowich et al . identified 151 articles that included females and males and that analyzed data disaggregated by sex or with sex as fixed factor or covariate .", "Working with that list of 151 articles , we asked the following questions for each: First , was a sex difference reported ?", "If so , what statistical approaches were used to support the claim ?", "We focused in particular on studies with factorial designs in which the authors reported that the effect of one factor , for example treatment , depended on sex .", "Next , we asked whether data from males and females were kept separate throughout the article , and if they were pooled , whether the authors tested for a sex difference before pooling .", "Finally , we noted whether the authors used the term ‘sex’ or ‘gender’ , particularly in the context of preclinical ( non-human animal ) studies ." ], [ "Results pertaining to Question 1 are shown in Figure 1A .", "Comparing the sexes , either statistically or by assertion , was common , occurring in 80% of the articles .", "A positive finding of a sex difference was reported in 83 articles , or 57% .", "Of the articles reporting a sex difference , 41 ( 49% of the 83 articles ) mentioned that result in the title or the abstract .", "Thus , in our sample of articles in which data were reported by sex , a sex difference was reported in more than half of the articles and in half of those , the difference was treated as a major finding by highlighting it in the title or abstract .", "In 44% of articles , a sex difference was neither stated nor implied .", "These results are broken down by discipline in Figure 1B .", "The sexes were most commonly compared in the field of Endocrinology ( 93% ) and least often in the field of Neuroscience ( 33% ) .", "In the field of Reproduction , the sexes were compared 89% of the time and in 100% of those cases , a sex difference was mentioned in the title or abstract .", "Sex differences were least likely to be emphasized in the title or abstract in the fields of General Biology and Neuroscience ( 11% each ) .", "Although a sex difference was claimed in a majority of articles ( 57% ) , not all of these differences were supported with statistical evidence .", "In more than a quarter of the articles reporting a sex difference , or 24/83 articles , the sexes were never actually compared statistically .", "In these cases , the authors claimed that the sexes responded differentially to a treatment when the effect of treatment was not statistically compared across sex .", "This issue is explored in more detail under Question 2 , below .", "Finally , we noted at least five articles in which the authors claimed that there was no sex difference , but did not appear to have tested statistically for one .", "For each article , we asked whether it contained a study with a factorial design in which sex was one of the factors .", "This design is common when researchers are interested in testing whether the sexes respond differently to a manipulation such as a drug treatment ( Figure 2A ) .", "Below , we use the term ‘treatment’ to refer to any non-sex factor in a factorial design .", "Such factors were not limited to treatment , however; they also included variables such as genotype , season , age , exposure to stimuli , etc .", "Hypothetical results of a study with such a design are shown in Figure 2B .", "In order to draw a conclusion about whether responses to treatment differed between females and males , the effect of the treatment must be compared across sex .", "Although there are several ways of making such a comparison ( see Cumming , 2012; Gelman and Stern , 2006 ) , it is typically done by testing for an interaction between sex and treatment .", "If the interaction is significant , then a claim can be made that the sexes responded differently to the treatment .", "Comparing the treated and control groups within each sex , in other words disaggregating the data by sex and testing for effects of treatment separately in females and males , does not test whether the sexes responded differently; that is , it does not test whether the magnitude of the response differs between females and males ( Gelman and Stern , 2006; Makin and Orban de Xivry , 2019; Maney , 2016; Nieuwenhuis et al . , 2011; Radke et al . , 2021 ) .", "The results pertaining to Question 2 are shown in Figure 2C-F .", "Out of the 147 articles we analyzed , 92 ( 63% ) contained at least one study with a factorial design in which sex was a factor ( Figure 2C ) .", "Regardless of whether a sex difference was claimed , we found that the authors explicitly tested for interactions between sex and other factors in only 27 of the 92 articles ( 29% ) .", "That is , authors tested statistically for a sex difference in the responses to other factor ( s ) less than one-third of the time .", "Testing for interactions with sex varied by discipline ( Figure 2D ) .", "Authors were most likely to test for and report the results of interactions in the field of Behavioral Physiology ( 54% of relevant articles ) and least likely in the fields of Physiology ( 0% ) and Reproduction ( 0% ) .", "Of the studies with a factorial design , 58% reported that the sexes responded differently to one or more other factors .", "The language used to state these conclusions often included the phrase ‘sex difference’ but could also include ‘sex-specific effect’ or that a treatment had an effect ‘in males but not females’ or vice versa .", "Of the 53 articles containing such conclusions , the authors presented statistics showing a significant interaction , in other words appropriate evidence that females and males responded differently , in only 16 ( 30%; Figure 2E , blue color in first column ) .", "In an additional article , the authors presented statistical evidence that the interaction was non-significant , yet claimed a sex-specific effect nonetheless .", "In five other articles , the authors mentioned testing for interactions but presented no results or statistics ( e . g . , p values ) for those interactions .", "In the remainder of articles containing claims of sex-specific effects , the authors took one of two approaches; neither approach included testing for interactions .", "Instead , authors proceeded to what would normally be the post hoc tests conducted after finding a significant interaction .", "In 24 articles ( 45% of articles with claims of sex-specific effects ) , authors reported the effect of treatment within each sex and , reaching different conclusions for each sex ( e . g . , finding a p value below 0 . 05 in one sex but not the other ) , inappropriately argued that the response to treatment differed between females and males ( see Figure 2B ) .", "In seven other articles claiming a sex-specific effect ( 13% ) , the sexes were compared within treatment; for example , authors compared the treated males with the treated females , not considering the control animals .", "Neither approach tests whether the treatment had different effects in females and males .", "Thus , a substantial majority of articles containing claims of sex-specific effects ( 70% ) did not present statistical evidence to support those claims ( Figure 2E , red color in first column ) ; further , in the majority of articles without such evidence ( 24/37 ) , the sexes were never compared statistically at all .", "The omission of tests for interactions was related to whether researchers were claiming sex differences or not .", "Among the articles that were missing tests for interactions and yet contained conclusions about the presence or absence of sex-specific effects ( 41 articles ) , those claims were in favor of sex differences 88% of the time , compared with only 12% claiming that the responses in females and males were similar .", "Of all of the articles claiming similar responses to treatment , authors tested for interactions in the majority of cases ( 67%; Figure 2E blue color in second column ) .", "The prevalence of reporting sex-specific effects is broken down by discipline in Figure 2F .", "The field with the lowest percentage of sex-specific effects was Behavior ( 18% ) , and that field also had the highest rate of backing up such claims with statistical evidence ( 71% ) .", "The field most likely to contain claims of sex-specific effects was Reproduction ( 67% ) , but this field was among three for which such claims were never backed up with statistical evidence ( 0% for Reproduction , Physiology , or Pharmacology ) .", "In this study we included only articles in which data were reported by sex as previously determined by Woitowich et al . , 2020 .", "Thus , any articles in which the sexes were pooled for all analyses were not included here .", "We assigned each of the 147 articles to one of three categories , as follows ( Figure 3A ) .", "In 31 ( 21% ) of the articles , data from males and females were analyzed separately throughout .", "In 62 ( 42% ) of the articles , males and females were analyzed in the same statistical models , but in those cases sex was included as a fixed factor or a covariate .", "In most cases when sex was a covariate , authors reported the results of the effect of sex rather than simply controlling for sex .", "In the remaining 54 ( 37% ) articles , the sexes were pooled for at least some of the analyses .", "Among the articles in which the sexes were pooled , the authors did so without testing for a sex difference almost half of the time ( 48%; Figure 3B ) .", "When authors did test for a sex difference before pooling , they sometimes found a significant difference yet pooled the sexes anyway; this occurred in 17% of the articles that pooled .", "When the sexes were pooled after finding no significant difference ( 35% of the articles that pooled ) , authors presented p values for the sex difference the majority of the time ( 11 out of 19 articles ) .", "Those p values ranged from 0 . 15 to >0 . 999 .", "We noted no effect sizes reported in the context of pooling .", "Across disciplines , pooling was most prevalent in Immunology ( 60% ) and least prevalent in General Biology ( 22% ) .", "Males and females were most likely to be kept separate in General Biology ( 56% ) and most likely to be included in statistical models in the field of Behavior ( 53% ) .", "When females and males were pooled , authors in the field of Immunology were least likely to have tested for a sex difference before pooling ( 33% ) and most likely to do so in Pharmacology ( 80% ) .", "Pooling after finding a significant difference was most common in the field of Reproduction ( 67% of articles that pooled ) .", "To refer to the categorical variable comprising male/female or man/woman ( all were binary ) , the term ‘sex’ was used exclusively in 69% of the articles ( Figure 4 ) .", "‘Gender’ was used exclusively in 9% , and both ‘sex’ and ‘gender’ were used in 19% .", "When both terms were used , they usually seemed to be used interchangeably .", "In 4% of the articles , neither term was used .", "Of the articles in which the term ‘gender’ was used , 20% of the time it referred to non-human animals , such as mice , rats , and pigs .", "In one case , both ‘sex’ and ‘gender’ were used to refer to non-human animals in the title .", "In another case , ‘gender’ was used to refer to human cells .", "The majority of articles on non-human species used ‘sex’ ( 85% ) ." ], [ "Woitowich et al . , 2020 , found that over the past decade , the proportion of biological studies that included both females and males has increased , but the proportion in which sex is treated as a variable has not .", "Here , we have taken a closer look at the studies determined by those authors to have reported data by sex , that is , to have conformed to NIH guidelines on SABV .", "We found that in this subset of studies , authors typically also compared the sexes either statistically or by assertion ( >80% of cases ) .", "Thus , the authors that complied with NIH guidelines to disaggregate data usually went beyond NIH guidelines to explicitly compare the sexes with each other .", "This finding is consistent with a larger analysis of articles in the field of Neuroscience from 2010 to 2014; when authors disaggregated data by sex , they usually proceeded to compare the sexes as well ( Will et al . , 2017 ) .", "It is important to note , however , that both Will et al . , 2017 , and Woitowich et al . , 2020 , found that data were not analyzed by sex in the majority of articles that included both sexes ( see Figure 1—figure supplement 1 ) .", "Thus , our current finding that the sexes were usually compared should be interpreted in the context of the subset of articles following NIH guidelines .", "In the set of articles analyzed here , sex differences were claimed in a majority and were often highlighted in the title or abstract .", "We therefore found little evidence that researchers—at least those who comply with NIH guidelines—are uninterested in sex differences .", "We cannot rule out the possibility , however , that the researchers following NIH guidelines are primarily those that are interested in sex differences .", "Testing whether the sexes respond differently to a treatment requires statistical comparison between the two effects , which is typically done by testing for a sex × treatment interaction .", "In our analysis , however , tests for interactions were done only 29% of the time ( Figure 2C and D ) .", "In the remaining 71% , the most common method for detecting differential effects of treatment was to compare qualitatively the conclusions drawn for each sex; that is , to assert that a p value below 0 . 05 for one sex but not the other ( Figure 2B ) represents a meaningful difference between the effects .", "But null hypothesis significance testing does not allow for such conclusions ( Cumming , 2012 ) .", "This error , and the frequency with which it is made , has been covered in multiple publications; for example Gelman and Stern , 2006 , titled their commentary “The difference between ‘significant’ and ‘not significant’ is not itself statistically significant . ”", "Makin and Orban de Xivry , 2019 , included the error in their ‘Top ten list of common statistical mistakes’ .", "In an analysis of 520 articles in the field of Neuroscience , Nieuwenhuis et al . , 2011 , found that the error was committed in about half of articles containing a factorial design .", "The current analysis showed that , even a decade later , the frequency of this error in the field of Neuroscience has not changed ( Figure 2D ) , at least when sex is one of the factors under consideration .", "The frequency of the error was high in most of the other disciplines as well , particularly Physiology and Reproduction , for which we found that authors never tested for interactions even though doing so was necessary to test their hypotheses about sex .", "Statements such as the following , usually made without statistical evidence , were common: ‘The treatment increased expression of gene X in a sex-dependent manner’; ‘Our results demonstrate that deletion of gene X produces a male-specific increase in the behavior’; ‘Our findings indicate that females are more sensitive to the drug than males’ .", "In some of these cases , the terms ‘sex-specific’ , ‘sex-dependent’ , or ‘sexual dimorphism’ were used in the title of the article despite a lack of statistical evidence supporting the claim .", "In many of these articles , some of which stated that finding a sex difference was the major goal of the study , the sexes were not statistically compared at all .", "Thus , a lack of statistical evidence for sex-specific effects did not prevent authors from asserting such effects .", "Authors failing to test for interactions were far more likely to claim sex-specific effects than not ( 88% vs . 12%; Supplementary file 1c ) ; they were also more likely to do so than were authors that did test for interactions ( 88% vs . 63%; Supplementary file 1c ) .", "Statistical analysis of these data showed that , in fact , sex-specific effects were reported significantly more often when no tests for interactions were reported ( χ2 = 5 . 84; p = 0 . 016 ) .", "Together , these results suggest a bias toward finding sex differences .", "In the absence of evidence , differences were claimed more often than not .", "A bias toward finding sex differences , where there are none , could artificially inflate the importance of sex in the reporting of biological data .", "Given that findings of sex × treatment interactions are rare in the human clinical literature , with false positives outnumbering false negatives ( Wallach et al . , 2016 ) , and given also that sex differences are often reported in the media and used to shape education and health policy ( Maney , 2014 ) , it is especially important to base conclusions from preclinical research on solid statistical evidence .", "The set of articles we analyzed was pre-screened by Woitowich et al . , 2020 , to include only studies in which sex was considered as a variable .", "Nonetheless , even in this sample , data were often pooled across sex for some of the analyses ( Figure 3A ) .", "In a majority of these articles , authors did not test for a sex difference before pooling ( Figure 3B ) .", "Thus , for at least some analyses represented here , the data were not disaggregated by sex , sex was not a factor in those analyses , and we do not know whether there might have been a sex difference .", "Even when authors did test for a sex difference before pooling , the relevant statistics were often not presented .", "Finding and reporting a significant sex difference did not seem to reduce the likelihood that the sexes would be pooled .", "Note that the original sample of 720 articles in the study by Woitowich et al . included 251 articles in which sex was either not specified or the sexes were pooled for all analyses ( Figure 1—figure supplement 1 ) .", "Thus , the issue is more widespread than is represented in the current study .", "Pooling is not consistent with the NIH mandate to disaggregate data by sex and can prevent detection of meaningful differences .", "We note further that effect sizes were generally not reported before pooling; in addition to p values , effect sizes would be valuable for any assessment of whether data from males and females can be pooled without masking a potentially important difference ( Beltz et al . , 2019; Diester et al . , 2019 ) .", "In their article on ‘Ten statistical mistakes… , ’ Makin and Orban de Xivry , 2019 , list another issue that is likely to be relevant to the study of sex differences: comparing multiple dependent variables across sex without correcting for multiple comparisons .", "The omission of such a correction increases the risk of false positives , that is , making a type I error , which would result in over-reporting of significant effects .", "This risk is particularly important for researchers trying to comply with SABV , who may feel compelled to test for sex differences in every measured variable .", "In the current study , we found this issue to be prevalent .", "For example , we noted articles in which researchers measured expression of multiple genes in multiple tissues at multiple time points , resulting in a large number of comparisons across sex .", "In one such study , authors made 90 separate comparisons in the same set of animals and found five significant differences , which is exactly the number one would expect to find by chance .", "Although opinions vary about when corrections are necessary , omitting them when they are clearly needed is likely contributing to over-reporting of sex differences broadly across disciplines .", "We found that a large majority of studies on non-human animals used ‘sex’ to refer to the categorical variable comprising females and males .", "In eight articles , we noted usage of the word ‘gender’ for non-human animals .", "This usage appears to conflict with current recommendations regarding usage of ‘gender’ , that is , gender should refer to socially constructed identities or behaviors rather than biological attributes ( Clayton and Tannenbaum , 2016; Holmes and Monks , 2019; Woitowich and Woodruff , 2019 ) .", "We did not , however , investigate the authors’ intended meaning of either term .", "Although definitions of ‘gender’ vary , the term might be appropriate for non-human animals under certain circumstances , such as when the influence of social interactions is a main point of interest ( Cortes et al . , 2019 ) .", "Operational definitions , even for the term ‘sex’ , are important and , in our experience conducting this study , almost never included in publications .", "As others have done ( e . g . , Duchesne et al . , 2020; Cortes et al . , 2019; Holmes and Monks , 2019; Johnson et al . , 2009 ) , we emphasize the importance of clear operational definitions while recognizing the limitations of binary categories .", "The categorization of each article into a particular discipline was defined exclusively by the journal in which it appeared , in order to be consistent with the original categorizations of Beery and Zucker , 2011 , and Woitowich et al . , 2020 .", "For most disciplines , fewer than a dozen articles were in our starting sample; for Neuroscience and Reproduction , only nine .", "As a result , after we coded the articles , some categories contained few or no articles in a given discipline ( see Supplementary file 1c ) .", "The within-discipline analyses , particularly the pie charts in Figure 3B , should therefore be interpreted with caution .", "Firm conclusions about whether a particular practice is more prevalent in one discipline than another cannot be drawn from the data presented here .", "As is the case for any analysis , qualitative or otherwise , our coding was based on our interpretation of the data presentation and wording in the articles .", "Details of the statistical approach were sometimes left out , leaving the author’s intentions ambiguous .", "Although our approach was as systematic as possible , a small number of articles may have been coded in a way that did not completely capture those intentions .", "We believe our sample size , particularly in the overall analyses across disciplines , was sufficient to reveal the important trends .", "SABV has been hailed as a game-changing policy that is already bringing previously ignored sex-specific factors to light , particularly for females .", "In this study , we have shown that a substantial proportion of claimed sex differences , particularly sex-specific effects of experimental manipulations , are not supported by sufficient statistical evidence .", "Although only a minority of studies that include both sexes actually report data by sex ( Woitowich et al . , 2020 ) , our findings suggest that when data are reported by sex , critical statistical analyses are often missing and the findings likely to be interpreted in misleading ways .", "Note that in most cases , our findings do not indicate that the conclusions were inaccurate; they may have been supported by appropriate statistical analyses .", "Our results emphasize the need for resources and training , particularly those relevant to the study designs and analyses that are commonly used to detect sex differences .", "Such training would benefit not only the researchers doing the work , but also the peer reviewers , journal editors , and program officers who have the power to hold researchers to a higher standard .", "Without better awareness of what can and cannot be concluded from separate analysis of males and females , SABV may have the undesired effect of reducing , rather than enhancing , rigor and reproducibility ." ], [ "Because we were interested in the frequency with which sex differences were found , we first identified articles in which the sexes were explicitly compared .", "We counted as a comparison any of the following: ( 1 ) sex was a fixed factor in a statistical model; ( 2 ) sex was included as a covariate in a statistical model and a p value for the effect of sex was reported; ( 3 ) a p value for a comparison of means between males and females was presented; ( 4 ) the article contained wording suggestive of a comparison , for example , ‘males were larger than females’ .", "We also included articles with wording suggestive of a sex difference in response to a treatment , for example , ‘the treatment affected males but not females’ or ‘the males responded to treatment , whereas the females did not’ , or ‘the treatment had a sex-specific effect’ .", "Similarly , we included here articles with language referring to a non-difference , for example , ‘we detected no sex differences in size’ or ‘the response to treatment was similar in males and females’ .", "Articles in which sex was included as a covariate for the purposes of controlling for sex , rather than comparing the sexes , were not coded as having compared the sexes ( see Beltz et al . , 2019 ) .", "When the sexes were compared but no results of those comparisons , for example , p values , were reported , that omission was noted and the article was coded accordingly .", "Each article in which the sexes were compared was then further coded as either reporting a sex difference or not , and if so , whether a sex difference was mentioned in the title or abstract .", "If mentioned in the title or abstract , the sex difference was coded as a ‘major finding’; otherwise , sex differences mentioned in the body of the paper , figures , or tables were coded as ‘minor’ .", "We looked for studies with a 2 × 2 factorial design ( Figure 2A ) in which sex was one of the factors .", "Sex did not need to be explicitly identified as a fixed factor; we included here all studies comparing across levels of one factor that comprised females and males with each of those levels .", "In some cases that factor was a manipulation , such as a drug treatment or a gene knockout .", "Non-sex factors also included variables such as age , season , presentation of a stimulus , etc .", "For simplicity , we refer to the other factor as ‘treatment’ .", "Any article containing at least one such study was coded as having a factorial design .", "The other articles were coded as containing no comparisons across sex or as containing only group comparisons across sex .", "The latter category included studies with sex as a covariate of interest in a model such as a multiple regression , if the authors were not making any claims about potential interactions between sex and other variables .", "For studies with a factorial design , we further coded the authors’ strategy of data analysis .", "First , we noted whether authors tested for an interaction between sex and treatment; that is , they tested whether the effect of treatment depended on sex .", "We coded as 'yes' one study in which the magnitude of the differences between treated and control groups was explicitly compared across sex .", "For articles containing tests for interactions , we noted the outcome of that test and the interpretation .", "Articles containing no tests for interactions were assigned to one of several sub-categories in the following order ( coded as the first category on this list for which the description was met for any analysis in the article ) : tested for effects of treatment within sex , tested for effects of sex within at least one level of treatment , or tested for main effects of sex only .", "Within each of those categories we further coded the outcome/interpretation , for example , sex difference or no sex difference .", "Any articles containing statements that the sexes responded differently to treatment or that the response was ‘sex-specific’ were coded as reporting a sex-specific effect .", "We also noted when authors reported an absence of such a result .", "Articles not comparing across sex at all , with statistical evidence or by assertion , were coded accordingly .", "We assigned articles to one of three categories: analyzed males and females separately throughout , included sex in the statistical model for at least some analyses ( with the rest analyzed separately ) , or pooled for at least some analyses .", "The second category , included sex in the model , included articles in which AIC or similar statistic was used to choose among models that included sex , although sex may not have been in the model ultimately chosen .", "This category did not distinguish between analyses including sex as a fixed factor vs . a covariate; this distinction is noted where relevant in Supplementary file 1b .", "Any article containing pooled data was coded as pooled , even if some analyses were conducted separately or with sex in the model .", "For articles that pooled , we further noted whether the authors tested for a sex difference before pooling and , if so , whether p values or effect sizes were reported .", "We searched the articles for the terms ‘sex’ and ‘gender’ and noted whether the authors used one or the other , both , or neither .", "Terms such as ‘sex hormones’ or ‘gender role’ , which did not refer to sex/gender variables in the study , were excluded from this assessment .", "For the articles using ‘gender’ , we further noted when the term was used for non-human animals .", "To visualize the data , we used river plots ( Weiner , 2017 ) , stacked bar graphs , and pie charts based on formulae and data presented in Supplementary file 1c ." ] ]
[ "As part of an initiative to improve rigor and reproducibility in biomedical research , the U . S . National Institutes of Health now requires the consideration of sex as a biological variable in preclinical studies .", "This new policy has been interpreted by some as a call to compare males and females with each other .", "Researchers testing for sex differences may not be trained to do so , however , increasing risk for misinterpretation of results .", "Using a list of recently published articles curated by Woitowich et al . ( eLife , 2020; 9:e56344 ) , we examined reports of sex differences and non-differences across nine biological disciplines .", "Sex differences were claimed in the majority of the 147 articles we analyzed; however , statistical evidence supporting those differences was often missing .", "For example , when a sex-specific effect of a manipulation was claimed , authors usually had not tested statistically whether females and males responded differently .", "Thus , sex-specific effects may be over-reported .", "In contrast , we also encountered practices that could mask sex differences , such as pooling the sexes without first testing for a difference .", "Our findings support the need for continuing efforts to train researchers how to test for and report sex differences in order to promote rigor and reproducibility in biomedical research ." ]
[ "Biomedical research has a long history of including only men or male laboratory animals in studies .", "To address this disparity , the United States National Institutes of Health ( NIH ) rolled out a policy in 2016 called Sex as a Biological Variable ( or SABV ) .", "The policy requires researchers funded by the NIH to include males and females in every experiment unless there is a strong justification not to , such as studies of ovarian cancer .", "Since then , the number of research papers including both sexes has continued to grow .", "Although the NIH does not require investigators to compare males and females , many researchers have interpreted the SABV policy as a call to do so .", "This has led to reports of sex differences that would otherwise have been unrecognized or ignored .", "However , researchers may not be trained on how best to test for sex differences in their data , and if the data are not analyzed appropriately this may lead to misleading interpretations .", "Here , Garcia-Sifuentes and Maney have examined the methods of 147 papers published in 2019 that included both males and females .", "They discovered that more than half of these studies had reported sex differences , but these claims were not always backed by statistical evidence .", "Indeed , in a large majority ( more than 70% ) of the papers describing differences in how males and females responded to a treatment , the impact of the treatment was not actually statistically compared between the sexes .", "This suggests that sex-specific effects may be over-reported .", "In contrast , Garcia-Sifuentes and Maney also encountered instances where an effect may have been masked due to data from males and females being pooled together without testing for a difference first .", "These findings reveal how easy it is to draw misleading conclusions from sex-based data .", "Garcia-Sifuentes and Maney hope their work raises awareness of this issue and encourages the development of more training materials for researchers ." ]
2021
[ "Introduction", "Results", "Phosphorylation of USP14 regulates global protein degradation", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system
elife-10510-v2
[ [ "The ubiquitin–proteasome system ( UPS ) , a major degradative mechanism in eukaryotic cells , is involved in the degradation of short-lived proteins as well as misfolded and damaged proteins ( Komander and Rape , 2012 ) .", "The 26S proteasome specifically targets and degrades proteins conjugated to ubiquitin .", "Regulation of protein deubiquitination by deubiquitinating enzymes ( DUBs ) is recognized as an important regulatory step in the UPS .", "Ubiquitin-specific protease-14 ( USP14 ) , a DUB reversibly associated with the proteasome , negatively regulates the activity of proteasomes by trimming ubiquitin chains on proteasome-bound substrates ( Borodovsky et al . , 2001; Koulich et al . , 2008; Lee et al . , 2010 ) .", "Purified recombinant USP14 is largely inactive and can be highly activated when in association with proteasome ( Hu et al . , 2005; Koulich et al . , 2008; Lee et al . , 2010 ) .", "However , a significant fraction of USP14 is present intracellularly in a proteasome-free state ( Koulich et al . , 2008 ) , and it is not clear if and how proteasome-free USP14 might serve a significant physiological function .", "Akt , a serine/threonine-specific protein kinase and an important intracellular signaling transducer for growth factors such as insulin , is involved in regulating cell proliferation , metabolism , transcription , migration , and apoptosis ( Manning and Cantley , 2007 ) .", "The activity of Akt is regulated by PI ( 3 , 4 , 5 ) P3 , a lipid product of the phosphoinositide 3-kinases ( PI3Ks ) .", "The intracellular levels of PI ( 3 , 4 , 5 ) P3 are negatively regulated by phosphatases such as SHIP1/2 and phosphatase and tensin homolog ( PTEN ) .", "The latter , a phosphoinoside phosphatase , is encoded by a tumor suppressor gene that is mutated in human cancers at high frequency ( Cantley and Neel , 1999 ) .", "Akt has been reported to mediate the phosphorylation of many substrates that in turn regulate cell proliferation , metabolism , transcription , migration , and apoptosis .", "However , very little is known about its role in the UPS , and furthermore no mechanistic link between Akt and UPS has been elucidated .", "In this study , we report that USP14 is an Akt substrate and that this phosphorylation activates the DUB activity of USP14 both in vitro and in cells .", "We also demonstrate that phosphorylation of USP14 is critical for Akt to control UPS and consequentially global protein degradation via the UPS .", "Our study reveals a novel mechanistic connection between activated Akt and the UPS through regulating of USP14 phosphorylation , which can impact global proteostasis in PTEN-negative cancer cells ." ], [ "Two forms of USP14 have been determined crystallographically: the inactive free form and an adduct between Ub-aldehyde ( Ubal ) and USP14 , which provides insight into the catalytically active state ( Hu et al . , 2005 ) .", "The key difference between these two structures is in the position of the blocking loops , BL1 and BL2 , which project over the catalytic cleft of USP14 and block the access of the C-terminal residues of ubiquitin in the inactive form ( Figure 1A ) .", "In Ubal-modified USP14 , BL1 and BL2 are rearranged , thus exposing the cleft .", "In particular , Ser432 , located within BL2 , shifts its position over a distance of 3–5 Å between the two states ( Hu et al . , 2005 ) ( Figure 1B ) . 10 . 7554/eLife . 10510 . 003Figure 1 . Structural basis of ubiquitin-specific protease-14 ( USP14 ) activation by phosphorylation of Ser432 . ( A ) Detailed view of blocking loop 2 ( BL2 ) , which occludes the active site of USP14 ( PDB access code 2AYN ) .", "The BL2 loop , which contains Ser432 , is shown in stick model , in the apo form .", "( B ) Combined ribbon representation and stick model showing a comparison of the conformations of the BL2 loop contained in the apo form ( blue , PDB access code 2AYN ) and in the USP14- Ub-aldehyde ( Ubal ) adduct ( orange , PDB access code 2AYO ) .", "In this drawing , the Ser432 and Cys114 residues are shown in stick model , and the bound Ubal ( a ubiquitin derivative in which the C-terminal carboxylate is replaced by an aldehyde ) in the complex is drawn in green .", "( C ) A surface charge potential representation ( contoured at ± 7 kT/eV; blue/red ) of USP14 ( PDB accession 2AYN ) showing that the S432 residue is very close to a highly negatively charged patch mainly formed by the acidic E188 , D199 , and E202 residues .", "When S432 is phosphorylated , the negatively charged phosphate group may induce a repulsive force , thereby relieving inhibition of the catalytic activity of USP14 .", "( D ) USP14 domain organization and sequence alignment of the Akt phosphorylation site within USP14 orthologs from different species .", "Two BLs ( BL1 and BL2 ) covering the USP14 active site are shown .", "The Akt phosphorylation site in USP14 from different species as predicted by Scansite .", "( E ) S432 is the major phosphorylation site in USP14 .", "HEK293T cells were treated as in Figure 1—figure supplement 1B , followed by electrospray ionization mass spectrometry ( ESI-MS ) analysis .", "Spectral counts were determined by ESI-MS .", "( F ) Akt phosphorylates USP14 in vitro .", "Bacterially expressed and purified USP14 was incubated with active Akt in the presence of ATP .", "Reaction products were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) , and phosphorylated species were detected by a phospho-Ser antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 00310 . 7554/eLife . 10510 . 004Figure 1—figure supplement 1 . Akt phosphorylates ubiquitin-specific protease-14 ( USP14 ) .", "( A ) Akt interacts with USP14 .", "HEK293T cells were transfected with indicated plasmids for 24 hr .", "The cell lysates were collected for co-immunoprecipitation and western blotting analysis .", "( B ) Schematic representation of mass spectrometry assay to determine USP14 phosphorylation sites by Akt .", "( C ) Four phosphorylation sites of USP14 were determined by mass spectrometry .", "( D ) The representative MS/MS spectrum of phosphorylated tryptic peptide ‘SSSphosSGHYVSWVK’ of human USP14 protein .", "The peptide sequence ‘SSSphosSGHYVSWVK’ containing phosphorylated S432 was identified by shotgun analysis using mass spectrometry when USP14 was coexpressed with Myr-Akt in HEK293T cells .", "Fragmentation ion of the amide bond of the peptide result in formation of “b” ion and “y” ion series corresponding to the N- and C-terminal fragments respectively .", "Representative ions with phosphorylation and H2O loss were manually labeled in red on the spectrum . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 004 Since Ser432 residue is located very close to a highly negatively charged patch ( Figure 1C ) , we reasoned that when Ser432 residue was phosphorylated , the negatively charged phosphate group might induce a repulsive force , thereby inducing rearrangement of the BL2 loop and removing the inhibitory effect of this loop on the activity of USP14 .", "The amino acid sequences around Ser432 are highly evolutionarily conserved among USP14 orthologs ( Figure 1D ) and Ser432 is predicted to be an Akt substrate by Scansite ( http://scansite3 . mit . edu/#home ) .", "We therefore tested the possibility that USP14 might be a substrate of activated Akt .", "We first examined the interaction between USP14 and Akt using a co-immunoprecipitation assay .", "As shown in Figure 1—figure supplement 1A , when USP14 and Akt were overexpressed in HEK293T cells , their interaction was readily detectable .", "To test whether Akt could phosphorylate USP14 , we overexpressed USP14 and an activated Akt ( Myr-Akt ) in HEK293T cells , and performed a quantitative phosphoproteomic analysis ( Figure 1—figure supplement 1B ) .", "We identified four phosphorylation sites on USP14 when it was expressed alone: Ser143 , Ser230 , Thr235 , and Ser432 ( Figure 1—figure supplement 1C , D ) .", "Notably , the phosphorylation levels of two of the four sites , Ser143 and Ser432 , were increased considerably in cells expressing activated Akt ( Figure 1E ) .", "To examine whether USP14 is a direct substrate for Akt , we conducted an in vitro kinase assay using activated recombinant Akt and purified recombinant USP14 expressed in Escherichia coli .", "We found that co-incubation of USP14 and Akt led to modification of USP14 as detected by a pan phospho-Ser antibody ( Figure 1F ) , suggesting that USP14 is a substrate for Akt .", "To determine if Ser143 and Ser432 were indeed phosphorylated by Akt , we used this pan phospho-Ser antibody as above and found phosphorylation of wild type ( WT ) USP14 , but not of S143A/S432A mutant USP14 , after incubating with activated Akt in a kinase assay ( Figure 2A ) .", "To differentiate the relative importance of Ser143 and Ser432 as phosphorylation sites by Akt , we overexpressed activated Akt ( Myr-Akt ) in HEK293T cells with WT , S143A , S432A , or double S143A/S432A ( AA ) mutants .", "We found that S143A mutant showed partially reduced phosphorylation as compared to that of WT , whereas phosphorylation of the USP14 S432A mutant was significantly decreased and that of AA double mutant was completely eliminated ( Figure 2B ) .", "These results suggested S432 as a major and S143 as a minor phosphorylation site of Akt . 10 . 7554/eLife . 10510 . 005Figure 2 . Ubiquitin-specific protease-14 ( USP14 ) is phosphorylated at Ser432 by activated Akt .", "( A ) In vitro phosphorylation of USP14 at S432 by Akt .", "Bacterially expressed and purified wild type USP14 or AA mutant incubated with active Akt in the presence of ATP .", "Reaction products were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) , and phosphorylation was detected by the phospho-Ser antibody .", "( B ) Akt phosphorylates USP14 at S432 in vivo .", "Western blot analysis of whole cell lysate and immunoprecipitates derived from HEK293T cells transfected with wild type USP14 , USP14 S143A , USP14 S432A , and USP14 S143A/S432A ( AA ) constructs using the phospho-Ser antibody .", "L . E . , long exposure .", "( C ) Immunoprecipitation ( IP ) and IB analysis of HEK293T cells transfected with HA-USP14 and Myr-Akt and preincubated with or without λ-phosphatase as indicated .", "( D ) Inhibition of Akt decreased exogenous USP14 phosphorylation .", "HEK293T cells were transfected with Myc-USP14 for 20 hr then treated with 1 μM MK2206 or deprived of serum for another 4 hr before harvest .", "( E ) In vitro kinase assay to detect Akt phosphorylation of USP14 by phospho-Ser432-specific antibody and phos-tag-containing gels .", "Bacterially expressed and purified wild type USP14 or S432A mutant was incubated with active Akt in the presence of ATP .", "The reaction products were resolved by SDS-PAGE , and USP14 phosphorylation was detected using an antibody that specifically recognizes Ser432 phosphorylation of USP14 or determined by differential migration on phos-tag gels .", "( F ) In vivo detection of endogenous USP14 Ser432 phosphorylation by anti-p-Ser432-specific antibody .", "Western blot analysis of immunoprecipitates derived from H4 cells transfected with or without Myr-Akt plasmids using the anti-p-Ser432-specific antibody .", "( G , H )", "Phosphorylation of endogenous USP14 S432 upon stimulation with insulin-like growth factor ( IGF-1 ) or epidermal growth factor ( EGF ) .", "HEK293T cells were serum-starved and pretreated with Akt inhibitor MK2206 ( 1 μM ) for 30 min before stimulation with IGF-1 ( 100 ng/mL ) for 30 min ( G ) or EGF ( 100 ng/mL ) for 1 hr ( H ) .", "The cell lysates were immunoprecipitated with USP14 antibody and western-blotted with anti-p-S432 antibody .", "( I ) Phosphorylation of endogenous USP14 S432 in Pten knockout cells with high activity of Akt .", "Lysates from mouse embryonic fibroblasts ( MEFs ) with indicated genotypes were immunoprecipitated with USP14 antibody and then Western blotted with p-S432 antibody .", "The differential migration of phospho-USP14 on phos-tag-containing gels was determined as shown in the bottom panel . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 00510 . 7554/eLife . 10510 . 006Figure 2—figure supplement 1 . Ubiquitin-specific protease-14 ( USP14 ) is phosphorylated at Ser432 by Akt .", "( A ) Akt phosphorylates USP14 at S432 in vivo .", "Western blotting analysis of whole cell lysate and immunoprecipitates derived from HEK293T cells transfected with wild type USP14 , USP14 S143A , USP14 S432A , and USP14 S143A/S432A ( AA ) constructs using an Akt phosphorylation-consensus motif ( R××S/T ) antibody .", "( B , C )", "Inhibition of Akt decreases USP14 S432 phosphorylation levels .", "H4 cells were treated with different concentration of Akt inhibitors MK2206 ( B ) or AZD5363 ( C ) as indicated for 4 hr .", "The cell lysates were collected for immunoprecipitation and western blotting analysis .", "( D ) Inhibition of phosphoinositide 3-kinases ( PI3K ) decreases USP14 S432 phosphorylation levels .", "H4 cells were treated with different concentration of PI3K inhibitors GDC0941 or Wortmannin as indicated for 4 hr .", "The cell lysates were collected for immunoprecipitation and western blotting analysis .", "( E ) ERK1/2 inhibition has no effect on USP14 S432 phosphorylation .", "H4 cells were treated with different concentration of ERK1/2 inhibitor U0126 as indicated for 4 hr .", "The cell lysates were collected for immunoprecipitation and western blotting analysis .", "( F ) Pten–/–mouse embryonic fibroblast ( MEF ) cells were treated with 1 μM Akt inhibitors for 4 hr , then cell lysates were collected for immunoprecipitation and western blotting analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 006 The phosphorylation of USP14 by Akt was further confirmed using an Akt phosphorylation-consensus motif ( R××S/T ) antibody ( Figure 2—figure supplement 1A ) .", "The reactivity of USP14 with pan phospho-Ser antibody was eliminated after incubation with lambda phosphatase ( Figure 2C ) .", "Notably , the phosphorylation levels of USP14 were decreased in cells when treated with MK2206 , an inhibitor of Akt ( Figure 2D ) , or when serum deprived , a condition known to inactivate endogenous Akt ( Zhang et al . , 2015 ) ( Figure 2D ) .", "To further verify the phosphorylation of USP14 S432 by Akt , we developed a phospho-Ser432-specific antibody .", "Phosphorylation of S432 can be detected after incubation of WT , but not S432A mutant USP14 , with recombinant activated Akt in a kinase reaction ( Figure 2E ) .", "This was further confirmed by using phos-tag electrophoresis which can specifically retard the migration of phosphorylated protein species ( Kinoshita et al . , 2009 ) ( Figure 2E ) .", "Expression of Myr-Akt also led to S432 phosphorylation of endogenous USP14 ( Figure 2F ) .", "Treatment with either MK2206 or AZD5363 , two structurally unrelated Akt inhibitors , led to decrease of USP14 S432 phosphorylation levels ( Figure 2—figure supplement 1B , C ) .", "Moreover , treatment with PI3K inhibitors , either Wortmannin or GDC0941 , but not ERK1/2 inhibitor U0126 , also significantly decreased the phosphorylation levels of USP14 S432 ( Figure 2—figure supplement 1D , E ) .", "In addition , we tested growth factors such as insulin-like growth factor ( IGF-1 ) or epidermal growth factor ( EGF ) , both of which are known to promote activation of Akt .", "We found that the treatment of IGF-1 or EGF resulted in phosphorylation of USP14 S432 , which was blocked in cells pretreated with MK2206 ( Figure 2G , H ) .", "Finally , USP14 S432 is dramatically more phosphorylated in PTEN knockout mouse embryonic fibroblasts ( MEFs ) , which carry high levels of Akt activity , than that of WT MEFs as determined by western blotting using the phospho-USP14 ( S432 ) antibody and phos-tag electrophoresis ( Figure 2I ) , and the phosphorylation of USP14 S432 was blocked by Akt inhibitors ( Figure 2—figure supplement 1F ) .", "From these results , we conclude that Ser432 of USP14 is a major phosphorylation site by Akt .", "Because bacterially expressed and purified USP14 protein exhibits very low catalytic activity ( Lee et al . , 2010 ) , we tested whether Akt-mediated phosphorylation might activate the DUB activity of USP14 .", "We compared the activity of recombinant USP14 in a Ub-AMC ( ubiquitin-7-amido-4-methylcoumarin , a fluorogenic substrate ) hydrolysis assay in the presence or absence of Akt .", "Bacterially expressed and purified USP14 ( Figure 3—figure supplement 1 ) showed trace hydrolyzing activity towards Ub-AMC as reported ( Lee et al . , 2010 ) , while USP14 incubated with Akt showed high activity ( Figure 3A ) .", "To validate Akt-mediated activation of USP14 in cells , we coexpressed USP14 and Myr-Akt in HEK293T cells .", "USP14 immunoprecipitated from cells coexpressing activated Akt showed higher activity in Ub-AMC assay than that expressed alone ( Figure 3B ) .", "On the other hand , USP14 isolated from HEK293T cells incubated with Akt inhibitor MK2206 showed reduced activity in Ub-AMC assay ( Figure 3C ) .", "Moreover , USP14 isolated from HEK293T cells stimulated with IGF-1 showed higher activity , which was suppressed when cells were pretreated with MK2206 ( Figure 3D ) .", "To determine the specific contribution of Ser432 , we compared the activity of USP14 S432A mutant protein in Ub-AMC assay with that of WT in the presence of Akt , and found that the stimulating effect of Akt on the hydrolyzing activity of USP14 was largely blocked by S432A mutation ( Figure 3E ) , but not by S143A mutation ( Figure 3—figure supplement 2B ) . 10 . 7554/eLife . 10510 . 007Figure 3 . Phosphorylation of ubiquitin-specific protease-14 ( USP14 ) by Akt activates USP14 DUB activity .", "( A ) Akt activates USP14 DUB activity in vitro .", "USP14 protein ( 1 μg ) was incubated with or without active Akt ( 1 μg ) in kinase assay buffer in a total volume of 50 μL for 1 hr at 30°C , then the reaction mixtures were subjected to Ub-AMC assay .", "RFU , relative fluorescence units .", "( B , C )", "Akt activates USP14 in cells .", "USP14 was immunoprecipitated from HEK293T cells coexpressed with activated Akt ( B ) or treated with 10 μM MK2206 for 4 hr ( C ) and then eluted with HA-peptide following Ub-AMC hydrolysis assay .", "( D ) Activation of USP14 by stimulating cells with IGF-1 .", "HEK293T cells were serum-starved and pretreated with or without Akt inhibitor MK2206 ( 1 μM ) for 30 min before stimulation with IGF-1 ( 100 ng/mL ) for 30 min .", "USP14 was then immunoprecipitated and eluted with HA-peptide .", "The activity of USP14 was determined using Ub-AMC hydrolysis assay .", "( E ) USP14 activation by Akt is blocked by S432A mutation .", "Ub-AMC hydrolysis assay of wildde type USP14 or S432A mutant in the presence or absence of active Akt .", "( F ) Ub-AMC hydrolysis assay of bacterially expressed and purified wild type USP14 or S432E mutant .", "( G ) Lineweaver–Burk analysis of USP14 S432E , obtained by measuring the initial rates at varying Ub-AMC concentrations ( see Figure 3—figure supplement 2E for reference ) .", "( H ) The activity of phospho-mimetic USP14 mutant can be further stimulated by the presence of proteasome .", "Ub-AMC hydrolysis assay of wild type USP14 or S432E mutant in the presence or absence of Ub-VS-treated human proteasome ( VS-proteasome ( see Lee et al . , 2010 ) ; 1 nM ) .", "Ptsm , 26S proteasome . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 00710 . 7554/eLife . 10510 . 008Figure 3—figure supplement 1 . Purification of ubiquitin-specific protease-14 ( USP14 ) recombinant protein .", "( A , B )", "A representative curve of USP14 recombinant protein purification before ( A ) and after ( B ) cleavage by 3C protease on size-exclusion chromatography .", "Bacterially expressed USP14 forms oligomer and dimer , only monomer was used for further experiments .", "Trx-tag was removed to get tag-free USP14 protein for in vitro kinase assay or Ub-AMC and ubiquitin cleavage assay .", "( C ) One dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) and Coomassie Brilliant Blue ( CBB ) staining of purified USP14 protein before and after cleavage by 3C protease . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 00810 . 7554/eLife . 10510 . 009Figure 3—figure supplement 2 . Phosphorylation of USP14 by Akt activates USP14 DUB activity .", "( A ) Validation of Ub-AMC assay on immunoprecipitated USP14 from HEK293T cells .", "Wild type or inactive mutant USP14 was immunoprecipitated from transfected HEK293T cells and then eluted with HA-peptide following Ub-AMC hydrolysis assay .", "( B–D )", "Ser143 phosphorylation has limited effect on USP14 activity .", "S143A mutant had similar levels of hydrolyzing activity as that of WT USP14 in the presence of active Akt ( B ) .", "Double E mutant ( S143E/S432E ) showed similar levels of hydrolyzing activity as that of S432E single mutant ( C ) .", "S143E mutant has minor effect on USP14 activity compared with S432E mutant ( D ) .", "( E ) Linear kinetics ( R2 > 0 . 99 ) of Ub-AMC hydrolysis .", "( F ) Distribution of total and phosphorylated USP14 specific in glycerol gradient centrifugation .", "Soluble lysates of Pten–/– mouse embryonic fibroblast ( MEF ) cells , prepared as described in Materials and methods , were subjected to glycerol density gradient centrifugation .", "Gradient fractions were collected and subjected to western blotting with the indicated antibodies .", "Anti-RPN11 was used as a control for proteasome . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 009 To further characterize the effect of Ser432 phosphorylation , we expressed and purified recombinant S432E USP14 protein , which mimics the phosphorylation state of USP14 , from E . coli ( Figure 3—figure supplement 1 ) and analyzed its activity by Ub-AMC assay .", "Interestingly , we found that USP14 S432E mutant protein alone showed high levels of Ub-AMC hydrolyzing activity ( Figure 3F ) .", "Consistent with S432 as the major phosphorylation site by Akt , double E mutant ( S143E/S432E ) showed almost the same levels of hydrolyzing activity as that of S432E single mutant and S143E mutation had no significant impact on the activity of USP14 ( Figure 3—figure supplement 2C , D ) .", "To determine its enzyme kinetics , we incubated USP14 S432E mutant protein with increasing amounts of Ub-AMC ( Figure 3—figure supplement 2E ) and determined the Km value ( Km = 26 μM ) from the slope of a Lineweaver–Burk plot ( Figure 3G ) .", "We characterized the distributions of p-S432 USP14 and total USP14 with that of proteasome in Pten–/– MEFs using glycerol gradient centrifugation ( Koulich et al . , 2008 ) .", "We found that the majority of p-S432 USP14 was distributed in the fractions with lower molecular weight proteins and distinguishable from the fractions where larger protein complexes , such as proteasomes , were localized .", "On the other hand , unphosphorylated USP14 was found in the fractions where larger molecular weight complexes , such as proteasome , are known to be localized ( Figure 3—figure supplement 2F ) .", "Thus , S432 phosphorylated and unphosphorylated USP14 might be distributed differently in the cells .", "We next determined whether phospho-mimetic mutant of USP14 could be further activated by interacting with proteasome .", "Interestingly , we found that the Ub-AMC hydrolytic activity of S432E mutant could be further activated when incubated with proteasome in vitro ( Figure 3H ) .", "Taken together , these results suggest that S432 phosphorylation and interaction with proteasome may be two different regulatory mechanisms for USP14 .", "To assess the impact of USP14 phosphorylation on its selectivity towards different types of ubiquitin linkages , we incubated USP14 WT and S432E mutant protein with diubiquitin species of K48 , K63 , and linear linkages .", "Conversion to monomeric Ub was monitored via sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) followed by western blotting .", "We observed significantly increased hydrolytic activity of S432E mutant , as compared to that of WT , towards both Lys48 and Lys63 diubiquitin , while linear diubiquitin was not readily cleaved by WT or mutant USP14 ( Figure 4A , B and Figure 4—figure supplement 1A ) .", "Similarly , immunoprecipitated USP14 from cells showed significant activity toward both Lys48 and Lys63 diubiquitin , but not linear diubiquitin ( Figure 4—figure supplement 1B , C ) .", "In contrast , S432A mutant immunoprecipitated from cells showed lower activity towards both Lys48 and Lys63 diubiquitin than that of WT ( Figure 4C ) . 10 . 7554/eLife . 10510 . 010Figure 4 . Phosphorylation stimulates ubiquitin-specific protease-14 ( USP14 ) activity towards both K48 and K63 ubiquitination .", "( A ) Dimeric Ub cleavage assay .", "USP14 S432E cleavage of Lys 48 and Lys 63 Ub chain linkages was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) .", "( B ) Cleavage of Lys 48 , Lys 63 and linear dimeric Ub chain types in the presence of USP14 S432E was measured over time and analyzed using SDS-PAGE .", "Quantification of the amount of dimer remaining from the data was shown below .", "( C ) Dimeric Ub cleavage analysis of immunoprecipitates derived from HEK293T cells transfected with wild type HA-USP14 or S432A mutant plasmids . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 01010 . 7554/eLife . 10510 . 011Figure 4—figure supplement 1 . Phosphorylation of ubiquitin-specific protease-14 ( USP14 ) promotes both K48 and K63 deubiquitination activity .", "( A ) USP14 S432E does not cleave linear di-Ub .", "USP14 S432E cleavage of linear dimeric Ub chain types and analyzed using SDS-PAGE .", "Cleavage of Lys48 dimeric Ub chain types was used as a positive control .", "( B , C )", "USP14 immunoprecipitated from transfected HEK293T cells and eluted with HA-peptide has high deubiquitination activity towards both K48 and K63 di-Ub but not linear di-Ub . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 011 Since USP14 is a negative regulator of the UPS ( Koulich et al . , 2008; Lee et al . , 2010 , 2011 ) and we found USP14 can be phosphorylated and activated by Akt , we reasoned that Akt-mediated activation of USP14 might lead to inhibition of the UPS and generally enhance the stability of many proteins .", "To this end , we generated a stable cell line expressing GFP-CL1 ( also known as GFPu ) , an engineered ubiquitin-dependent proteasome substrate widely used as a reporter for UPS activity ( Bence et al . , 2001; Kelly et al . , 2007; Li et al . , 2013; Liu et al . , 2014 ) ( Figure 5—figure supplement 1A–C ) .", "Treatment of cells with Akt inhibitors or serum deprivation or PI3K inhibitor , all of which can block Akt activity ( Zhang et al . , 2015 ) , led to reduced level of GFP-CL1 as detected by both western blotting and fluorescence microscopy ( Figure 5A–C and Figure 5—figure supplement 1D ) .", "Conversely , the expression of activated Akt ( Myr-Akt ) led to increased levels of GFP-CL1 protein .", "Treatment of WT H4 cells with IGF-1 or EGF also led to increased levels of GFP-CL1 protein ( Figure 5D–G and Figure 5—figure supplement 1E ) .", "In contrast , in USP14 knockout H4 cells ( generated using CRISPR/Cas9 technology , Figure 5—figure supplement 2A–D ) , the expression of Myr-Akt did not affect the levels of GFP-CL1 ( Figure 5H ) .", "From these results , we conclude that Akt negatively regulates the UPS in an USP14-dependent manner . 10 . 7554/eLife . 10510 . 012Figure 5 . Akt regulates ubiquitin–proteasome system ( UPS ) function through phosphorylation of ubiquitin-specific protease-14 ( USP14 ) .", "( A ) Inhibition of Akt promotes UPS function .", "H4-GFP-CL1 cells were treated with different concentrations of MK2206 as indicated for 4 hr .", "The cells were then harvested and subjected to western blotting analysis using indicated antibodies .", "( B ) H4-GFP-CL1 cells were treated as in ( A ) and Figure 5—figure supplement 1D .", "Images of the cells were collected using an ArrayScan HCS 4 . 0 Reader .", "The average GFP intensity in 2 , 000 cells from each indicated sample was determined .", "Data are displayed as mean ± SD of the GFP intensity per cell .", "**p<0 . 01 , ***p<0 . 001 ( C ) Inhibition of Akt or phosphoinositide 3-kinase ( PI3K ) promotes UPS function .", "H4-GFP-CL1 cells were treated with Akt inhibitor AZD5363 ( 1 μM ) or PI3K inhibitor GDC0941 ( 1 μM ) as indicated for 4 hr .", "The cells were then harvested and subjected to western blotting analysis using indicated antibodies .", "p-GSK3β ( S9 ) was blotted to indicate the inhibition of Akt .", "( D ) Activation of Akt inhibits UPS .", "H4-GFP-CL1 cells were transfected with Myr-Akt for 24 hr .", "The cells were then harvested and subjected to western blotting analysis using indicated antibodies .", "( E ) IGF-1 stimulation inhibits UPS function .", "H4-GFP-CL1 cells were serum-starved and pretreated with Akt inhibitor MK2206 ( 1 μM ) for 30 min before stimulating with IGF-1 ( 100 ng/mL ) for 30 min .", "The cells were then imaged and quantified as in ( B ) .", "( F ) H4-GFP-CL1 cells were treated as in ( D ) and Figure 5—figure supplement 1E .", "Then cells were imaged and quantified as in ( B ) .", "( G ) EGF stimulation inhibits UPS function .", "H4-GFP-CL1 cells were serum-starved and pretreated with Akt inhibitor MK2206 ( 1 μM ) for 30 min before stimulation with EGF ( 100 ng/mL ) for 1 h .", "The cells were then harvested and subjected to western blotting analysis using indicated antibodies .", "( H ) Akt regulates UPS function through USP14 .", "Myr-Akt was transfected into either wild type or Usp14–/– H4 cells stably expressing GFP-CL1 for 24 hr .", "The cells were then harvested and subjected to western blotting analysis using indicated antibodies .", "( I ) Akt regulates UPS function through phosphorylation of USP14 .", "Myr-Akt was transfected into either wild type USP14 or USP14 AA reconstitution cell lines stably expressing GFP-CL1 for 24 hr .", "The cells were then imaged and quantified as in ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 01210 . 7554/eLife . 10510 . 013Figure 5—figure supplement 1 . Regulation of UPS by Akt .", "( A , B , C )", "Validation of GFP-CL1 assay .", "A schematic representation of GFP-CL1 assay .", "CL1 degron ( ACKNWFSSLSHFVIHL ) was added to the C-terminal of GFP as a UPS activity reporter ( A ) .", "H4-GFP-CL1 cells were treated with 10 μM MG132 for 4 hr .", "Images of the cells were collected using an ArrayScan HCS 4 . 0 Reader .", "The average GFP intensity in 2 , 000 cells from each indicated sample was determined .", "The data are displayed as means ± SD of the GFP intensity per cell ( B ) .", "H4-GFP-CL1 cells were treated with different concentration of MG132 as indicated for 4 hr , and then cells were harvested and subjected to western blotting analysis using indicated antibodies ( C ) .", "( D ) H4-GFP-CL1 cells were serum-starved overnight and harvested and subjected to western blotting analysis using indicated antibodies .", "( E ) H4-GFP-CL1 cells were serum-starved overnight before stimulation with IGF-1 for 30 min .", "Then the cells were harvested and subjected to western blotting analysis using indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 01310 . 7554/eLife . 10510 . 014Figure 5—figure supplement 2 . Regulation of UPS by Akt depends on phosphorylation of USP14 . ( A , B , C , D ) Generation of USP14 knockout H4 cell line using CRISPR/Cas9 system .", "A schematic of the Cas9/sgRNA/oligo targeting site in the exon2 of Usp14 .", "The sgRNA coding sequence is underlined and labeled in red .", "The protospacer-adjacent motif ( PAM ) sequence is underlined ( A ) .", "The deleted sequences in the Usp14–/– cell lines are presented .", "The number of sequences analyzed is indicated right ( B ) .", "Sequencing analysis of Usp14–/– cell lines .", "The arrow indicates the missing sequences ( TCAAG ) ( C ) .", "Western blotting analysis of USP14 expression in WT cells and Usp14–/– cells ( D ) .", "( E ) Akt regulates UPS function through phosphorylation of USP14 .", "An expression vector for Myr-Akt was transfected into either wild type USP14 or USP14 AA reconstitution cell lines stably expressing GFP-CL1 and incubated for 24 hr .", "Then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies .", "( F ) GFP-cODC assay was verified .", "H4-GFP-cODC cells were treated with 10 μM MG132 for 4 hr , and then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies .", "( G ) H4-GFP-cODC cells were treated with 10 μM MK2206 for 4 hr or transfected with an expression vector for Myr-Akt as indicated for 24 hr .", "Then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 014 We next tested the importance of USP14 phosphorylation for Akt to regulate UPS .", "We found that in contrast to USP14 WT reconstituted H4 cells , USP14 AA mutant reconstituted H4 cells showed no increase in the accumulation of GFP-CL1 in response to the expression of activated Akt ( Figure 5—figure supplement 2Eand Figure 5I ) .", "As a control , we found that the expression of Akt had no effect on a ubiquitin-independent substrate of the proteasome , C-terminal ornithine decarboxylase-GFP ( GFP-cODC ) ( Hoyt et al . , 2005; Kelly et al . , 2007; Lee et al . , 2010 ) ( Figure 5—figure supplement 2F , G ) , suggesting that Akt does not inhibit the UPS through a general inhibition of the proteasome itself .", "Taken together , these data show that phosphorylation of USP14 by Akt is important for this kinase to negatively regulate the UPS in a ubiquitin-dependent manner ." ], [ "To further understand the physiological roles of Akt-mediated USP14 phosphorylation and subsequently activation , we sought to study the impact of USP14 phosphorylation on global protein degradation .", "Since the loss of USP14 accelerates cellular proteolysis ( Koulich et al . , 2008; Lee et al . , 2010 ) , we performed a quantitative proteomic analysis to determine the levels of proteins in WT H4 cells , H4 USP14-KO cells , and H4 USP14-KO cells complemented with WT USP14 , S143A/S432A ( AA ) , or S143D/S432D ( DD ) mutants .", "Using an isobaric tandem mass tag ( TMT ) labeling approach , our mass spectrometry analysis identified 18 , 400 peptides with high confidence ( q<0 . 01 ) , corresponding to 3 , 648 proteins with a minimum of two peptides from each protein .", "A total of 2 , 763 proteins , which were quantified in at least two replicates , were subjected to further analysis .", "We found the global protein patterns of H4 USP14-KO cells were similar to those of H4 USP14 KO-AA cells , but distinct from those of WT H4 cells .", "We identified a common set of 87 proteins that were reduced in H4 KO cells as compared to H4 WT cells or to H4 KO cells complemented with WT USP14 ( KO-WT ) ( Figure 6 , Lane1-2 ) .", "The levels of these proteins were also significantly reduced in H4 KO-AA cells ( Figure 6 , Lane 3 ) .", "Importantly , the levels of this set of 87 proteins in H4 KO-DD cells were significantly higher than that of H4 KO-AA cells ( Figure 6 , Lane 4 ) . 10 . 7554/eLife . 10510 . 015Figure 6 . Phosphorylation of ubiquitin-specific protease-14 ( USP14 ) regulates global protein degradation . The quantitative analysis of proteome change in USP14 knockout or USP14 mutant cells were performed by tandem mass tag ( TMT ) -isobaric labeling followed by shotgun analysis .", "The heat map was plotted based on the set of 87 proteins that are down-regulated greater than or equal to 1 . 2-fold in H4 KO cells compared to H4 WT cells or to H4 KO cells complemented with WT USP14 ( KO-WT ) .", "The log base 2 of average ratios was plotted as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 01510 . 7554/eLife . 10510 . 016Figure 6—figure supplement 1 . Western blotting analysis of mTOR expression in WT cells and USP14 mutant-reconstituted cells . The cell lysates were harvested and subjected to western blotting analysis using indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 10510 . 016 To verify that the identified changes in protein abundance were due to proteasomal degradation , we treated H4 KO-AA cells with proteasome inhibitor MG132 and analyzed the protein level change of these 87 proteins .", "We found that the levels of these proteins increased significantly in MG132-treated KO-AA cells compared to that of control KO-AA cells ( Figure 6 , Lane 5 ) , suggesting that these proteins were indeed subject to an increased rate of proteasome degradation with expression of non-phosphorylatable USP14 .", "Interestingly , the top hit on this list of 87 proteins that were differentially regulated upon the loss of USP14 is mTOR , a central established regulator of cellular metabolism and tumorigenesis .", "We confirmed the role of USP14 on the levels of mTOR by western blotting .", "We found that the levels of mTOR were reduced in H4 KO and H4 KO cells complemented with USP14 AA mutant , but restored upon the expression of USP14 DD mutant ( Figure 6—figure supplement 1 ) .", "Taken together , our results suggest that phosphorylation of USP14 may provide a mechanism for Akt to regulate global protein degradation through the proteasome , which in turn may control key cellular pathways involved in regulating metabolism and tumorigenesis ." ], [ "We report here a new mode of USP14 regulation involving its phosphorylation by the protein kinase Akt .", "The activity of USP14 is induced 800-fold by association with the proteasome ( Lee et al . , 2010 ) .", "Remarkably , Akt-dependent phosphorylation of USP14 elevates the catalytic activity of proteasome-associated USP14 beyond this level .", "Thus , Akt not only activates USP14 by a different mechanism than the proteasome , but it can cooperate with the proteasome to achieve more aggressive removal of ubiquitin from proteasome-docked substrates .", "Akt activation of USP14 has marked effects on protein turnover in cells , highlighting the physiological significance of the activity state of USP14 .", "USP14 and its ortholog from yeast ( Ubp6 ) can suppress degradation through both deubiquitination and a noncatalytic activity ( Bashore et al . , 2015; Hanna et al . , 2007; Lee et al . , 2010 ) .", "The ability to rapidly adjust the rate of proteasomal degradation by responding to external signals , including growth factors , nutritional demands , and stress , is critical for maintaining cellular survival and preventing the toxicity associated with protein misfolding and accumulation of toxic aggregates .", "By regulating the rate of proteasomal degradation through phosphorylating USP14 , Akt may exert controls over the entire proteome of short-lived proteins as well as cellular protein quality .", "Since Akt is dramatically activated in PTEN-deficient cancer cells , the control of USP14 phosphorylation by Akt may provide a mechanism for cancer cells with PTEN loss , one of the most common cancer mutations , to control global intracellular proteostasis by regulating protein degradation through proteasomes .", "Furthermore , since Akt can be activated by a wide range of growth factors , such as insulin , EGF , IGF , and fibroblast growth factor ( FGF ) , through their respective receptors , regulation of USP14 by Akt-mediated phosphorylation may provide a general mechanism for growth factors to control global proteostasis during cell growth .", "Our results do not exclude the possibility that other kinases that are activatable by growth factors and activated in PTEN-deficient conditions can also mediate the phosphorylation of USP14 .", "The precise control of the UPS allows timely and selective degradation of surplus and/or aberrant proteins which is essential for normal cellular physiology .", "Dys-regulation of UPS may disrupt cellular proteostasis and lead to the inappropriate accumulation of target proteins to compromise cellular and tissue homeostasis .", "Since UPS is critically involved in the degradation of cellular short-lived proteins which frequently serve in mediating intracellular signaling process , the regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a mechanism to control multiple signaling process , raising the possibility that control of short-lived proteins might serve as an active process that has impact on cellular signaling , rather than as a degradative process per se .", "In this regard , regulation of UPS by Akt has been proposed to contribute to type II diabetes mediated by inflammation .", "Stimulation of adipocytes by TNFα has been shown to lead to reduction of Akt ( Medina et al . , 2005 ) .", "Thus , a reduction of Akt might promote UPS to lead to global proteomic changes to contribute to the pathological consequence in diabetes ." ], [ "All cell lines were maintained at 37°C with 5% CO2 .", "HEK293T cells were cultured in DMEM ( Gibco ) with 10% ( vol/vol ) FBS ( Gibco ) and 1% penicillin/streptomycin .", "H4 and H4-Usp14-/- cells were maintained in DMEM supplemented with 10% ( vol/vol ) FBS , 1% penicillin/streptomycin , and 1×sodium pyruvate ( Invitrogen ) .", "Commercial antibodies used for western blotting analysis include: anti-Phospho-Akt ( Ser473 ) ( #3787 , 1:1000 dilution ) , anti-Phospho-Akt Substrate ( R××pS/pT ) ( #9614 , 1:1000 dilution for WB and 1:250 dilution for IP ) , anti-USP14 ( rabbit , #11931 , 1:1000 dilution ) , anti-FLAG ( #2368 , 1:1000 dilution ) were from Cell Signaling Technology .", "Anti-phosphoserine ( #61-8100 , 1:1000 dilution for WB and 1:250 dilution for IP ) was from Invitrogen .", "Anti-Akt ( sc-8312 , 1:1000 dilution ) and anti-USP14 ( mouse , sc-393872 , 1:1000 dilution ) were from Santa Cruz Biotechnology .", "Anti-Myc ( 16286-1-AP , 1:1000 dilution ) , anti-HA ( 51064-2-AP , 1:1000 dilution ) , and anti-GAPDH ( 60004-1-Ig , 1:10000 dilution ) were from Proteintech .", "Anti-Tubulin ( PM054 , 1: 10000 dilution ) was from MBL .", "Recombinant active Akt protein ( #14-276 ) was from Millipore .", "Mouse monoclonal Anti-FLAG ( F1804 , 1:500 for IP ) , anti-Myc ( A7470 ) , and anti-HA Affinity Gel ( E6779 ) were from Sigma-Aldrich .", "IU1 ( S7134 ) , MK2206 ( S1078 ) , AZD5363 ( S8019 ) , GDC0941 ( S1065 ) , Wortmannin ( S2758 ) , U0126 ( S1102 ) , and MG132 ( S2619 ) were from Selleckchem .", "Phospho-USP14 S432 antibodies were generated by Proteintech .", "Briefly , synthetic peptides corresponding to phosphorylated S432 epitope ( N'-Cys-THQGRSSs ( phospho ) SGHYVSW ) of USP14 were used to immunize rabbits and the antisera were affinity purified .", "The cDNAs for USP14 and a constitutively active form of Akt , N-terminally myristoylation signal ( MGSSKSKPK ) -attached Akt ( Myr-Akt ) , were cloned into pcDNA3 . 1 using Phanta Max Super-Fidelity DNA Polymerase ( Vazyme Biotech Co . , Ltd ) and ClonExpressTM II cloning kit ( Vazyme Biotech Co . , Ltd ) .", "GFP-CL1 was derived by adding the CL1 degron ( ACKNWFSSLSHFVIHL ) to the C-terminal of GFP and cloned into pMSCV to generate retroviral transfer vector .", "Mutagenesis was performed using MutExpressTM II mutagenesis kit ( Vazyme Biotech Co . , Ltd ) .", "The cells were transfected with plasmid DNA using PolyJet DNA In Vitro Transfection Reagent ( Signagen Laboratories ) according to the manufacturer’s instructions .", "The USP14 wild type or mutant DNA was cloned into pET-32M vector ( an in-house modified version of pET32a vector containing a N-terminal Trx-tag and His6-tag ) .", "Recombinant proteins were expressed in BL21 ( DE3 ) E . coli cells .", "The bacterial cultures were grown at 37°C until OD600 nm reached 0 . 6–0 . 8 , and USP14 expression was then induced overnight with 0 . 2 mM IPTG at 16°C .", "The cells were harvested in binding buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 500 mM NaCl , 5 mM imidazole ) containing protease inhibitors and lysed by the NANO homogenizer machine ( FBE , Shanghai ) .", "The lysate was then clarified by centrifugation at 18 , 000 × g for 30 min .", "His6-tagged proteins were purified by Ni2+-NTA agarose ( Qiagen ) affinity chromatography .", "Each recombinant protein was further purified by size-exclusion chromatography .", "The terminal tag of each recombinant protein was cleaved by 3C protease overnight at 4°C and further removed by size-exclusion chromatography .", "Recombinant USP14 or USP14 mutant protein ( 1 μg ) was incubated with 1 μg active Akt , 0 . 2 mM ATP , and kinase assay buffer ( Cell Signaling ) in a total volume of 50 μl for 1 hr at 30°C .", "The reaction mixtures were subjected to Ub-AMC assay by the addition of 50 μl 2×Ub-AMC buffer .", "Alternatively , the kinase reaction was stopped by the addition of 50 μl 2×sample buffer , and resolved by SDS-PAGE , followed by blotting with phospho-specific antibodies .", "Pten–/– MEFs cells were lysed in buffer A ( 20 mM Tris-HCl ( pH 7 . 6 ) , 20 mM NaCl , 1 mM β-mercaptoethanol , 1 mM ATP , and 5 mM MgCl2 ) .", "After 10 min at 4°C , cells were disrupted with 50 passages through a 27-gauge needle .", "Lysates were centrifuged at 16 , 000 × g for 10 min , supernatants were supplemented with 10% glycerol .", "Density gradient centrifugation was conducted in 10–40% linear glycerol gradients .", "Gradients contained 50 mM Tris-HCl ( pH 7 . 6 ) , 20 mM NaCl , 1 mM dithiothreitol , 1 mM ATP , and 5 mM MgCl2 .", "Samples were centrifuged at 55 , 000 × g for 3 hr .", "Fractions were collected for further analysis .", "For UPS reporter cell line , H4 cells and Usp14-/- H4 cells were infected with retroviral particles expressing GFP-CL1 or GFP-cODC , and then selected with 1 μg/ml puromycin to generate stable cell lines .", "For reconstitution lines , Usp14-/- H4 cells were infected with lentiviral particles expressing HA-USP14 ( WT or mutant ) .", "Usp14 was knocked out from H4 cells using the CRISPR/Cas9 system ( Jinek et al . , 2013 ) , with a guide RNA spanning exon 2 .", "The guide RNA was individually cloned into the pX330 vector and transfected into H4 cells .", "Transfected cells were sorted by fluorescence-activated cell sorting using green fluorescent protein .", "Single colonies were screened using PCR to confirm the expected genomic deletion and western blot to confirm the loss of USP14 protein expression .", "Ub AMC-conjugated proteins were purchased from Boston Biochem .", "Assays were carried out in a flat-bottom , low-flange 384-well plate in a 40 μl reaction .", "Enzymes and substrates were prepared in Ub-AMC assay buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 1 mM EDTA , 1 mM ATP , 5 mM MgCl2 , 1 mM DTT , and 1 mg/ml ovalbumin ) .", "The reaction was initiated by adding of Ub-AMC and measured at Ex345/Em445 using an Envision plate reader ( PerkinElmer ) .", "For determination of KM , USP14-S432E was incubated with the indicated concentrations of Ub-AMC .", "Lineweaver–Burk analysis was carried out using a linear regression fit of the data with the equation 1ν = KMVmax1[S] + 1Vmax to calculate KM .", "For ubiquitin cleavage assays , hydrolysis reactions were carried out at 30°C in reaction buffer , with a constant enzyme concentration of 400 nM USP14 and USP14 S432E in a 50 μl reaction volume .", "Aliquots of 10 μl were taken at the indicated times and added to 10 μl SDS loading buffer to stop the reaction .", "The USP14 protein was subject to trypsin digestion on beads after immunoprecipitation experiment .", "The enrichment of phosphorylated peptides by TiO2 was performed with tryptic USP14 peptides .", "The enriched phosphorylated peptides were analyzed on Orbitrap Fusion mass spectrometer ( Thermo Scientific ) .", "The activation type of HCD was performed for MS2 .", "Protein identification was performed by Thermo Proteome discoverer ( v1 . 4 ) with Sequest HT .", "The precursor mass tolerance was set at 10 ppm , and the fragment mass tolerance was set at 0 . 1 Da .", "The cysteine carboxyamido methylation was set as a static modification , and phosphorylated serine , threonine , and tyrosine residues were set as variable modifications .", "The peptide false-positive rate was controlled to be less than 1% .", "PhosphoRS site probability analysis was performed .", "Quantitative analysis of proteome changes in USP14 knockout or USP14 mutant cells was performed by TMT-isobaric labeling followed by shotgun analysis .", "The cell lysate of WT , Usp14–/– , Usp14–/– reconstituted with WT USP14 ( KO-WT ) , Usp14–/– reconstituted with AA mutant USP14 ( KO-AA ) , Usp14–/– reconstituted with DD mutant USP14 ( KO-DD ) , and KO-AA cells treated with 10 μM MG132 for 4 hr were digested with trypsin and labeled with 126 , 127 , 128 , 129 , 130 , 131-TMT labeling reagent ( Thermo Scientific ) , respectively , according to the manufacturer’s instruction .", "Equal amount of peptides with each TMT tag were mixed , and the resulting mixture of peptides was subjected to fractionation using off-line high pH reverse phase chromatography .", "Six fractions were collected and subsequently analyzed on Orbitrap Fusion mass spectrometer .", "Three replicates were performed .", "Protein identification and quantification was done by Thermo Proteome discoverer ( v1 . 4 ) .", "The peptide false-positive rate was controlled to be less than 1% .", "The peak integration tolerance was set at 10 ppm .", "Only unique peptides were used for protein quantitation .", "An isobaric tag purity correction was performed .", "The labeling efficiency was measured and was greater than 99% .", "The quantitation normalization based on protein median was performed .", "The average ratios of each protein from three replicates were used for analysis .", "Cells were fixed with 4% paraformaldehyde and stained with 3 μg/ml DAPI ( Sigma ) .", "Images data were collected with an ArrayScan HCS 4 . 0 Reader with a 203 objective ( Cellomics ArrayScan VTI ) for DAPI-labeled nuclei and GFP-tagged intracellular proteins .", "Error bars for microscopy were presented as the standard deviation of triplicate samples .", "Error bars for Western blotting analysis represent the standard deviation between densitometry data from three biological replicates .", "Student’s t-test was used as statistical analysis by using GraphPad Prism ." ] ]
[ "Regulation of ubiquitin-proteasome system ( UPS ) , which controls the turnover of short-lived proteins in eukaryotic cells , is critical in maintaining cellular proteostasis .", "Here we show that USP14 , a major deubiquitinating enzyme that regulates the UPS , is a substrate of Akt , a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors .", "We report that Akt-mediated phosphorylation of USP14 at Ser432 , which normally blocks its catalytic site in the inactive conformation , activates its deubiquitinating activity in vitro and in cells .", "We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation .", "Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN , we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells ." ]
[ "Proteins are the workhorses of cells .", "These molecules provide structure , transmit messages and carry out many other essential tasks .", "When proteins have fulfilled their purpose , or become damaged , they must be removed through a garbage disposal-like molecular machine in cells called the proteasome .", "A breakdown in the proteasome may lead to diseases in humans such as cancers and neurodegeneration .", "Cells have a system that can identify and mark proteins for destruction , and another system that counteracts this process and spares proteins from destruction .", "Precise regulation of these two systems helps ensure a healthy balance in cells .", "One enzyme that can spare proteins from the proteasome is called USP14 .", "Previously , this enzyme is known to be switched on when it connects with the protein disposal machinery to control which proteins get destroyed .", "But , many of the USP14 enzymes in cells are not associated with this proteasome machinery and it was unclear if and how these ‘free’ enzymes might be important for the cell .", "Now , Xu et al . report a new mechanism that can switch on USP14: another enzyme called Akt can switch on USP14 by adding a phosphate group to a specific site in USP14 .", "Akt is an important signaling molecule that is activated in many tumor cells to promote the growth and multiplication of cells .", "Xu et al . discovered that by controlling USP14 activity , Akt can control the activity of the protein disposal machinery that in turn regulates the levels of many other proteins .", "These findings suggest that abnormal activity of USP14 in tumor cells with elevated Akt activity may contribute to cancer formation ." ]
2015
[ "Introduction", "Results", "Discussion", "Conclusion", "Materials and methods" ]
[ "neuroscience" ]
Using an achiasmic human visual system to quantify the relationship between the fMRI BOLD signal and neural response
elife-09600-v2
[ [ "Functional magnetic resonance imaging ( fMRI ) based on the blood oxygenation level dependent ( BOLD ) signal has provided unprecedented insights into the workings of the human brain .", "The quantitative relationship between neural signals and the fMRI BOLD response is not precisely known and remains an active area of investigation .", "Most studies using the BOLD signal to infer brain activity rely on analytical methods ( e . g . , the general linear model ) that assume a linear relationship between the BOLD signal and neural response , despite noticeable deviations from linearity ( Boynton et al . , 1996 ) .", "The BOLD signal is indirectly related to local neural response through mechanisms associated with oxygen metabolism and blood flow ( Davis et al . , 1998; Hoge et al . , 1999; Thompson et al . , 2003; Griffeth and Buxton , 2011 ) .", "The neural response that is associated with information processing is itself multi-faceted .", "It comprises several interacting components , including subthreshold and suprathreshold electrical activities , the transport , release and reuptake of neurotransmitters , and various maintenance activities .", "Each of these components has its own metabolic and hemodynamic consequences .", "The common extracellular measurements of neural response include single- and multi-unit spiking activities and local field potential ( LFP ) .", "While seminal studies have demonstrated a close relationship between the BOLD signal and these extracellular measurements of neural response ( Logothetis et al . , 2001; Mukamel et al . , 2005 ) , the quantitative nature of this relationship has not been sufficiently characterized .", "More importantly , since the relationship between these extracellular measurements and the intracellular components of neural activity is complex , the measured relationship between the BOLD signal to any specific extracellular components ( e . g . , power in the gamma band of LFP ) may not reflect the relationship between the BOLD signal and the totality of neural response .", "Most applications of fMRI , particularly in human neuroscience , sidestep any need for explicitly estimating neural activity and instead rely on establishing a direct relationship between the BOLD response and the stimulus condition .", "The general approach is to assume the BOLD responses evoked at different times and in different stimulus conditions sum linearly .", "Boynton and colleagues ( 1996 ) studied how the BOLD signal varied with the contrast and duration of stimulus presentation in the striate cortex and found that the system is approximately linear , in the sense that the BOLD response evoked by a 12 s stimulus was well approximated by summing the responses from two consecutive 6-s stimulations , even though predictions based on stimulations of much shorter durations ( e . g . , 3 s ) failed to accurately predict the long-duration stimulus response .", "While this and similar studies ( Cohen , 1997; Dale and Buckner , 1997; Heckman et al . , 2007 ) have clearly noted the lack of linearity , their general message of an approximately linear system has nevertheless been used to justify the broad application of the general linear model ( GLM ) in fMRI data analyses .", "While the neural response is not explicitly involved in this type of analysis , it is always in the background — any nonlinearity observed in the BOLD response , e . g . , in surround suppression or adaptation ( Grill-Spector and Malach , 2001; Kourtzi and Huberle , 2005; Larsson and Smith , 2012 ) is often attributed to the underlying nonlinear neural response .", "The implicit assumption in common practice is that the relationship between the BOLD response and the neural response is essentially linear , a view that is widespread ( Logothetis and Wandell , 2004 ) but under-examined .", "An extensive set of biophysical models has been proposed to express either the steady-states ( Davis et al . , 1998; Griffeth and Buxton , 2011 ) or the dynamics of the BOLD response ( Buxton et al . , 1998; Mandeville et al . , 1999; Feng et al . , 2001; Toronov et al . , 2003; Blockley et al . , 2009; Kim and Ress , 2016 ) in terms of more basic physiological components , such as blood flow , blood volume , oxygen saturation , and oxygen extraction fraction in different vascular compartments .", "These biophysical models are foundational in our understanding of the BOLD signal , yet they do not provide any explicit and quantitative linkage between the neural response and the physiological components that are the inputs to these models .", "Friston et al . ( 2000 ) ( see also Stephan et al . , 2007 ) , proposed a linkage between the evoked neural response and the blood-flow parameter of the Balloon model by Buxton et al . ( 1998 ) .", "While the resulting model is a powerful tool for inferring effective connectivity between brain regions from the BOLD signal , direct empirical support for this specific linkage is limited .", "How could we empirically determine the quantitative relationship between the BOLD signal and the neural response , and do so when the constituents of the neural response are not comprehensively defined ?", "A condition known as achiasma or non-decussating retinal-fugal fibre syndrome may provide an excellent model system for this purpose .", "This congenital condition prevents the normal crossing of optic nerve fibers from the nasal hemi-retina to the brain hemisphere contralateral to the eye ( Apkarian et al . , 1994; 1995 ) .", "The result is a full representation of the entire visual field ( as opposed to only half the visual field ) in each cerebral hemisphere ( Williams et al . , 1994; Victor et al . , 2000; Hoffmann et al . , 2012; Davies-Thompson et al . , 2013; Kaule et al . , 2014 ) .", "Specifically , the representations of the two visual hemifields are superimposed in the low-level visual areas ( V1-V3 ) ipsilateral to each eye , such that two points in the visual field located symmetrically across the vertical meridian are mapped to the same point on the cortex ( Hoffmann et al . , 2012 ) .", "In other words , there are two pRFs for every point on this person’s low-level visual cortex .", "The two pRFs are symmetrically located across the vertical meridian .", "Prior to the current study , it was not known if these pRFs were represented by one or two neural populations , or if these neural populations interacted .", "In the current study , we found that the two pRFs are each represented by an independent population of neurons .", "The result is an in-vivo system with two independent populations of spatially intermingled neurons that share the same local control of blood vasculature .", "Because their population receptive fields ( pRFs ) do not overlap , an experimenter can independently stimulate each population by presenting a stimulus to its respective receptive field .", "Such a system is ideal for characterizing the relationship between neural and BOLD responses .", "Even though we may not know the constituents of the neural response , it will be reasonable to assume that the local neural response evoked by presenting identical stimuli to both pRFs , thereby activating both neuronal populations equally , is twice the neural response evoked by presenting the stimulus to just one of the pRFs .", "Measuring BOLD responses under these conditions allows us to not only directly test for linearity between the BOLD signal and neural response but also quantify the relationship between them , up to an arbitrary scaling factor .", "This approach does not require us to know the constituents of neural activity , and it is non-invasive .", "To determine the relationship between neural response and the corresponding fMRI BOLD signal , we measured BOLD responses in the cortical areas V1-V3 of our achiasmic subject to luminance-defined stimuli .", "We presented stimuli of different contrasts to either one or both of the pRFs .", "From this data set , we used a model-free non-parametric method to infer the quantitative relationship between the BOLD signal ( B ) and neural response ( Z ) .", "We found that the resulting B vs . Z function is well approximated by a power function with an exponent close to 0 . 5 .", "The exponent stayed the same for short and long stimulus durations .", "We successfully cross-validated this result by comparing the inferred neural responses from this and twelve other fMRI studies to the single-unit responses obtained from non-human primates in similar contrast-response experiments ." ], [ "Our primary research subject ( S ) was a 24-year-old achiasmic Caucasian male .", "S was diagnosed with isolated foveal hypoplasia .", "His uncorrected visual acuity was 0 . 7 logMAR for the left eye and 0 . 5 logMAR for the right eye ( best-corrected 0 . 5 and 0 . 3 logMAR , respectively ) .", "He has a congenital nystagmus .", "His nystagmus ( peak-to-peak ) amplitude was less than 2 . 1° ( 95 percentile ) horizontally and negligible ( 0 . 7° ) vertically .", "The horizontal nystagmus followed a saw-tooth waveform with a right-forward fast phase and a ( horizontal ) frequency of between 0 . 6–2 . 3 Hz ( mode at 2 . 3 Hz ) during tasks performed for the current study .", "He was right-eye-preferred and suppressed his left eye ( self-report ) .", "A Single Cover Test revealed left-eye esotropia .", "He could not achieve binocular fusion and had no measurable stereoacuity .", "We confirmed his absence of an optic chiasm using MRI ( Figure 1A ) .", "We obtained high-resolution monocular hemifield retinotopic maps for each of his eyes .", "The retinotopy was well defined and of high quality .", "Within the retinotopically defined visual areas V1-V3 , the subject’s retinotopic representation of the ipsilateral visual field is a mirror image of its contralateral field representation ( Figure 1B ) .", "The two are superimposed on the hemisphere ipsilateral to the stimulated eye .", "Two points placed in the subject’s visual field symmetrically across the vertical meridian are mapped to the same point on the cortex .", "This left-right mirror mapping is established at the level of individual voxels – 3x3x3 mm3 ( Figure 1C ) .", "Additional tests with vertically-reflected stimuli showed nearly identical BOLD signal modulation for individual voxels in V1-V3 ( Figure 1—figure supplement 1 ) .", "These findings were consistent with previous reports on human achiasma ( Hoffmann et al . , 2012 ) . 10 . 7554/eLife . 09600 . 003Figure 1 . Retinotopy of the achiasmic subject S .", "( A ) The MRI image of S shows the lack of the optic chiasm .", "( B ) S has a well-defined retinotopy , but unlike normal retinotopic representations , both left and right visual fields are mapped to the same hemisphere ipsilateral to the stimulated eye ( data from right-eye stimulation are shown ) .", "Eccentricity and polar angle maps are organized orderly for both visual hemifields .", "Visual area boundaries were identified at where the polar angle reversed .", "( C ) A schematic of the two population receptive fields ( pRFs ) of a fMRI voxel .", "For individual voxels in V1-V3 , eccentricities ( upper panels ) and polar angles relative to the lower vertical meridian ( lower panels ) of each of the two pRFs are plotted against each other .", "Most values fall close to the identity line , demonstrating that the two pRFs of each voxel are at mirrored locations across the vertical meridian . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 00310 . 7554/eLife . 09600 . 004Figure 1—figure supplement 1 . fMRI BOLD response evoked with ROI-defining stimuli .", "( A ) The ROI-defining stimuli in the backward-slash ( Stimulus A ) and forward-slash ( Stimulus B ) configurations .", "The corresponding areas with significant modulation ( FDR<0 . 05 ) are depicted on the flattened visual cortex of the right hemisphere of the achiasmic subject , who viewed the stimuli with his right eye .", "These stimuli were used to define the ROIs for the analyses of the adaptation experiment ( Figure", "2 ) and the BOLD summation experiment ( Figure 3 ) .", "White lines delineate the borders of the retinotopically defined visual areas .", "Black contours outline the boundaries of the ROIs used in the experiments , corresponding to the middle 2 degrees ( diameter ) of the larger ( 4-degree diameter ) outer Gabor patches .", "( B ) BOLD response amplitudes of Stimulus A versus those of Stimulus B for randomly selected voxels in areas V1-V3 .", "The stimuli evoked nearly identical responses in each voxel , suggesting that ( 1 ) each voxel has two pRFs and ( 2 ) the neural populations underlying each pRF are nearly identical . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 004 The slight but systematic deviation from perfect homotopy in terms of eccentricity ( Figure 1C , top row ) is likely due to his asymmetric nystagmus waveform ( with a leftward slow phase ) , which may have biased the time-averaged gaze position slightly towards the left of fixation .", "For those experiments that aimed to measure the interactions between the stimuli presented to both of the hemifields , we designed the experiments to be insensitive to this and other deviations from perfect gaze control or homotopy .", "Whenever appropriate , we made one of the stimuli ( mask ) much larger in size than the other ( probe ) to ensure that the probe was always projected to a cortical region that also represented the mask .", "When two stimuli had to be of the same size , we analyzed only the cortical regions associated nominally with the middle regions of the stimuli where overlaps were mostly guaranteed .", "We also conducted supplementary analyses to validate that the fMRI voxels used in the primary analysis were jointly activated by both stimuli .", "Retinotopic mapping showed that each fMRI voxel in the visual cortex of S has two pRFs .", "We next tested if the neural populations that underlie these pRFs interact with each other .", "Behaviorally , S does not show any confusion between visual hemifields .", "Most tellingly , S is an avid reader , which strongly suggests that the co-localized neural populations do not interact functionally ( otherwise , the text on the left of fixation would mask the text on the right of fixation ) .", "To quantify possible interactions between the pRFs , we measured whether the contrast threshold for S to detect a Gabor grating could be affected by placing a high-contrast flickering checkerboard symmetrically across either the vertical or horizontal meridian from the target location .", "Placing the checkerboard mask symmetrically across the vertical meridian causes it to be projected to the same cortical location as the target in the visual cortex of S , while placing it across the horizontal meridian does not ( Figure 2A ) .", "S performed this sensitive threshold task monocularly with his right eye in front of a calibrated CRT screen .", "We found that the placement of the flickering checkerboard did not affect the detection threshold , and that the threshold is within the normal range without masking ( Figure 2B ) .", "This and other experiments performed by us ( Figure 2—figure supplement", "1 ) and others ( Victor et al . , 2000; Hoffmann and Dumoulin , 2015 ) have failed to demonstrate any anomalous interactions between the two pRFs , strongly suggesting that the two underlying neural populations are functionally independent . 10 . 7554/eLife . 09600 . 005Figure 2 . Behavioral and physiological evidence of independence between the co-localized neural populations .", "( A ) Schematic of the psychophysical experiment of contrast detection with a contralateral mask .", "The 45° target Gabor patch was presented in either the lower-right ( shown ) or upper-left quadrant .", "A high-contrast flickering checkerboard mask was presented at the mirrored location from the target quadrant , across either the vertical or horizontal meridian .", "S was to identify which one of the two temporal intervals the target appeared in .", "( B ) Contrast detection thresholds were essentially the same for the two mask-target arrangements .", "Error bars denote ± SE across blocks .", "( C ) Design of the fMRI adaptation experiment .", "Each block of trials was preceded with 20 s of pre-adaptation .", "Each trial began with a 5 s presentation of the adapting stimulus ( ‘top-up’ adaptation ) , followed by one of the four test stimuli .", "Relative to the adapting stimulus , the test stimulus could either be at the same or mirrored location , and could have either the same or orthogonal orientation .", "Attention was controlled with a demanding central fixation task .", "( D ) Time courses of fMRI BOLD responses in V1-V3 to the four test conditions .", "Shaded error band denote ± SE across trials .", "The responses evoked by the test stimuli presented at the mirrored location relative to the adaptor , regardless of orientation , did not differ significantly from those evoked by the test stimuli at the same location as the adaptor but with orthogonal orientation . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 00510 . 7554/eLife . 09600 . 006Figure 2—figure supplement 1 . Results from supplementary psychophysical experiments implicating two groups of non-interacting neurons .", "( A ) Contrast detection task with a contralateral noise mask .", "The target Gabor patch was presented in either the lower-right ( shown ) or upper-left quadrant .", "An isotropic noise mask of the same spatial frequency band as the target was presented at the mirrored location of the target across either the vertical or horizontal meridian .", "In the cross-vertical-meridian arrangement ( blue ) , but not in the cross-horizontal-meridian arrangement ( red ) , both the target and the mask projected to the same cortical locations in V1-V3 of the achiasmic subject S . S was to identify which one of the two temporal intervals contained the target .", "Contrast detection thresholds were essentially identical for the two mask positions .", "Error bars denote ± SE across different blocks .", "( B ) Letter identification task: S’s task was to identify the target ( center ) letter presented at an eccentricity of 10 deg and flanked with the 4 tumbling E’s .", "The target letter was randomly drawn from the set of 10 letters ( C , D , H , K , N , O , R , V , S and Z ) .", "The stimuli appeared for 150 ms at a given location on the CRT monitor .", "There were three conditions: ( 1 ) the flankers and target on the same side , ( 2 ) the flankers and target on symmetrically opposite sides across the vertical meridian , and ( 3 ) target only .", "When the flankers and the target were on the same side ( Condition 1 ) , letter identification performance was severely impaired by the flankers -- the well-known crowding effect .", "When the flankers and target were on the opposite sides ( Condition 2 ) , even though the cortical representations of the flankers and target were equally close as in Condition 1 , no crowding effect was found , and performance was identical to the target-only condition .", "These behavioral experiments show that even though the cortical representations of left and right visual fields overlap in V1-V3 in achiasma , the neuronal populations that represent these visual fields do not interact functionally . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 006 To test if the two neural populations are physiologically independent , we measured long-duration fMRI adaptation effects ( Fang et al . , 2005; 2007 ) .", "We first established that identical stimuli presented separately to either of the pRFs yielded nearly identical BOLD responses ( Figure 1—figure supplement 1 ) .", "We then measured the effect of adaptation when S was monocularly performing a highly demanding fixation task .", "The adapting and testing patterns were four counter-flickering Gabors ( Figure 2C ) .", "Relative to the adaptors , the test patterns could be either at the same spatial locations or at the mirrored locations across the vertical meridian .", "The orientation of the test stimulus could be either the same as or orthogonal to the adaptors .", "As expected , when the adaptors and test patterns were at the same spatial locations , we observed strong BOLD adaptation when they were of matching orientation and a large release from adaptation when they were of orthogonal orientations .", "The critical conditions are when the adapting and testing patterns were at different spatial locations but projected to the same points on the cortex of S . In these conditions , regardless of orientation , we found a large release from adaptation equal or greater in amplitude than in the same-location cross-orientation condition .", "We take this result to mean that the co-localized neural populations that underlie the two pRFs on either side of the vertical meridian do not interact physiologically with each other .", "Our results thus far suggest that each fMRI voxel in the visual cortex of S contains two independent populations of neurons , each with a distinct pRF .", "To infer the relationship between neural response and the BOLD signal , we conducted a 'pure' BOLD summation experiment with S in which stimulus summation was absent .", "We stimulated each neural population separately with spatially disjoined stimuli A and B that were mirror images of each other ( Figure 3A ) ; we also stimulated them together by presenting A and B simultaneously ( A+B ) .", "Each stimulus consisted of four counter-flickering black-and-white checkerboards .", "The Weber contrast of the stimuli ranged from 0 . 05 to 1 . 0 in four equal log steps for a total of 5 contrast levels .", "In separate experiments , a stimulus was presented for either 1 s or 6 s , followed by a 16 s blank .", "We measured the full peristimulus time course of the evoked BOLD response and its amplitude .", "In V1-V3 and across the contrast levels , the BOLD response to the combined stimulus A+B was significantly higher than what could be produced by doubling the contrast of stimulus A or B [paired t-test; V1: t ( 19 ) = -4 . 48 , p=2 . 57x10-4; V2: t ( 19 ) = -2 . 96 , p=0 . 008; V3: t ( 19 ) = -4 . 30 , p=3 . 85x10-4] ( Figure 3C ) but significantly lower than the sum of the BOLD responses to stimuli A and B [paired t-test; V1: t ( 24 ) = 2 . 53 , p=0 . 018; V2: t ( 24 ) = 6 . 20 , p=2 . 11x10-6; V3: t ( 24 ) = 6 . 07 , p=2 . 87x10-6] ( Figure 3B ) .", "This finding shows that BOLD summation , in the absence of nonlinearity associated with neuronal summation ( assuming that the A+B stimulus simultaneously excited two independent populations of neurons ) , is itself nonlinear , with a compressive nonlinearity .", "The compressive nonlinearity was not due to response saturation since the 1-s stimulation duration yielded essentially the same nonlinearity as the 6-s simulation .", "This result implies a nonlinear relationship between neural and BOLD responses , and challenges the prevailing linearity assumption . 10 . 7554/eLife . 09600 . 007Figure 3 . BOLD summation in the absence of neural nonlinearity associated with stimulus summation , with the 6-s stimuli .", "( See Figure 3—figure supplement 1 for results obtained with the 1-s stimuli , which are qualitatively identical . ) ( A ) The stimuli used in the BOLD summation experiment .", "The full stimulus display subtended 24° ( w ) x 19° ( h ) .", "Stimulus types A and B are single-sided stimuli , while type A+B is a double-sided stimulus .", "BOLD responses associated with the outer checkerboard discs were extracted from the corresponding ROIs .", "These outer discs were of diameter 4° and centered at an eccentricity of 7° .", "( B ) Estimated peristimulus time courses from V1-V3 for the three stimulus types at five different contrast levels .", "Red and magenta represent responses ( lines ) and ± SE ( bands ) to the single-sided stimuli , and green represents responses to the double-sided stimuli .", "Black dashed lines represent the predictions of linear BOLD summation , which overestimated the measured responses ( green bands ) .", "Gray bars on the abscissa indicate the duration of the stimulus ( 6 s ) .", "( C ) The contrast response functions of V1-V3 as defined by the amplitudes of the time courses .", "The amplitude of a time course was taken to be the average response between 7–9 s post-stimulus onset when the response typically reached its peak .", "The red lines represent the average single-sided response amplitudes as a function of luminance contrast .", "The green lines represent the average double-sided response amplitudes .", "The gray bands represent the predicted responses ( 68 . 2% confidence interval ) evoked with the double-sided stimulus under the assumption of linear BOLD summation ( i . e . the summed response to conditions A and B ) , and the magenta bands represent the prediction of contrast summation – the equivalent contrast of a double-sided stimulus being twice that of the corresponding single-sided stimulus .", "The measured double-sided responses were significantly lower than the predictions of linear BOLD summation and higher than that of contrast summation . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 00710 . 7554/eLife . 09600 . 008Figure 3—figure supplement 1 . fMRI BOLD summation with a 1-s stimulus duration .", "( A ) Estimated peri-stimulus BOLD time courses from V1-V3 for the three stimulus types at five contrast levels .", "Red and magenta curves represent , respectively , the BOLD responses ( lines ) and ± SE ( bands ) to the single-sided stimuli , and the green curves represents BOLD responses to the double-sided stimuli .", "Black dashed lines represent the predictions of linear BOLD summation , which overestimated the measured responses ( green bands ) .", "Gray bars on the abscissa indicate the duration of the stimulus presentation ( 1 s ) .", "( B ) The contrast response functions of V1-V3 as defined by the amplitudes of the time courses .", "The amplitude of a time course was taken to be the estimated response at 5 s post stimulus onset , when the response typically reached its peak .", "The red lines represent the averaged response amplitudes to the single-sided stimuli as a function of luminance contrast .", "Green lines represent the responses to the double-sided stimuli .", "The dark bands represent the predicted response amplitudes ( and 68 . 2% confidence intervals ) evoked with the double-sided stimuli assuming linear BOLD summation , while the magenta bands represent the prediction of contrast summation .", "Neither of these predictions fits the data .", "These results are qualitatively identical to those obtained with the 6 s stimulus ( Figure 3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 00810 . 7554/eLife . 09600 . 009Figure 3—figure supplement 2 . Results of the 6-s BOLD summation experiment with and without removing the global noise components . The experiment with a 1 s stimulus duration does not have a sufficient signal-to-noise ratio to yield reliable results with conventional deconvolution analysis ( using finite-impulse-response basis ) .", "The GLM denoising method of Kay et al . ( 2013 ) was used to estimate and remove the most prominent principle components of the noise that were shared across voxels .", "To confirm that this denoising method did not lead to any systematic bias , we applied the same denoising method to the data obtained with the 6 s stimulus duration experiment , for which conventional deconvolution analysis is applicable ( Figure 3 ) .", "The red and magenta colors represent single-sided responses and the green color represents double-sided responses ( see also Figure 3 ) .", "Solid lines , which are identical to those in Figure 3 , represent time courses estimated using the conventional deconvolution analysis without adding the estimated global noise components as regressors of no interest ( see methods ) .", "Square symbols represent the time courses inferred with the global noise components removed by representing them as regressors of no interest .", "The two methods yielded virtually identical results for the 6-s stimulus . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 00910 . 7554/eLife . 09600 . 010Figure 3—figure supplement 3 . Results of the 1-s and 6-s BOLD summation experiments obtained from the corresponding retinotopically-defined V1 ROI in the left hemisphere of the achiasmic subject . Red and magenta represent responses ( lines ) and ± SE ( bands ) to the single-sided stimuli , and green represents responses to the double-sided stimuli .", "Since the achiasmic subject viewed the stimuli with only his right eye , there was no stimulus-evoked response in the early visual areas of the left hemisphere .", "If there were any anticipatory and endogenous response , we should be able to observe the response in the left hemisphere .", "We found no such response – the peri-stimulus time courses from the left hemisphere do not deviate significantly from zero . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 010 We defined the amplitude of a BOLD response to be the peak of the BOLD time course .", "We sought to express BOLD amplitude ( B ) as a function of neural response ( Z ) , where Z refers to the local aggregate of neural response with unspecified constituents .", "At each cortical region of interest , the BOLD summation experiment provided five levels of Z , corresponding to the five levels of luminance contrast for the single-sided stimuli ( A , B ) .", "( The single-sided stimuli A and B resulted in nearly identical time courses ( Figure 3B , see also Figure 3—figure supplements 2 and Figure 4—figure supplement 2 on robustness ) , indicating that the inputs from the left and right visual fields were balanced .", "We therefore use their averages in the analysis .", ") For each level of Z evoked by a single-sided stimulus , the level of neural response evoked by the corresponding double-sided stimulus ( A+B ) is doubled under the assumptions of co-localization and independence , which we have empirically validated .", "We therefore have five pairs of BOLD measurements that correspond to Zi and 2Zi ( Figure 4A , left column ) .", "We do not know the values of Zi , but we can reasonably assume that these levels of neural response were ordered by stimulus contrast: Z1≤…≤Z5 .", "We proceed to 'stitch' the five pairs of measurement to form a continuous function without assuming any specific functional form , except that the resulting function should be monotonic and smooth .", "Without loss of generality , we can set Z1 to 1 .", "We are then left with four unknowns 1≤Z2≤…≤Z5 .", "We estimated the four ordered values Z2≤…≤Z5 such that the ten data point ( Z1 . B1 , 1 ) , … , ( Z5 . B1 , 5 ) , ( 2Z1 . B2 , 1 ) , … , ( 2Z5 . B2 , 5 ) can be best described , in the least-squares sense , by a monotonic and smooth function ( see Methods ) .", "This procedure amounts to shifting horizontally on a log-scaled abscissa a pair of data points { ( Zi . B1 , i ) , ( 2Zi . B2 , i ) } relative to other pairs until all five pairs ( ten data points ) fall on a smooth monotonic curve .", "Applying this stitching procedure to BOLD amplitude data resulted in three BvZ functions ( Figure 4B , left column ) for V1 , V2 , and V3 respectively .", "While the stitching procedure did not a priori assume a specific functional form , it is clear from Figure 4B that the resulting BvZ functions can be well fitted by a power-law function ( Figure 4B ) : B=kZγ , where k is an arbitrary scaling factor related to the unit of Z ( R2 = 0 . 997 , 0 . 992 , and 0 . 988 for V1 , V2 , and V3 , respectively ) .", "The indeterminacy of k reflects the fact that we do not know the constituents of neural response .", "For our purpose , the critical parameter is the exponent ( γ ) .", "This power-law relationship between neural and fMRI BOLD responses is also evident from the parallel lines in Figure 4A – there was no significant interaction between sided-ness ( single vs . double ) and contrast levels .", "An alternative approach for estimating γ by first determining the applicability of a power-law function is described in Figure 4—figure supplement 1; it resulted in essentially the same set of values for γ . 10 . 7554/eLife . 09600 . 011Figure 4 . fMRI BOLD signal as a function of neural response .", "( A ) Five pairs of BOLD response amplitudes evoked in V1-V3 with the single- and double-sided stimulations , each with two stimulus durations , 6-s ( left column ) and 1-s ( right column ) .", "If the neural response to a single-sided stimulus is Zi , then the neural response to the corresponding double-sided stimulus will be 2Zi , given our empirical determinations of co-localization and independence of the neuronal populations in an achiasmic visual cortex .", "( B ) The BOLD vs . neural response ( BvZ ) functions for V1-V3 as inferred by the stitching procedure for the two stimulus durations .", "The inferred functions can be well fitted with power-law functions ( i . e . straight lines in log-log coordinates ) .", "These functions are nonlinear , with a log-log slope significantly shallower than unity ( the background gray lines ) .", "( C ) The exponents ( γ ) of the power-law fit of the BvZ functions for V1-V3 .", "Error bars denote 95% CI .", "The red line indicates γ = 0 . 5 .", "γ estimated from V2 and V3 ( γ ~ 0 . 5 ) were not significantly different , while that obtained from V1 was biased upward , due to a violation of the co-localization assumption ( see Discussion ) required for inferring the BvZ function using the summation experiment .", "We thus inferred the ( true ) BvZ function of V1-V3 using the average γ estimated from V2 and V3 only . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 01110 . 7554/eLife . 09600 . 012Figure 4—figure supplement 1 . Alternative derivation of the BvZ function .", "( A ) BOLD amplitudes evoked with a double-sided stimulus were linearly proportional to that evoked with the corresponding single-sided stimulus of the same contrast .", "There were 5 runs per data point for the 6-s condition and 9 runs for the 1-s condition .", "For each of the visual areas V1-V3 , a linear function provides a good fit the data points [6 s: R2≥0 . 90; 1 s: R2≥0 . 85] , and the intercepts are not significantly different from zero [nested model comparison for each visual area; 6 s: F ( 1 , 3 ) <0 . 37 , p>0 . 59; 1 s: F ( 1 , 3 ) <0 . 27 , p>0 . 64] .", "( B ) Equivalently , contrast had no significant effect on the ratio of BOLD amplitude of a double-sided condition to that of the corresponding single-sided condition [nested model comparison for each visual area , 6 s: F ( 1 , 3 ) <0 . 53 , p>0 . 52; 1 s: F ( 1 , 3 ) <0 . 16 , p>0 . 72] .", "Performing a one-way ANOVA on the BOLD ratios obtained from individual runs also failed to find any significant effect of contrast in any of the visual areas [6 s: F ( 4 , 20 ) <0 . 37 , p>0 . 8; 1 s: F ( 4 , 40 ) <1 . 76 p>0 . 15] .", "Assuming that the underlying neural responses to the double-sided stimuli is twice those of the corresponding single-sided stimuli , these results imply that the relationship between neural and fMRI BOLD responses follows power law: B = kZγ , where γ can be determined from the ratio of BOLD amplitude evoked by the double-sided stimuli to that evoked by the single-sided ones: γ = log2 ( B2 , i/B1 , i ) .", "The estimated values of γ from the BOLD ratios are 0 . 75 ( V1 ) , 0 . 54 ( V2 ) , 0 . 53 ( V3 ) from the 6 s presentation and 0 . 55 ( V1 ) , 0 . 48 ( V2 ) , 0 . 44 ( V3 ) from the 1 s presentation .", "These values are very close to those estimated using the stitching procedure ( Figure 4C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 01210 . 7554/eLife . 09600 . 013Figure 4—figure supplement 2 . Robustness of BOLD summation results .", "( A ) BOLD amplitudes of every voxel in each ROI ( V1-V3 ) as evolved by the two versions of the single-sided stimulus ( Stim A and B of Figure", "3 ) in the 6-s experiment .", "The voxels responded approximately equally to both versions of the stimulus , indicating that any imperfect homotopy and/or gaze control did not significantly affect the stimuli's ability to equally co-activate the voxels .", "Red dots represent voxels in the ROIs that were strongly responsive ( uncorrected p<0 . 001 ) to both versions of the stimulus .", "Reanalyzing the data using only these strongly responsive voxels yielded essentially the same results as in Figure 4 when all the voxels with the ROIs were used ( right vs . left column , respectively , of B–D . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 013 We found that for a stimulus duration of 6 s , γ = 0 . 76 [95% CI: 0 . 66 , 0 . 87] , 0 . 54 [0 . 48 , 0 . 62] , and 0 . 55 [0 . 46 , 0 . 67] for V1 , V2 , and V3 , respectively .", "If hemodynamics are essentially the same in V1-V3 , then the exponent γ will be the same across these areas .", "The exponents were indeed statistically indistinguishable between V2 and V3; however , the value for V1 was outside the 95% confidence intervals of the values for V2 and V3 and vice versa ( Figure 4C , left column ) .", "We believe that the measured γ value obtained in V1 was inflated towards unity ( linearity ) because the neural populations associated with the two pRFs are not truly co-localized in V1 ( see Discussion ) .", "However , the assumption of co-localization is needed for inferring the BvZ function from the summation experiment .", "Given the good agreement in the γ value between V2 and V3 , the true value of γ in V1 is likely close to those estimated from V2 and V3 .", "We thus combine the measurements from areas V2 and V3 to obtain the BvZ function with an exponent equal to 0 . 54 ± 0 . 05 for the 6-s stimulus .", "We believe this BvZ function is sufficiently general and applicable for at least the occipital lobe .", "Changing stimulus duration changes the time course of the evoked neural response .", "If the BvZ function describes a general relationship between the neural response and fMRI BOLD signal , then the value of γ should be relatively constant across stimulus durations .", "This is indeed the case .", "Reducing the stimulus duration from 6 s to 1 s resulted in only small changes to the exponent of the BvZ function , with γ = 0 . 56 [0 . 51 , 0 . 62] , 0 . 49 [0 . 44 , 0 . 54] , and 0 . 43 [0 . 37 , 0 . 50] for V1 , V2 , and V3 , respectively ( Figure 4 , right column ) .", "As with the 6-s stimulus , γ estimated from V1 is again significantly higher that those from V2 and V3 , while those from V2 and V3 are not significantly different from each other .", "Combining data from V2 and V3 yield γ = 0 . 47 ± 0 . 03 .", "Hence , a sixfold change in stimulus duration resulted in very little change in the nonlinearity .", "Combining data from the two stimulus durations , we have γ ~ 0 . 5 .", "Spike rate is one of the most common measures of neural response , and the BOLD response has been related to spike rate ( Heeger et al . , 2000; Heeger and Ress , 2002; Logothetis and Wandell , 2004 ) .", "To cross-validate our finding and to make contact with the broader literature , we used the inferred BvZ function ( with γ inferred from V2 and V3 ) to estimate the neural response Z from the BOLD amplitude data of the single-sided conditions in the BOLD summation experiment , which were typical contrast response measurements .", "The inferred neural activity in V1 for both the 6-s and 1-s stimuli matched extremely well with the average primate V1 contrast response function measured in terms of single-unit spiking activity by Albrecht ( 1995 ) ( Figure 5A ) .", "Contrary to earlier reports based on the same single-unit data ( Heeger et al . , 2000 ) , linearly scaling our BOLD amplitude data does not fit the single-unit spiking data .", "The nonlinearity in our data cannot be attributed to anticipatory and other endogenous responses that might be induced by the task structure ( Sirotin and Das , 2009 ) ( Figure 3—figure supplement 3 ) .", "This is because our subject was engaged in a demanding central fixation task ( orientation discrimination ) that was asynchronous with the blocked contrast stimuli . 10 . 7554/eLife . 09600 . 014Figure 5 . Comparisons between neural response inferred from the BvZ function ( B = kZγ ) and single-unit spiking activity .", "( A ) Neural contrast response functions .", "The black dots and line are the average single-unit firing rate as a function of luminance contrast recorded in macaque V1 ( replotted from Albrecht , 1995 ) and the best fitting Naka-Rushton function ( Naka and Rushton , 1966 ) , respectively .", "Red open and filled circles represent the BvZ-inferred neural contrast responses in V1 of the achiasmic subject , computed from our 6-s and 1-s single-sided BOLD data sets ( from Figure 3 and Figure 3—figure supplement", "1 ) and matched to single-unit firing rates with a single scaling constant to align the data point at contrast = 1 .", "Gray open and filled triangles represent the linearly scaled BOLD contrast responses from the same data sets .", "The BvZ-inferred neural responses are in excellent agreement with the single-unit spiking data , whereas linearly scaled BOLD responses are not .", "( B ) fMRI BOLD contrast responses from 21 published data sets ( a-u; see Table", "1 ) were individually matched to the single-unit spiking responses function of ( A ) , assuming a power-law function to convert BOLD response to spike rate .", "The fits were good , with R2 ranging from 0 . 73 to 0 . 99 .", "The distribution of the best-fitting exponents ( γ ) is shown in ( C ) .", "The median of the distribution ( 0 . 48 ) is close to the exponent ( 0 . 5 , red dashed vertical line ) of the BvZ function inferred from the achiasmic subject .", "Asterisk ( * ) marks studies in which subjects attended to the stimuli used to obtain the contrast response functions .", "Underline ( _ ) marks studies that measured and analytically discounted any task-related baseline response from the contrast response functions .", "( D , E )", "An otherwise identical analysis as in ( B , C ) but using the single-unit ( spike rate ) contrast response function obtained from Heeger et al . ( 2000 ) instead of the single-unit contrast responses function used in ( A ) from Abrecht ( 1995 ) .", "( Note that some data points are out of the ordinate range in B and D; they are omitted from the plots . ) DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 014 The BvZ function obtained from our achiasmic subject , with γ~0 . 5 , appears to represent the typical neurovascular coupling in humans .", "We arrived at this conclusion by considering 21 datasets from 12 published studies ( Table", "1 ) on the contrast response function measured with fMRI BOLD in human V1 .", "We assume the BvZ function to follow a power-law relationship , as we have found with the achiasmic subject , but allowed the exponent ( γ ) to be a free parameter .", "γ = 1 represents a linear relationship between BOLD response and spike rate .", "We used two single-unit contrast response functions ( spike rate vs . contrast ) for this meta-analysis .", "One of the functions ( Figure 5B ) was from Albrecht ( 1995 ) , which was an average of 19 monkey V1 neurons , each tested with their respective optimal stimuli .", "The other ( Figure 5D ) was from Heeger et al . ( 2000 ) , which was averages of the predicted response ( Geisler and Albrecht , 1997 ) of over 300 neurons to the specific stimulus used in Boynton et al . ( 1999 ) ( Geisler , personal communication ) .", "For each BOLD data set , we estimated the value of γ such that the resulting BvZ function , when applied to a single-unit contrast response function , resulted in a predicted BOLD response that best matched the BOLD data set in terms of an error-weighted least-squares fit .", "With respect to the two neuronal contrast response functions , the bulk of the distribution of γ lies mid-range between 0 and 1 ( Figure 5B–E ) .", "The overall picture given by this set of nearly two dozens experiments is that BOLD response is nonlinearly related to spike rate .", "The interquartile range of both distributions includes γ ~ 0 . 5 , the exponent of the BvZ function independently estimated from our pure BOLD summation experiments with subject S . In fact , γ ~ 0 . 5 is near the median of one distribution ( Figure 5C ) and the mode of the other distribution ( Figure 5E ) . 10 . 7554/eLife . 09600 . 015Table 1 . Data sources of Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 09600 . 015Legend Publication Figure # ( Condition ) Subjects γ ( Figure 5B ) γ ( Figure 5D ) R2 ( Figure 5B ) R2 ( Figure 5D ) aPestilli et al . , 2011 Figure 4B ( Focal Cue – non Target ) Human0 . 500 . 640 . 770 . 86b*Pestilli et al . , 2011 Figure 4B ( Focal Cue - Target ) Human0 . 150 . 210 . 930 . 94c*Pestilli et al . , 2011 Figure 4B ( Distributed Cue –Target ) Human0 . 240 . 340 . 790 . 88d*Pestilli et al . , 2011 Figure 4B ( Distributed Cue – non Target ) Human0 . 250 . 370 . 850 . 93e*Avidan et al . , 2002 Figure 2B ( Faces ) Human0 . 610 . 720 . 960 . 93f*Avidan et al . , 2002 Figure 2B ( Objects ) Human1 . 061 . 240 . 910 . 89g*Buracas & Boynton 2007 Figure 1Human0 . 320 . 400 . 930 . 98h*Boynton et al . , 1999 Figure 3 and Figure 4Human0 . 640 . 840 . 970 . 99iSchumacher et al . , 2011 Figure 4A ( gradient echo ) Human0 . 280 . 380 . 730 . 85jSchumacher et al . , 2011 Figure 4A ( spin echo ) Human0 . 280 . 360 . 860 . 93kSchumacher et al . , 2011 Figure 4C ( gradient echo ) Human0 . 560 . 660 . 970 . 98lSchumacher et al . , 2011 Figure 4C ( spin echo ) Human0 . 400 . 500 . 910 . 96m Li et al . , 2008 Figure 3 ( Unattended ) Human0 . 650 . 750 . 990 . 96n* Li et al . , 2008 Figure 3 ( Attended ) Human0 . 260 . 360 . 910 . 85oMoradi & Heeger 2009 Figure 2Human0 . 250 . 370 . 860 . 83p*Olman et al . , 2004 Figure 4A ( Natural Images ) Human0 . 340 . 500 . 970 . 97q*Olman et al . , 2004 Figure 4A ( Whitened Images ) Human0 . 480 . 680 . 990 . 97rPark et al . , 2008 Figure 9Human0 . 610 . 700 . 980 . 94sTootell et al . , 1998 Figure 5Human0 . 710 . 870 . 890 . 94t*Zenger-Landolt & Heeger 2003 Figure 9 ( Without Surround ) Human0 . 750 . 830 . 850 . 93uLogothetis et al . , 2001 Figure 5B ( BOLD ) Monkey0 . 650 . 680 . 750 . 88Asterisk ( * ) marks indicate studies in which subjects attended to the stimuli used to obtain the contrast response functions . Underline ( _ ) marks indicate studies that measured and analytically discounted any task-related baseline response from the contrast response functions ." ], [ "The quantitative relationship between the fMRI BOLD signal and neural response depends on the interacting hemodynamic quantities that are linked to neuronal activity: cerebral blood flow ( CBF ) , cerebral blood volume ( CBV ) and cerebral metabolism rate of oxygen ( CMRO2 ) ( Davis et al . , 1998; Hoge et al . , 1999; Logothetis et al . , 2001; Thompson et al . , 2003; Mukamel et al . , 2005 ) .", "It also depends on the origin of the MR signal ( e . g . , intra- vs . inter-vascular space , capillaries vs . pial vessels ) , which in turn depends on the pulse sequence and magnetic field strength used to make the measurement ( Griffeth and Buxton , 2011 ) .", "The BOLD signal is inherently measurement-dependent , and there is not a 'canonical' BOLD signal .", "For the common applications of fMRI in human neuroscience , it is arguably more important to understand and quantify at the net-effect level , rather than component-level , the relationship between the measured BOLD signal and the evoked neural response .", "A common approach for quantifying the neural-BOLD relationship is to manipulate a stimulus and measure the evoked electrophysiological and hemodynamic responses either simultaneously ( Brinker et al . , 1999; Ngai et al . , 1999; Logothetis et al . , 2001; Devor et al . , 2003; Sheth et al . , 2004; Hoffmeyer et al . , 2007; Huttunen et al . , 2008; Magri et al . , 2011 ) or in separate experiments ( Hewson-Stoate et al . , 2005; Nangini et al . , 2008; Liu et al . , 2010 ) .", "The hemodynamic signal measured can be either the fMRI BOLD signal itself or its physiological components ( CBF , CBV , CMRO2 ) .", "Several studies suggested that the BOLD signal or one of its components was linearly correlated with neuronal firing rates ( Smith et al . , 2002 ) , synchronized synaptic activity as manifested in local field potentials ( LFP ) ( Ngai et al . , 1999; Logothetis et al . , 2001; Martindale et al . , 2003 ) , electroencephalographic ( EEG ) ( Brinker et al . , 1999; Arthurs et al . , 2000 ) , magnetoencephalographic ( MEG ) ( Ou et al . , 2009 ) , or intracranial electrocorticography ( ECoG ) ( Siero et al . , 2013 ) activities .", "However , some of these studies ( Ngai et al . , 1999; Nangini et al . , 2008 ) may not have had sufficient statistical power for detecting departures from linearity .", "Other studies had a limited stimulation range , lacking measurements near the response threshold ( Logothetis et al . , 2001 ) , or limited sampling points within a range ( Smith et al . , 2002 ) .", "As a result , their findings of linearity may not be general .", "More importantly , a majority of the linear fits ( Ngai et al . , 1999; Logothetis et al . , 2001; Schumacher et al . , 2011 ) had a significant non-zero intersect , which would require a paradoxically non-zero change in the BOLD signal when the evoked neural response is zero .", "Absent of other factors , such a non-zero intersect implies that the underlying response function is actually nonlinear .", "Other studies have suggested that the relationship between the BOLD signal and neural activity is nonlinear ( Devor et al . , 2003; Sheth et al . , 2004; Hewson-Stoate et al . , 2005; Hoffmeyer et al . , 2007; de Zwart et al . , 2009; Liu et al . , 2010; Magri et al . , 2011; Kay et al . , 2013 ) .", "Magri et al . ( 2011 ) , for example , found that the visually evoked fMRI BOLD responses as a function of band-limited neural signals ( LFP and MUA ) are sublinear .", "Others ( Devor et al . , 2003; Sheth et al . , 2004; Hewson-Stoate et al . , 2005 ) reported supra-linear relationships between neural activity and hemodynamic responses measured with intrinsic signal optical imaging and spectroscopy , or equivalently a linear relationship between the luminance contrast of a stimulus and the optical signal ( Lu and Roe , 2007 ) ( since the relationship between luminance contrast and neural activity is generally sublinear ) .", "This discrepancy could be due to the nonlinear relationships between the BOLD signal and its physiological components ( CBF , CBV , and CMRO2 ) that were measured .", "Biophysical models relating the BOLD response to its constituents ( Buxton et al . , 1998; Davis et al . , 1998; Hoge et al . , 1999; Griffeth and Buxton , 2011 ) , which are generally nonlinear , are needed to resolve this discrepancy .", "Several studies ( Huettel and McCarthy , 2000; Liu et al . , 2010 ) used temporal summation to investigate the relationship between neural response and the BOLD signal .", "This is a popular non-invasive approach .", "The results , however , can be confounded with neural adaptation ( Huettel and McCarthy , 2000 ) .", "To avoid neural adaptation , the summation stimuli must be temporally separated with a sufficiently large lag time , thus precluding these methods from gauging the true ( simultaneous ) response nonlinearity .", "Our approach is unique in that it allows a simple additive mixing of neural response across any time interval , including zero lag , to make a net-effect level quantification of the underlying relationship between the measured fMRI BOLD signal and the evoked neural response .", "Our method bypasses any nonlinearity between the stimulus and neural response .", "It also sidesteps the need to define the constituents of neural response ( e . g . , spiking activity , different frequency bands of LFP ) .", "These unique abilities stem from the configuration of the visual cortex in human achiasma: that there are two identical but independent populations of neurons that are co-localized on the cortex , sharing the same local control of blood supply .", "Each of these neuronal populations is associated with a distinct population receptive field .", "If stimulating one population results in some level of neural response , then for most reasonable definitions of 'neural response' , stimulating both populations by placing identical stimuli in each of the two population receptive fields will double the local neural response ( because two populations of neurons , instead of just one population , are responding ) .", "This notion of 'neural response' does not differentiate the specific components of neural response , such as spikes or LFP , which are themselves related in a complex manner .", "Rather , it is a holistic quantity ( Z ) that describe the totality of neural response , a notion that is perhaps more useful for most cognitive neuroscience investigations using fMRI .", "The fact that we succeeded in using the inferred BvZ functions to predict BOLD contrast-response functions from single-unit contrast-dependent spiking activity demonstrates the validity of our approach .", "While these results may suggest that Z is more related to spikes than to other neural response components nonlinearly related to spikes , more studies will be needed to quantify the link between the totality of neural response of a neural population and its individual components .", "The balance of the components that make up Z ( e . g . , spikes , LFP's ) may depend on the stimulus , brain area , and task .", "We found that each voxel in the low-level visual cortex of the achiasmic subject S has two non-interacting population receptive fields ( pRFs ) .", "The empirical evidence we reported here suggests that there are two independent neuronal populations co-localized in each voxel of V1-V3 that subserve these two population receptive fields .", "Williams et al . ( 1994 ) found that in achiasmic Belgian sheepdogs , retinal axons originating from the nasal hemi-retina , which would normally cross the midline , instead innervated ipsilateral LGN and occupied those layers that would otherwise receive inputs from the contralateral eye .", "Alternating layers of the LGN in achiasma thus form mirror-image maps of the visual field .", "If axon guidance between LGN and V1 during development is typical in achiasma , then what would develop into the ocular dominance columns of V1 in a normal visual system will instead become visual-field dominance columns in achiasma , as suggested by Victor et al . ( 2000 ) .", "Preliminary measurements with 7T fMRI showed results consistent with the presence of visual-field dominance columns in V1 ( Olman et al . , 2014 ) .", "We expect the organization of visual-field dominance columns to be mostly within Layer 4 of V1 , which receives direct hemifield-segregated inputs from LGN ( Williams et al . , 1994 ) .", "Downstream from Layer 4 , we expect this columnar organization to dissolve into finely intermingled but non-interacting unilateral ( and monocular ) neurons , for the following reason .", "In a normally developed visual system , binocular neurons receive inputs from monocular neurons in the neighboring ocular dominance columns .", "However , in achiasma , such 'binocular' neurons would stay monocular and unilateral .", "This is because the inputs from neighboring visual-field dominance columns would not correlate , as they represent unrelated locations in the left and right visual fields .", "Neurons that received inputs from neighboring columns would likely suppress or eliminate the inputs from one of the visual fields , chosen at random and without preference , since the inputs from the two visual fields are of equal strength ( Figure 1—figure supplement 1 , Figure 3B , Figure 3—figure supplement 1 , Figure 4—figure supplement 2 ) .", "Hence , downstream from Layer 4 of V1 , neurons that represented different hemifields of the visual space would be functionally independent but finely intermingled .", "In other words , we speculate a coarse segregation , at the scale of the ocular dominance columns , of the unilateral neuronal populations in Layer 4 of V1 .", "The intermixing of the unilateral neurons would become finer downstream from V1 Layer 4 , starting with neurons in the superficial and deep layers of V1 .", "This may explain why the γ value of the BvZ function observed in V1 was higher than those found in V2 and V3 – the coarsely segregated neuronal populations in V1 Layer 4 , which would not fully share the local vascular system , lead to a more linear summation ( higher value of γ ) .", "In V1 , Layer 4 tends to have a larger contribution to the measured BOLD signal because of its dense vasculature but contributions from superficial and deep layers are also significant ( Polimeni et al . , 2010 ) .", "We postulate that neurons representing the two hemifields would be more intermingled in the superficial and deep layers than those in Layer 4 .", "These neurons would likely be more complex and less linear in their response properties to visual stimulation than neurons in Layer 4 ( Movshon et al . , 1978a , b ) .", "Their contribution to the BOLD signal relative to those in Layer 4 may be significantly reduced with long-duration simulation because of adaptation .", "The summation function thus appeared more linear in the 6-s condition than in the 1-s condition .", "It is important to point out that the summation function cannot be used to infer the BvZ function if the conditions of co-localization and non-interaction are not met .", "The condition of co-localization was not met in V1 , and we could not use the summation function of V1 to infer its BvZ function .", "We do not think that the relationship between neural and BOLD responses depends significantly on stimulus duration .", "The fact that the BvZ functions inferred from V2 and V3 , where the co-localization condition was met , provides an excellent fit to the V1 single-unit data ( Figure 5A ) further suggests that the inferred BvZ function is applicable in at least V1-V3 ." ], [ "We have shown that the unique organization of the low-level visual cortex in human achiasma provides a versatile in vivo model for non-invasive quantification of the relationship between the evoked neural and fMRI BOLD responses .", "The presence of two independent neuronal populations with non-overlapping receptive fields at the same cortical location allows for the independent control of two separate sources of the local neural activity via stimulus presentation .", "By measuring the fMRI BOLD responses associated with a doubling of the local neural response , we found that the amplitude of the evoked BOLD response is proportional to the sum-total of the evoked neural response raised to a power around 0 . 5 ." ], [ "The achiasmic subject detected a Gabor target on a calibrated and gamma-linearized CRT display using only his right eye .", "The display had 10 bits of luminance resolution after gamma linearization by means of an analog video attenuator ( Li et al . , 2003 ) and custom software ( https://github . com/usc-tlab/LinearFineContrast . git ) .", "In separate blocks of trials , the target was presented in the upper-left or lower-right quadrant against a uniform gray field with a luminance of 30 . 4 cd/m2 .", "A high contrast ( Weber contrast = 1 . 0 ) task-irrelevant flickering checkerboard was displayed at a mirror-symmetric location from the target , either about the vertical meridian , such that the target and the flickering checkerboard would activate the same set of cortical regions , or about the horizontal meridian , such that the target and checkerboard would activate two different sets of cortical regions .", "The target was a 45° oriented Gabor with a center spatial frequency of 1 cycle/degree and a space constant ( 2σ ) of 1 . 4° .", "The task-irrelevant flickering checkerboard was of size 10° by 10° with 0 . 5° by 0 . 5° checks and flickered at 4 Hz in a square pattern .", "A two-interval-alternative-forced-choice paradigm ( judging which interval contained the target ) , controlled with the adaptive procedure QUEST ( Watson and Pelli , 1983 ) , was used to measure the contrast threshold for a 75% correct detection rate .", "Each trial consisted of two temporal intervals ( target-present and target-absent in random order ) separated by a 500 ms blank .", "In each interval , the flickering checkerboard appeared for its entire duration of 600 ms . The Gabor target appeared for the last 150 ms of the target-present interval .", "During both intervals , a beep was presented 450 ms after the interval onset to reduce temporal uncertainty about target .", "The subject identified the target-present interval with a button press .", "A new trial began 500 ms after the subject had responded .", "Feedback was given after each response .", "For each target location , the subject completed 4 blocks of 50 trials .", "The fMRI data were collected using a 3-Tesla Siemens TIM Trio scanner with a 32-channel head coil using a T2*-weighted echo planar imaging sequence ( TE/TR/flip angle = 25 ms/1 s/60° ) .", "19 slices with 3x3x3 mm3 isotropic voxels were prescribed to be perpendicular to the calcarine sulcus , covering the occipital pole .", "A high-resolution T1-weighted anatomical data set ( 3D MPRAGE; 1x1x1 mm3 isotropic voxels , TE/TR/flip angle/TI = 2 . 98 ms/2300 ms/9°/900 ms ) was collected in the same session before the functional runs and used to assist prescription of the functional slices .", "Eye movement was monitored online using an MRI compatible ASL 504 eye tracker with long-range optics .", "We analyzed the recording to estimate nystagmus amplitude and frequency during tasks and to determine if there were any stimulus-dependent biases in fixation .", "Additional offline measurements of eye movements were performed with a EyeLink 1000 Tower Mount monocular eye tracker .", "Data analysis was performed using BrainVoyager QX ( Brain Innovation , Maastricht , The Netherlands ) and custom-built Matlab ( MathWorks , Massachusetts , USA ) code .", "The anatomical volume obtained in the retinotopic mapping session was segmented and inflated to generate a model of the cortical surface .", "Functional volumes were preprocessed , which included 3D motion correction , linear trend removal , and high-pass ( > 0 . 0118 Hz ) filtering .", "The functional images were aligned to one another and to the anatomical volume .", "The first 15–22 s of each scan ( retinotopy experiment: 15 s , long-duration adaptation experiment: 22 s , BOLD summation experiments: 22 s for 6-s presentation and 17 s for 1-s presentation ) were discarded to ensure equilibrium in longitudinal magnetization and subject state .", "Rotating wedges and expanding half-rings made up of flickering ( 4 Hz ) radially scaled color checker-board patterns , presented to one eye at a time , were used to identify the retinotopic visual areas in subject S . For polar angle mapping , a 45° wedge with a radius of 8 . 5° rotated ( jumped ) counterclockwise by 11 . 25° every second , so that it swept the whole visual field in 32 s .", "For eccentricity mapping , the half rings were presented in the subject’s right or left visual field in alternating blocks .", "They expanded in equal logarithmic steps from the center of display , where the subject fixated , and took 20 s to reach the maximum radius of 8 . 5° .", "The fixation of the display changed from ‘+’ to ‘×’ or vice versa randomly between 5 and 10 s ( uniform distribution ) ; the subject pressed a button as soon as a change of the fixation mark was detected .", "A common set of regions of interest ( ROIs ) in V1-V3 was used to analyze data from both the adaptation and BOLD summation experiments .", "The stimuli used for the adaptation experiments ( Gabor patches , see next subsection ) , set at 100% contrast , were also used to define these ROIs during scans independent of the main experiments .", "The stimuli consisted of four Gabor patches arranged in configurations resembling a forward or backward slash ( Figure 2C ) .", "Each scan consisted of 13 fixation-only blocks interleaved with 12 stimulus blocks .", "The fixation-only blocks lasted for 16 s and the stimulus blocks lasted for 6 s .", "One stimulus configuration was presented in each stimulus block , and configurations alternated between blocks .", "During the stimulus block , the Gabor patches counter-flickered at 1 Hz and alternated their orientations ( horizontal or vertical ) at 0 . 5 Hz .", "The subject attended the fixation mark to perform a change-detection task at fixation .", "A general linear model ( GLM ) assuming a stereotypical hemodynamic response function was used to identify the ROIs .", "The ROIs for backward- and forward-slash arrangements were defined separately , corresponding to the areas that responded more strongly to the ROI-defining stimulus than the blank interval ( False Discovery Rate ( FDR ) < 0 . 05 ) and were within areas V1-V3 at the expected eccentricity ( based on retinotopy scans ) .", "Since the two sets of ROIs ( one for each stimulus arrangement ) were very similar ( Figure 1—figure supplement 1 ) , we defined the stimulus response areas as the overlap of the two sets at FDR < 0 . 05 .", "Because subject S has a nystagmus ( horizontal amplitude generally less than ± 2 . 1° , peak-to-peak ) , we further restricted the ROIs to include , based on retinotopic coordinates , only the responsive voxels that responded to the central 2° of the two outer patches , which were 4° in diameter and centered at an eccentricity of 7° .", "The restricted stimulus response areas were further partitioned into ROIs in V1-V3 based on the subject’s retinotopy .", "The adaptor and test stimuli consisted of four luminance-contrast defined Gabor patches in two spatial configurations , resembling either a backward ( upper-left to lower-right ) slash or a forward ( upper-right to lower-left ) slash ( Figure 2C ) .", "The two outer Gabor patches were at an eccentricity of 7° , with a carrier frequency of 1 cycle/degree and a space constant 2σ of 1 . 4° .", "The two inner Gabor patches were centered at an eccentricity of 3 . 5° , with a carrier frequency of 2 cycles/degree and a space constant of 0 . 7° .", "The Weber contrast of the carrier was 1 . 0 against a gray background of 156 cd/m2 .", "The carrier of all four Gabor patches could be oriented either horizontally or vertically .", "Across different scans , the adaptor stimulus could be in one of four possible conditions: either in a forward- or backward-slash configuration , and oriented either horizontally or vertically .", "Relative to those of the adaptor stimulus , the four Gabor patches of the test stimulus were presented either at the same or different ( mirrored ) spatial location , with either the same or orthogonal orientation .", "There were 16 scans , with each scan consisting of one adaptor stimulus and 51 trials .", "A scan began with 20 s of pre-adaptation .", "Each trial started with a 5 s top-up adaptation period , followed by one of the five event types: four types of test stimulus ( 2 configurations by 2 orientations ) or a blank interval .", "In a test trial , after 0 . 4 s of a blank interval following the top-up adaptation period , the test stimulus was presented for 0 . 3 s , followed by a 0 . 3-s blank .", "In a blank trial , no stimulus was presented for 1 s after top-up adaptation .", "The central fixation mark was present for all trial types ( including the blank trial ) .", "At random intervals uniformly distributed between 3 and 7 s , the fixation mark randomly changed from ‘+’ to ‘×’ for about 400 ms before returning to ‘+’ .", "The subject was instructed to attend to the fixation mark throughout a scan and press a key as soon as he detected a change of the fixation mark .", "The trial types were counterbalanced to ensure that the frequency of the immediately preceding trial type was equal , and the same for each of the 5 trial types .", "For each experimental scan , the fMRI voxel data were % -transformed ( y ( t ) −y¯/y¯×100% ) and averaged within each ROI .", "ROI-averaged data were concatenated across scans , and the results were deconvolved against an indicator function formed by placing a Dirac delta function at the onset of the stimulus interval of the same trial type .", "Motion correction parameters were entered as regressors of no interest in the analysis to capture the noise introduced by head motion .", "The deconvolution analysis resulted in one response time course for each non-blank trial type , depicting the BOLD response evoked by the test stimulus .", "The BOLD summation experiment had three types of stimuli .", "Stimulus types A and B each consisted of 4 circular patches of black-and-white checkerboard arranged in either a backward-slash ( A ) or a forward-slash ( B ) configuration .", "For stimulus type A+B , the stimuli of type A and B were presented simultaneously , resulting in 8 checkerboard patches on the display .", "All patches in a stimulus were of the same contrast .", "We tested five Weber contrast levels from 0 . 05 to 1 . 0 in equal logarithmic steps against a uniform gray field of 156 cd/m2 .", "The outer patches were centered at an eccentricity of 7° and had radius 2° .", "The inner patches were centered at an eccentricity of 3 . 5° and had radius 1° .", "Each scan tested a specific contrast level .", "Within a scan , 19 stimulus blocks of 6 s each ( or 1 s in a separate experiment ) were interleaved with 19 16-s blank blocks .", "For each stimulus block , a stimulus of given type ( A , B , or A+B ) was presented , counter-flickering at 2 Hz .", "For each scan , the stimulus blocks were arranged in an otherwise random sequence with the constraint that each of the three stimulus types was preceded by every stimulus type equally often .", "To encourage and maintain fixation throughout a scan , the central fixation mark changed from ‘+’ to ‘×’ for about 400ms and back at random intervals , uniformly distributed between 3 s and 7 s .", "When the fixation was changed to ‘×’ from ‘+’ , it was further tilted by ± 5° .", "The subject was to attend the fixation mark and report the direction of tilt as soon as the ‘ ×’ appeared by pressing one of two response keys .", "The time course of the evoked BOLD response for each stimulus condition was inferred using deconvolution .", "For the experiment with the 6-s stimulus duration , the deconvolution procedure was essentially identical to that used for the long-duration adaptation experiment .", "For the experiment with the 1-s stimulus duration , the signal-to-noise ratio ( SNR ) of the data was low .", "To improve SNR , we used a GLM-based denoising method described in Kay et al . ( 2013 ) .", "The method assumes that physiological noise across measured voxels in the brain is correlated in the sense that it resides in a low-dimensional space .", "The method estimates this space in terms of its principle components from the voxels that are not driven by the stimulus .", "These components are then entered into the GLM design matrix for a given experiment as regressors of no interest in order to estimate and reduce the influence of noise from each voxel .", "To ensure that this method does not introduce confounds , we further tested this method using data from the 6-s experiment , which are of high SNR , and found that results were essentially the same as the ones obtained with the generic deconvolution method ( Figure 3—figure supplement 2 ) .", "Given n pairs of BOLD response amplitudes B1 , i , B2 , i , i=1…n , corresponding to the n ( n=5 contrast levels in our experiment ) of single-sided and double-sided measurements , we sought to estimate n-1 parameters , 1≤Z2≤…≤Zn , such that the 2n points { ( 1 , B1 , 1 ) , ( 2 , B2 , 1 ) , ( Z2 , B1 , 2 ) , ( 2Z2 , B2 , 2 ) , … , ( Zn , B1 , n ) , ( 2Zn , B2 , n ) }are maximally consistent , in the least squares sense , with a monotonic and smooth function .", "The multiplier 2 represents our assumption that the double-sided condition generated twice the neural response of the corresponding single-sided condition .", "In our case , the candidate monotonic and smooth function turned out to be a general cubic function restricted to be monotonically ascending .", "This is based on the following consideration .", "To be model-neutral , we first considered the entire family of cubic spline functions with k control points ( k≥2 ) that are constrained to be monotonic .", "A cubic spline function with k control points has 2k degrees of freedom .", "We iteratively estimated the n-1 values of Zi and the 2k parameters of the cubic spline function by minimizing the normalized chi-square , which is the sum of squared errors between the 2n points and the cubic spline function , divided by the degrees of freedom of the residual .", "The degrees of freedom of the residual are n+1−2k , and k has to be at least 2 .", "In our case , n=5 , meaning that the residual would have no degrees of freedom for k>2 .", "As a result , we considered only the family of cubic spline functions with two control points , situated at ( 1 , B1 , 1 ) and ( 2Z5 , B2 , 5 ) , respectively .", "The parameter estimation started with an arbitrary choice of 1≤Z2 , … , ≤Zn , which was iteratively optimized by using the fminsearch function of MATLAB ( The MathWorks ) .", "In the inner loop , we used the SLM MATLAB toolbox by John D’Errico ( D'Errico , 2009 ) to fit a monotonic cubic spline function to the 2n points { ( 1 , B1 , 1 ) , ( 2 , B2 , 1 ) , ( Z2 , B1 , 2 ) , ( 2Z2 , B2 , 2 ) , … , ( Zn , B1 , n ) , ( 2Zn , B2 , n ) } .", "We computed normalized chi-square , which we used as the objective function for fminsearch to choose the next set of Zi until convergence ." ] ]
[ "Achiasma in humans causes gross mis-wiring of the retinal-fugal projection , resulting in overlapped cortical representations of left and right visual hemifields .", "We show that in areas V1-V3 this overlap is due to two co-located but non-interacting populations of neurons , each with a receptive field serving only one hemifield .", "Importantly , the two populations share the same local vascular control , resulting in a unique organization useful for quantifying the relationship between neural and fMRI BOLD responses without direct measurement of neural activity .", "Specifically , we can non-invasively double local neural responses by stimulating both neuronal populations with identical stimuli presented symmetrically across the vertical meridian to both visual hemifields , versus one population by stimulating in one hemifield .", "Measurements from a series of such doubling experiments show that the amplitude of BOLD response is proportional to approximately 0 . 5 power of the underlying neural response .", "Reanalyzing published data shows that this inferred relationship is general ." ]
[ "When a part of the brain becomes active , more oxygen-rich blood flows to it to keep its neurons supplied with energy .", "This flow of blood can be measured using a technique called functional magnetic resonance imaging ( fMRI ) .", "Yet , it was not known exactly how the magnitude of the signal recorded from the oxygenated blood flow – dubbed the BOLD ( blood oxygenation level dependent ) signal – relates to the level of neural activity .", "In most people , the brain area that processes fundamental visual information – called the visual cortex – receives signals from both eyes , sent via the optic nerves .", "The two eyes’ optic nerves are bridged together with a structure called the optic chiasm , which ensures that each side of the brain gets input from both eyes for one side of the visual field .", "However , in rare cases , a person may lack an optic chiasm , and instead each side of the brain processes information about both sides of the visual field seen by one eye .", "This condition is known as achiasma .", "Bao et al . have now used fMRI and behavioral experiments to study the brain activity of a volunteer who lacks an optic chiasm .", "This revealed that each half of the visual field stimulates different neurons in the same brain hemisphere of an achiasmic visual cortex .", "The two sets of neurons do not interact with each other , but they do share the same local blood supply .", "Moreover , these sets of neurons are organized in such a way as to preserve normal vision , and can be controlled independently using visual stimulation .", "If both sets of neurons are stimulated with the same visual input at the same time , they together trigger twice as much neural activity as when just one set is stimulated .", "This also causes an increased BOLD signal as more blood flows to that region of the brain .", "Bao et al . were therefore able to infer a mathematical relationship between neural activity and the BOLD signal .", "This revealed that the magnitude of the BOLD signal is proportional to the square root of the underlying neural activity .", "Reanalyzing previously published BOLD data from other fMRI studies of healthy humans and monkeys supports this conclusion .", "Bao et al . ’s study provides scientists with a human model for noninvasively studying the origins and neural underpinnings of fMRI measurements , which may change how we analyze and interpret brain-imaging results in the future .", "The biggest challenge that researchers will likely face is in recruiting individuals with this rare condition of achiasma ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes
elife-20417-v2
[ [ "The currently accepted view that plasma membranes of eukaryotic cells are in a state of flux due to rapid internalization and recycling of membrane proteins received its first experimental support in a prescient 1976 paper by Ralph Steinman and his colleagues ( Steinman et al . , 1976 ) .", "Steinman ( later awarded a Nobel Prize for the discovery of dendritic cells ) observed that a surface area equivalent to the entire plasma membrane was internalized every 2 hr in L-cells , which implied a rate of turnover for the bulk plasma membrane much faster than that of individual membrane proteins known at that time .", "His non-intuitive conclusion was that the majority of internalized membrane was returned to the cell surface in the form of small vesicles: “A plausible morphologic mechanism for membrane recycling is that it involves the production of tiny vesicles capable of returning large amounts of surface membrane , with very little content , to the cell surface” Steinman’s conjecture of membrane recycling through small vesicles was soon validated with the discovery of receptor-mediated endocytosis and of the return of endosomal receptors to the plasma membrane following separation from ligands ( Pearse and Bretseher , 1981 ) .", "Membrane recycling is now recognized as a fundamental property of nucleated cells required for diverse functions , including cell migration , cytokinesis , receptor signaling , and synaptic transmission .", "Much progress has been made in elucidating the molecular components required for the highly selective sorting and trafficking of Steinman’s 'tiny vesicles' , now termed endosomes .", "These include identification of numerous small GTPases , as well as of phosphoinositide lipids , together with their effectors and regulators , which collaborate to determine the endosomal identity ( Balla , 2013; Jean and Kiger , 2012 ) .", "In addition , diverse molecular motors that promote both long-range and local endosomal transport have been identified ( Granger et al . , 2014 ) .", "Despite these remarkable findings , it is not clear how the individual activities associated with endosomes are integrated to promote a highly regulated and precise delivery of particular membrane proteins to specific cellular locations in polarized cells .", "One plausible mechanism is through scaffolding proteins capable of simultaneously recruiting and modulating the activity of motor and signaling complexes at endosomal membranes .", "For instance , the molecular scaffolds JNK interacting proteins 1 and 3 ( JIP1 and JIP3 ) promote axonal transport through binding both protein kinases and the small G protein Arf6 on intracellular membranes .", "Moreover , JIP1 and JIP3 regulate kinesins directly and dynein indirectly through interaction with the dynactin complex ( Fu and Holzbaur , 2013 ) .", "The kinesin motor Kif16B provides another example , which , through direct binding of both PI3P lipids and the small G protein Rab14 , promotes the transport of FGFR2-endosomes to the fast-growing ends of microtubules during early embryonic development ( Hoepfner et al . , 2005; Ueno et al . , 2011 ) .", "AnkB is a member of the vertebrate ankyrin family of plasma membrane-organizing proteins that , in contrast to Ankyrin-G ( AnkG ) and Ankyrin-R ( AnkR ) , associates with intracellular organelles ( He et al . , 2013; Lorenzo et al . , 2014 ) .", "AnkB promotes axonal transport and growth through coupling dynactin to organelles containing PI3P lipids ( Lorenzo et al . , 2014 ) .", "AnkB is broadly expressed , suggesting it may coordinate organelle transport in multiple cellular contexts .", "Here , we report that AnkB , through binding to PI3P lipids , dynactin , and the Rab GTPase-regulating protein RabGAP1L , functions as a master integrator of endosomal transport , which promotes polarized trafficking of PI3P-positive endosomes bearing α5β1-integrin to the leading edge of migrating fibroblasts ." ], [ "AnkB promotes fast axonal transport by coupling the motor adaptor dynactin to PI3P-positive organelles in neurons ( Lorenzo et al . , 2014 ) .", "Therefore , we hypothesized that AnkB also contributes to the long-range transport of PI3P-positive organelles in other cell types .", "We selected primary cultures of mouse embryonic fibroblasts ( MEFs ) because these cells express 220 kDa AnkB ( Figure 1A ) , and are a standard laboratory model that have been extensively studied with respect to organelle transport .", "We first examined the dynamics of AnkB-positive organelles by tracking the motility of vesicles expressing 220 kDa AnkB-mCherry in AnkB null ( Ank2-/- ) MEFs isolated from postnatal day 0 ( PND0 ) Ank2-/- mice ( Scotland et al . , 1998 ) .", "It is important to note that over-expression of 220 kDa AnkB is lethal for MEFs , and it was critical to express 220 kDa AnkB-mCherry in an AnkB null background .", "We observed that a subset of WT AnkB-mCherry-associated organelles exhibited fast long-range motility , with a net velocity greater than 4 µm/s and a persistence greater than 0 . 5 , which is consistent with long-range , fast microtubule-based transport ( Figure 1B ) . 10 . 7554/eLife . 20417 . 003Figure 1 . AnkB is a PI3P-lipid effector in MEFs .", "( A ) AnkB immunoblot ( IB ) of whole cell lysate from WT and Ank2-/- MEFs .", "( B ) Representative tracks of WT AnkB-mCherry vesicles in Ank2-/- MEFs and mean velocity and persistence of WT AnkB-mCherry and DD1320AA AnkB-mCherry vesicles .", "Scale bar , 10 µm .", "Tracks were plotted in an XY coordinate system assuming ( 0 , 0 ) as initial position .", "( C ) Molecular surface representation of the ZU5N-ZU5C-UPA-DD .", "The DD1320 site critical for binding to dynactin 4 is pointed by red arrow .", "Basic residues on the PI3P-binding surface are colored in yellow .", "The R1194 site critical for PI3P binding is circled and pointed in red .", "( D ) Images show the localization of the PI3P biosensor GFP-2×FYVE to WT AnkB-mCherry vesicles in Ank2-/- MEFs .", "R1194A AnkB-mCherry was found diffusely distributed in the cytoplasm .", "Scale bar , 10 µm .", "( E ) Percentage of double mCherry and GFP-positive vesicles .", "Data in ( B ) and ( E ) represent mean ± SD for three independent experiments .", "***p<0 . 001 , two-tailed t-test .", "N = 26 ( B ) , 12 ( E ) .", "( F–G )", "Quantitative analysis of localization of WT AnkB-mCherry ( F ) and GFP-2xFYVE ( G ) on different organelles .", "Results are expressed as the ratio of co-localized vesicles over either total AnkB-mCherry or GFP-2xFYVE vesicles .", "Data represent mean ± SD for three independent experiments .", "N = 10 , 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 00310 . 7554/eLife . 20417 . 004Figure 1—figure supplement 1 . Distribution of AnkB and PI3P lipids on multiple organelles .", "( A ) Representative tracks of DD1320AA AnkB-mCherry vesicles in Ank2-/- MEFs ( left ) .", "Scale bar , 10 µm .", "Tracks were plotted in an XY coordinate system assuming ( 0 , 0 ) as initial position ( right ) .", "( B and D )", "Representative images of live Ank2-/- MEFs showing the localization of WT AnkB-mCherry to different organelles ( B ) and Pearson's co-localization coefficient ( D ) .", "Organelle markers used include Rab5-GFP ( early endosomes ) , LAMP1-GFP ( lysosomes ) , Rab11-GFP ( recycling endosomes ) , Mito-GFP ( mitochondria ) , TGN38-GFP ( Golgi ) .", "Scale bar , 10 µm .", "( C and E )", "Representative images of fixed WT MEFs showing the localization of the PI3P biosensor GFP-2×FYVE to different organelles ( C ) and Pearson's co-localization coefficient ( E ) .", "Organelle markers detected using antibodies against endogenous proteins included EEA1 ( early endosomes ) , LAMP1 ( lysosomes ) , Rab11 ( recycling endosomes ) , Mito-Track ( mitochondria ) , golgin-97 ( Golgi ) .", "Data represent mean ± SD for three independent experiments .", "N = 16 . Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 004 To examine the role of AnkB’s association with dynactin in organelle motility in MEFs , we tracked the motion of vesicles expressing the mutant DD1320AA AnkB-mCherry protein , unable to associate with the dynactin complex ( Ayalon et al . , 2011 ) .", "DD1320AA AnkB-mCherry-positive organelles showed shorter-range motility , reduced net velocity ( velocity < 4 µm/s , persistence < 0 . 5 ) , and completely lacked a population with fast long-range motion ( velocity > 4 µm/s , persistence > 0 . 5 ) , which is otherwise observed in Ank2-/- MEFs expressing WT AnkB-mCherry ( Figure 1B and Figure 1—figure supplement 1A ) .", "Hence , these results demonstrate that AnkB provides long-range motility to a subset of organelles by coupling them through dynactin to either dynein or kinesin motors .", "AnkB harbors a highly conserved basic pocket within the second ZU5 ( ZU5C ) domain that specifically binds PI3P lipids and is required for AnkB’s association with axonal cargos ( Figure 1C , yellow surface ) ( Wang et al . , 2012; Lorenzo et al . , 2014 ) .", "To examine if AnkB also associates with organelles through PI3P lipids in MEFs , we labeled PI3P lipids using the GFP-2xFYVEEEA1 domain ( Schink et al . , 2013 ) .", "Live microscopy revealed that over 70% of WT AnkB-mCherry-positive vesicles detected were PI3P-positive , and around 45% of PI3P-positive organelles were associated with WT AnkB-mCherry ( Figure 1D–F and Figure 1—figure supplement 1D ) .", "In sharp contrast , mutant R1194A AnkB-mCherry , which cannot bind PI3P lipids ( Lorenzo et al . , 2014 ) ( Figure 1C red circle ) , failed to localize to PI3P-positive organelles , and was instead diffusely distributed throughout the cytoplasm ( Figure 1D–E ) .", "We next sought to uncover the identity of AnkB-positive structures in live MEFs by coexpressing WT AnkB-mCherry and organelle markers in Ank2-/- MEFs .", "Although WT AnkB-mCherry expressed was preferentially localized to Rab5-positive early endosomes , it also exhibited partial overlap with Rab11-positive recycling endosomes and LAMP1-positive lysosomes .", "Moreover , we detected a restricted association of AnkB-mCherry with puncta at mitochondria ends .", "In contrast , we observed almost no localization of AnkB-mCherry to either Golgi or ER membranes ( Figure 1F and Figure 1—figure supplement 1B , D ) .", "We also examined the association of PI3P lipids with multiple organelles in fixed MEFs using the GFP-2xFYVEEEA1 probe and antibodies against endogenous organelle-specific proteins .", "As expected ( Gillooly et al . , 2000; Di Paolo and De Camilli , 2006 ) , PI3P lipids were enriched in endo-lysosomal membranes ( Figure 1—figure supplement 1C , E ) , which closely resembled AnkB’s subcellular distribution pattern in MEFs ( Figure 1—figure supplement 1B , D ) .", "Collectively , these results demonstrate that AnkB associates with multiple organelles through PI3P lipids and promotes their long-range transport through interaction with the dynactin motor protein adaptor complex .", "Rab GTPases regulate endosomal identity and trafficking through the recruitment of effector molecules , and cycle between active ( GTP-bound ) and inactive ( GDP-bound ) states , which are respectively determined by GDP/GTP exchange factors ( RabGEFs ) and GTPase activating proteins ( RabGAPs ) ( Frasa et al . , 2012 ) .", "Therefore , we asked whether AnkB interacts with Rab GTPases or their regulators .", "AnkB is a multipartite protein with an N-terminus membrane-binding domain ( MBD ) containing twenty-four ankyrin repeats , a supermodule structure ( Zu5N-Zu5C-UPA domains ) that binds β-spectrins , dynactin , and PI3P lipids , a death domain ( DD ) , and an intrinsically unstructured C-terminal regulatory domain , which engages in intramolecular interactions ( Wang et al . , 2012; Bennett and Lorenzo , 2016 ) ( Figure 2A ) .", "Among these domains , only the death domain ( named due to structural similarity to death domains of apoptosis-related proteins ) has no known partners .", "Interestingly , a yeast-two-hybrid ( Y2H ) screen using the death domain of human AnkB ( AnkB DD ) ( Figure 2A ) and a normalized universal mouse cDNA library identified ten individual positive clones , all sharing the last 65 C-terminal residues of RabGAP1L ( Figure 2B ) . 10 . 7554/eLife . 20417 . 005Figure 2 . RabGAP1L binds to the death domain of AnkB .", "( A , B )", "Schematic representation of the domain organization of 220 kDa AnkB ( A ) and 93 kDa RabGAP1L ( B ) .", "( C ) Co-immunoprecipitation ( Co-IP ) of endogenous AnkB and RabGAP1L ( top ) , AnkG and RabGAP1L ( bottom ) from WT MEF lysates .", "( D ) Proximity ligation assay .", "Red dots indicate cellular sites of interaction between AnkB and RabGAP1L , blue shows nuclear staining .", "( E ) Co-IP of WT AnkB-HA and RabGAP1L-GFP ( left ) , E1537K AnkB-HA and RabGAP1L-GFP ( middle ) , and WT AnkB-HA and KK783EE RabGAP1L-GFP ( right ) from HEK293 cells expressing corresponding plasmids .", "( F ) Kymographs of WT AnkB-mCherry and RabGAP1L-GFP ( top ) , WT AnkB-mCherry and KK783EE RabGAP1L-GFP ( middle ) , and E1537K AnkB-mCherry and WT RabGAP1L-GFP ( bottom ) motion in Ank2-/- MEFs .", "White arrowheads indicate vesicles showing AnkB-mCherry and RabGAP1L co-transport .", "( G ) Molecular surface representation of AnkB death domain ( AnkB DD ) and RabGAP1L TBC-C-terminal domain ( TBC-Ct ) .", "Residues critical for interaction are highlighted by yellow circles .", "( H ) Co-localization of either WT AnkB-mCherry with WT RabGAP1L-GFP , E1537K AnkB-mCherry with WT RabGAP1L-GFP , or WT AnkB-mCherry with KK783EE RabGAP1L-GFP in Ank2-/- MEFs .", "Data represent mean ± SD from three independent experiments .", "***p<0 . 001 , one-way ANOVA with Tukey post-test .", "N = 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 00510 . 7554/eLife . 20417 . 006Figure 2—figure supplement 1 . Validation of the RabGAP1L antibody and AnkB/RabGAP1L interaction .", "( A ) Immunoblot of whole cell lysates from WT and Ank2-/- MEFs using a house-made antibody against RabGAP1L polypeptides .", "( B ) Sequence alignment of the death domains of human AnkR , AnkB , and AnkG .", "Red boxes indicate divergent charged residues between AnkB and AnkG death domains .", "Red asterisk denotes the E1537 AnkB residue required for binding to RabGAP1L .", "( C ) Y2H analysis of interaction between mutant AnkB death domain and RabGAP1L TBC-C-terminal domain ( residues E507-L815 ) .", "Individual mutations within the AnkB DD tested are shown .", "( D and F )", "Sequence alignment shows the conservation of E1537 AnkB ( D ) and KK783 RabGAP1L ( F ) sites among multiple species .", "( E ) Immunoblot of whole cell lysates from transfected HEK293 cells used in immunoprecipitation studies in Figure 2E .", "( G ) Representative images of Ank2-/- MEFs expressing either WT AnkB-mCherry and WT RabGAP1L-GFP ( left ) , E1537K AnkB-mCherry and WT RabGAP1L-GFP ( center ) , or WT AnkB-mCherry and KK783EE RabGAP1L-GFP ( right ) used to generate kymographs shown in Figure 2F .", "Scale bar , 10 µm .", "( H ) Immunofluorescence staining of AnkB ( red ) and RabGAP1L ( green ) in WT MEFs . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 006 RabGAP1L belongs to the Tre2–Bub2–Cdc16 ( TBC ) domain-containing family of Rab-specific GTPase-activating proteins ( TBC/RabGAPs ) ( Fukuda , 2011 ) , which regulate intracellular membrane trafficking in multiple cellular contexts ( Fuchs et al . , 2007; Haas et al . , 2005; Patino-Lopez et al . , 2008 ) .", "Specifically , RabGAP1L , via a catalytic site on the TBC domain , inactivates Rab22A by promoting its GDP-bound configuration ( Itoh et al . , 2006; Frasa et al . , 2012 ) .", "In addition , RabGAP1L contains an N-terminal phosphotyrosine-binding ( PTB ) domain and a kinesin-like domain of unknown function ( Hidaka et al . , 2000 ) ( Figure 2B ) .", "Co-immunoprecipitation ( co-IP ) and co-localization experiments using affinity-purified antibodies against AnkB and RabGAP1L , which recognize single polypeptides of 220 kDa and 93 kDa in MEFs , respectively ( Figure 1A and Figure 2—figure supplement 1A ) , showed that endogenous AnkB and RabGAP1L interact in cells ( Figure 2C top ) .", "Interestingly , the AnkG death domain ( AnkG DD ) , which is 65% homologous with the AnkB DD ( Figure 2—figure supplement 1B ) , did not interact with RabGAP1L in Y2H assays ( data not shown ) .", "Similarly , full-length endogenous AnkG did not co-immunoprecipitate with RabGAP1L from MEF lysates ( Figure 2C bottom ) .", "Thus , AnkB either gained the ability to bind to RabGAP1L after the divergence of AnkB and AnkG in early vertebrate evolution , or AnkG lost this activity .", "We performed a proximity ligation assay ( PLA ) as well as immunofluorescence assay to further assess the association of endogenous AnkB and RabGAP1L , and to identify the sites of their interaction in MEFs .", "PLA produces a positive signal when putative binding partners are within less than 40 nm of each other , which allows the DNA labels of antibodies against these proteins to form double strands ( Söderberg et al . , 2006 ) .", "Consistent with the Y2H and co-IP results , we observed strong PLA labeling of cytoplasmic organelles in WT MEFs and complete loss of signal in Ank2-/- MEFs ( Figure 2D ) .", "Immunofluorescent staining of endogenous AnkB and RabGAP1L also re-confirmed their co-localization on a subset of cytoplasmic organelles ( Figure 2—figure supplement 1H ) .", "We next sought to identify AnkB and RabGAP1L residues critical for their interaction , which could also serve as critical controls in cellular assays .", "The lack of association between RabGAP1L and AnkG suggested that divergent sites in the sequences of AnkB DD and AnkG DD may mediate the AnkB DD-RabGAP1L interaction .", "Binding assays between RabGAP1L and AnkB with alanine mutations in seven of its DD charged residues diverging from AnkG’s DD ( Figure 2—figure supplement 1B , red box ) showed that the E1537A substitution significantly weakened the interaction with RabGAP1L , while the reverse-charge mutant E1537K blocked their association ( Figure 2—figure supplement 1C ) .", "The E1537 site , highly conserved among vertebrate AnkB proteins , resides on the surface of the crystal structure of AnkB DD ( Figure 2G and Figure 2—figure supplement 1F ) .", "Within the AnkB DD interacting portion of RabGAP1L , the highly conserved , positively charged residues 783KKLKK ( Figure 2—figure supplement 1D ) provided potential candidates for interaction with AnkB DD .", "We found that reversing the charge of the surface exposed 783KK residues to EE ( Figure 2G ) abolishes RabGAP1L binding to AnkB DD in Y2H assays ( data not shown ) .", "Co-immunoprecipitation from HEK293 cell lysates of full length WT , but not E1537K , AnkB-HA with WT RabGAP1L-GFP; as well as of WT , but not KK783EE , RabGAP1L-GFP with WT AnkB-HA ( Figure 2E and Figure 2—figure supplement 1E ) corroborated these results .", "Time-lapse video microscopy assessing the dynamic localization of AnkB-mCherry and RabGAP1L-GFP co-expressed in Ank2-/- MEFs revealed that AnkB co-transports with RabGAP1L .", "Kymograph analysis confirmed that both proteins were co-localized and co-transported on motile vesicles ( Figure 2F top , 2 hr and Figure 2—figure supplement 1G ) .", "In contrast , E1537K AnkB-mCherry , which does not bind RabGAP1L , still localizes to vesicles , but no longer co-transports with RabGAP1L-GFP ( Figure 2F bottom , 2 hr and Figure 2—figure supplement 1G ) .", "Furthermore , the KK783EE RabGAP1L-GFP mutant that abrogated interaction with AnkB also eliminated RabGAP1L-GFP and AnkB-mCherry vesicular co-localization and co-transport ( Figure 2F center , 2 hr and Figure 2—figure supplement 1G ) .", "We next asked whether RabGAP1L localizes to PI3P-positive organelles in an AnkB-dependent manner .", "While over 60% of RabGAP1L-mCherry vesicles detected in WT MEFs were associated with PI3P-positive vesicles , strikingly , less than 20% of RabGAP1L-mCherry localized to PI3P-positive compartments in Ank2-/-MEFs ( Figure 3A , D ) .", "This result is further confirmed by immunofluorescence detection of endogenous RabGAP1L in WT and Ank2-/-MEFs expressing PI3P indicator , GFP-2xFYVEEEA1 ( Figure 3—figure supplement 1A–B ) .", "Together , these results reveal a new protein-protein interaction between AnkB and RabGAP1L that recruits RabGAP1L to PI3P-positive organelles . 10 . 7554/eLife . 20417 . 007Figure 3 . AnkB promotes RabGAP1L targeting to Rab22A/PI3P-positive compartments and Rab22A dissociation from PI3P-positive endosomes .", "( A–C )", "Representative images of live WT and Ank2-/- MEFs expressing either RabGAP1L-mCherry and GFP-2xFYVE ( A ) , RabGAP1L-mCherry and GFP-Rab22A ( B ) , or mCherry-Rab22A and GFP-2xFYVE ( C ) .", "Scale bar , 10 µm .", "( D ) Quantitative data of the percentage of RabGAP1L that localize to GFP-2xFYVE labeled PI3P-positive vesicles in WT and Ank2-/- MEFs .", "( E ) Quantitative data of the percentage of RabGAP1L that localize to GFP-Rab22A-positive compartments in WT and Ank2-/- MEFs .", "( F ) Quantitative data of the percentage of Rab22A that localize to GFP-2xFYVE labeled PI3P-positive compartments in WT and Ank2-/- MEFs .", "Data represent mean ± SD for three independent experiments .", "***p<0 . 001 , two-tailed t-test .", "N = 10 , 11 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 00710 . 7554/eLife . 20417 . 008Figure 3—figure supplement 1 . Distribution of endogenous RabGAP1L to PI3P-positive and Rab22A-positive compartment .", "( A–B )", "Representative image of immunofluorescent staining of RabGAP1L in WT and Ank2-/- MEFs ( A ) , and quantitative data showing the percentage of RabGAP1L vesicles localize to 2XFYVE labeled PI3P organelles .", "( C–D )", "Representative image of immunofluorescent staining of RabGAP1L in WT and Ank2-/- MEFs ( C ) , and quantitative data showing the percentage of Rab22A vesicles positive for RabGAP1L ( D ) .", "( E–F )", "Pearson's coefficient analysis of co-localization of RabGAP1L with PI3P ( E ) and Rab22A ( F ) .", "( G ) Pearson's coefficient analysis of co-localization of Rab22A with PI3P in live cells .", "Data represent mean ± SD for three independent experiments .", "N = 18 ( B and D ) , 12 ( E–G ) .", "Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 008 RabGAP1L preferentially activates the GTPase activity of Rab22A ( Itoh et al . , 2006 ) .", "Rab22A is a vertebrate Rab GTPase closely related to Rab5 that localizes to early endosomes , where it interacts with the early endosomal antigen 1 ( EEA1 ) and the Rab5 guanine nucleotide exchange factor Rabex-5 ( Kauppi et al . , 2002; Zhu et al . , 2009 ) .", "Interestingly , Rab22A facilitates the formation of a specialized subset of early endosomes , implicated in endocytosis as well as recycling of both clathrin-dependent and clathrin-independent cargos ( Holloway et al . , 2013; Maldonado-Báez and Donaldson , 2013; Weigert et al . , 2004 ) .", "We evaluated whether RabGAP1L recruitment to Rab22A-positive organelles depends on AnkB .", "While in WT MEFs 40% to 60% of GFP-Rab22A-positive vesicles co-localized with RabGAP1L-mCherry , this co-localization was reduced to less than 15% in Ank2-/- MEFs ( Figure 3B , E ) .", "This result is further confirmed by immunofluorescence of endogenous RabGAP1L in WT and Ank2-/-MEFs expressing GFP-Rab22A ( Figure 3—figure supplement 1C–D ) .", "Inactivation of membrane-associated Rab GTPases ensures their dissociation from bound vesicles , which is critical for the transition between endosomal compartments during endosomal trafficking ( Frasa et al . , 2012 ) .", "Therefore , we speculated that AnkB , via the recruitment of RabGAP1L to PI3P-positive organelles , might promote inactivation of Rab22A and its dissociation from early endosomes .", "In line with previous reports of Rab22A association with early endosomes ( Kauppi et al . , 2002; Zhu et al . , 2009 ) , we found that in WT MEFs over 40% of Rab22A localized to PI3P-enriched membranes ( Figure 3C , F ) .", "Remarkably , loss of AnkB expression not only abrogated co-localization of RabGAP1L to Rab22A-positive organelles ( Figure 3B , E ) , but also resulted in an increased association of Rab22A with PI3P-positive compartments , especially within the perinuclear region ( Figure 3C , F ) .", "Thus , our data indicate that AnkB promotes dissociation of Rab22A from PI3P-enriched organelles through the recruitment of RabGAP1L .", "The interaction between RabGAP1L and AnkB prompted us to examine whether AnkB has a role in determining organelle transport polarity .", "To study the dynamics of AnkB-associated organelles in migrating MEFs , which are polarized cells with well-defined front and rear ends , we tracked their motion from the perinuclear region by expressing AnkB proteins tagged with mMaple3 , a photoconvertible molecule that switches from green to red fluorescent emission following blue light exposure ( Wang et al . , 2014a ) .", "Ank2-/- MEFs expressing either mMaple3-tagged WT or mutant E1537K AnkB were plated on tissue culture wells containing an insert , which was later removed to allow cell migration into the exposed region ( Figure 4A ) .", "The perinuclear region of migrating cells was pulsed with blue light resulting in conversion of about 70% of green AnkB-mMaple3 to red fluorescent signal ( Figure 4B , red inset ) .", "To dynamically track AnkB-mMaple3 particles , we monitored loss of red fluorescence , due to outward migration of photoconverted perinuclear vesicles , as well as gain of green fluorescence , due to entry of non-photoconverted inward-moving vesicles into the perinuclear region ( Figure 4B–E ) . 10 . 7554/eLife . 20417 . 009Figure 4 . AnkB-associated perinuclear endosomal organelles exhibit polarized transport in migrating MEFs .", "( A ) Schematic of the experimental design used in the combined photoconversion- wound-induced migration assay .", "Perinuclear AnkB-mMaple3 signal in Ank2-/- MEF that is migrating at the edge of the wound was converted from green to red fluorescence by blue light exposure .", "Green or red fluorescent signal were tracked for 16 min following photoconversion .", "For image representation and analysis cells were divided in front and rear halves determined based on their migratory direction .", "( B and D )", "Representative image of Ank2-/- MEF expressing WT AnkB-mMaple3 ( B ) or E1537K AnkB-mMaple3 ( D ) at t = 0 s ( pre-converted ) , 45 s ( perinuclear region converted ) , and 16 min ( tracking end point ) .", "Scale bar , 10 µm .", "( C and E )", "Quantification of red fluorescent intensity ( post-converted AnkB-mMaple3 ) and green fluorescent intensity ( pre-converted AnkB-mMaple3 ) in the perinuclear region .", "( F , I )", "Comparative analysis of green fluorescent gain ( F ) and red fluorescent loss ( I ) in the perinuclear region .", "( G , H )", "Percentage of red fluorescent loss and green fluorescent gain in perinuclear region at 16 min . ( J ) Quantification of red fluorescent intensity gain at either the front or rear cell ends .", "Intensities at each time point are normalized to the background intensity at t = 0 s .", "Data represent mean ± SD for three independent experiments .", "*p=0 . 011 , ***p<0 . 001 , two-tailed t-test .", "N = 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 00910 . 7554/eLife . 20417 . 010Figure 4—figure supplement 1 . Distribution of WT AnkB-mMaple3 and E1537K AnkB-mMaple3 in MEFs during a photoconversion assay .", "( A , B )", "Representative images of Ank2-/- MEFs expressing either WT AnkB-mMaple3 ( A ) or E1537K AnkB-mMaple3 ( B ) .", "For each genotype: Top row left panel shows the distribution of green AnkB-mMaple3 vesicles before photoconversion ( t = 0 s ) .", "Middle row left panel shows post-converted red perinuclear AnkB-mMaple3 vesicles ( t = 45 s ) .", "Bottom row left panel shows the distribution of post-converted red AnkB-mMaple3 vesicles 16 min after photoconversion .", "Scale bar , 8 µm .", "For each row , center and right panels show the higher magnification images of the rear and front cell ends . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 01010 . 7554/eLife . 20417 . 011Figure 4—figure supplement 2 . Global recycling of cell surface proteins , β3-integrin , and transferrin is not affected in Ank2-/- MEFs .", "( A ) Representative images show the internalization and recycling of biotin-labeled cell surface proteins in WT and Ank2-/- MEFs over a 30 min period .", "Biotin signal was detected with streptavidin-488 .", "Scale bar , 10 µm .", "( B ) Quantification of the percentage of recycled cell surface proteins in WT and Ank2-/- MEFs .", "Data represent mean ± SD for five independent experiments .", "Means from each experiment are shown .", "N = 15 . ( C ) Representative images of β3-integrin recycling in WT and Ank2-/- MEFs .", "The plasma membrane is labeled by WGA ( blue ) .", "Scale bar , 10 µm .", "( D ) Quantification of the percentage of recycled β3-integrins in WT and Ank2-/- MEFs within 30 min after initiation of recycling .", "Data represent mean ± SD for five independent experiments .", "Means from each experiment are shown .", "N = 15 . ( E ) Representative images of Trn recycling in WT and Ank2-/- MEFs .", "Scale bar , 10 µm .", "( F ) Quantification of the percentage of remaining intracellular Trn signal in WT and Ank2-/- MEFs within 30 min after initiation of recycling .", "Data represent mean ± SD for seven independent experiments .", "Means from each experiment are shown .", "N = 32 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 011 We found that the rate of entry of green vesicles from peripheral areas was equivalent for both WT and E1537K AnkB ( Figure 4C , E and G , green symbols ) .", "However , photoconverted red WT AnkB vesicles exited the perinuclear region at twice the rate of mutant E1537K AnkB ( Figure 4C , H–I , red symbols ) .", "Moreover , photoconverted perinuclear WT AnkB-mMaple3 red vesicles exhibited a biased transport toward the migrating front of the cell ( Figure 4J and Figure 4—figure supplement 1A ) .", "In contrast , red-converted E1537K AnkB-mMaple3 moved from the perinuclear region into both the front and the rear of cells at equivalent rates ( Figure 4J and Figure 4—figure supplement 1B ) .", "Taken together , these results demonstrate that AnkB , via interaction with RabGAP1L , coordinates the polarized transport of perinuclear PI3P-positive organelles to the migrating front of MEFs .", "We next sought to identify membrane protein ( s ) that are transported via the AnkB/RabGAP1L/Rab22A-mediated pathway in MEFs .", "Using a cell surface protein biotinylation assay ( Bitsikas et al . , 2014 ) , we found that AnkB deficiency did not cause global changes in either plasma membrane protein internalization or recycling , as shown by the similar rate and extent of internalization and recycling of biotinylated surface proteins to the plasma membrane in WT and Ank2-/- MEFs ( Figure 4—figure supplement 2A–B ) .", "We also found no difference between WT and Ank2-/- MEFs in the dynamics of internalization and recycling of known specialized endocytic cargos , including transferrin ( Trn ) and αvβ3-integrin ( Figure 4—figure supplement 2C–F ) .", "We next evaluated the role of AnkB in active transport of the fibronectin receptor α5β1-integrin , which , similar to AnkB , exhibits polarized delivery from cytoplasmic compartments to the leading edge of migrating fibroblasts ( Bretscher , 1989 , 1992 ) .", "Moreover , polarized transport of the fibronectin receptor is extensively regulated by GTPases and their adaptor proteins ( Caswell et al . , 2008 , 2009; Thapa et al . , 2012 ) .", "We tagged α5-integrin with mMaple3 and tracked its dynamics in migrating WT and Ank2-/- MEFs .", "In WT MEFs , photoconverted perinuclear α5-integrin-mMaple3 localized to vesicles that were predominantly transported towards the migrating cell front ( Figure 5A–B and G–I and Figure 5—figure supplement 1A ) .", "In contrast , in Ank2-/- MEFs , significantly fewer photoconverted perinuclear α5-integrin-mMaple3 exited the converted region , and showed no preference in transport directionality ( Figure 5C–D and G–I and Figure 5—figure supplement 1B ) .", "Thus , these results indicate that AnkB is required for the polarized transport of α5-integrin from the perinuclear region to the migrating front of MEFs . 10 . 7554/eLife . 20417 . 012Figure 5 . AnkB is required for polarized transport of α5-integrin towards the front end of migrating MEFs .", "( A and C )", "Same photoconversion- wound-induced migration assay was performed in WT and Ank2-/- MEFs expressing α5-integrin-mMaple3 .", "Representative images of WT MEFs ( A ) and Ank2-/- MEFs ( C ) expressing α5-integrin-mMaple3 at 0s ( pre-converted ) , 45 s ( perinuclear region converted ) and16 mins ( tracking end point ) .", "Scale bar , 10 µm .", "( B and D )", "Quantification of red fluorescent intensity ( post-converted α5-integrin-mMaple3 ) and green fluorescent intensity ( pre-converted AnkB-mMaple3 ) in the perinuclear region of WT ( B ) and an Ank2-/- ( D ) MEFs .", "( E , H )", "Comparative analysis of green fluorescent gain ( E ) and red fluorescent loss ( H ) in the perinuclear region .", "( F , G )", "Quantitative data of the percentage of red fluorescent loss and green fluorescent gain in the perinuclear region of WT and Ank2-/- MEFs at 16 min . ( I ) Quantification of red fluorescent intensity gain at either the front or rear cell ends .", "Intensities at each time point are normalized to the background intensity at t = 0 s .", "Data represent mean ± SD for six independent experiments .", "Mean from each of the six experiment is shown in E and H . **p=0 . 022 , two-tailed t-test .", "N = 13 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 01210 . 7554/eLife . 20417 . 013Figure 5—figure supplement 1 . Distribution of α5-integrin-mMaple3 in WT and Ank2-/- MEFs during a photoconversion assay .", "( A , B )", "Representative images of WT ( A ) and Ank2-/- ( B ) MEFs expressing mMaple3-tagged α5-integrin during a photoconversion assay .", "For each genotype: Top row left panel shows the distribution of green α5-integrin -mMaple3 vesicles before photoconversion ( t = 0 s ) .", "Middle row left panel shows post-converted red perinuclear α5-integrin-mMaple3 vesicles ( t = 45 s ) .", "Bottom row left panel shows distribution of post-converted red α5-integrin -mMaple3 vesicles 16 min after photoconversion .", "Scale bar , 8 µm .", "For each row , center and right panels show high magnification images of the rear and front cell ends . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 013 The fact that α5β1-integrin has a low degradation rate and is actively recycled in a long-loop microtubule dependent route led us hypothesize that a majority of photo-converted perinuclear α5-integrins belong to the recycling population ( Caswell et al . , 2009; Bridgewater et al . , 2012; Arjonen et al . , 2012; Böttcher et al . , 2012 ) .", "Therefore , to determine whether α5β1-integrin requires AnkB for polarized recycling , we followed the itinerary of internalized β1-integrin in WT and Ank2-/- MEFs by calculating the percentage of recycled β1-integrin at the cell surface at selected post-internalization times ( Figure 6A ) .", "After 60 min , WT MEFs recycled over 60% of internalized β1-integrins to the plasma membrane ( Figure 6B , D ) .", "In contrast , in Ank2-/- MEFs less than 20% of internalized β1-integrins recycled to the cell surface over the same period , and instead accumulated at the perinuclear region ( Figure 6B , D ) .", "The pool of labeled intracellular β1-integrins in Ank2-/- MEFs eventually did return to the plasma membrane after 3–6 hr ( data not shown ) , with a significantly slower recycling rate than in WT MEFs . 10 . 7554/eLife . 20417 . 014Figure 6 . An AnkB-mediated mechanism promotes α5β1-integrin recycling to the plasma membrane of migrating MEFs .", "( A ) Schematic representation of the β1-integrin recycling assay .", "Cell surface β1-integrins were labeled with anti-β1-integrin antibody conjugated with Alexa 488 at 4°C .", "Cells were incubated at 37°C for 30 min to allow internalization .", "Remaining cell surface labeling is reduced by acid wash and recorded as recycle time point 0 min .", "Cells were returned to 37°C incubation for indicated times following the wash with PBS and culture media .", "( B ) Representative images of WT and Ank2-/- MEFs at recycling points t = 0 min and t = 60 min .", "β1-integrins are shown in green , plasma membranes labeled with WGA-Alexa 633 are shown in blue .", "Yellow arrows indicate plasma membrane areas at the migrating front containing recycled β1-integrin in WT and in Ank2-/-MEFs expressing WT AnkB-mCherry .", "White arrows indicate the absence of recycled β1-integrin signal at the plasma membrane of Ank2-/-MEFs ( C ) Schematic representation of the domain organization of AnkB-mCherry constructs used in structural function experiments .", "The various mutation sites are marked in red .", "( D ) Quantitative data of β1-integrin recycling in WT , Ank2-/- , and Ank2-/- MEFs expressing WT or mutant AnkB-mCherry constructs .", "Data represents mean ± SD from five independent experiments .", "Individual data points indicate the mean from each experiment .", "N = 16 . ( E ) Images show prolonged association of Rab5 with β1-integrin vesicles in Ank2-/- MEFs .", "( F ) Pearson’s co-localization coefficient between Rab5 and internalized β1-integrin 10 min post recycling in WT and Ank2-/- MEFs .", "Data represents mean ± SD for three independent experiments .", "***p<0 . 001 , two-tailed t-test .", "N = 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 01410 . 7554/eLife . 20417 . 015Figure 6—figure supplement 1 . Structural-functional study of AnkB-mCherry protein rescue of β1-integrin recycling deficits in Ank2-/- MEFs , β1-integrin localization to Rab22A- and Rab5-positive early endosomes .", "( A ) Representative images of Ank2-/- MEFs expressing E1537K AnkB-mCherry ( top ) , R1194A AnkB-mCherry ( middle ) , and DD1320AA AnkB-mCherry ( bottom ) proteins taken at different recycling times .", "Yellow arrows indicate plasma membrane areas containing recycled β1-integrin .", "White arrows indicate the absence of recycled β1-integrin signal at the plasma membrane of non-rescued cells .", "( B ) Representative images of Ank2-/- MEFs expressing the ZU5-C-terminal portion ( ZU5-Ct ) of AnkB-mCherry at different recycling times .", "Yellow arrows indicate plasma membrane areas containing recycled β1-integrin molecules .", "( C ) Representative images of WT ( left ) and Ank2-/- ( right ) MEFs expressing mCherry-Rab22A and Alexa 488-labeled β1-integrins fixed 15 min after initiation of internalization .", "( D ) Quantification of mCherry-Rab22A and internalized β1-integrin co-localization 15 min after initiation of internalization in WT and Ank2-/- MEFs .", "Data represents mean ± SD from six independent experiments .", "Means from each experiment are shown .", "N = 18 .", "**p=0 . 002 , two-tailed t-test .", "( E ) An association of Rab5-RFP with β1-integrin vesicles in WT and Ank2-/- MEFs 10 min after initiation of internalization . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 01510 . 7554/eLife . 20417 . 016Figure 6—figure supplement 2 . Knock down of RabGAP1L or replacement with GAP-deficient RabGAP1L affects cell viability .", "( A ) RabGAP1L immunoblot of whole cell lysates from RabGAP1L shRNA knockdown or control cells at Day0 and Day1 post-treatment with Doxycycline .", "( B ) Representative images of control MEFs , RabGAP1L knockdown MEFs , RabGAP1L knockdown MEFs rescued with WT or GAP-deficient R584A RabGAP1L .", "( C ) Growth curve of control and RabGAP1L knockdown MEFs .", "( D ) Survival rate of control MEFs , RabGAP1L knockdown MEFs , RabGAP1L knockdown MEFs rescued with WT or GAP-deficient R584A RabGAP1L .", "The survival is scored as indicated in the graph .", "Scale bar , 10 µm .", "Data represent mean ± SD for three independent experiments , N = 19 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 01610 . 7554/eLife . 20417 . 017Figure 6—figure supplement 3 . Overexpression of WT or constitutively active Rab22A impairs receptor recycling .", "( A–B )", "Representative images ( A ) and quantitative data ( B ) of β1-integrin recycling within 60 min in control WT MEFs , WT MEFs expressing mCherry-WT Rab22A or constitutively active Q64L Rab22A .", "( C ) Quantitative data showing the percentage of endocytosed β3-integrin and transferrin remained intracellular after 30 min of recycling .", "Data represent mean ± SD for three independent experiments .", "N = 15 . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 017 Integration of phosphoinositide lipids , GTPases , and motors is essential for efficient recycling of endocytic cargoes , including α5β1-integrin ( Jović et al . , 2007 , 2009; Thapa et al . , 2012 ) .", "Therefore , we addressed the requirement for AnkB interactions with PI3P lipids , dynactin , and RabGAP1L for α5β1-integrin recycling using a structure-function rescue approach .", "Expression of WT AnkB-mCherry fully restored the rate and extent of β1-integrin recycling to the leading edge of migrating Ank2-/- MEFs ( Figure 6B–D ) .", "In marked contrast , neither E1537K AnkB-mCherry ( lacking RabGAP1L binding ) nor R1194A AnkB-mCherry ( lacking PI3P binding ) rescued β1-integrin recycling deficits in Ank2-/- MEFs ( Figure 6C , D and Figure 6—figure supplement 1A ) .", "DD1320AA AnkB-mCherry ( unable to bind dynactin ) ( Figure 6C ) partially rescued β1-integrin recycling to about 50% of the WT levels ( Figure 6D and Figure 6—figure supplement 1A ) .", "These results indicate that AnkB’s binding to PI3P lipids and RabGAP1L are essential steps in β1-integrin recycling , while its association with the dynactin complex increases its efficiency .", "AnkB’s MBD is comprised of 24 ANK repeats folded as a solenoid with a peptide-binding groove that mediates binding to multiple membrane proteins ( Bennett and Lorenzo , 2016 ) .", "Thus , we hypothesized that AnkB’s MBD facilitates β1-integrin recycling via direct interaction with α5β1-integrin or other associated adaptor proteins .", "To our surprise , expression of a truncated AnkB-mCherry construct lacking the MBD ( Zu5-Ct AnkB-mCherry ) was sufficient to restore β1-integrin recycling to the leading edge of Ank2-/- MEFs ( Figure 6C , D and Figure 6—figure supplement 1B ) .", "Based on these findings , we concluded that integrin recognition through ANK repeats is not required for AnkB-dependent β1-integrin recycling .", "Lastly , we investigated whether AnkB is required for either the initial sorting of β1-integrin to early endosomes or for subsequent endosomal maturation steps .", "The localization of internalized β1-integrin to Rab22A- or Rab5-positive early endosomes , both in WT and Ank2-/- MEFs ( Figure 6—figure supplement 1C–E ) , suggests that the initial sorting to early endosomes is independent of AnkB .", "However , β1-integrin exhibited increased co-localization with Rab5-positive early endosomes and with Rab22A in Ank2-/- MEFs following initiation of recycling ( Figure 6E , F and Figure 6—figure supplement 1D ) .", "These results indicate that AnkB , through recruitment of RabGAP1L , facilitates the dissociation of Rab22A from β1-integrin-containing endocytic vesicles to allow their transition from Rab5-positive early endosomes to Rab5-negative recycling endosomal compartments .", "Spatio-temporal regulation of Rab and Arf GTPase activities is critical for controlling the endosomal recycling of α5β1-integrins ( Caswell et al . , 2008; Pellinen et al . , 2006; Li J et al . , 2007 ) .", "To directly address the requirement of GAP activity of RabGAP1L in β1-integrin recycling , we generated shRNA mediated RabGAP1L knock-down cells .", "However , cells with knock-down of RabGAP1L or replacement with GAP-deficient RabGAP1L , the R584A mutant that abolish the IxxDxxR arginine finger motif ( Pan et al . , 2006; Frasa et al . , 2012 ) , exhibited altered morphology and global defects in cell growth ( Figure 6—figure supplement 2 ) .", "These results suggest that RabGAP1L also plays important roles though its GAP activity in cellular events other than integrin trafficking and cell migration .", "Rab21 provides a similar example and is required for β1-integrin trafficking as well as cell adhesion and cytokinesis ( Pellinen et al . , 2006 , 2008; Mai et al . , 2011 ) .", "To test if Rab22A activity is responsible for α5β1-integrin recycling downstream of the AnkB-RabGAP1L pathway , we performed β1-integrin recycling assay in WT MEFs over-expressing either mCherry-tagged WT Rab22A or constitutively active mutant Q64L Rab22A .", "Interestingly , WT MEFs over-expressing WT Rab22A showed a reduced rate of β1-integrin recycling .", "The deficiency was further increased in MEFs expressing Q64L Rab22A ( Figure 6—figure supplement 3A–B ) .", "Moreover , we noticed that WT MEFs expressing Q64L Rab22A not only affected internalization of β1-integrins , but also of transferrin and β3-integrins , whose recycling is not affected in the loss of ankB ( Figure 6—figure supplement 3C ) .", "These results suggest that hyper-activation of Rab22A disrupts general recycling of multiple receptors while the AnkB-RabGAP1L mediated regulation specifically tunes the Rab22A activity on selected PI3P-positive organelles bearing β1-integrin .", "Fibroblasts rely on the efficient recycling of α5β1-integrin adhesion receptors to the plasma membrane for directional migration ( Caswell et al . , 2008; Mai et al . , 2011; Jović et al . , 2007; Zech et al . , 2011 ) , and β1-integrin is required for haptotaxis along fibronectin gradients ( De Franceschi et al . , 2015; King et al . , 2016 ) .", "Considering that AnkB promotes α5β1-integrin recycling to the leading edge of migrating MEFs , we next investigated the possibility that AnkB loss impairs directional migration based on a linear fibronectin gradient .", "Using a microfluidic chamber system-based haptotaxis assay , we tracked the position of individual WT and Ank2-/- MEFs migrating on a linear gradient of fibronectin during a 24 hr interval , which allowed us to calculate the forward migration index ( FMI ) , overall velocity , and persistence of motion ( Wu et al . , 2012 ) ( Figure 7A ) .", "Typically , fibroblasts haptotaxing on fibronectin gradients display an average FMI above 0 . 1 , with a 95% confidence interval ( CI ) above 0 .", "In contrast , a FMI of 0 with 95% CI crossing 0 is considered as not haptotaxing ( Wu et al . , 2012 ) .", "While WT MEFs , similar to control IA32 fibroblasts , exhibited haptotaxis towards higher concentrations of fibronectin , Ank2-/- MEFs migrated in a random pattern ( Figure 7B , C ) .", "Interestingly , Ank2-/- MEFs exhibited no difference in the overall velocity or persistence of the cell migration , suggesting that the cell motility machinery is fully functional in the absence of AnkB ( Figure 7D , E ) .", "The finding that AnkB is required for recycling of α5β1-integrin , but not of αvβ3-integrin , is consistent with reports that fibroblasts rely primarily on β1-integrins for establishing fibronectin haptotaxis ( King et al . , 2016 ) . 10 . 7554/eLife . 20417 . 018Figure 7 . An AnkB-RabGAP1L complex is required for haptotaxis of MEFs on a fibronectin gradient .", "( A ) Schematic of a microfluidic chamber system-based haptotaxis assay and analysis .", "( B ) Rose plot showing the distribution of the tracking end point of cells migrate on a linear gradient of fibronectin .", "( C ) Mean FMI of WT MEFs , control IA32 fibroblasts , Ank2-/- MEFs and Ank2-/- MEFs expressing WT AnkB-GFP or E1537K AnkB-GFP with 95% confidence interval ( 95% CI ) .", "Mean FMI with 95% CI crossing 0 is considered as not haptotaxing .", "( D , E )", "Mean velocity ( D ) and persistence ( E ) of the motility of WT , Ank2-/- , and Ank2-/- MEFs expressing WT AnkB-GFP or E1537K AnkB-GFP .", "Data represent mean ± SD from four independent experiments .", "*p=0 . 0238 , 0 . 04 .", "N = 98 , 156 , 116 , 70 , 78 .", "one-way ANOVA with Tukey post-test . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 018 Interestingly , rescue experiments in Ank2-/- MEFs expressing either WT or E1537K AnkB-GFP ( lacking RabGAP1L binding ) showed that expression of WT AnkB-GFP restored the haptotatic migration pattern in Ank2-/- MEFs while E1537K AnkB-GFP did not rescue the impaired haptotactic response ( Figure 7B , C ) .", "Together , these data indicate that the AnkB-RabGAP1L interaction , which is required for polarized recycling of β1-integrin , is also required for efficient fibroblast migration along a fibronectin gradient .", "In future studies , it will be important to characterize the role of the AnkB-RabGAP1L pathway in cell migration in a more physiological relevant 3D micro-environment and to evaluate its role in cancer cell metastasis ( Caswell et al . , 2007 , 2008 ) ." ], [ "We report the discovery of a new pathway required for polarized membrane transport of specialized cargos .", "It was previously reported that AnkB promotes fast axonal transport through recruiting dynactin to PI3P-positive organelles ( Lorenzo et al . , 2014 ) .", "We demonstrate that in fibroblasts AnkB functions as a PI3P effector associated with early endosomes , and describe a new interaction of AnkB with RabGAP1L .", "Moreover , we show that AnkB recruits RabGAP1L to PI3P-positive organelles , where it inactivates Rab22A , and identify α5β1-integrin as a specialized cargo that depends on AnkB/RabGAP1L for efficient recycling to the leading edge of migrating fibroblasts ( Figure 8 ) .", "Thus , our results establish AnkB as a key nodal element in the protein circuitry required for α5β1-integrin recycling and for directional fibroblast migration on fibronectin gradients . 10 . 7554/eLife . 20417 . 019Figure 8 . Model of AnkB-mediated mechanism for endosomal transport .", "( A ) Model of AnkB-mediated mechanism for the recruitment of RabGAP1L to PI3P/Rab22A-positive endosomal compartments , which is critical for the maturation and recycling of α5β1-integrin containing endosomes . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 01910 . 7554/eLife . 20417 . 020Figure 8—figure supplement 1 . AnkB and RabGAP1L co-localize at the corpus callosum in the CNS and costameres in skeletal muscle .", "( A ) Immunofluorescent staining of endogenous AnkB ( red ) and RabGAP1L ( green ) in WT PND30 mice brain .", "Scale bar , 20 mm . ( B ) Proximity ligation of AnkB and RabGAP1L in WT PND30 mice brain .", "Red: positive ligation/interaction .", "Blue: DAPI .", "Yellow arrow points to corpus callosum .", "Scale bar , 20 mm . ( C ) Immunofluorescent staining of endogenous AnkB ( red ) and RabGAP1L ( green ) in WT PND30 mice skeletal muscle ( SKM ) .", "( D ) Proximity ligation of AnkB and RabGAP1L in WT PND30 mice skeletal muscle .", "Red: positive ligation/interaction .", "Blue: DAPI .", "Scale bar: 20 µm , zoom: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 20417 . 020 An AnkB/RabGAP1L-based pathway with roles in polarized long-range organelle transport likely first appeared in jawed vertebrates over 400 million years ago as a result of neofunctionalization of duplicated genes .", "Human AnkB , RabGAP1L , and Rab22A all have homologues in ghost sharks and zebrafish with a high level of sequence similarity , including nearly complete conservation of their AnkB-RabGAP1L binding sites .", "In contrast , residues required for interaction between AnkB and RabGAP1L are highly divergent in the closest homologues of Drosophila melanogaster and C . elegans .", "Human AnkB and its paralogue AnkG exhibit over 70% sequence conservation , with only a few localized regions of divergence .", "One highly divergent site between these two ankyrins is an unstructured peptide connecting the MBD and the first ZU5 domain , which through intramolecular inhibition , prevents interactions between AnkB and membrane partners ( He et al . , 2013 ) .", "Here , we identify an additional divergent site within AnkB’s DD that allows AnkB , but not AnkG , to bind to RabGAP1L .", "AnkB , likely gained RabGAP1L-binding activity after divergence of AnkB and AnkG proteins in early vertebrate evolution , although , alternatively , AnkG may have lost this function .", "Similarly , RabGAP1L differs from its paralogue RabGAP1 primarily in the C-terminal residues required to bind AnkB .", "A functional prediction , based on the recent evolution of the AnkB/RabGAP1L-based pathway , is that it will engage a subset of cargos with specialized roles in vertebrate physiology .", "In support of this idea , Rab22A , an AnkB/RabGAP1L substrate identified in this study , participates in recycling of other specialized cargos , including the Menkes copper transporter , the epidermal growth factor receptor ( EGFR ) , and the major histocompatibility complex class I ( Holloway et al . , 2013; Maldonado-Báez and Donaldson , 2013; Weigert et al . , 2004 ) .", "Interestingly , Rab22A shares 70% sequence identity with Rab22B/Rab31 , also implicated in the endosomal transport of specialized cargos such as EGFR and the p75 neurotrophin receptor ( Baeza-Raja et al . , 2012; Chua and Tang , 2014 ) .", "It will be of interest to determine whether Rab22B is a RabGAP1L substrate , and whether it also depends of AnkB for its inactivation .", "Likewise , it will be important to identify the full complement of membrane-associated proteins engaged by the AnkB/RabGAP1L pathway .", "As an initial step in further elucidating the physiological role ( s ) of the AnkB/RabGAP1L interaction , we performed a proximity ligation assay as well as immunofluorescent staining of total AnkB and RabGAP1L in brain and skeletal muscle tissues from PND 30 mice ( Figure 8—figure supplement 1 ) .", "We detected a strong PLA signal in the corpus callosum in the CNS and costameres in skeletal muscle ( Figure 8—figure supplement 1B , D ) .", "Interestingly , AnkB is required for preservation of the corpus callosum and assembly of costameres ( Scotland et al . , 1998; Lorenzo et al . , 2014; Ayalon et al . , 2008 ) .", "The cellular role of the AnkB/RabGAP1L interaction remains to be further characterized in more specialized mice models such as E1537K AnkB knock-in mice lacking RabGAP1L-binding activity .", "An AnkB mutation eliminating interaction with the dynactin complex impairs but does not abolish α5β1-integrin recycling to the plasma membrane ( Figure 6D ) .", "The residual α5β1-integrin transport may operate through alternative mechanisms to recruit motor proteins to AnkB-associated organelles , and facilitate their traffic from the perinuclear compartment to the plasma membrane .", "One potential candidate is the kinesin-3 family member KIF16B , which binds directly to PI3P lipids and promotes outward transport of Rab5-associated early endosomes to the cell surface ( Hoepfner et al . , 2005 ) .", "Inactivation of Rab22A by RabGAP1L through its TBC domain likely contributes to the maturation of early endosomes .", "GTP-bound Rab22A directly associates with the Rab5 GEF Rabex-1 , which activates Rab5 ( Zhu et al . , 2009 ) .", "Alternatively , conversion of Rab22A to its GDP form through the AnkB-mediated recruitment of RabGAP1L would be expected to reduce Rab5 activation , thus promoting loss of early endosome identity .", "Another possibility is that RabGAP1L perform functions independent of GAP-activity , similar to p120RasGAP , which competes with , instead of inactivating , Rab21in binding to integrin α cytoplasmic tails to promote integrin recycling ( Mai et al . , 2011 ) .", "However , whether and how loss of Rab22A association with early endosomes lead to the polarized transport of organelles preferentially to the front of migrating fibroblasts remains to be elucidated .", "The regulation of GTPases and their adaptor proteins also involves kinases ( Stenmark , 2009; Frasa et al . , 2012 ) .", "Specifically , previous studies had shown that Diacylglycerol kinase α ( DGKα ) is required for Rab-coupling protein ( RCP ) -dependent integrin trafficking and Akt mediated phosphorylation of ACAP1 , a GAP for Afr6 , is required for integrin recycling ( Rainero et al . , 2012; Li et al . , 2005 ) .", "Although the kinase ( s ) that regulate RabGAP1L activity has not been identified , it remains to be a possible regulatory mechanism for sensing directional cues during haptotaxis ( Figure 8 ) .", "Integrin dynamics has been the focus of intense investigation with particular attention from investigators in the fields of cancer cell metastasis , and angiogenesis ( De Franceschi et al . , 2015; Caswell et al . , 2007; Dozynkiewicz et al . , 2012; Paul et al . , 2015; Tian et al . , 2012 ) .", "Interestingly , Rab22A also contributes to exosome biogenesis and shedding from primary tumor cells and tumor metastasis ( Wang et al . , 2014b ) .", "Our discovery of an AnkB/RabGAP1L pathway as a key regulator of Rab22A localization and activity , and of α5β1-integrin polarized transport , offers new insights into the molecular circuitry underlying fibroblast and endothelial cell migration and tumor metastasis , as well as potential new targets for regulation .", "Long-range transport and targeting of integrins underlies neurite outgrowth and neuronal migration ( Anton et al . , 1999; Condic , 2001; Condic and Letourneau , 1997; Wu and Reddy , 2012 ) .", "Moreover , proper levels and dynamics of α5β1-integrin complexes , shown to localize to nerve growth cones , are required in multiple stages of brain development and synaptic function ( Bi et al . , 2001; Graus-Porta et al . , 2001; Marchetti et al . , 2010; Yanagida et al . , 1999 ) .", "Intriguingly , the neuron-specific 440 kDa AnkB isoform is exclusively targeted to axons and enriched at axonal growth cones and might play a role in axonal guidance ( Kunimoto , 1995 ) .", "It is , thus , conceivable that similar to its roles in migrating fibroblasts , an AnkB/RabGAP1L pathway might facilitate the 440 kDa AnkB-mediated axonal growth cone behavior , axonal guidance and synaptogenesis ." ], [ "All animal care and procedures were approved by the Institutional Animal Care and Use Committee of Duke University .", "AnkB KO mice ( Scotland et al . , 1998 ) were generated by targeted disruption of the endogenous Ank2 gene by homologous recombination .", "In brief , a clone containing 17 kb of the Ank2 gene isolated from a 129SVJ genomic λ DNA library was modified to introduce a NotI site-flanked cassette containing a neomycin resistance gene , an in-frame HA epitope , and a stop codon within an exon in the spectrin-binding domain of AnkB .", "Male C57BL/6 chimeras containing the targeting construct were crossed to C57BL/6 females to assure germline transmission .", "Heterozygous carriers ( Ank2+/− ) were maintained in a mixed 129SVJ/C57BL/6 genetic background and used to generate WT ( Ank2+/+ ) and AnkB KO mice ( Ank2−/− ) littermates .", "AnkB-2xHA , AnkB-mCherry , DD1320AA AnkB-mCherry , and R1194A AnkB-mCherry clones have been previously described ( Lorenzo et al . , 2014 ) .", "Full-length EB1-GFP , LAMP1-GFP , Rab5-RFP , Rab11-GFP , and TGN38-GFP were purchased from Addgene ( Addgene , Cambridge , MA ) .", "The pN1-DEST-mMaple3 vector used as backbone for the mMaple3-tagged clones was generated from the pN1-DEST-mCherry vector .", "In brief , the AgeI-MfeI mCherry sequence was replaced with a 5’-AgeI- mMaple3-MfeI-3’ fragment ( cloned from Zyxin-mMaple3 , a generous gift from Xiaowei Zhuang , Harvard University , MA ) .", "AnkB-mMaple3 and α5-integrin-mMaple3 clones were then generated by LR recombination ( Invitrogen , Carlsbad , CA ) of either AnkB-pENTER or α5-integrin-pENTER with pN1-DEST-mMaple3 .", "AnkB ZU5N-ZU5C-UPA-DD-Ct-mCherry was generated by replacing the GFP sequence from ZU5N-ZU5C-UPA-DD-Ct-GFP with the mCherry sequence using the PmeI and NotI sites .", "ΔDD AnkB-mCherry , E1537K AnkB-mCherry mutants were generated by site-directed mutagenesis .", "mMaple3-2×FYVEEEA1 was generated by replacing the GFP sequence in the GFP-2×FYVEEEA1 clone , a generous gift from P . De Camilli ( Yale University , CT ) , with the mMaple3 sequence using the AgeI and XhoI sites .", "The mouse RabGAP1L cDNA sequence was subcloned into pENTER using the D-TOPO approach .", "mCherry- and GFP-tagged RabGAP1L derivatives were generated by LR recombination .", "The mouse Rab22A cDNA was subcloned into the SalI and BamHI sites of pEGFP-C1 to generate GFP-Rab22A .", "Then , the GFP sequence was removed and replaced by a 5’-AgeI- mMaple3- AccIII-3’AgeI fragment .", "MBP-RabGAPL1 ( 1–235 ) -6xHis was generated by LR recombination between RabGAP1L ( 1-235 ) -pENTER and the pMAL-c4G-DEST vector .", "Similarly , pGBKT7-AnkB DD and pGBKT7-AnkG DD clones were generated by LR recombination between AnkB DD-pENTER or AnkG DD-pENTER and the pGBKT7-DEST vector .", "pGADT7-RabGAP1L ( E507-L815 ) were generated by LR recombination between RabGAP1L ( E507-L815 ) -pENTER and pGADT7-DEST vector .", "All mutations were introduced by site-directed mutagenesis .", "An affinity-purified antibody against RabGAP1L was generated by immunization of rabbits with a purified peptide containing amino acids 1–235 of mouse RabGAP1L ( see generation of anti-RabGAP1L antibody ) .", "Rabbit affinity-purified antibodies against total AnkB ( C-terminal domain ) , β2-spectrin ( spectrin repeats 4–9 ) , GFP , sheep anti-AnkB ( C-terminal domain ) and goat anti-AnkG ( C-terminal domain ) were all generated in our laboratory ( Ayalon et al . , 2011 ) .", "Other primary antibodies include , mouse anti-HA and chicken anti-GFP ( Aves Labs , Tigard , OR ) , rabbit anti-Rab5 and anti-TGN38 ( Cell Signaling Technology , Danvers , MA ) , mouse anti-LAMP1 ( Developmental Studies Hybridoma Bank , University of Iowa , IA ) , rabbit anti-Rab11 ( Invitrogen ) , rat anti-EEA1 and rabbit anti-golgin97 ( Abcam , Cambridge , UK ) , mouse anti-α-tubulin and mouse anti-sheep/goat IgG ( Thermo , Waltham , MA ) .", "Alexa488-anti-mouse/rat CD29 ( Biolegend , San Diego , CA ) , Alexa633-WGA and Alexa568-tranferrin ( Life Technologies , Carlsbad , CA ) were used in internalization and recycling assays .", "Secondary antibodies used includes: Alexa Fluor 488 or 568–conjugated donkey anti–mouse , anti–rabbit , anti-chicken , anti-rat , anti-sheep or anti–goat purchased from Invitrogen .", "A His-MBP-RabGAP1L ( residues 1–235 ) protein was purified from bacterial cultures by a two-step affinity purification protocol using nickel and amylose beads .", "MBP and His tags were removed by subsequent treatment with precision protease and affinity chromatography using GST beads .", "Purified RabGAP1L peptides were used as antigen for rabbit immunization .", "Serum from immunized rabbits was collected and the anti-RabGAP1L antibody purified using in-tandem ovalbumin- , MBP- , and antigen ( RabGAP1L 1–235 ) -affinity columns .", "The eluted antibody was mixed 1:1 ( volume ) with glycerol and stored at −20°C .", "Primary fibroblasts were isolated from postnatal day zero ( PND0 ) Ank2−/− and WT pups and cultured by standard methods .", "Passage one or two MEFs were electroporated with plasmids using an ECM 830 Square Wave Electroporation System830 ( BTX , Harvard Apparatus , Holliston , MA ) following the standard protocol recommended by the manufacturer .", "Cells were either imaged live or fixed with 4% PFA for further examination .", "Cells expressing fluorescently-tagged proteins were cultured on fibronectin-coated MatTek dishes and imaged using a Zeiss LSM 780 inverted confocal microscope equipped with temperature and CO2 controls .", "Individual cells were selected for either one-frame or time-lapse imaging ( 1 frame/2 s , 60 frames ) .", "Tracks of individual vesicles were generated using the manual tracker function of ImageJ .", "Velocity ( µm ) = displacement/time and Persistence = displacement/total distance .", "Total protein homogenates from MEFs were prepared in PBS containing 150 mM NaCl , 0 . 32 M sucrose , 2 mM EDTA , 0 . 1% Triton X-100 , 0 . 1% sodium deoxycholate , 0 . 1% SDS , and protease inhibitors ( 10 µg/ml AEBSF , 30 µg/ml benzamidine , 10 µg/ml pepstatin , and 10 µg/ml leupeptin; EMD Millipore , Billerica , MA ) .", "Samples were centrifuged at 100 , 000 g for 30 min , and the supernatants were precleared with protein A/G Dynabeads ( EMD Millipore ) and subjected to immunoprecipitation using antibodies against AnkB , AnkG , RabGAP1L , or control IgG .", "Immunoprecipitation samples were resolved by SDS-PAGE and Western blotting , and signal detected using the Odyssey CLx imaging system .", "For coimmunoprecipitation experiments , 5 × 106 HEK293 cells were plated in 10 cm dishes and transfected with 4 µg of each plasmid using Lipofectamine 2000 ( Invitrogen ) according to the manufacturer’s instructions .", "Cells were harvested 72 hr after transfection and lysed in 0 . 5% Triton X-100 in lysis buffer ( 10 mM sodium phosphate , 0 . 32 M sucrose , 2 mM EDTA , and protease inhibitors ) .", "Cell lysates were centrifuged at 100 , 000 g for 30 min , and the soluble fraction was collected and pre-cleared by incubation with protein A/G Dynabeads .", "Coimmunoprecipitation experiments were performed using protein A/G Dynabeads and mouse anti-HA or rabbit anti-GFP antibodies .", "Immunoprecipitation samples were resolved by SDS-PAGE and Western blot as described in the previous paragraph .", "The Y2H screen was performed using the Matchmaker Gold Yeast-Two-Hybrid System and the Mouse Universal Normalized cDNA library ( Clontech , Mountain View , CA ) .", "The screen was performed following the recommendations of the manufacturer .", "The GAL4 DNA-BD/bait construct was prepared by ligating a PCR fragment containing the AnkB death domain ( aa1485-1563 , accession no . NM_020977 . 3 ) into the pGBKT7 vector ( Clontech ) .", "The yeast strain Y2HGold was maintained on YPD agar plates on SD –TRP ( tryptophan ) plates when transfected with the GAL4 DNA-BD/AnkB DD bait construct .", "To confirm the expression of the bait and prey plasmids , cells were grown on SD-Leu/Trp plates .", "The library was transformed into the AH 109 yeast strain ( Takara Bio Inc . , Mountain View , CA ) .", "The Y2H screen was performed under high-stringency growth conditions as recommended by the manufacturer .", "Yeast cells coexpressing either AnkB DD or AnkG DD ( baits ) together with either the C-terminal sequence of RabGAP1L cloned in pGADT7 , or the empty pGADT7vector ( prey ) were grown on plates lacking leucine , tryptophan , histidine , and adenine ( -Leu , -Trp , -His , and -Ade ) and supplemented with 125 ng/ml Aureobasidin A . Same conditions were used to assess interactions between AnkB DD mutant baits and the RabGAP1L C-terminal domain .", "PLA was performed using the commercial Duolink kit ( Sigma-Aldrich , St . Louis , MO ) following the manufacturer’s recommendations .", "PFA-fixed cells or deparaffinized tissue sections were incubated overnight with a pair of primary antibodies , each produced in different species , against the putative interacting partners .", "Duolink minus- and plus-probes were used to detect antibody-labeled proteins .", "Cells were transfected with plasmids encoding WT or E1537K AnkB-mMaple3 or α5-integrin-mMaple3 and plated on fibronectin-coated MatTek dishes with a silicone insert .", "Inserts were removed 48 hr post-plating to allow cell migration towards the exposed region .", "Migrating cells at the edges of the dish were selected for photoconversion and imaged by time-lapse video microscopy .", "In brief , the perinuclear region of mMaple3-expressing cells was stimulated with blue light ( λ = 405 nm ) to convert mMaple3 particles from green to red fluorescence .", "Photoconverted cells were continuously imaged for 16 min ( 1frame/15 s , 64 frames ) .", "The fluorescent intensity ( FI ) of red and green particles within the perinuclear region was determined using the Volocity software ( Perkin Elmers , Waltham , MA ) .", "Retrograde transport of non-converted mMaple3-tagged vesicles towards the perinuclear region , expressed as percentage of green fluorescent gain , was calculated as: [FI at 16 min – FI at 45 s ( post-conversion time ) ) / ( FI at 0 s – intensity at 45", "s ) ] .", "The anterograde transport of mMaple3-tagged vesicles was expressed as percentage of red fluorescent loss and calculated as [ ( intensity at 45 s– intensity at 16 min ) / ( intensity at 45 s – intensity at 0", "s ) ] .", "The transport of converted mMaple3 vesicles from the perinuclear region is expressed as the gain of fluorescent intensity at the front or rear cell ends at each time point .", "Cells were washed with room temperature PBS , fixed for 15 min at room temperature in a 4% paraformaldehyde solution in PBS , and permeabilized with 0 . 2% vol/vol Triton X-100 in PBS for 10 min .", "Samples were then blocked for 60 min in blocking buffer ( 3% BSA , 0 . 2% Tween-20 in PBS ) and incubated overnight with primary antibodies in blocking buffer at 4°C .", "Cells were washed three times with PBS , incubated with fluorescent-labeled secondary antibody conjugates in blocking buffer at room temperature , washed three times with PBS , and mounted in Pro-Long Gold mounting media ( Life Technologies , Carlsbad , CA ) .", "Cells were rinsed twice with ice cold PBS and labeled with 0 . 2 mg/ml sulfo-NHS-SS biotin ( Thermo ) in PBS at 4°C for 30 min .", "The remaining sulfo-NHS-SS biotin was quenched with 50 mM Tris pH 8 . 0 in PBS , and the cells were washed two more times with PBS .", "To allow endocytosis , pre-warmed medium was added to the cells and the cultures were incubated at 37°C for various time points before fixation with 4% PFA .", "After 30 min of endocytosis , remaining surface exposed biotin labels were removed by incubating the cells 2 × 5 min in cold 100 mM MESNa buffer ( 50 mM Tris , 100 mM NaCl , 1 mM EDTA , 0 . 2 wt/vol BSA , pH 8 . 6 ) .", "Cells were returned to a 37°C incubator to allow the recycling of biotin-labeled internalized proteins .", "Cells were fixed at various time points and the biotin-labeled proteins detected by Streptavidin-Alexa 488 ( Life Technologies ) .", "Cells were incubated with either Alexa 488-anti-mouse/rat β1-integrin , Alexa 488-anti-mouse/rat β3-integrin ( BioLegend ) , or Alexa 568-transferrin ( Life Technologies ) in DMEM containing 0 . 5% FBS ( DMEM-FBS ) at 4°C for 30 min followed by washes with ice-cold PBS and DMEM-FBS .", "To allow endocytosis , fluorescently-labeled cultures were incubated at 37°C for 30 min .", "The remaining surface-associated fluorescence was quenched by brief acid wash ( 0 . 5% acetic acid , 0 . 5 M NaCl , pH 3 . 0 ) .", "Following internalization cells were washed with PBS and , either fixed ( recycling time 0 min ) , or incubated at 37°C for the indicated times before fixation ( recycling time 15 min , 30 min , 60 min ) .", "The percentage of recycled proteins at a given time point", "( t ) is expressed as: fluorescent intensity on the plasma membrane at time point", "( t ) /fluorescent intensity in cytoplasm at time point ( 0 ) .", "The percentage of remaining intracellular transferrin at a given time point is expressed as a ratio of the fluorescent intensity remaining in the cytoplasm at time point", "( t ) /fluorescent intensity in cytoplasm at time point ( 0 ) .", "The PLKO-BFP-Tet-on vector was generated as described in He et al . ( 2013 ) .", "The production of lentivirus with vectors inserted with the shRNA hairpin targeting mouse RabGAP1L and the subsequent infection of cultured cells were performed following a standard protocal ( He et al . , 2013 ) .", "Hairpins used were: luciferase control ( 5′-GGAGATCGAATCTTAATGTGC-3′ ) and mouse RabGAP1L ( 5′-TGGAACAGGCTTGCAATATT -3′ ) .", "2 × 104 cells were plated in matak plate and treated with 4 µg/ml doxycycline the next day ( Day1 ) .", "Cells were transfected with RabGAP1L rescue plasmids 8 hr later and the number of cells were counted every 24 hr .", "The haptotaxis assay was performed as previously described ( Wu et al . , 2012 ) .", "WT or Ank2-/- MEFs were plated on microfluidic chambers containing a linear fibronectin gradient .", "Ank2-/- MEFs transfected with WT AnkB-GFP or E1537K AnkB-GFP were sorted , and GFP-positive cells were pooled and plated on microfluidic chambers as described above .", "Cells were allowed to migrate for 24 hr on the chamber , and then were manually tracked allowing the calculation of FMI , velocity and persistence of migration , as described in Figure 7A .", "Rose plots of directional migration on normalized polar coordinates were generated using a MATLAB script described in King et al . , 2016 .", "Each experiment was repeated using at least three lines of mouse embryonic fibroblast isolated from three individual PND0 WT ( Ank2+/+ ) and AnkB KO ( Ank2-/- ) littermates , which is considered as biological replicates .", "Each experiment was done three or more times , which is considered as technical replicates .", "All of the data were combined for statistical analysis unless otherwise stated .", "GraphPad Prism ( GraphPad Software , La Jolla , CA ) was used for statistical analysis .", "Statistical differences were determined by unpaired Student’s t-test or by repeated measures one-way ANOVA , followed by a post hoc Tukey’s test .", "Results are presented as mean ± SD .", "Significance was considered as p≤0 . 05 .", "Exact p-values were shown when p>0 . 001 ." ] ]
[ "Endosomal membrane trafficking requires coordination between phosphoinositide lipids , Rab GTPases , and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport .", "Here we report that ankyrin-B ( AnkB ) , through integrating all three systems , functions as a critical node in the protein circuitry underlying polarized recycling of α5β1-integrin in mouse embryonic fibroblasts , which enables persistent fibroblast migration along fibronectin gradients .", "AnkB associates with phosphatidylinositol 3-phosphate ( PI3P ) -positive organelles in fibroblasts and binds dynactin to promote their long-range motility .", "We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like ( RabGAP1L ) and recruits it to PI3P-positive organelles , where RabGAP1L inactivates Rab22A , and promotes polarized trafficking to the leading edge of migrating fibroblasts .", "We further determine that α5β1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling .", "Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5β1-integrin and likely of other specialized endocytic cargos ." ]
[ "The membranes that surround animal and other eukaryotic cells are in a state of flux .", "Small fluid-filled sacs known as vesicles form from the membrane and move into the cell , while other vesicles are returning with cargos of chemicals .", "These processes allow cells to rapidly adjust the composition of their surfaces for different activities , such as migrating to other parts of the body .", "Like rock climbers , migrating cells need to hang onto nearby surfaces as they move and so vesicles deliver sticky “adhesion” proteins to the front of migrating cells .", "At least three different kinds of molecule are involved in delivering vesicles to a particular end of the cell .", "Fat molecules known as phosphoinositide lipids act as markers to identify different vesicles , while proteins called GTPases determine which direction the third type of molecules ( known as molecular motors ) will move the vesicles across the cell .", "However , it is still not clear how these molecules work together to transport specific cargos to the front of cells during migration .", "Now , Qu et al . discover that a protein called ankyrin-B coordinates the directional transport of specific vesicles within migrating cells .", "Biochemical experiments in cells isolated from mouse embryos show that ankyrin-B recognizes vesicles containing specific phosphoinositide lipids and attaches to them .", "Ankyrin-B then recruits molecular motors and another protein called RabGAP1L , which regulates the activity of GTPases , to direct the movements of the vesicles .", "Microscopy experiments reveal that this machinery is essential to transport cell adhesion proteins and other specific cargos to the front of migrating cells .", "The next step following on from this work will be to examine how ankyrin-B and RabGAP1L behave in cells that are still inside the body of the animal .", "Future experiments will identify other cargos that use this machinery to reach the cell membrane and investigate how cells recognize and select these cargos for transport ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "neuroscience" ]
Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity
elife-38080-v3
[ [ "Investigations of the gene regulatory networks involved in cell-type specification during embryonic development have been protein-centric for decades .", "However , given the prevalence of high-throughput sequencing analyses of mammalian genomes , it is now appreciated that non-coding RNAs ( ncRNAs ) account for at least 50~80% of transcriptomes ( Pauli et al . , 2011; Rinn and Chang , 2012 ) .", "Regulatory ncRNAs can be broadly classified based on their size ( Mattick , 2009 ) .", "Short RNA species ( ~20–30 nucleotides [nt] ) , such as microRNAs ( miRNAs ) , have emerged as pivotal modulators of development and disease through mediation of translational repression or mRNA degradation ( Esteller , 2011 ) .", "Long non-coding RNAs ( lncRNAs; >200 nt ) are gaining prominence for their roles in many cellular processes , from chromatin organization to gene expression regulation during embryonic development ( Kung et al . , 2013; Rinn and Chang , 2012; Rutenberg-Schoenberg et al . , 2016 ) .", "Thus , it is not surprising that lncRNAs were recently found to be associated with an array of diseases including cancers , as well as cardiovascular and neurological disorders ( Briggs et al . , 2015 ) .", "Accumulating evidence supports that lncRNAs can induce cis- and trans-acting gene silencing .", "For example , the lncRNA Airn directly represses the paternally-expressed Igf2r gene in cis for the maintenance of ESC differentiation ( Pauler et al . , 2005 ) and Xist lncRNA triggers in cis inactivation of the X chromosome ( Lee , 2009 ) .", "The human lncRNA HOTAIR , which is expressed from the caudal HOXC locus , acts in trans to target the HOXD cluster for gene silencing ( Li et al . , 2013; Rinn et al . , 2007 ) .", "Approximately 20% of lncRNAs are associated with polycomb repressive complex 2 ( PRC2 ) ( Zhao et al . , 2010; Khalil et al . , 2009 ) , which is comprised of many subunits and functions to deposit histone H3K27 trimethylation ( H3K27me3 ) and to suppress gene expression ( Di Croce and Helin , 2013; Margueron and Reinberg , 2011; Simon and Kingston , 2013 ) .", "Although some evidence indicates that lncRNAs might serve as scaffolds for PRC2 assembly and guide PRC2 to specific genomic targets , whether the interaction is specific and necessary in development or disease contexts is still unclear ( Cifuentes-Rojas et al . , 2014; da Rocha et al . , 2014; Davidovich and Cech , 2015; Davidovich et al . , 2015; Davidovich et al . , 2013; Kaneko et al . , 2014a; Kaneko et al . , 2014b ) .", "Therefore , it is imperative to demonstrate that the specific interactions of lncRNAs with the PRC2 complex are functionally important and have specific regulatory targets to direct development or induce disease in vivo .", "We used spinal motor neuron ( MN ) differentiation as a paradigm to assess these interactions .", "Although spinal cord development is one of the best characterized processes in the central nervous system ( CNS ) ( Alaynick et al . , 2011; Catela et al . , 2016; Chen et al . , 2011; Mazzoni et al . , 2013a; Mazzoni et al . , 2013b; Narendra et al . , 2015; Philippidou and Dasen , 2013 ) , how lncRNAs are involved in its transcription factor-driven gene regulatory networks is unclear ( Briscoe and Small , 2015 ) .", "MN differentiation into subtypes is mediated by the mutually exclusive expression of Hox transcription factors , which is programmed according to the body segment along the rostrocaudal ( RC ) axis .", "For example , segmental identity of MNs is defined by the mutually exclusive expression of Hox6 , Hox9 and Hox10 ( Dasen et al . , 2003; Lacombe et al . , 2013 ) .", "In each segment , MNs are grouped into different columns according to their innervating targets .", "For instance , within the brachial Hox6on segment , MNs are further grouped into axial muscle projecting MNs ( Lhx3on , MMC ) and forelimb-innervating MNs ( Foxp1on , LMC ) .", "Finally , another set of mutually exclusive Hox proteins , such as Hox5 and Hox8 expression in the Foxp1on LMC , further controls the rostral and caudal motor pool identity , which directs motor pools to either innervate proximal or distal muscles in the forelimb ( Catela et al . , 2016; Dasen et al . , 2005 ) .", "In the spinal cord , polycomb proteins control the exclusion of certain Hox protein expression at specific RC positions and maintain this repression in differentiated cells .", "Depletion of the polycomb repressive complex 1 ( PRC1 ) component Bmi1 at brachial level causes ectopic expression of Hoxc9 and subjects LMC neurons to a thoracic preganglionic column ( PGC ) fate .", "Conversely , elevation of Bmi1 represses Hoxc9 at thoracic level and subjects PGC neurons to an LMC fate ( Golden and Dasen , 2012 ) .", "These observations suggest that specific Hox repression may be maintained in MNs by distinct PRC1 activity levels , programmed along the RC axis .", "Recently , it was shown that during MN differentiation , Hox chromatin is demarcated into discrete domains controlled by opposing RC patterning signals ( i . e . , retinoic acid ( RA ) , Wnt , and fibroblast growth factors ( FGFs ) ) that trigger rapid and domain-wide clearance of H3K27me3 modifications deposited by PRC2 ( Mazzoni et al . , 2013b ) .", "More specifically , RA activates retinoic acid receptors ( RARs ) and binds to the Hox1~5 chromatin domains , which is followed by synchronous domain-wide removal of H3K27me3 to acquire cervical spinal identity .", "At the tailbud , a gradient of Wnt and FGF signals induces expression of the Cdx2 transcription factor that binds and clears H3K27me3 from the Hox1~Hox9 chromatin domains , thereby establishing brachial or thoracic segmental identity ( Mazzoni et al . , 2013b ) .", "Together , these findings indicate that epigenetic regulation of Hox clusters is critical to initiate and maintain patterns of Hox expression and that cross-repressive interactions of combinations of Hox proteins later consolidate the diversification of postmitotic MNs .", "However , the underlying mechanism that demarcates the histone modifiers at a molecular level is still unclear .", "Although many lncRNAs are known to regulate these histone modifiers , whether lncRNAs are directly involved in MN fate determination remains to be established .", "We found that lncRNAs in the imprinted Dlk1-Dio3 locus are highly enriched in postmitotic MNs .", "The Dlk1-Dio3 locus contains three protein-coding genes ( Dlk1 , Rtl1 , and Dio3 ) from the paternally inherited allele , and multiple lncRNAs and small ncRNAs are derived from the maternally inherited allele , including Meg3 , Rian ( containing 22 box C/D snoRNAs ) , as well as the largest miRNA mega-cluster in mammals ( anti-Rtl1 , which contains the miR-127/miR-136 cluster of 7 miRNAs , and Mirg that within the miR-379/miR-410 cluster ) .", "Interestingly , all of the ncRNAs are regulated by a common cis-element and epigenetic control , resulting in a presumable large polycistronic transcription unit ( Das et al . , 2015; Lin et al . , 2003; Seitz et al . , 2004 ) .", "Although the Dlk1-Dio3 locus is well known to play crucial roles in stem cells ( Lin et al . , 2007; Lin et al . , 2003; Qian et al . , 2016 ) , we unexpectedly found that expressions of Meg3 and other lncRNAs from the Dlk1-Dio3 locus are also all enriched in postmitotic MNs .", "However , whether this locus functions during neural development had not been explored previously .", "Here , we show that lncRNAs in the imprinted Dlk1-Dio3 locus shape postmitotic MNs by inhibiting progenitor and non-neural genes , and they also control MN subtype identity by regulating Hox expression .", "Our results provide strong evidence for the critical function of lncRNAs during MN development , emphasizing their physiological functions during embryonic development ." ], [ "As epigenetic landscape remodelling and the cell fate transition during MN differentiation are well characterized ( Chen et al . , 2011; Li et al . , 2017; Tung et al . , 2015 ) , we took advantage of an ESC differentiation approach that can recapitulate MN development to systematically identify cell lncRNAs during this differentiation process .", "Firstly , an ESC line harbouring the MN transgenic reporter Hb9::GFP was harnessed into MNs ( Wichterle et al . , 2002 ) , and we then sequentially collected RA-induced nascent neural epithelia ( Hoxa1on , NE at day 2 ) , MN progenitors ( Olig2on , pMN at day 4 ) , and postmitotic MNs ( Hb9::GFPon , postmitotic MNs at day 7 ) by fluorescence-activated cell sorting ( FACS ) .", "Simultaneously , spinal interneurons ( INs ) derived from [smoothened agonist; SAG]low conditions were collected and Hb9::GFPoff cells were sorted at day 7 as controls ( Figure 1A ) .", "Next , we performed strand-specific RNA-seq across libraries preserving non-polyadenylated transcripts while removing ribosomal RNAs , since many lncRNAs are non-polyadenylated ( Yin et al . , 2012; Zhang et al . , 2012 ) , and carried out de novo transcriptome assembly ( Qian et al . , 2016 ) to discover novel lncRNAs that might be specifically enriched during MN development ( detailed in the Materials and methods and summarized in Figure 1—figure supplement 1A; Supplementary file 1 ) .", "Several known markers for each cell type during MN differentiation were accurately recovered , corroborating the high quality and specificity of our RNA-seq data ( Figure 1B ) .", "Our approach yielded 10 , 177 lncRNAs , 752 of which ( 7 . 39% ) were previously unidentified from the Ensemble mm10 database .", "We also found that 4295 ( 77 . 78% ) of our identified lncRNAs overlapped with recently reported spinal MN-related lncRNAs , which were discovered by poly A+-enriched RNA-seq approaches ( Amin et al . , 2015; Narendra et al . , 2015 ) ( Figure 1—figure supplement 1B ) .", "Finally , we removed minimally expressed transcripts ( TMM normalized read count <10 in all samples ) , which left 602 expressed lncRNAs ( Supplementary file 2 ) .", "Based on stage-specific scores ( see Materials and methods ) , 70 stage-signature lncRNAs during the ESC~MNs differentiation process were uncovered ( ESC , NE , pMN , MN , and IN in Figure 1C ) .", "Compared to protein-coding genes , both annotated lncRNAs and novel lncRNAs ( newly identified in our de novo transcriptome assembly ) had higher cell-type specificity ( Figure 1D , Kolmogorov-Smirnov test , p=1 . 41 × 10−9 and p=3 . 53 × 10−7 , respectively; see Materials and methods ) , implying that lncRNAs might play specific roles in each cell type during ESC~MN differentiation .", "To identify developmentally up-regulated lncRNAs , we compared day 4 pMNs vs . day 7 postmitotic MNs ( Figure 2A ) .", "Furthermore , to retrieve cell-type-specific lncRNAs , we performed a pairwise comparison of day 7 postmitotic MNs against day 7 INs ( Figure 2B ) .", "We identified 117 lncRNA candidates from our analysis as being postmitotic MN-enriched .", "We further selected several MN-lncRNAs that manifested high normalized reads from RNA-seq data and verified their MN-specific expression by qPCR ( Figure 1—figure supplement 1C ) .", "Interestingly , the lncRNAs Meg3 , Rian , and Mirg , which are transcribed from the imprinted Dlk1-Dio3 locus on mouse chromosome 12qF , all manifested strong enrichment in postmitotic MNs ( simplified schematic locus depicted in Figure 2C , detailed locus information in Figure 2—figure supplement 1A ) .", "Since these lncRNAs are conserved amongst placental mammals ( Ogata and Kagami , 2016 ) , we chose to characterize their functions in greater detail .", "To investigate why Meg3-Rian-Mirg are highly enriched in postmitotic MNs , we examined the binding landscape of MN-specific transcription factors ( i . e . , Lhx3 and Isl1 ) , histone modifications ( H3K4me3 and H3K27ac ) , and chromatin accessibility ( ATAC-seq ) across the Meg3-Rian-Mirg locus from previously published studies ( Figure 2D ) ( Mazzoni et al . , 2013a; Narendra et al . , 2015; Rhee et al . , 2016 ) .", "Within this locus , we uncovered an MN-specific active chromatin region that possesses enhancer/promoter characteristics with direct MN-specific transcription factor binding ( Figure 2D ) .", "Furthermore , overexpression of MN-TFs in a maternally-inherited intergenic differentially methylated region deletion ( IG-DMRmatΔ ) ESC line , which leads to simultaneous silencing of all maternally-expressed lncRNAs in the Meg3-Rian-Mirg locus but leaves the MN-TF binding site intact ( Figure 2—figure supplement 1A ) ( Lin et al . , 2007; Lin et al . , 2003 ) , can robustly induce Meg3 expression ( Figure 2—figure supplement 1B ) .", "Therefore , we suggest that MN-TFs bind and directly activate Meg3 during ESC~MN differentiation .", "We further performed Meg3 in situ hybridization and immunostaining of the adjacent sections along the RC axis from E10 . 5~12 . 5 .", "We found that Meg3 expression: ( 1 ) is enriched in the mantle zone of the developing spinal cord during development and is gradually enriched in postmitotic MNs ( Isl1/2on cells ) after E12 . 5; ( 2 ) has no preference for columnar MN subtypes , as revealed by Foxp1 ( LMC-MNs ) and Lhx3 ( MMC-MNs ) immunostaining; and ( 3 ) exhibits rostral high ( brachial and thoracic ) and caudal low ( lumbar ) asymmetry after E12 . 5 ( Figure 2E and F ) .", "Why does Meg3 exhibit strong enrichment in the brachial spinal cord ?", "Given that previous reports indicate that rostral Hox genes enriched in the brachial spinal cord are mediated by an RA gradient ( Mazzoni et al . , 2013b; Novitch et al . , 2003 ) , we hypothesized that Meg3 might also be induced by RA .", "To examine this possibility , we checked if there is any RA-driven binding to RAR sites near the Meg3 promoter ( Figure 2G ) ( Mahony et al . , 2011 ) .", "Interestingly , we found that RA treatment results in novel binding of RAR directly to the Meg3 promoter , as well as subsequent recruitment of the basal transcription complex ( Pol2-S5P in Figure 2H ) .", "Moreover , we observed that Meg3 is induced after the addition of RA in IG-DMRmatΔ ESCs after 8 hr ( Figure 2—figure supplement 1C ) , indicating that RA/RAR activation triggers the strong Meg3 expression in rostral brachial MNs .", "Finally , to characterize the abundance and subcellular localization patterns of Meg3 at a cellular level , we designed a set of single molecule RNA FISH probes specific to Meg3 and examined their expression in ESC~MNs .", "We observed a speckled pattern of Meg3 expression enriched in the nucleus , suggesting it has a potential function in gene regulation ( Figure 2—figure supplement 1D ) .", "Furthermore , qPCR of subcellular-fractionated RNAs from ESC~MNs validated that Meg3 is not only enriched in the nucleus , but that it is also chromatin-associated ( Figure 2—figure supplement 1E; Gapdh as cytoplasmic marker , Rnu1 ( U1 snRNA ) as nuclear marker , and Kcnq1ot1 as a chromatin-associated RNA control ) .", "Together , these findings suggest that lncRNAs in the Dlk1-Dio3 locus are postmitotic MN-enriched , and that they are directly activated by MN-TFs and RA/RAR .", "At a cellular level , Meg3 is highly enriched in MN nuclei and is chromatin-associated , indicating a potential function in chromatin regulation .", "While several previous reports have revealed that interactions between Jarid2 and ncRNAs regulate PRC2 recruitment to chromatin , including lncRNAs in the Dlk1-Dio3 locus of ESCs ( da Rocha et al . , 2014; Kaneko et al . , 2014a; Kaneko et al . , 2014b ) , the roles of PRC2/Jarid2 in postmitotic cells are less clear .", "Unlike the PRC2 complex , Jarid2 is known to have diverse cell-type-specific functions ( Landeira and Fisher , 2011 ) .", "Surprisingly , we found that expression of Jarid2 was reactivated in postmitotic MNs , pointing to a possible specific regulation in this cell type ( Figure 3—figure supplement 1A upper panel ) ( Takeuchi et al . , 1995 ) .", "Moreover , compared to several known lncRNAs that interact with PRC2 complex , Meg3 and Rian manifested much more abundant expressions in the postmitotic MNs ( Figure 3—figure supplement 1A lower panel ) .", "This prompted us to examine if lncRNAs in the Dlk1-Dio3 locus bind to the PRC2 complex and maintain postmitotic MN fate by controlling the H3K27me3 landscape .", "To test this hypothesis , we first demonstrated that immunoprecipitation ( IP ) of endogenous PRC2 complex components ( i . e . , Ezh2 and Suz12 ) , as well as the PRC2 cofactor Jarid2 , from ESC~MNs specifically retrieves Meg3 , Rian and Mirg RNA , whereas the nuclear ncRNA Rnu1 and the lncRNA Malat1 were not captured by Ezh2 , Jarid2 , or Suz12 ( Figure 3A ) .", "Given that Rian and Mirg are further processed to snoRNAs and miRNAs ( Lin et al . , 2003 ) and that Meg3 is known to regulate pluripotency ( Stadtfeld et al . , 2010 ) , imprinting ( Das et al . , 2015 ) , and PRC2 function ( Zhao et al . , 2010 ) , we focused on biochemical characterization of Meg3 .", "Several Meg3 isoforms have previously been documented , so we scrutinized across the entire Meg3 locus ( ~31 kb ) and found that two isoforms , Meg3v1 and Meg3v5 ( Ensemble mm10 database ) , are predominantly expressed in Hb9::GFPon MNs ( Figure 3—figure supplement 1B , C ) .", "Moreover , Meg3v1 and Meg3v5 isoforms account for more than 99% of Meg3 transcripts than other isoforms during ESC~MN differentiation .", "( Figure 3—figure supplement 1D ) ( Kaneko et al . , 2014b; Zhou et al . , 2007 ) .", "Interestingly , Meg3v1 and Meg3v5 have mutually exclusive exon sequences ( Figure 3—figure supplement 1E ) , raising the possibility that the two isoforms might exert different functions .", "However , both purified biotinylated Meg3v1 and Meg3v5 RNA retrieved Ezh2 from cell nuclear extracts of ESC~MNs ( Figure 3B; GFP RNA was used as a negative control ) .", "These results suggest that these Meg3 isoforms directly interact with PRC2/Jarid2 complexes and might facilitate association of the PRC2 complex with Jarid2 .", "As the PRC2 complex and Jarid2 are known to interact in a non-stoichiometric manner ( Pasini et al . , 2010; Peng et al . , 2009 ) , we further examined if Meg3 facilitates the interaction between PRC2 complex and Jarid2 .", "To test this possibility , we performed IP with Ezh2 ( a core component of PRC2 ) to retrieve Jarid2 from ESC~MNs ( Figure 3C ) .", "We first verified that Meg3 knockdown ( KD ) did not affect the protein abundance of Ezh2/Jarid2 , but we did observe that it undermined the interaction between Ezh2 and Jarid2 , suggesting that Meg3 facilitates this interaction ( Figure 3C and Figure 3—figure supplement 1F ) .", "We then investigated if the two Meg3 isoforms have differing abilities to facilitate Ezh2/Jarid2 binding by generating two locus-defined Tet-ON-inducible Meg3 ESCs ( Figure 3D , iMeg3v1 and iMeg3v5 ) .", "Upon doxycycline induction , both Meg3 isoforms were induced ~20–50 fold , yet the abundance of Ezh2/Jarid2 remained unaffected ( Figure 3E and F ) .", "Meg3v5 overexpression in ESC~MNs significantly increased the binding of Ezh2 and Jarid2 , whereas Meg3v1 overexpression Figure 3E; Figure 3F had a minimal effect ( Figure 3G ) , indicating that Meg3v5 is a strong facilitator of the binding of the PRC2 complex and Jarid2 .", "Accordingly , we suggest that Meg3 , and particularly the Meg3v5 isoform , facilitates the binding of the PRC2 complex and Jarid2 in postmitotic MNs .", "To test if the binding of Ezh2/Jarid2 by the lncRNAs in the Dlk1-Dio3 locus is important to maintain the epigenetic landscape in postmitotic MNs , we systematically analyzed genome-wide H3K27me3 profiles of control and IG-DMRmatΔ ESC~MNs by ChIP-seq ( chromatin immunoprecipitation-sequencing ) ( Figure 4A ) .", "To overcome the complication of concomitant up-regulation of paternal coding genes in IG-DMRmatΔ ESCs ( Lin et al . , 2007; Lin et al . , 2003 ) , we further established two retrovirus-based short hairpin RNAs ( shRNAs ) targeting Meg3 and used a knockdown approach to prevent impairment of DMR sites .", "Both shRNAs reduced the expression of Meg3 by an average of ~90% compared to endogenous levels in ESC~MNs ( Figure 4—figure supplement 1A ) .", "As negative controls , we performed independent infections with retroviruses containing scrambled shRNA with no obvious cellular target RNA .", "We selected two stable Meg3 KD ESCs ( referred to as H6 and K4 hereafter ) that had the best ESC morphology for further experiments .", "Verification by qPCR indicated that the expressions of two other lncRNAs , Rian and Mirg , from the maternal allele of the Dlk1-Dio3 imprinted locus were all significantly down-regulated in Meg3 KD MNs , whereas paternal genes were unaffected ( Figure 4—figure supplement 1A ) .", "This finding is consistent with a previous report indicating that Meg3-Rian-Mirg probably represents a single continuous transcriptional unit ( Das et al . , 2015 ) .", "We then performed H3K27me3 ChIP-seq of control and Meg3 KD MNs .", "We observed a trend of global down-regulation of H3K27me3 in both independent experiments of Meg3 KD MNs , most likely a reflection of compromised Ezh2/Jarid2 interaction ( Figure 4—figure supplement 1B ) .", "Since the response to PRC2 activity change in a given cell type might be context-dependent ( Davidovich et al . , 2013 ) , we sought to identify relevant genes in MNs regulated by Dlk1-Dio3 locus-derived lncRNAs based on the loss of H3K27me3 .", "To achieve this , we profiled gene transcriptomes of control , IG-DMRmatΔ , and Meg3 KD ESC~MNs .", "Next , we compared the co-upregulated genes between IG-DMRmatΔ and Meg3 KD ESC~MNs , together with H3K27me3 landscape upon the loss of ncRNAs in the Dlk1-Dio3 locus ( Figure 4A and Figure 4—figure supplement 1C ) .", "This approach revealed 585 genes in MNs that displayed down-regulation of the H3K27me3 epigenetic landscape and concomitant up-regulation of gene expression upon loss of the Meg3 lncRNAs ( Figure 4A ) .", "Gene ontology ( GO ) analysis of these genes revealed significant enrichment for RC patterning and progenitor genes , and strikingly so for homeodomain Hox genes ( Figure 4—figure supplement 1D; false discovery rate ( FDR ) q-value ≤0 . 05 ) .", "Subsequently , we observed that MN progenitor ( Pax6 and Irx3 ) and majority of caudal Hox genes ( Hox8~13 ) were up-regulated with a concomitant down-regulation of the H3K27me3 epigenetic landscape ( Figure 4B and C ) .", "We corroborated this finding by generating a third Meg3 KD ESC line ( I6 ) and confirming that all Meg3 KD ESC~MNs exhibited imbalanced expression of 3' and 5' Hox genes across entire Hox clusters ( Figure 4—figure supplement 1E ) .", "Thus , for both Meg3 KD and IG-DMRmatΔ ESC~MNs , dysregulation of progenitor and caudal Hox gene expression is apparent , likely due to loss of the robustness of the epigenetic landscape of postmitotic MNs .", "If Meg3 scaffolds PRC2/Jaird2 to maintain the silenced H3K27me3 epigenetic landscape in progenitor and caudal Hox genes of postmitotic MNs , we predicted that ( 1 ) the binding patterns of Ezh2/H3K27me3 would display concordant tendency in MNs; and ( 2 ) the bindings of Ezh2/Jarid2 to the gene loci of progenitor and caudal Hox genes in MNs would be compromised upon the loss of Meg3 .", "Consistent with our prediction , we verified that ( 1 ) the epigenetic landscapes of H3K27me3 in the progenitor and caudal Hox genes uncovered here are concordant with Ezh2 enrichment revealed by a previous study that used the same ESC~MN differentiation approach to generate cervical Hoxa5on MNs ( Figure 4—figure supplement 2A , B ) ( Narendra et al . , 2015 ) ; ( 2 ) Upon Meg3 KD , the bindings of Ezh2/Jaird2 to progenitor ( i . e . , Pax6 and Irx3 ) and caudal Hox ( i . e . , Hoxc8 ) genes were concomitantly reduced ( Figure 4—figure supplement 2C–E ) .", "Taken all together , these results suggest that Meg3 bridges the PRC2/Jarid2 complex to perpetuate the rostral MN cell fate by silencing epigenetic state of MN progenitor and caudal Hox genes .", "To corroborate the observed phenotype of IG-DMRmatΔ ESC~MNs , we further scrutinized the MN phenotype in IG-DMRmatΔ embryos .", "Consistent with previous studies , IG-DMRmatΔ embryos died soon after E16 ( Lin et al . , 2007 ) , so we analyzed MN phenotypes from E10 . 5~E14 . 5 in this study .", "We first verified that the expression of Meg3 is still lacking in the developing spinal cord of E14 . 5 IG-DMRmatΔ embryos ( Figure 5—figure supplement 1A ) .", "Ventral neuronal progenitor patterning was not affected in the IG-DMRmatΔ embryos revealed by Olig2 and Nkx2 . 2 ( Figure 5—figure supplement 1B , C ) .", "We then checked the dorsal progenitor proteins Pax6 and Irx3 .", "Compared to the control littermates , we observed a significant increase in the percentage of Pax6on ( 45% penetrance , n = 5/11 ) , and Irx3on cells ( 100% penetrance , n = 8/8 ) for postmitotic MNs ( Is11/2on ) along the entire RC axis of the ventral spinal cord in the IG-DMRmatΔ embryos ( Figure 5A and C , only the cervical segment is shown; quantifications shown in Figure 5B and D ) .", "However , Hb9on and Isl1 ( 2 ) on MNs were comparable between the control and IG-DMRmatΔ embryos at E10 . 5 ( Figure 5E and F ) .", "Although dorsal progenitor genes were aberrantly up-regulated in the postmitotic MNs , production of MNs remained relatively unaffected , suggesting that the generation of MNs is still intact with the co-expressed progenitor/postmitotic genes in the IG-DMRmatΔ embryos ( Figure 5G ) .", "This outcome is consistent with the postmitotic expression of Meg3 and its function to maintain the silenced epigenetic state of progenitor genes .", "Next , we checked if the expression of Hox proteins is affected in IG-DMRmatΔ embryos .", "We first assessed how loss of Dlk1-Dio3 locus-derived lncRNAs affected the specification of segmental MNs , marked by brachial ( Hoxc6 ) , thoracic ( Hoxc9 ) , and lumbar ( Hoxd10 ) Hox levels .", "We observed comparable numbers of cells expressing respective Hox proteins at each segmental level between control and mutant embryos ( Figure 5—figure supplement 1D , E ) .", "Columnar identities of axial ( Lhx3on ) and limb-innervating MNs ( Foxp1on ) were also largely unaffected in the IG-DMRmatΔ embryos ( Figure 5—figure supplement 1F , G ) .", "To further examine MN subtype diversification within the limb-innervating MNs , we checked the Hox proteins involved in pool specification ( Catela et al . , 2016; Dasen et al . , 2005 ) .", "Whereas reciprocal expression of Hoxa5 and Hoxc8 was maintained along the RC axis in the Hox6on LMC MNs of control embryos , Hoxc8 was expanded rostrally into Hoxa5on territory in IG-DMRmatΔ embryos , along with a significant increase of the Hoxc8-mediated downstream motor pool genes , Pea3 and Scip ( n = 5 embryos in Figure 6A and C for rostral brachial segments and Figure 6B and D for caudal brachial segments; quantification in Figure 6E and F ) .", "The reduction of Hoxa5 was not attributable to apoptosis , as cCasp3on cells were comparable in both control and IG-DMRmatΔ mutant embryos ( data not shown ) .", "Taken together , the switching of Hoxa5on to Hoxc8on in MNs of IG-DMRmatΔ embryos is not transient , which leads to a concomitant change in motor pool fate ( Figure 6G ) .", "To further examine the impact of switching the MN pool subtype identity of LMC-MNs , we assessed the potential trajectory and target selectivity of motor axons in wild type control and IG-DMRmatΔ embryos ( Liau et al . , 2018 ) .", "We bred IG-DMRmatΔ mutants to a transgenic line of Hb9::GFP mice in which all motor axons are labeled with GFP and then analyzed the overall pattern of limb innervation .", "First , the images of motor nerves from light sheet microscopy were converted into panoramic 3D images ( upper panel in Figure 7A; Videos 1 and 2 ) .", "The overall trajectory of each motor nerve was reconstructed by Imaris ( lower panel in Figure 7A ) and this conversion enabled semi-automatic calculation of the number of motor nerve terminals in each skeletal muscle , as well as comparison of the extent of motor axon arborization between skeletal muscles ( see Materials and methods for details ) .", "Under higher magnification with better resolution , we observed the terminal arbors of suprascapular ( Ss ) nerves of scapulohumeralis posterior muscles and axillary ( Ax ) nerves were significantly eroded and reduced ( Figure 7B and C ) , consistent with the caudalized switch from Hoxa5 to Hoxc8 expression within LMC neurons .", "Concomitantly , increased arborization complexity of distal muscle-innervating nerves , including posterior brachial cutaneous ( PBC ) nerves were manifested in the IG-DMRmatΔ embryos ( Figure 7D , quantification in 7E , n = 6 , p<0 . 01 , Mann–Whitney U test ) .", "Thus , the IG-DMRmatΔ mutants displayed deficiencies in peripheral innervation of MNs , which might be a consequence of dysregulation of Hox proteins and/or other axon arborization genes in the absence of lncRNAs from the Dlk1-Dio3 locus .", "As our current and previous results indicated that the expressions of most lncRNAs in the Meg3-Rian-Mirg locus are reduced upon Meg3 KD and in IG-DMRMatΔ ( Figure 4—figure supplement 1A ) ( Lin et al . , 2003 ) , it remains puzzling if these ncRNAs work independently or synergistically in this locus to regulate MNs .", "To further parse this question , we generated two single lncRNA RianΔ/Δ and MirgΔ/Δ ESCs respectively by using CRISPR-Cas9 mediated approaches ( Figure 8A ) .", "The design of the targeted deletion regions of Meg3 and Rian followed two previous studies ( Han et al . , 2014; Labialle et al . , 2014 ) , which led to a 23 kb deletion in the RianΔ/Δ ESC and a 20 kb deletion in the MirgΔ/Δ ESC ( Figure 8A ) .", "We first verified that the paternal gene ( i . e . , Dlk1 ) is not affected in either RianΔ/Δ or MirgΔ/Δ ESCs .", "In the RianΔ/Δ ESC , expressions of Meg3 and Mirg were relatively unaffected , whereas that of Rian was compromised .", "Conversely , only expression of Mirg was impaired significantly in the MirgΔ/Δ ESCs , but expressions of Meg3 and Rian manifested minimal changes in that cell line ( Figure 8B ) .", "Upon differentiation , Meg3 KD ESC~MNs showed ectopic up-regulated expressions of progenitor and caudal Hox genes ( Figure 4 and Figure 8C ) .", "In contrast , expression of Hoxa5 remained unchanged in the RianΔ/Δ and MirgΔ/Δ ESC~MNs , and Pax6 and Hoxc8 expressions were also unaltered , with similar expression levels to controls ( Figure 8C ) .", "These results indicate that Meg3 might be the major regulatory lncRNA responsible for the observed MN phenotype displayed by the IG-DMRmatΔ embryos ( Figure 8D ) ." ], [ "Although lncRNAs derived from the Dlk1-Dio3 locus are highly expressed in the CNS , their functions during neural development are largely unknown ( Wang et al . , 2012; Zhang et al . , 2003; Zhou et al . , 2012 ) .", "Upon KD of Meg3 ( a Dlk1-Dio3 locus-derived lncRNA ) , we uncovered that:", "1 ) many adjacent progenitor genes were significantly up-regulated; and", "2 ) the rostral Hox genes were significantly down-regulated , with a concomitant increased expression of caudal Hox genes in ESC~MNs .", "This phenotype was recapitulated in IG-DMRmatΔ embryos , in which Hoxc8 expression is expanded in otherwise Hoxa5on MNs .", "Several reports have identified that certain lncRNAs can shape the Hox epigenetic landscape by cis and trans modulation ( Dasen , 2013; Rinn et al . , 2007; Wang et al . , 2011 ) .", "In addition , we recently uncovered that a novel trans Hox-miRNA circuit can filter Hox transcription noise and prevents precocious protein expression to confer robust individual MN identity ( Li et al . , 2017 ) .", "Here , we have now added the Meg3 imprinted lncRNA to that list as a novel trans-acting lncRNA that maintains the Hox epigenetic landscape , most likely by recruiting Jarid2 to the PRC2 complex .", "Given that Meg3 is also highly expressed in ESCs ( Kaneko et al . , 2014a; Mo et al . , 2015 ) , we plan to generate a targeted Meg3 floxed allele mouse line in the future that will allow us to specifically knockout Meg3 in MNs and recover the potential function of Meg3 in cell-type specific contexts .", "Why do MNs deploy lncRNA-mediated strategy to maintain postmitotic cell fate by inhibiting progenitor genes and regulating Hox boundaries ?", "The dynamic role of lncRNAs in modulating PRC2 function is well documented; ranging from recruitment , complex loading and activity control to gene targeting ( Davidovich and Cech , 2015; Kretz and Meister , 2014 ) .", "A recent study of Drosophila Hox genes revealed that epigenetic H3K27me3 chromatin modification functions as a legitimate carrier of epigenetic memory ( Coleman and Struhl , 2017 ) , providing compelling evidence for a physiologically significant role of chromatin modification in epigenetic inheritance .", "Nonetheless , the epigenetic memory carried by H3K27me3 in a postmitotic cell may still be overridden by H3K27me3-opposing demethylases ( Coleman and Struhl , 2017 ) .", "Our RNA-seq data revealed that two prominent H3K27me3 demethylases , Kdm6a ( Utx ) and Kdm6b ( Jmjd3 ) , are reactivated in postmitotic MNs ( data not shown ) .", "Although the function of H3K27me3 demethylase reactivation in postmitotic MNs remains unknown , these findings raise the possibility that the epigenetic memory of the H3K27me3 landscape in postmitotic MNs might still need to be ‘actively’ maintained to counterbalance H3K27me3 demethylase activity .", "Enrichment of postmitotic MN lncRNAs might therefore bridge and scaffold the PRC2/Jarid2 interaction and activity to maintain MN epigenetic memory by repressing progenitor genes and also carve MN subtype identity by repressing caudal Hox genes ( Figure 8D ) .", "Although we observed a complete 100% penetrance of Hoxa5-c8 cell fate switch in the rostrobrachial segments and an increase co-expression of Irx3onHb9on cells in the IG-DMRmatΔ embryos ( Figures 5 and 6 ) , ectopic Pax6 was manifested at partial penetrance ( 45% , 5 in the 11 mutants ) and several caudal Hox protein ( including Hox9 and", "10 ) were not shown to display significant change in vivo ( Figure 5 and Figure 5—figure supplement 1 ) , consistent with the finding that removal of Ezh2 from MN progenitors has no detectable impact on segmental Hox expression in the spinal cord ( Hoxc6 and Hoxc9 ) ( Golden and Dasen , 2012 ) .", "This result also suggests that a compensatory PRC1-mediated function in vivo might make up for the loss of Meg3-mediated epigenetic maintenance in segmental MNs ( Golden and Dasen , 2012; Mazzoni et al . , 2013b ) .", "Our results are not entirely unexpected as many potent miRNA/lncRNA KO mice reflect either only partial phenotype penetrance ( Li et al . , 2013; Li et al . , 2016; Medeiros et al . , 2011 ) , or more severe phenotypes upon genetic or environmental stresses ( Williams et al . , 2009 ) .", "Moreover , many miRNAs/TFs function as repressors to silence progenitor/neighboring interneuron genes ( Chen et al . , 2011; Shirasaki and Pfaff , 2002 ) , they may also constitute a coherent loop with lncRNAs to safeguard the terminal postmitotic cell fate with a fail-safe control .", "A previous study reported that hypomorphic Suz12-/- ESCs maintained with a low amount of H3K27me3 can differentiate into MNs , albeit with a significant increase in the expression of caudal Hoxc6 and Hoxa7 compared to wild-type cells ( Mazzoni et al . , 2013b ) .", "Interestingly , another study found that several PRC2 mutant ESC lines that maintain varying levels of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to MNs , but only a subset of the genes that function to specify other lineages were not repressed in these cells ( Thornton et al . , 2014 ) .", "This outcome might not be surprising since other epigenetic marks , such as DNA methylation , might safeguard gene expression throughout differentiation ( Manzo et al . , 2017 ) .", "In this study , up-regulated genes in spinal MNs upon loss of Meg3-Rian-Mirg exhibited 50% concordance ( 3953/7474 ) with the up-regulated genes in Suz12-/- spinal MNs ( data not shown ) .", "Together , these results strongly endorse the critical function of PRC2/lncRNA in perpetuating the postmitotic cell fate of cervical Hoxa5on MNs .", "Given that lncRNAs such as Hotair are proposed to scaffold the PRC2 complex and guide it to specific genome loci ( Rinn et al . , 2007; Rinn and Chang , 2012; Tsai et al . , 2010 ) , it is tantalizing to hypothesize that Meg3 might manifest the dual functions of scaffolding the PRC2/Jarid2 complex and guiding it to specific loci in different cell contexts .", "This scenario could partially explain why only subsets of genes in an MN context are particularly sensitive to the loss of ncRNAs from the Dlk1-Dio3 locus .", "Inspired by the salient Hox phenotype exhibited in the IG-DMRmatΔ mutants , we envisage using Meg3 as a paradigm to decipher the Meg3-protein-DNA interactome by ChIRP-seq/ChIRP-MS , thereby allowing us to decipher the detailed targeting mechanism of PRC2/Jarid2 involved in maintaining the epigenetic landscape during embryonic development ( Chu et al . , 2015 ) .", "Interestingly , Hotair -/- mice also show partial penetrance of homeotic transformation and increased expression of Meg3 ( Li et al . , 2013 ) .", "This finding prompts us to test in the future the hypothesis that trans-acting lncRNAs might have an unexpected redundant role for PRC2 complex scaffolding and targeting despite having no primary sequence conservation .", "Generating compound lncRNA mutants that can scaffold the PRC2/Jarid2 complex will shed light on this topic .", "The role of lncRNAs in modulating PRC2 function is well documented , but very dynamic; from recruitment , complex loading and activity control to gene targeting ( Davidovich and Cech , 2015; Kretz and Meister , 2014 ) .", "Mammalian PRC2 binds thousands of RNAs in vivo , and it is a good system for studying the recruitment of chromatin modifying factors by RNA .", "Recent studies suggest that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and propose a mechanism by which lncRNAs contribute to PRC2 recruitment ( da Rocha et al . , 2014; Kaneko et al . , 2014a; Kaneko et al . , 2014b ) .", "Other studies have provided an alternative working model , whereby the JARID2 and PRC2 sub-complex have different RNA-binding affinities and RNA binding to EZH2 inhibits its methyltransferase activity in a concentration- and binding affinity-dependent manner .", "Surprisingly , the binding of RNA attenuates the methyltransferase activity of EZH2 , which allows JARID2 to relieve the repressive effect of RNA on PRC2 catalysis ( Cifuentes-Rojas et al . , 2014; Davidovich et al . , 2015; Davidovich et al . , 2013 ) .", "Both these models differ but are not contradictory , as both models emphasize the role of RNA in the PRC2/JARID2 complex , albeit with different binding affinities and modes of action .", "Interestingly , a recent report further uncovered that MEG3 binds to chromatin sites with GA/GT-rich sequences through RNA–DNA triplex formation ( Iyer et al . , 2017 ) .", "This finding raises the possibility that the GA-rich motif alone is not sufficient in all cell types ( Chu et al . , 2011 ) .", "Future systematic Meg3 ChIRP-seq analyses , together with Ezh2/Jarid2 CLIP-seq , at all stages of ESC~MN differentiation might aid in identifying the context-dependent/independent lncRNA-mediated PRC2 targeting strategy .", "There have been multiple efforts to dissect the functions of the maternally-expressed lncRNAs in the Meg3-Rian-Mirg locus over the past decade ( McMurray and Schmidt , 2012; Steshina et al . , 2006; Takahashi et al . , 2009; Zhou et al . , 2010 ) , but definitive results remain elusive .", "The difficulty is mainly attributable to two major hurdles; namely that", "1 ) there are many DMRs that control imprinting status in upstream , promoter , and exon regions of Meg3 , and", "2 ) Meg3 might function in cis to regulate its imprinting status ( Matsubara et al . , 2015; Ogata and Kagami , 2016 ) .", "Specifically , two Meg3 knockout mouse lines have previously been generated either by deletion of the first five exons plus approximately 300 bp of the adjacent upstream promoter region of Meg3 ( ~5 . 9 kb ) ( Zhou et al . , 2010 ) or by deletion of ~10 kb that includes the Meg3-DMR region plus the first five exons of Meg3 ( Takahashi et al . , 2009 ) .", "Both of these Meg3 KO lines also manifested loss of maternal Rian and Mirg lncRNA expression .", "However , whereas the ~5 . 9 kb Meg3 KO line ( Zhou et al . , 2010 ) exhibited perinatal lethality , the ~10 kb Meg3 KO line ( Takahashi et al . , 2009 ) presented a much milder phenotype in that the mice were born alive and lived up to 4 weeks after birth .", "We also found that expression of most , if not all , lncRNAs in the Meg3-Rian-Mirg locus are reduced upon Meg3 KD and in IG-DMRMatΔ mutants .", "Therefore , it remains technically challenging to obtain a specific Meg3 KO without abrogating the expression levels of downstream lncRNAs .", "Although we have further shown here that Meg3 acts as a scaffold for the PRC2/Jarid2 complex , it is still possible that Rian and Mirg could independently and/or synergistically function with Meg3 to contribute to the Hox-mediated MN subtype switching we observed in the IG-DMRMatΔ mutants .", "To investigate this possibility , we instead generated two independent targeted deletion ESC lines of Meg3 downstream lncRNAs , RianΔ/Δ and MirgΔ/Δ .", "Consistent with previous results ( Han et al . , 2014; Labialle et al . , 2014 ) , deletions of these downstream lncRNAs show less profound phenotype than upstream Meg3 .", "Our results reveal that the progenitor gene Pax6 and caudal Hox genes in these two KO ESC~MNs are relatively unaffected when compared to Meg3 KD .", "Interestingly , Mirg KO embryos seem to have a less severe phenotype , and no observed homeotic transformation has been reported ( Labialle et al . , 2014 ) .", "Furthermore , our results indicate that Mirg does not bind to Ezh2/Suz12/Jarid2 ( Figure 3A ) .", "Thus , it is likely that Meg3 might be the major contributor to MN subtype specification via epigenetic regulation .", "In this study , we did not delete lncRNA Rtl1as in the Dlk1-Dio3 locus , as the deletion of Rtl1as simultaneously compromises paternal Rtl1 expression ( da Rocha et al . , 2008 ) .", "Therefore , we still can not completely rule out the possible synergistic ncRNA effects accounting for MN phenotype we observed in the IG-DMRMatΔ embryos .", "Since lncRNAs are emerging as important modulators of gene regulatory networks and as epigenetic regulators of gene expression , we are endeavoring to systematically generate individual lncRNA KO embryos in the Meg3-Rain-Mirg locus by a CRISPR-Cas9-mediated approach .", "We anticipate that a detailed map of individual and synergetic lncRNA/miRNA functions during neural development attributable to this imprinted locus will be uncovered in the near future .", "Consistent with the imprinting status of the DLK1-DIO3 locus in humans , epimutations ( hypermethylations ) and microdeletions affecting IG-DMR and/or MEG3-DMR of maternal origin result in a unique human phenotype manifested as a small bell-shaped thorax , coat-hanger-like appearance of the ribs , abdominal wall defects , placentomegaly and polyhydramnios .", "One hallmark of patients with this disease , termed ‘Kagami-Ogata syndrome’ ( KOS ) ( Kagami et al . , 2015; Ogata and Kagami , 2016 ) , is that nearly all of them display delayed gross motor development .", "It is currently unknown why epimutations and microdeletions of maternal IG-DMR give rise to this phenotype .", "In our IG-DMRmatΔ embryos , we previously observed extra ossification at the sites where the 6th to 8th ribs attach to the sternum , similar to the malformed thorax in KOS patients ( Lin et al . , 2007 ) .", "Interestingly , this phenotype was also observed in Hox5 mutant mouse embryos ( McIntyre et al . , 2007 ) .", "Here , we uncovered that two isoforms of the Meg3 imprinted lncRNA are enriched in embryonic MNs and confer the fidelity of the epigenetic landscape for the Hoxa5-Hoxc8 boundary of MN subtypes .", "Loss of Meg3 in vitro and in vivo abrogates the Hoxa5on MNs in the brachial region , with a concomitant increase of ectopic Hoxc8on subtypes .", "This switch leads to erosion of Hoxa5on motor axon arborization in the proximal muscles .", "As Meg3 expression is also highly enriched in somites , we suggest that impairment of the Hox boundary mediated by Meg3 in the spinal cord and ribs might account for the bell-shaped thorax and motor deficit in KOS patients , potentially identifying a new therapeutic target for KOS patients .", "In addition to the roles of the Meg3 imprinted lncRNA uncovered by our study , other reports have also emphasized Meg3 as being important for proper growth and development and to be a putative tumor suppressor that activates p53 and inhibits cell proliferation ( Takahashi et al . , 2009; Zhang et al . , 2010; Zhou et al . , 2007 ) .", "Moreover , aberrant repression of Meg3 and other maternally-expressed lncRNAs from the DLK1-DIO3 imprinting cluster is present in several induced pluripotent stem cell ( iPSC ) lines .", "This scenario might lead to failure of these iPSCs to form viable mice ( Stadtfeld et al . , 2010 ) or to efficiently differentiate into neural lineage cells ( Mo et al . , 2015 ) , raising the possibility that Meg3 might be involved in a broad spectrum of developmental processes and disease contexts .", "Thus , our exploration of Meg3 may also suggest new avenues for treating other diseases , such as cancers , as well as in elucidating the reprogramming mechanism of iPSCs ." ], [ "ESCs were cultured and differentiated into spinal MNs as previously described ( Wichterle et al . , 2002; Wichterle et al . , 2009 ) .", "Cells were trypsinized and collected for FACS at day seven to purify GFPon neurons for qPCR analysis and strand-specific RNA-seq when required .", "All cell lines used in this study are subject to regular mycoplasma test .", "The IG-DMRmatΔ mouse strain is described in Lin et al . ( 2003 ) .", "Female mice carrying the deletion were mated with wild type C57BL6/J male mice to generate embryos with the maternally-inherited deletion .", "Mice were mated at the age of 8~12 weeks and the embryo stage was estimated as E0 . 5 when a copulation plug was observed .", "Embryos were analyzed between E9 . 5~E13 . 5 .", "All of the live animals were kept in an SPF animal facility , approved and overseen by IACUC Academia Sinica .", "The Meg3 HuSH-29 shRNA plasmids ( Origene , cat . No . TG501330 ) and non-effective scrambled sequence ( TR20003 ) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs .", "We used two different shRNA sequences to knockdown Meg3 .", "Additionally , stable infected ESCs were selected by puromycin .", "Single ESC clones with good morphology and only presenting knockdown efficiencies >90% were picked for further expansion and characterization .", "ESCs or embryoid bodies were harvested for total RNA isolation by Trizol ( Thermo Scientific ) .", "For qPCR analysis , total RNA from each sample was reverse transcribed with Superscript III ( Thermo Scientific ) .", "One-tenth of the reverse transcription reaction was used for subsequent qPCR reactions , which were performed in triplicate with three independent experimental samples on a LightCycler480 Real Time PCR instrument ( Roche ) using SYBR Green PCR mix ( Roche ) for each gene of interest .", "Gapdh was used as a control for normalization .", "For GeneChip expression analysis , RNA was purified and amplified using the Qiagen RNAeasy kit and a one-color Low Input Quick Amp Labeling Kit ( Agilent Genomics ) and hybridized to a SurePrint G3 Mouse GE 8×60K Microarray .", "Differentially-expressed genes were defined by ranking all probes according to Moderated t-test and a fold-change threshold ≥2 ( p<0 . 001 ) .", "We followed a previously published protocol to perform ChIP-seq for ESC~MNs ( Mazzoni et al . , 2011; Narendra et al . , 2015 ) .", "Four million cells were freshly dissociated from day 7 ESC~MNs by trypsin and fixed in 10 mM HEPES pH 7 . 6 , 1% formaldehyde , 15 mM NaCl , 0 . 15 mM EDTA and 0 . 075 mM EGTA for 15 min at room temperature .", "After fixation , cells were quenched with 1 . 25 M glycine .", "After an ice-cold PBS wash and low-speed centrifugation , nuclear extracts were suspended with ice-cold shearing buffer ( SDS included ) containing protease inhibitor and sheared using a Covaris M220 system to an average chromatin size of 200 bp .", "Chromatin was diluted with 2X IP buffer ( 2% NP-40 , 200 mM NaCl in 10 mM Tris-HCl pH 8 , 1 mM EDTA ) .", "Anti-H3K27me3 antibody was added to each ChIP ( antibodies are listed in the resource table ) .", "Each ChIP reaction was performed in a rotator at 4°C overnight , followed by washing in wash buffer ( 25 mM HEPES pH 7 . 6 , 1 mM EDTA , 0 . 1% N-Lauryl sarcosine , 1% NP-40 , and 0 . 5 M LiCl ) at room temperature .", "Cross-linking was reversed at 65°C for 16 hr with 5 M NaCl .", "Proteinase K was added to digest for another 2 hr at 56°C and DNA was extracted using the ChIP DNA Clean and Concentrator system ( Zymo Research ) .", "We treated 1% of the input in parallel .", "Libraries were prepared according to the Illumina protocol and sequenced using an Illumina NextSeq Sequencing System .", "Immunohistochemistry was performed on 15 μm cryostat sections as described ( Chen et al . , 2011 ) .", "Primary antibodies used in this study are detailed in the resource table .", "Whole-mount antibody staining was performed as described ( Dasen et al . , 2008 ) , and GFP-labeled motor axons were visualized in projections of a Zeiss Lightsheet Z . 1 microscope ( 400–600 μm ) .", "Unless indicated otherwise , immunohistological data shown in figures are representative of n>3 analyzed mutants .", "Images for control animals are from age-matched littermates .", "In situ hybridizations were performed as described previously ( Chen et al . , 2007; Chen et al . , 2011 ) and in the Materials and Methods .", "We followed a previously published protocol to extract subcellular fractions of RNA ( Gagnon et al . , 2014 ) .", "We used TRIzol ( Thermo Fisher Scientific ) to extract RNA and perform reverse transcription ( RT ) with hexamer primers .", "Gapdh ( mRNA in cytoplasm ) , Rnu1 ( snRNA in nucleus ) , and Kcnq1ot1 ( a known chromatin-associated lncRNA ) were used as quality controls to verify fractionation .", "In vitro-transcribed biotin-labelled RNAs were generated by the Biotin RNA Labeling Mix ( Roche ) and T7 RNA polymerase ( Promega ) .", "Templates were treated with RNase-free DNase I ( Promega ) and the reaction mix was purified with Oligo Clean and Concentrator ( D4060 , Zymo Research ) .", "Biotinylated RNA ( 3 μg ) was heated to 65°C for 10 min and then slowly cooled down to 4°C .", "After that , RNA structure buffer ( 10 mM Tris pH 7 , 0 . 1 M KCl , 10 mM MgCl2 ) was added and the mix was shifted to room temperature for 20 min to allow proper secondary structure formation .", "Folded RNA was then mixed with 1 mg of ESC protein nuclear extract in RIP buffer ( 500 mM NaCl , 10 mM HEPES pH 7 . 5 , 25% glycerol , 1 mM EDTA , and protease inhibitor ) and incubated at 4°C for one hour .", "Twenty µL Dynabeads M-280 Streptavidin ( Invitrogen ) were added to each binding reaction and further incubated at room temperature for one hour .", "Beads were washed briefly five times and boiled in SDS buffer , and the retrieved protein was detected by standard Western blot analysis .", "For each IP , cells were harvested from a 10 cm dish and washed twice with ice cold PBS .", "Cell pellets were allowed to swell in twice the volume of cytoplasmic lysis buffer ( 50 mM NaCl , 10 mM HEPES-pH 7 . 5 , 500 mM sucrose , 1 mM EDTA and protease inhibitors ) .", "Samples were incubated on ice for 10 min , followed by centrifugation at 2 , 000 rpm for 10 min .", "The cloudy supernatant cytoplasmic fraction was removed .", "After washing twice ( 50 mM NaCl , 10 mM HEPES-pH 7 . 5 , 25% glycerol , 1 mM EDTA and protease inhibitors ) , the cell pellets were resuspended in the same volume of high salt buffer ( 500 mM NaCl , 10 mM HEPES-pH 7 . 5 , 25% glycerol , 1 mM EDTA and protease inhibitors ) , and rotated for 30~60 min at 4°C .", "Then cell pellets were centrifuged at 14 , 000 rpm for 10 min at 4°C .", "The supernatant was incubated overnight at 4°C with antibody and pre-cleared Protein-G beads ( depending upon the antibody ) to immunoprecipitate endogenous protein against the specific antibody used .", "We collected 10% of cleared supernatant as input .", "Subsequently , IP-protein beads were washed three times with PBS and 0 . 01% Tween-20 , each for 5 min at 4°C .", "IP-proteins and their interacting partners were eluted from beads in 6X reducing loading buffer at 70°C for 15 min .", "Finally , samples were cooled down to room temperature and spun briefly to collect condensation .", "Standard Western blot procedures were applied using anti-Jarid2 ( Novus Biologicals , NB100-2214 ) or anti-Ezh2 ( Millipore , 17–662 ) antibodies .", "Blots were developed using HRP-conjugated anti-rabbit or -mouse antibodies , depending on the species of the primary antibody .", "Signals were developed and filmed by enhanced SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo , 34096 ) .", "All exposures were done using hyper film .", "ESC~MNs were cultured and harvested on slides .", "Cells were fixed in 4% paraformaldehyde for 10 min at room temperature , permeabilized for 5 min on ice in PBS with 0 . 5% Triton X-100 , and then rinsed in 70% EtOH for subsequent RNA FISH .", "Slides and coverslips were kept in 70% EtOH at 4°C until staining .", "Slides were then washed in wash buffer ( 10% deionized formamide in 2X SSC ) for 5 min and incubated in a dark room at 37°C for at least 4 hr with 1 μL of probe stock solution and 100 μL of hybridization buffer ( 1 g dextran sulfate , 1 mL 20X SSC , 1 mL deionized formamide ) .", "Meg3 smFISH probes were purchased from Stellaris .", "Images were captured with a Delta Vision microscopy system .", "RIP was performed with the RNA-Binding Protein Immunoprecipitation Kit ( 17–700 , Millipore ) according to the manufacturer’s protocol with some modifications .", "ESC~MNs were dissociated at a concentration of 2 million cells/mL and treated with 0 . 3% formaldehyde in ice-cold PBS for 10 min at 37°C .", "Glycine/PBS was added to a final concentration of 0 . 125 M and each sample was incubated for 5 min at room temperature .", "After crosslinking , ten million cells were washed twice with cold PBS and then suspended in 100 μL RIP lysis buffer ( with the addition of protease inhibitor and RNase inhibitor ) .", "The lysate was incubated on ice for 5 min and centrifuged at 14 , 000 rpm for 10 min at 4°C .", "Ezh2 , Jarid2 , and Suz12 antibodies were added for respective IP reactions and then incubated in RIP buffer ( 0 . 5 M EDTA/RNase inhibitor ) for 3 hr to overnight at 4°C .", "Samples were washed at least five times with RIP washing buffer .", "RIP beads were resuspended in RIPA buffer ( RIP washing buffer +10% SDS +protease K ) to reverse crosslinking at 56°C for 30 min .", "RNA samples were extracted and qPCR was performed as described above .", "Isolated proteins before proteinase K treatment were collected from the beads and verified by Western blot analysis .", "Data on retrieved RNAs was calculated from the RT/input ratio for each experiment .", "All statistical analyses were generated with GraphPad Prism 6 ( GraphPad Software ) .", "The values are shown as mean ± SD , as indicated .", "Student’s t-tests were used for comparisons between experimental samples and controls .", "Statistical significance was defined as * p<0 . 05 and ** p<0 . 01 by Student’s t-test .", "Adapter contamination in the paired-end reads was removed using PEAT ( Li et al . , 2015 ) , and the trimmed reads were aligned to the mm10 genome with STAR ( Dobin et al . , 2013 ) .", "The standard GTF-formatted transcript annotation was defined by GENCODE ( version M9 ) ( Harrow et al . , 2012 ) , which includes many evidence-based lncRNAs .", "We used this annotation to aid the junction read alignment in STAR , the output of which was submitted to Cufflinks ( Trapnell et al . , 2010 ) for de novo transcript assembly with the option ‘library type; first-strand’ to allow strand-specific alignments .", "We followed a strategy for novel lncRNA identification similar to that suggested by a previous report ( Qian et al . , 2016 ) , by which only transcripts that were longer than 200 bp , had no overlap with any known genes , and consisted of more than one exon were regarded as novel lncRNAs .", "We pooled these novel lncRNAs along with all known genes annotated in GENCODE and used HTseq ( Anders et al . , 2015 ) to calculate the read count aligned onto each transcript .", "This procedure was repeated for all RNA-seq samples in this study .", "The read counts of all transcripts among different samples were normalized using a TMM algorithm with the trimming option M = 30% and A = 5% ( Robinson and Oshlack , 2010 ) .", "A general comparison of different normalization algorithms can be found in Lin et al . ( 2016 ) .", "We calculated the specificity score of each transcript among the samples at different stages according to the Jensen–Shannon definition for tissue specificity scores ( Cabili et al . , 2011; Trapnell et al . , 2010 ) .", "The transcripts were split into three groups—namely protein coding genes , annotated lncRNAs , and novel lncRNAs—for which specificity score distributions were plotted and compared .", "Reads were trimmed by PEAT and aligned to the mm10 genome using Bowtie2 ( Langmead and Salzberg , 2012 ) .", "Following a similar flow analysis described in our previous work ( Chen et al . , 2013; Yildirim et al . , 2011 ) , all alignments were extended downstream to span an exact 150 bp-long region .", "Extensions that exceeded the ends of chromosomes were clipped .", "The extended alignments were input into the genomecov functionality supported in the BEDTools suite ( Quinlan and Hall , 2010 ) to generate read coverage profiles at a base-pair resolution .", "The coverage for each chromosomal position was normalized according to the mappable read count .", "Each sample was averaged and binned to reveal major trends .", "To identify possible differentially-enriched histone marks among stages or treatments , we used MACS 1 . 4 ( Feng et al . , 2011 ) to call peaks ( p-value<10−5 ) in each ChIP-seq sample with the corresponding input library and then overlapping peaks were merged using MAnorm ( Shao et al . , 2012 ) to reveal loci with a significant change between two samples .", "The 3D images acquired with a Zeiss Lightsheet Z . 1 microscope were subjected to analyses in Imaris 8 . 4 . 0 ( Bitplane , Zurich , Switzerland ) for quantification of axon arborization .", "Regions of interest were segmented for detection of individual neurons .", "Motor nerve terminals were semi-automatically traced using the filament tracer wizard from a defined starting point .", "The AutoPath ( no loops ) algorithm was selected .", "Seed points detected from background signals were manually removed .", "Disconnected segments were removed by indicating the maximum gap length , and background subtraction was applied for noise removal .", "The ‘Filament No . Dendrite Terminal Points’ tool automatically calculated the number of motor nerve terminals ." ] ]
[ "The mammalian imprinted Dlk1-Dio3 locus produces multiple long non-coding RNAs ( lncRNAs ) from the maternally inherited allele , including Meg3 ( i . e . , Gtl2 ) in the mammalian genome .", "Although this locus has well-characterized functions in stem cell and tumor contexts , its role during neural development is unknown .", "By profiling cell types at each stage of embryonic stem cell-derived motor neurons ( ESC~MNs ) that recapitulate spinal cord development , we uncovered that lncRNAs expressed from the Dlk1-Dio3 locus are predominantly and gradually enriched in rostral motor neurons ( MNs ) .", "Mechanistically , Meg3 and other Dlk1-Dio3 locus-derived lncRNAs facilitate Ezh2/Jarid2 interactions .", "Loss of these lncRNAs compromises the H3K27me3 landscape , leading to aberrant expression of progenitor and caudal Hox genes in postmitotic MNs .", "Our data thus illustrate that these lncRNAs in the Dlk1-Dio3 locus , particularly Meg3 , play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes and they shape MN subtype identity by regulating Hox genes ." ]
[ "When a gene is active , its DNA sequence is ‘transcribed’ to form a molecule of RNA .", "Many of these RNAs act as templates for making proteins .", "But for some genes , the protein molecules are not their final destinations .", "Their RNA molecules instead help to control gene activity , which can alter the behaviour or the identity of a cell .", "For example , experiments performed in individual cells suggest that so-called long non-coding RNAs ( or lncRNAs for short ) guide how stem cells develop into different types of mature cells .", "However , it is not clear whether lncRNAs play the same critical role in embryos .", "Yen et al . used embryonic stem cells to model how motor neurons develop in the spinal cord of mouse embryos .", "This revealed that motor neurons produce large amounts of a specific group of lncRNAs , particularly one called Meg3 .", "Further experiments showed that motor neurons in mouse embryos that lack Meg3 do not correctly silence a set of genes called the Hox genes , which are crucial for laying out the body plans of many different animal embryos .", "These neurons also incorrectly continue to express genes that are normally active in an early phase of the stem-like cells that make motor neurons .", "There is wide interest in how lncRNAs help to regulate embryonic development .", "With this new knowledge of how Meg3 regulates the activity of Hox genes in motor neurons , research could now be directed toward investigating whether lncRNAs help other tissues to develop in a similar way ." ]
2018
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Reverse-correlation analysis of navigation dynamics in Drosophila larva using optogenetics
elife-06225-v2
[ [ "To successfully navigate their environments , animals transform sensory inputs into motor outputs in patterns that strategically orient themselves towards improving conditions .", "The navigational strategies of insect larvae represent a long-standing paradigm for studying the mechanisms of animal orientation ( Loeb , 1918; Mast , 1938 ) .", "The small size and simple nervous system of the Drosophila larva , combined with its powerful genetic toolbox and recent advances in optical neurophysiology and anatomical reconstruction of circuit structure and connectivity , opens the possibility of understanding the neural encoding of animal navigation from sensory inputs to motor outputs without gaps ( Saalfeld et al . , 2012 ) .", "To accomplish this , a quantitative framework to describe navigation decision-making is needed .", "Such a framework can then be used to dissect the function of the neurons and circuits in charge of processing sensory information .", "Drosophila larva navigation involves the regulation of transitions between two basic motor states , runs during which the animal moves forward using rhythmic peristaltic waves and turns during which the larva sweeps its head back and forth until it selects the direction of a new run ( Luo et al . , 2010; Gomez-Marin et al . , 2011; Gomez-Marin and Louis , 2012 ) ( Figure 1A ) .", "Attractive and repulsive responses can be estimated by the tendency of the larva to aggregate near or avoid an environmental stimulus ( Kreher et al . , 2008 ) .", "Attractive and repulsive responses can also be observed in the movement patterns of individual larvae ( Louis et al . , 2007; Luo et al . , 2010; Gershow et al . , 2012 ) .", "When the larva encounters improving conditions over time , it lowers the likelihood of ending each run with a turn , thereby lengthening runs in favorable directions .", "When the larva encounters improving conditions during each head sweep of a turn , it increases the likelihood of starting a new run , thereby starting more runs in favorable directions .", "Thus , subjecting the larva to an attractant tends to suppress transitions from runs to turns and stimulate transitions from turns to runs; subjecting the larva to a repellant has the opposite effects . 10 . 7554/eLife . 06225 . 003Figure 1 . Experimental method for reverse-correlation analysis using optogenetics .", "( A ) Larvae navigate by alternating between two basic motor states: runs and turns .", "The navigation strategy of the animal can be characterized by finding the mathematical functions , fr → t and ft → r that represent the stimulus dependence of transition rates .", "( B ) Schematic of experimental setup .", "Larvae crawl on a 22 × 22 cm agar plate .", "Dark-field illumination is provided by lateral infrared LED bars , and animal movements are recorded with a CCD camera equipped with an infrared long-pass filter .", "Optogenetic illumination is provided by a matrix of red 625-nm LEDs from above .", "( C ) We made extracellular recordings in the olfactory organ of the Drosophila larvae .", "Here , we show the rasters of the spikes induced by CsChrimson activation of the Or45a-expressing olfactory receptor neuron ( ORN ) .", "We used 3 different pulse widths: 0 . 2 , 0 . 5 , and 1 s , all of them with the same intensity used for behavior experiments ( 1 . 9 W/m2 ) .", "The red bar in the top of each raster represents the period during which red lights were ON .", "Each vertical line in the raster represents one spike .", "( D ) Analogous to figure ( C ) , we measured induced spiking of Or42a .", "The red bar in the top of each raster represents the period during which red lights were ON .", "Each vertical line in the raster represents one spike .", "( E ) Mean stimulus history before each run-to-turn transition and ( F ) turn-to-run transition exhibited by Orco>CsChrimson larvae subjected to random ON/OFF optogenetic stimulation .", "The stimulus history for each motor state transition is aligned ( dotted line ) and averaged by assigning +1 to the LED ON state and −1 to the LED OFF state .", "Data represent mean ( black line ) ± one SEM ( gray shaded region ) for 2018 transitions exhibited by 135 larvae .", "20 event-triggered stimulus histories are shown in the raster to illustrate the random binary stimulus pattern used in our experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 00310 . 7554/eLife . 06225 . 004Figure 1—figure supplement 1 . Optogenetic activation of OK6-Gal4 motor neurons . To test if our experimental setup robustly activates CsChrimson , we expressed it in motor neurons using the OK6-Gal4 driver ( Sanyal , 2009 ) .", "Effective activation would result in most muscles of the larvae contracting simultaneously and not allowing larvae to crawl .", "Consistent with that , during illumination 100% of the 85 larvae tested stopped crawling during illumination and slowly recovered motility afterwards .", "The figure shows the mean speed ( black line ) ± SEM ( grey shaded area ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 004 Much progress has been made in understanding the molecular and cellular organization of the chemosensory system of the Drosophila larva , but how specific chemosensory neurons relay information to guide navigational movements remains poorly understood .", "( Kreher et al . , 2005; Vosshall and Stocker , 2007; Kreher et al . , 2008; Kwon et al . , 2011 ) .", "One challenge of studying chemotaxis is that it is difficult to provide sensory input to behaving animals with the flexibility , receptor specificity , and precision needed to build computational models of chemosensory-guided navigation .", "The recent development of a red-shifted version of channelrhodopsin , CsChrimson , which is activated at wavelengths that are invisible to the larva's phototaxis system , now allows us to specifically manipulate the activity of neurons in behaving animals with reliability and reproducibility ( Klapoetke et al . , 2014 ) .", "Here , we sought a mathematical characterization of the navigation dynamics evoked by optogenetic activation of different sets of neurons .", "We focus on the navigation driven by chemosensory inputs .", "Although the organization of the chemosensory periphery is well-defined , the quantitative mapping from sensory activity to behavioral dynamics has not yet been determined .", "To do this , we engineered a high-throughput experimental setup capable of recording the run and turn movements of freely moving larvae subjected to defined optogenetic activation of selected chemosensory neurons .", "By measuring large numbers of animals responding to defined random patterns of optogenetic stimulation , we were able to collect enough data to use reverse-correlation analysis to connect optogenetic activation patterns of sensory neurons with motor patterns ( Ringach and Shapley , 2004 ) .", "We used this information to build linear–nonlinear ( LN ) models that accurately predict behavioral dynamics in response to diverse patterns of optogenetic activation of sensory neurons ( Geffen et al . , 2009 ) .", "We used our method to study how the optogenetic activation of olfactory receptor neurons ( ORNs ) and different sets of gustatory receptor neurons ( GRNs ) map to navigational movements .", "Analysis of gustatory neurons allowed us to investigate the navigational responses evoked by individual GRNs and their combinations .", "We find that compact LN models that connect optogenetic activation to behavioral responses are nonetheless sufficient to describe or predict navigational behavior and should facilitate future studies to elucidate the circuit mechanisms that shape sensorimotor transformations ." ], [ "We can characterize the navigation strategy of the Drosophila larva by identifying the mathematical functions that describe transitions between two basic motor states: running and turning ( Figure 1A ) .", "We sought these functions ( fr → t , ft → r ) for defined patterns of chemosensory stimulation delivered via optogenetics .", "We used transgenic animals that express the red-shifted channelrhodopsin CsChrimson in selected olfactory and gustatory neurons using the UAS-Gal4 system ( Brand and Perrimon , 1993 ) .", "In our setup , we followed the movements of large numbers of late second-instar larvae navigating the surface of a 22 cm × 22 cm agar plate under dark-field illumination provided by infrared LEDs ( Figure 1B ) .", "The entire plate was subjected to spatially uniform optogenetic illumination from above using a matrix of 625 nm red LEDs , a wavelength chosen to activate CsChrimson while invisible to the larva's photosensory system ( Keene and Sprecher , 2012; Klapoetke et al . , 2014 ) .", "We tuned our light intensity ( 1 . 9 W/m2 ) to a level where negligible behavioral response is detected in wild-type animals crossed with UAS-CsChrimson fed with 0 . 5 mM all-trans-retinal .", "We made sure that this light intensity is strong enough to activate CsChrimson by testing it with a well-studied motor neuron line ( Figure 1—figure supplement 1 ) .", "To obtain direct evidence that optogenetic illumination in our behavioral setup activates sensory neurons , we used electrophysiology .", "We made extracellular recordings of the dorsal organ ( DO ) of individual larvae expressing CsChrimson in specific ORNs and recorded the responses to red light activation pulses of 0 . 2 , 0 . 5 , and 1 s of the same intensity used in the behavioral experiments .", "We found that optogenetic activation of the ORN-expressing Or45a reliably and reproducibly induced spike trains during exposure to red light ( Figure 1C ) .", "Similar results were obtained using larvae expressing CsChrimson in the ORN-expressing Or42a ( Figure 1D ) .", "These results confirm direct correspondence between ON/OFF pulses of CsChrimson activation and induced spiking in single sensory neurons .", "To map the input–output relationships with optogenetic interrogation of chemosensory neurons , we used reverse-correlation methods viewing the whole animal as a transducer .", "We subjected larvae to random patterns of optogenetic stimulation and collected the statistics of all behavioral responses exhibited by the freely moving larvae .", "We used the simplest white process for reverse-correlation , a Bernoulli process where we assigned −1 for lights OFF and +1 for lights ON , and calculated the mean stimulus history that preceded each run-to-turn or turn-to-run transition ( Figure 1E , F ) .", "These event-triggered stimulus histories represent how the animal uses optogenetic activation patterns of specific neurons to regulate each motor state transition and are proportional to the linear filter components of fr → t and ft → r ( see ‘Materials and methods’ ) .", "When freely crawling larvae encounter increasing chemoattractant concentrations during runs , they decrease the likelihood of initiating a turn .", "When they encounter increasing chemoattractant concentrations during the head sweep of a turn , they increase the likelihood of starting a new run .", "The Or42a receptor is activated by a number of volatile chemoattractants including ethyl butyrate and ethyl acetate ( Louis et al . , 2007; Kreher et al . , 2008; Asahina et al . , 2009 ) .", "Genetically modified animals in which the Or42a-expressing ORN is the only functional ORN are capable of climbing olfactory gradients towards these attractants .", "These observations strongly suggest that the Or42a-expressing ORN mediates attractant responses .", "To test our system , we subjected Or42a>CsChrimson larvae to random optogenetic stimulation .", "We found that run-to-turn transitions coincided with a decrease in the probability of optogenetic activation ( Figure 2A , left ) , whereas turn-to-run transitions coincided with an increase ( Figure 2A , right ) .", "These patterns are consistent with an attractive response to Or42a activation .", "Importantly , the full shape of the event-triggered stimulus histories informs us about how the temporal optogenetic activation patterns of Or42a regulate each type of navigational movement .", "Methods that measure the tendency of larvae to aggregate near chemoattractants or net movement up chemoattractant gradients provide information about the overall tendency to navigate but not about the discrete decision-making processes that drive navigation . 10 . 7554/eLife . 06225 . 005Figure 2 . ORNs evoked navigation strategy .", "( A ) Event-triggered stimulus histories for run-to-turn ( left panel ) and turn-to-run ( right panel ) transitions exhibited by Or42a>CsChrimson larvae subjected to random optogenetic stimulation as described in Figure 1 .", "Consistent with an attractive response , the likelihood of optogenetic activation falls before a run-to-turn transition and rises before a turn-to-run transition .", "In run-to-turn transitions , crawling speed begins to fall before the initiation of turning movements ( green traces ) .", "The mean beginning of deceleration averaged over all animals is flagged by the red dot ( ± STD ) .", "The units of normalized speed are standard deviations away from the mean crawling speed during runs .", "Data represent mean ( black line ) ± one SEM ( grey shaded region ) for 2752 transitions exhibited by 124 larvae .", "( B ) Event-triggered stimulus histories exhibited by Or45a>CsChrimson larvae .", "Consistent with a repulsive response , the likelihood of optogenetic activation increases before a run-to-turn transition and decreases before a turn-to-run transition .", "Data represent mean ( black line ) ± one SEM ( gray shaded region ) for 3313 transitions exhibited by 119 larvae .", "The mean beginning of deceleration averaged over all animals is flagged by the red dot ( ± STD ) .", "( C ) Control larvae event-triggered averages .", "Event-triggered averages of control larvae were uncorrelated with red light illumination patterns .", "Data represent mean ( black line ) ± one SEM ( gray shaded region ) for 4677 transitions exhibited by 121 larvae .", "The mean beginning of deceleration averaged over all animals is flagged by the red dot ( ± STD ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 005 Random optogenetic activation of all the ORNs via expression of UAS-CsChrimson with the Orco olfactory-receptor-coreceptor driver ( previously called Or83b ) mediated an attractive response similar to the one shown with the Or42a driver alone ( Figure 1E , F ) .", "This is consistent with most ORNs in the Drosophila larva being thought to mediate attractant responses ( Kreher et al . , 2008; Mathew et al . , 2013 ) .", "One exception is the Or45a-expressing ORN , which has recently been shown to mediate an aversive response in an optogenetic setup; larvae that express channelrhodopsin in Or45a-expressing neurons will avoid an illuminated region of an agar plate ( Bellmann et al . , 2010 ) .", "A role for the Or45a-expressing neurons in repellency is also consistent with the observation that they are the only ORNs that detect octyl acetate , a chemical repellant ( Cobb and Dannet , 1994; Kreher et al . , 2008 ) .", "We sought the linear filters of this olfactory repellant response in our setup by quantifying the movements of Or45a>CsChrimson larvae subjected to random optogenetic stimulation ( Figure 2B ) .", "Run-to-turn transitions in Or45a>CsChrimson larvae coincided with an increase in the probability of optogenetic illumination and turn-to-run transitions coincided with a decrease , consistent with repellant behavior .", "For comparison , we calculated event-triggered stimulus histories using larvae heterozygous for UAS-CsChrimson and with the same genetic background as our Gal4 lines ( w1118 × UAS-CsChrimson ) subjected to random illumination ( Figure 2C ) .", "These control larvae were raised in the same conditions and fed the same food as larvae used for all other experiments ( ‘Materials and methods’ ) .", "These larvae showed no correlations between the probability of illumination and motor state transitions .", "Their motor state transitions were random and spontaneous .", "In our setup , we flag turn-to-run transitions by the resumption of peristaltic forward movement and run-to-turn transitions as the onset of head-sweeping behavior .", "However , the decision to finish a run may begin at an earlier point , when the animal first begins to slow down .", "We measured the crawling speeds of larvae before flagged run-to-turn transitions and found that runs decelerate ∼1 s before the onset of head-sweeping behavior ( Figure 2A–C ) .", "For both repellant and attractants , an increase and decrease in the probability of optogenetic illumination , respectively , coincides with the beginning of run deceleration ( Figure 2A , B ) .", "The average deceleration time was 1 s for all experiments conducted in this study ( Student's t-test , p < 0 . 01 ) .", "A satisfactory model of navigation should be able to predict behavioral responses to various stimulus waveforms .", "We asked whether we could use our measurements of event-triggered stimulus histories to build such a model .", "A simple and widely used formalism is the LN model .", "In LN models , the linear filter component is proportional to our measurement of the event-triggered stimulus history ( Geffen et al . , 2009 ) .", "First , the stimulus waveform is passed through this linear filter to make an initial prediction of the behavioral response .", "Linear estimates have common problems , such as taking negative values and failing to account for saturation .", "To correct these problems , the linear prediction is then scaled with a static nonlinear function .", "This static nonlinearity can be calculated by comparing a linear prediction with experimental measurements .", "We used larvae with CsChrimson in Or45a-expressing neurons to test an LN model in predicting behavior .", "We calculated the static nonlinearity for both run-to-turn and turn-to-run transitions by comparing linear predictions obtained with the event-triggered stimulus histories shown in Figure 2B with experimental measurements ( Figure 3A ) .", "Next , we implemented the linear filter and static nonlinearity in an LN model ( Figure 3B ) to predict the behavioral response of these larvae to different inputs , using step increases in optogenetic illumination as well as defined trains of pulses of different widths .", "We found remarkably good agreement in these predictions to both stimulus types ( Figure 3C ) .", "We note that the LN prediction begins to fail to account for the turn-to-run transitions at long times following a step increase in optogenetic illumination .", "Turns typically last <4 s , which limits the length of stimulus history that can be used in a linear filter , which thus puts a ∼4-s upper bound on the length of stimulus response that can be predicted .", "Taken together , our results show that LN models governing stimulus-evoked transitions between motor states can be used to predict larval chemotaxis behavior with high accuracy ( Figure 3C ) .", "The LN model was also successful in predicting the behavior of Or42a-expressing neurons and other chemosensory neurons ( Figure 3—figure supplement 1; Figure 3—figure supplement 2; the procedure for detailed calculations are described in ‘Materials and methods’ ) . 10 . 7554/eLife . 06225 . 006Figure 3 . Linear-nonlinear ( LN ) models of behavior .", "( A ) Estimating the static nonlinear function for run-to-turn and turn-to-run transitions exhibited by Or45a>CsChrimson larvae .", "Linear prediction using the event-triggered stimulus histories from Figure 2B was compared with the experimental measurements that generated the stimulus histories .", "The static nonlinearity for the run-to-turn transition is fitted using least squares estimation of a sigmoidal function ( R2 = 0 . 8792 ) .", "The static nonlinearity for the turn-to-run transition is fitted with a line ( R2 = 0 . 5041 ) .", "( B ) Schematic representation of the LN model of navigation .", "Linear filters are convolved with the input signal , and the result is scaled according to the static nonlinear function fitted to estimate the probability rates for switching from one motor state to the other .", "( See ‘Materials and methods’ ) .", "( C ) LN model predictions ( blue lines ) of behavioral responses to step changes in optogenetic illumination ( left panels ) and defined random flicker ( right panels ) .", "Predictions are made using the linear filter measured in Figure 2B and the static nonlinearity measured in Figure 3A .", "Experimental measurements to compare with prediction ( black dots ) represent data from N = 120 for the step response prediction and N = 240 larvae for the flicker response prediction . DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 00610 . 7554/eLife . 06225 . 007Figure 3—figure supplement 1 . LN models of Gr21a and Gr10a .", "( A ) Estimating the static nonlinear function for run-to-turn and turn-to-run transitions exhibited by Gr21a>CsChrimson larvae ( left panels ) .", "Linear prediction using the event-triggered stimulus histories from Figure 3A was compared with the experimental measurements that generated the stimulus histories .", "The static nonlinearity for the run-to-turn transition is fitted using least squares estimation of a sigmoidal function ( R2 = 0 . 9494 ) .", "The static nonlinearity for the turn-to-run transition is fitted with a line ( R2 = 0 . 5082 ) .", "LN model predictions ( blue lines ) and linear filter predictions ( green lines ) of behavioral responses to defined random flicker ( right panels ) .", "Predictions are made using the linear filter measured in Figure 3A and the static nonlinearity showed in the left panel .", "Experimental measurements to compare with prediction ( black dots ) represent data from N = 156 larvae .", "( B ) Estimating the static nonlinear function for run-to-turn transitions exhibited by Gr10a>CsChrimson larvae ( left panel ) .", "Linear prediction using the event-triggered stimulus histories from Figure 5 was compared with the experimental measurements that generated the stimulus histories .", "The static nonlinearity for the run-to-turn transition is fitted using least squares estimation of a sigmoidal function with ( R2 = 0 . 6221 ) .", "At the resolution power employed in this study , the turn-to-run linear filter could not be distinguished from noise .", "LN model predictions ( blue lines ) and linear filter predictions ( green lines ) of behavioral responses to defined random flicker ( right panels ) .", "Predictions are made using the linear filter measured in Figure 5 and the static nonlinearity showed in the left panel .", "Experimental measurements to compare with prediction ( black dots ) represent data from N = 183 larvae . DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 00710 . 7554/eLife . 06225 . 008Figure 3—figure supplement 2 . LN models of Or42a and Gr2a .", "( A ) Estimating the static nonlinear function for run-to-turn and turn-to-run transitions exhibited by Or42a>CsChrimson larvae ( left panels ) .", "Linear prediction using the event-triggered stimulus histories from Figure 2A was compared with the experimental measurements that generated the stimulus histories .", "The static nonlinearity for the run-to-turn transition is fitted using least squares estimation of a sigmoidal function with ( R2 = 0 . 7617 ) .", "The static nonlinearity for the turn-to-run transition is fitted with a sigmoid with ( R2 = 0 . 625 ) .", "LN model predictions ( blue lines ) and linear filter predictions ( green lines ) of behavioral responses to defined random flicker ( right panels ) .", "Predictions are made using the linear filter measured in Figure 2A and the static nonlinearity showed in the left panel .", "Experimental measurements to compare with prediction ( black dots ) represent data from N = 207 larvae .", "( B ) Estimating the static nonlinear function for run-to-turn transitions exhibited by Gr2a>CsChrimson larvae ( left panel ) .", "Linear prediction using the event-triggered stimulus histories from Figure 4C was compared with the experimental measurements that generated the stimulus histories .", "The static nonlinearity for the run-to-turn transition is fitted using least squares estimation of a sigmoidal function with ( R2 = 0 . 6752 ) .", "LN model predictions ( blue lines ) and linear filter predictions ( green lines ) of behavioral responses to a step decrease ( right panels ) .", "Predictions are made using the linear filter measured in Figure 4C and the static nonlinearity showed in the left panel .", "Experimental measurements to compare with prediction ( black dots ) represent data from N = 195 larvae . DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 008 The dynamics of behavioral responses are shaped by the linear filter component of LN models , while the static nonlinearity only provides saturation and instantaneous scaling .", "To test if optogenetic activation of different chemosensory neurons could produce behavioral responses with distinct dynamics , we undertook a search for different linear filters measured by event-triggered stimulus histories using reverse-correlation .", "The Gr21a receptor senses carbon dioxide , a powerful Drosophila repellant ( Faucher et al . , 2006; Gershow et al . , 2012 ) .", "We measured the event-triggered stimulus histories of Gr21a>CsChrimson larvae subjected to random optogenetic stimulation .", "We found that run-to-turn transitions coincided with an increase in the probability of optogenetic illumination from baseline , whereas turn-to-run transitions coincided with a decrease ( Figure 4A ) .", "These patterns are consistent with a repellant response .", "However , the linear filter associated with Gr21a for run-to-turn transition revealed important differences in shape and timing of stimulus history as compared with the filter for Or45a .", "The run-to-turn transition in both cases was preceded by a positive lobe in the probability of optogenetic activation lasting ∼2 s .", "This positive lobe was itself preceded by a pronounced negative lobe lasting ∼1 . 5 s for Gr21a but not for Or45a . 10 . 7554/eLife . 06225 . 009Figure 4 . Distinct navigation dynamics .", "( A ) Event-triggered stimulus histories exhibited by Gr21a>CsChrimson larvae .", "Linear filters of Gr21a neurons .", "Consistent with a repulsive response , the likelihood of optogenetic activation increases before a run-to-turn transition and decreases before a turn-to-run transition .", "Data represent mean ( black line ) ± one SEM ( gray shaded region ) for 4680 transitions exhibited by 90 larvae .", "The mean beginning of deceleration averaged over all animals is flagged by the red dot ( ± STD ) .", "( B ) LN prediction and experimental measurements of different repellant responses to step changes in optogenetic illumination .", "Faster adaptation to baseline is observed in the case of the Gr21a-expressing neurons than Or45a .", "Step responses were measured with 115 Gr21>CsChrimson larvae and 120 Or45a>CsChrimson larvae; each larva was subjected to 30 steps of optogenetic activation .", "( z-test substantiate significant difference in the dynamics of the cyan and black curves see Figure 4—figure supplement 1A ) .", "( C ) Event-triggered stimulus histories exhibited by Gr2a>CsChrimson larvae .", "Consistent with an attractive response , the likelihood of optogenetic activation decays before a run-to-turn transition and raises before a turn-to-run transition .", "Data represent mean ( black line ) ± one SEM ( gray shaded region ) for 3672 transitions exhibited by 128 larvae .", "The mean beginning of deceleration averaged over all animals is flagged by the red dot ( ± STD ) .", "( D ) Linear prediction and experimental measurements of different attractant responses to step changes in optogenetic illumination .", "Faster responses and adaptation to baseline are observed in the case of the Gr2a than Or42a .", "Step responses were measured with 195 Gr2a>CsChrimson larvae and 117 Or42a>CsChrimson larvae; each larva was subjected to 30 steps of optogenetic activation .", "( z-test substantiate significant difference in the dynamics of the cyan and black curves see Figure 4—figure supplement 1B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 00910 . 7554/eLife . 06225 . 010Figure 4—figure supplement 1 . Statistical analysis of behavioral dynamics . Each dot shown in Figure 4B , D is a probability but is also the mean of the distribution of larvae undergoing a run-to-turn transition if we consider each larvae undergoing this transition as 1 and larvae not undergoing this transition as 0 .", "Then , we have a distribution for each point in the cyan and black curves shown; each of those distributions can be compared with the distribution before the light stimulus is presented .", "Since we have sufficient data such that np >= 5 and n ( 1 − p ) >= 5 in all cases ( n is the number of samples , and p is the probability of undergoing a transition ) , we conducted a z-test to compare the distribution at each time point after opotgenetic stimulation with the baseline distribution .", "We show the p-values for each point in the case of Gr21a and Or45a in ( A ) .", "All the p-values lower than 0 . 05 are shown; we note that Gr21a larvae become significantly different than their baseline behavior 0 . 5 s before than Or45a larvae , in addition , Or45a larvae stay at values different from their baseline behavior for at least 0 . 75 s longer than Gr21a larvae .", "In the case of Or42a and Gr2a , we conducted the same analysis ( B middle ) and obtained that Gr2a behavior becomes significantly different than baseline at least 1 . 75 s before Or42a .", "However , in the case of Or42a , adaptation is only partial: Or42a larvae reach steady-state values of P ( r → t ) at different levels when red lights are ON or when red lights are OFF because of that we also computed the z-test between Or42a after lights are turn OFF as compared to the distribution at the steady-state condition with lights ON .", "With this consideration , we obtained a very small difference between Gr2a and Or42a rising time ( B bottom ) .", "As observed , the distribution of Or42a larvae stayed at values significantly higher than the value of P ( r → t ) with lights ON . DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 010 How do differences in the shape and timing of linear filters translate into behavioral responses with different dynamics ?", "To explore this question , we compared the prediction and experimental measurement of stepwise activation of Or45a- and Gr21a-expressing neurons ( Figure 4B ) .", "Biphasic linear filters—such as that associated with Gr21a and also seen in other sensory systems like the Escherichia coli chemotactic response—contribute to adaptation following transient stimulation ( Block et al . , 1982 ) .", "A step increase in stimulation with repellants will cause a transient increase in the probability of run-to-turn transition .", "We predicted and confirmed differences in the adaptive return to baseline behavior for Gr21a and Or45a .", "The probability of run-to-turn transition returns to baseline faster in the case of Gr21a .", "Since each point represents a distribution of binary values ( larvae transitioning from running to turning and larvae not transitioning ) , we used a z-test to identify regions where P ( r → t ) is significantly higher than baseline with p < 0 . 05 .", "We found that P ( r → t ) of Gr21a larvae reach values significantly higher than baseline at least 0 . 5 s earlier than Or45a larvae .", "In addition , Or45a larvae stay at elevated values of P ( r → t ) for at least 0 . 75 s longer than Gr21a larvae ( Figure 4—figure supplement 1A ) .", "We also asked whether differences in behavioral dynamics caused by different linear filters might be found in attractant responses .", "Gr2a is expressed in the A1 and A2 GRNs of the DO as well as in two unidentified neurons in the terminal organ ( Kwon et al . , 2011 ) .", "The role of the Gr2a receptor is not known , but it is part of the subfamily of Gr68a , which has been identified as a pheromone receptor in the adult fly ( Bray and Amrein , 2003 ) .", "We calculated the event-triggered stimulus histories of Gr2a>CsChrimson larvae and found that run-to-turn transitions coincided with a decrease in optogenetic activation , consistent with an attractant response ( Figure 4C ) .", "Interestingly , the linear filter associated with Gr2a was distinct from that of Or42a .", "In Or42a>CsChrimson , the run-to-turn transition was preceded by a single negative lobe lasting ∼2 s .", "In Gr2a>CsChrimson larvae , the negative lobe was itself preceded by a positive lobe .", "As we did for repellants ( Figure 4B ) , we asked whether the response dynamics to step decrease in optogenetic stimulation were distinct .", "We predicted and confirmed differences in behavioral dynamics .", "The most noticeable feature is that Or42a larvae reach different steady states of P ( r → t ) for lights ON or OFF; this creates differences in step-response dynamics .", "We conducted a z-test to identify regions where P ( r → t ) is significantly higher than baseline with p < 0 . 05 .", "Since the steady-state P ( r → t ) for Or42a larvae is different for lights ON and lights OFF , we conducted the z-test with both values ( Figure 4—figure supplement 1B ) .", "Because Or42a larvae start at a lower P ( r → t ) , they take at least 1 . 75 s longer than Gr2a larvae for P ( r → t ) to become significantly higher than the lights OFF steady-state P ( r → t ) .", "Comparison with the steady-state P ( r → t ) for lights ON confirms that the steady-state P ( r → t ) for lights OFF is significantly higher with p < 0 . 05 ( Figure 4—figure supplement 1B ) .", "We note that unlike the linear filters for run-to-turn transitions , the linear filters for turn-to-run transitions showed a similar shape for all Gal4 drivers that we used in this study .", "These filters only showed some variation in amplitude ( Figures 1 , 2 , 4 , 5 ) . 10 . 7554/eLife . 06225 . 011Figure 5 . Reverse-correlation analysis of bitter-sensing gustatory receptor neurons ( GRNs ) .", "Event-triggered stimulus histories exhibited by GrX>CsChrimson larvae using a set of GAL4 drivers that express in different subsets of GRNs .", "The cellular identities describing each expression pattern are taken from Kwon et al . ( 2011 ) .", "Each measurement represents 3270 to 4016 transitions taken from 87 to 134 larvae .", "Curves represent mean ( black line ) ± one SEM ( gray shaded region ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 01110 . 7554/eLife . 06225 . 012Figure 5—figure supplement 1 . Statistical analysis of Gr9a and Gr94a triggered average . The triggered averages of Gr9a and Gr94a showed very weak response .", "Because of this , we tested whether their observed behavior is significantly different than the control .", "Each point of the triggered averages is a distribution , thus , we compared the distributions of each point with the corresponding one in the control with a t-test .", "In the case of Gr9a , it was only significantly different than the control for 0 . 75 s of the 2 s prior to the transition ( panel A , bottom; the bottommost plot is a zoomed version of the middle plot ) .", "We obtained that Gr94a is significantly different than the control with p < 0 . 05 1 . 75 s before the transition ( panel B , bottom ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 01210 . 7554/eLife . 06225 . 013Figure 5—figure supplement 2 . Normalized speed of Gr lines . The normalized speed of each Gr line is shown .", "Normalized speed is computed using standard score .", "In all cases , the slowdown initiation happens 1 s prior to the transition ( t-test p < 0 . 01 ) .", "Red dots flag the slowdown initiation mean location ( average of when the individual tracks start slowing down changing the derivative of speed to negative ) , and the red bar is 2 standard deviations .", "The grey shaded regions near the normalized speed value represent the SEM , in some cases , it is difficult to see because of the large number of samples used: each measurement represents 3270 to 4016 transitions taken from 87 to 134 larvae . DOI: http://dx . doi . org/10 . 7554/eLife . 06225 . 013 The molecular and cellular organization of the chemosensory system of the Drosophila larva is numerically simple .", "The 21 ORNs contained in the larval DO together express 25 members of the Or family of odorant receptors and the Orco coreceptor ( Fishilevich et al . , 2005; Kreher et al . , 2005 ) .", "In contrast , 10 GRNs distributed in the DO and terminal organ—named A1 , A2 , B1 , B2 , and C1-C6—together express 28 members of the Gr family of gustatory receptors .", "Whereas most ORNs express a single Or , GRNs can express multiple Grs and each Gr can be expressed in multiple GRNs ( Kwon et al . , 2011 ) .", "Thus , using larvae expressing CsChrimson under the control of different Grx-Gal4 drivers enabled us to assess the contribution of selected GRNs to behavior .", "The C1 neuron expresses 17 receptors , some of which are found in other neurons ( e . g . , Gr32a , which is also found in B2 ) and some of which are specific to C1 ( e . g . , Gr9a ) .", "Most Grs are thought to respond to repulsive bitter compounds because they express the bitter markers Gr33a and Gr66a ( Kwon et al . , 2011 ) , suggesting that C1 is a broadly tuned mediator of repellant responses .", "Consistent with this hypothesis , optogenetic activation of C1 with random stimuli using Gr9a>CsChrimson larvae evoked a weak repellant response where the run-to-turn transition coincided with a slight increase in the probability of optogenetic illumination ( Figure 5A ) ( this response was significantly different than the control with p < 0 . 05 , see Figure 5—figure supplement 1A ) .", "The crawling speeds of larvae before flagged run-to-turn transitions triggered by optogenetic activation of GRNs is shown in Figure 5—figure supplement 2 .", "Optogenetic activation of C1 together with B2 using Gr32a>CsChrimson larvae evoked a much stronger repellant response ( Figure 5B ) .", "Optogenetic activation of specifically the B2 neuron using Gr10>CsChrimson larvae evoked a repellant response ( Figure 5C ) .", "Optogenetic activation of C1 together with C4 using Gr39a . b>CsChrimson larvae generated a strong repellant response ( Figure 5D ) .", "One possibility is that co-activation of narrowly tuned GRNs that express fewer Grs potentiates the repellant response of the broadly tuned C1 GRN; however , this interpretation should be taken with caution since different Gal4 drivers may induce different spiking rates upon optogenetic activation with CsChrimson .", "We found that optogenetic activation of the C2 neuron alone using Gr94a>CsChrimson larvae evoked a weak attractive response ( Figure 5E ) ( this response was significantly different than the control with p < 0 . 05 , see Figure 5—figure supplement 1B ) .", "This is surprising because the C2 neuron also expresses the bitter receptors Gr33a and Gr66a , which should drive repellant responses , although these receptors are also found in other neurons .", "One possibility is that the attractant response driven by C2 is inverted when additional gustatory neurons are recruited .", "This hypothesis is supported by our observation that co-activation of C1 and C2 using Gr39a . a>CsChrimson larvae exhibited a much stronger repellant response than activation of C1 alone ( Figure 5F ) .", "Co-activation of C1 , C2 , and C4 using Gr59d>CsChrimson larvae also exhibited a strong repellant response ( Figure 5G ) .", "The strongest repellant response was observed by co-activating C1-C4 , B1 , and B2 using Gr66a>CsChrimson larvae ( Figure 5H ) ." ], [ "A fundamental step towards understanding how animal navigation is encoded in neural circuits is the development of a quantitative framework that accurately describes behavioral dynamics .", "To take this step with the Drosophila larva , we combined optogenetics with high-resolution behavioral analysis and reverse-correlation techniques to build LN models that provide an accurate estimate of the decision-making processes that guide navigation during optogenetically induced chemotaxis .", "LN models separate time dependencies and instantaneous scaling into two modules , the linear filter and static nonlinearity , respectively .", "We find that the LN model is capable of accounting for diverse dynamics across attractant and repellant responses in both the gustatory and olfactory systems .", "For example , LN models accurately predicted the differences in response speed and adaptation when different GRNs and ORNs were activated .", "One reason for the diversity of dynamics is that the Drosophila larva chemosensory system , in addition to encoding attractant and repellant responses , is also capable of shaping the dynamics of behavioral responses in ecologically important ways .", "For example , the priorities given to specific chemicals encountered in the environment might not only be measured in terms of their relative degrees of attraction or repulsion but also in the speed of the behavioral response that they trigger or the speed of adaptation .", "We note that some of the observed differences in behavioral dynamics might be caused by using different transgenic lines and different Gal4 drivers with different potencies .", "It would thus be useful to confirm the differences in behavioral dynamics that are suggested by our optogenetic manipulations with direct stimulation of each GRN and ORN and quantitative behavioral analysis in defined environments using cell-specific odorants and tastants .", "Navigational dynamics evoked by specific sets of gustatory neurons have remained elusive because of the lack of chemicals that are specific to individual GRNs .", "Our reverse-correlation analysis using optogenetic activation with CsChrimson allowed us to determine not only the valence ( attraction or repulsion ) of navigation mediated by different combinations of GRNs but also the dynamics of the evoked behavior .", "Although little is known about the circuits downstream of the GRNs , our analysis of sensorimotor transformations serves as a reference to determine how these circuits organize navigational decision-making .", "Although chemotactic navigation behavior involves just two motor states ( running and turning ) , it is possible , in principle , to extend reverse-correlation analysis to a larger number of possible behavioral states .", "Vogelstein et al presented recently a study where they used optogenetic pulses to trigger different subsets of neurons throughout the larval brain ( Pfeiffer et al . , 2008; Vogelstein et al . , 2014 ) .", "They identified 29 statistically different behavioral states , likely because they were able to interrogate circuits for a much wider variety of larval behaviors than navigation .", "It would be useful to apply reverse-correlation methods such as ours to examine transitions between this rich set of behavioral states to identify how specific neurons mediate a broader range of behavioral decisions than navigation up or down stimulus gradients .", "The wiring diagram of the Drosophila larva nervous system is likely to be the next whole animal connectome that will be reconstructed ( Cardona et al . , 2010 ) .", "Powerful genetic tools are making it possible to target specific neurons throughout the Drosophila nervous system with cellular resolution ( Pfeiffer et al . , 2008 ) .", "The new availability of powerful optogenetic tools for activating and inactivating neurons , particularly red-shifted molecules that are outside the spectrum of Drosophila vision , is making it possible to pinpoint the role of specific neurons in overall behavior ( Chuong et al . , 2014; Klapoetke et al . , 2014 ) .", "An essential step in building whole nervous system models of behavior that incorporate wiring and dynamics is computational modeling .", "Bringing together computational modeling of behavior with new tools for behavioral and physiological analysis , such as those described here , should open the door to a thorough understanding of behavioral circuits from sensory input to motor output in the small but surprisingly sophisticated nervous system of the Drosophila larva ." ], [ "All larvae were raised in the dark at 22°C and fed yeast with 0 . 5 mM all-trans-retinal .", "All GrX-Gal4 lines were previously described ( Weiss et al . , 2011 ) .", "The UAS-CsChrimson flies were a gift of Vivek Jayaraman .", "Other lines were provided by the Bloomington Stock Center: Or42a-Gal4 ( BL#9970 ) , Or45a-Gal4 ( BL#9975 ) , Orco-Gal4 ( BL#23909 ) , Gr21a-Gal4 ( BL#23890 ) , Gr66a-Gal4 ( BL#28801 ) , and w1118 ( BL#5905 ) .", "Male Gal4 flies were crossed to UAS-CsChrimson virgins in small cages ( Genesee Scientific , San Diego , CA ) where eggs were laid on grape juice plates .", "Larvae were thoroughly washed in water , and late second-instar larvae were selected under a dissecting microscope .", "For spatial navigation assays , groups of 20–30 larvae were placed in the center of a ∼5-mm thick 22 × 22 cm agar ( Fisher Scientific , Pittsburgh , PA ) plate and allowed to freely move for 20 min .", "Animals were recorded with a CCD Mightex camera with a long-pass ( 740 nm ) infrared filter at 4 Hz .", "Light stimulation was produced with a custom built LED matrix assembled with SMD 5050 flexible LED strip lights of 12 V DC and 625 nm wavelength ( LEDlightninghut . com ) and controlled with an H-bridge driver and custom code written for a LabJack U3 controller .", "Random light sequences were synchronized with the acquisition of images of the camera .", "Illumination was at 850 nm wavelength with custom built LED bars .", "The selection of the wavelength of the infrared LEDs was to be far enough from the red LEDs in order to allow the selection of a long-pass filter to avoid the red LED illumination from affecting behavioral recordings .", "We mounted the infrared LEDs for dark-field illumination in opto-mechanic elements that allow adjusting the angle of the LED bars with respect to the behavioral arena .", "This was to avoid larval ‘shadows’ in the movies , which result in much lower efficiency of data acquisition .", "The red LEDs were connected in parallel to avoid the creation of a light gradient caused by voltage drop in each LED .", "We verified uniform light intensity at 1 . 9 W/m2 ± 0 . 06 .", "We followed previously described methods ( Kreher et al . , 2005 ) .", "In brief , action potentials of the ORNs were extracellularly recorded by placing a custom made tungsten recording electrode ( with a piezo manipulator ) through the cuticle into the dome of the DO of third-instar larvae .", "The larva was placed on its ventrum on a metal rod and immobilized by wrapping Parafilm around the rod and the body , exposing only the very anterior part of the larva containing the domes of the dorsal organs .", "A reference electrode , a drawn out borosilicate glass capillary filled with Ephrussi and Beadle solution , was previously inserted through the Parafilm into the larva's body .", "Light stimulation was generated with an LED at 627 nm ( Luxeonstar ) driven by a BuckPuck ( LUXdrive LEDdynamics ) and synchronized via a photocoupler relay ( Toshiba TLP597A ) with the data acquisition system ( Syntech IDAC-4 ) .", "The electrophysiological optogenetic experiments were conducted in a completely dark room , and the intensity of the light stimulus at the location of the larva's DO was set to 1 . 9 W/m2 .", "The image stacks recorded were processed using the MAGAT ( multiple animal gait and trajectory ) analyzer ( available online at https://github . com/samuellab/MAGATAnalyzer ) and analyzed using MATLAB ( Gershow et al . , 2012 ) .", "To produce the random stimulus , a Bernoulli process was used .", "This process is wide-sense stationary , produces independent binary values ( Lights ON or OFF ) at every instant , and its autocorrelation function is the Dirac delta function .", "The linear transformations for r → t and t → r transitions were estimated by the event-triggered averages multiplied by the mean t → r or r → t rates , respectively ( Sakai , 1992; Dayan and Abbott , 2001 ) .", "The convolution of the filters with the stimulus was computed numerically without fitting any function to the filter .", "The number of larvae used in the experiments of each figure can be found in the respective legends .", "We model navigational behavior as two alternating motor states: runs and turns .", "This allows the precise quantification of behavioral response as a time series of basic motor patterns .", "To activate CsChrimson , we use binary ON/OFF red light following a Bernoulli process ( see below for rationale for using this process ) .", "We assign −1 to OFF state and +1 to ON state .", "During a run , the behavioral response can be characterized as the likelihood of initiating a turn ( thereby ending the run ) .", "During a turn , the behavioral response is the likelihood of initiating a new run .", "Below , we describe our calculations for the run-to-turn transition .", "The same process was followed to make calculations for the turn-to-run transition .", "Our calculations follow standard reverse-correlation methods ( Dayan and Abbott , 2001 ) .", "First , we model the animal as a linear transducer .", "By definition , the probability of transition from run-to-turn , rT→R , is a weighted sum of stimulus history , s ( t ) : ( 1 ) rR→T ( t ) =∫0∞hR→T ( τ ) s ( t−τ ) dτ , where hR→T is linear filter or kernel .", "The Dirac delta function , δ ( t ) , is defined by: ( 2 ) ∫0∞h ( τ ) δ ( t−τ ) dτ=h ( t ) .", "When the stimulus is a Dirac delta function , the response is a direct measurement of the linear filter: ( 3 ) rR→T ( t ) =∫0∞hR→T ( τ ) δ ( t−τ ) dτ=hR→T ( τ ) .", "Alternatively , the first-order filter can be recovered by measuring the response to a random stimulus .", "The linear filter relates the cross-correlation of input and output ( Rrs ) and the autocorrelation of the input ( Rss ) by: ( 4 ) Rrs ( t ) =∫0∞h ( τ ) Rss ( t−τ ) dτ .", "For a Bernoulli process , as the one used here , the autocorrelation function ( Rss ) is a Dirac delta function ( δ ( t ) ) , therefore , Equation 4 becomes ( 5 ) Rrs ( t ) =∫0∞h ( τ ) δ ( t−τ ) dτ=h ( t ) .", "Thus , the cross-correlation of input and output represents a measurement of the linear filter .", "In our analysis , the relevant events in the output are the transitions from running to turning and vice versa .", "The average optogenetic activation signal that precedes each of these events is called the event-triggered average and can be written as shown below: ( 6 ) C ( τ ) =1〈n〉∫0TrR→T ( t ) s ( t−τ ) dt , Where 〈n〉 is the average number of turns per trial , and T is the duration of each trial .", "In our experiments , trials lasted 20 min .", "The cross-correlation function ( Rrs ) can be written: ( 7 ) Rrs ( τ ) =1T∫0TrR→T ( t ) s ( t+τ ) .", "From Equations ( 5 ) , ( 6 ) , ( 7 ) : ( 8 ) h ( τ ) =Rrs ( τ ) =〈n〉TC ( −τ ) .", "Thus , we estimated our linear filters by measuring the event-triggered average and multiplying by the average number of events in one trial divided by the duration of the trial .", "While many white processes could potentially be used for reverse-correlation analysis , we selected a Bernoulli process to minimize the introduction of nonlinear relationships between spiking and optogenetic activation .", "Using Gaussian white noise , for example , would require intensity modulation of the optogenetic stimulus .", "While it is possible that induced spiking scales linearly with light intensity for some Gal4 drivers , a nonlinear relationship might also occur .", "By using a Bernoulli process , we can use a uniform light intensity for optogenetic stimulation and need only assume a consistent level of spiking with optogenetic stimulation .", "This assumption was validated by direct electrophysiological measurement in two ORNs ( see Figure 1 ) .", "For a Bernoulli process , the random stimulation is confined to the timing of the ON or OFF state of the LEDs .", "Nonlinearities might still be introduced , for example , in delays in the onset or cessation of spiking with respect to the start or end of each illumination pulse .", "These effects pose an upper limit on the frequencies that can be used for random activation .", "To reduce the effect of these latencies while retaining the ability to characterize larval decision-making that occurs on the 1-s time scale , we used a stimulus frequency of 4 Hz .", "Because our method accurately predicts behavioral responses to trains of pulses of different widths as the one used in the right panel of Figure 3C , the top right panel of Figure 3—figure supplement 1A , the right panel of Figure 3—figure supplement 1B , or the top right panel of Figure 3—figure supplement 2A , nonlinearities owing to spike latencies are not likely to have significantly affected the construction of our models .", "To extract LN models and predictions from each 20 min movie of 20–30 animals in each experiment , we followed this workflow:Acquire behavioral movies and used the MAGAT Analyzer to segment trajectories . Identify all runs and turns and their initial frame and duration . Obtain all light patterns preceding the initiation of each run and turn . Using the light patterns , compute the triggered average for each transition using Equation 7 ( Figure 2 ) .", "Make predictions using the measured triggered average ( i . e . , convolution of Equation 9 with input signal ) .", "Compare with experimental measurements and fit sigmoidal function using least squares ( e . g . , Figure 3A ) .", "Assemble LN model and test using either subjecting a new set of larvae to defined trains of light pulses of different width ( e . g . , Figure 3C , right ) or step increases or decreases in illumination ( e . g . , Figure 3C , left ) ." ] ]
[ "Neural circuits for behavior transform sensory inputs into motor outputs in patterns with strategic value .", "Determining how neurons along a sensorimotor circuit contribute to this transformation is central to understanding behavior .", "To do this , a quantitative framework to describe behavioral dynamics is needed .", "In this study , we built a high-throughput optogenetic system for Drosophila larva to quantify the sensorimotor transformations underlying navigational behavior .", "We express CsChrimson , a red-shifted variant of channelrhodopsin , in specific chemosensory neurons and expose large numbers of freely moving animals to random optogenetic activation patterns .", "We quantify their behavioral responses and use reverse-correlation analysis to uncover the linear and static nonlinear components of navigation dynamics as functions of optogenetic activation patterns of specific sensory neurons .", "We find that linear–nonlinear models accurately predict navigational decision-making for different optogenetic activation waveforms .", "We use our method to establish the valence and dynamics of navigation driven by optogenetic activation of different combinations of bitter-sensing gustatory neurons .", "Our method captures the dynamics of optogenetically induced behavior in compact , quantitative transformations that can be used to characterize circuits for sensorimotor processing and their contribution to navigational decision making ." ]
[ "Living organisms can sense their surroundings and respond in appropriate ways .", "For example , animals will often move towards the smell of food or away from potential threats , such as predators .", "However , it is not fully understood how an animal's nervous system is setup to allow sensory information to control how the animal navigates its environment .", "Optogenetics is a technique that allows neuroscientists to control the activities of individual nerve cells in freely moving animals , simply by shining light on to them .", "Here , Hernandez-Nunez et al . have used optogenetics in fruit fly larvae to activate nerve cells that normally respond to smells and tastes , while the larvae's movements were tracked .", "Fruit fly larvae were chosen because they have a simple , but well-studied , nervous system .", "These larvae also move in two distinct ways: ‘runs’ , in which a larva moves forward; and ‘turns’ , during which a larva sweeps its head back and forth until it selects the direction of a new run .", "The data from these experiments were quantified using a specific type of statistical analysis called ‘reverse correlation’ and used to build mathematical models that predict navigational behavior .", "This analysis of the experiments allowed Hernandez-Nunez et al . to reveal how specific sensory nerve cells can contribute to pathways that control an animal's navigation—and an independent study by Gepner , Mihovilovic Skanata et al . revealed similar results .", "The approach of using optogenetics in combination with quantitative analysis , as used in these two independent studies , is now opening the door to a more complete understanding of the connections between the activity of sensory nerve cells and perception and behavior ." ]
2015
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers
elife-01695-v1
[ [ "Entry of cells into M phase of mitosis or meiosis requires the phosphorylation of thousands of sites on hundreds of proteins ( Dephoure et al . , 2008; Lindqvist et al . , 2009; Dulla et al . , 2010; Olsen et al . , 2010 ) .", "Many of these sites are substrates of cyclin-dependent kinases ( CDKs ) , most notably MPF ( M phase-promoting factor; CDK1-Cyclin B ) .", "CDK phosphosites are ‘proline-directed’ , being phosphorylated at serine or threonine followed by proline ( Nigg , 1993; Holmes and Solomon , 1996 ) .", "These M phase-specific phosphorylations must eventually be removed so that mitotic/meiotic cells can reset to interphase .", "PP2A-B55 ( heterotrimeric protein phosphatase 2A composed of a catalytic C subunit , a structural A subunit , and a B55-type regulatory subunit ) is the phosphatase responsible for removing many key CDK-generated phosphorylations at the conclusion of M phase ( Clarke et al . , 1993; Mayer-Jaekel et al . , 1994; Mochida and Hunt , 2007; Castilho et al . , 2009; Mochida et al . , 2009; Schmitz et al . , 2010 ) .", "The circuitry’s basic logic suggests that PP2A-B55 activity must be downregulated when cells go into M phase; if not , the phosphorylations catalyzed by MPF would be prematurely removed , preventing M phase entry ( Clarke et al . , 1993; Lee et al . , 1994 ) .", "Indeed , a regulatory module that turns off PP2A-B55 specifically during M phase has recently been characterized ( Figure 1 ) .", "In brief , MPF phosphorylates and thereby activates the Greatwall kinase ( Gwl ) ( Yu et al . , 2006; Blake-Hodek et al . , 2012 ) .", "Activated Gwl phosphorylates small proteins of the Endosulfine family ( Endos; [Gharbi-Ayachi et al . , 2010; Mochida et al . , 2010] ) at a unique , highly conserved site .", "Gwl-phosphorylated Endos ( pEndos ) binds to and inactivates PP2A-B55 , thus protecting a major class of CDK-governed phosphorylations from premature removal during M phase ( reviewed in Glover , 2012; Lorca and Castro , 2012 , 2013 and Hunt , 2013 ) .", "The Gwl→pEndos ⊣ PP2A-B55 pathway is critical for M phase entry and maintenance in frog egg extracts , starfish oocytes , and in many cell types in culture or within metazoan organisms ( Archambault et al . , 2007; Burgess et al . , 2010; Hara et al . , 2012; Lorca et al . , 2010; Voets and Wolthuis , 2010; Yu et al . , 2004 ) . 10 . 7554/eLife . 01695 . 003Figure 1 . Function of the Gwl → pEndos ⊣ PP2A-B55 module in cell cycle transitions . The major driver for transitions between interphase and M phase is the cyclic activation and degradation of MPF ( Cdk1-Cyclin B ) .", "When activated ( in part through a feed-forward autoregulatory loop involving the kinases Myt1 and Wee1 and the phosphatase Cdc25; not shown ) , MPF phosphorylates many substrates ( CDKSs ) that play key roles in M phase events .", "One such MPF substrate is the kinase Greatwall ( Gwl ) , which in its active form phosphorylates Endosulfine ( Endos ) .", "Phosphorylated Endos binds to and inhibits the phosphatase PP2A-B55 .", "This inhibition protects MPF substrates , including components of the autoregulatory loop , from premature dephosphorylation during M phase entry .", "During M phase exit , MPF is inactivated by degradation of its Cyclin B component ( not shown ) , and PP2A-B55 becomes reactivated to dephosphorylate many MPF substrates .", "M phase exit also requires the dephosphorylation and inactivation of both Gwl and Endos .", "Here , we show that PP2A-B55 catalyzes Endos dephosphorylation .", "The activities responsible for the inactivation of Gwl currently remain unknown . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 003 In this report , we explore how this M phase activation system is reversed when cells exit mitosis or meiosis .", "Not only must CDK-dependent phosphorylations be removed by newly reactivated PP2A-B55 , but also Gwl and pEndos must be inactivated at the end of M phase by the removal of their stimulatory phosphorylations ( Figure 1 ) .", "If Gwl were to remain active , it would continually keep Endos phosphorylated; PP2A-B55 would stay inactive and the system would remain in M phase because mitotic phosphorylations could not be removed .", "On the other hand , if Gwl alone were inactivated but pEndos were to stay phosphorylated , M phase would again be maintained .", "We focus here on the second part of this equation by identifying the phosphatase that dephosphorylates and turns off pEndos .", "At the onset of these investigations , we thought that pEndos inactivation could not be achieved solely by PP2A-B55 .", "Not only is the Gwl-phosphorylated pEndos site not proline-directed , as are CDK targets , but also a system based only on PP2A-B55 would be closed and futile: if PP2A-B55 is inactive during M phase , how could this ‘dead‘ enzyme turn itself back on ? Clearly , the ‘anti-Endos’ phosphatase ( s ) targeting Gwl-phosphorylated pEndos must instead be active during at least late M phase .", "As we will show , the expectation that anti-Endos is active during M phase has been borne out .", "However , counterintuitively , we found that almost all of this anti-Endos activity is contributed by PP2A-B55 .", "This surprising conclusion is made possible because the kinetic parameters of this enzyme for pEndos and for CDK-phosphorylated substrates are very different .", "Based on this divergence , we suggest a straightforward mechanism we call ‘inhibition by unfair competition’ that explains how pEndos acts both as an inhibitor and a substrate of PP2A-B55 , and how this phosphatase can simultaneously be ‘on’ for some substrates but ‘off’ for others .", "This mechanism may have broad implications for the control of other cellular protein phosphatases ." ], [ "We initiated our search for the anti-Endos phosphatase by asking if this activity has the basic characteristic predicted by the logic of the system: in contrast with the anti-CDKS activity of PP2A-B55 , which is off during M phase , the anti-Endos phosphatase should remain active during M phase to permit subsequent M phase exit .", "This question could be addressed readily using extracts of Xenopus eggs , which are prepared in an M phase state but can be induced to exit M phase by addition of Ca2+ ( Murray and Kirschner , 1989; Murray , 1991; Tunquist and Maller , 2003 ) .", "Figure 2A shows that in accordance with this prediction , considerable anti-Endos activity is indeed seen during M phase .", "The level is roughly half that seen in interphase; as will be explained below , we believe this difference results from competition between exogenous radiolabeled pEndos and endogenous unlabeled pEndos present in M phase but not interphase .", "As expected from previous studies ( Mochida and Hunt , 2007; Castilho et al . , 2009 ) , anti-CDKS activity ( i . e . , PP2A-B55 ) was completely blocked in M phase extracts and strongly induced by treatment with Ca2+ ( Figure 2A ) . 10 . 7554/eLife . 01695 . 004Figure 2 . Characterization of anti-Endos in extracts . In all parts of this figure , red circles depict anti-Endos , whereas blue squares represent anti-CDKS .", "( A ) Anti-Endos is present during M phase .", "Xenopus CSF ( M phase ) extracts were incubated at 22°C .", "At time t = 0 , Ca2+ was added to half of the extract to induce M phase exit; control extract without Ca2+ remained in M phase .", "At the indicated times , aliquots were assayed for anti-Endos and anti-CDKS as described in ‘Materials and methods’ .", "During M phase , anti-CDKS ( light blue squares ) is undetectable , whereas anti-Endos ( light red circles ) is active .", "As the extracts exit M phase ( interphase is achieved within 15–20 min of Ca2+ addition; [Yu et al . , 2006; Zhao et al . , 2008; Castilho et al . , 2009] ) , anti-CDKS activity ( dark blue squares ) is strongly induced , while anti-Endos ( dark red circles ) increases about twofold .", "( B–E )", "Drug sensitivities of phosphatase activities .", "Y-axis values represent the percentage of the phosphatase activity for the given combination of extract and substrate measured in the absence of the inhibitor .", "Anti-Endos and anti-CDKS have similar sensitivities to okadaic acid and fostriecin , but anti-Endos is substantially more resistant than anti-CDKS to tautomycetin and phosphomimetic Endos S68D .", "In B and C , green triangles represent dephosphorylation activity against CDK-phosphorylated Histone H3; in C , purple stars are activity against CDK-phosphorylated Histone H1v1 . 0 .", "In part C , the fostriecin resistant portions of the H3 phosphatase ( about 40% of the total ) and the H1v1 . 0 phosphatase ( about 80% of the total ) likely represent PP1 activity .", "The HeLa extracts examined in panels B–D were from asynchronous cells , the vast majority of which are in interphase .", "( F ) The specific activities of anti-CDKS and anti-H3 increase upon dilution of the extract , presumably because weakly binding inhibitors are titrated away , but the specific activity of anti-Endos increases at most only marginally upon dilution .", "The y-axis shows the phosphatase activity on the indicated substrates , normalized to the original volume of undiluted extract .", "In all panels , n = 1; biological and evolutionary replicates of the experiments in panels B–D are presented in Figure 2 figure supplements 1–5 . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 00410 . 7554/eLife . 01695 . 005Figure 2—figure supplement 1 . Anti-Endos is completely inhibited by okadaic acid and calyculin . A In all parts of this figure , red circles depict anti-Endos , and blue squares are anti-CDKS; in B and C green triangles represent dephosphorylation activity against Histone H3 .", "In all panels except part D , each symbol represents a single assay .", "( A and B )", "Biological replicates of the experiment shown in Figure 2B .", "( C ) Xenopus CSF extracts were untreated ( M phase ) or treated with Ca2+ for 30 min ( interphase ) and then assayed for phosphatase activity .", "As in Figure 2A , anti-CDKS is undetectable in CSF extracts .", "The sensitivity of anti-Endos to okadaic acid is similar in M phase and interphase extracts; in both cases , the IC50 for anti-Endos is about threefold higher than that for anti-CDKS in interphase .", "We presume this difference reflects the substantial fraction of anti-Endos in Xenopus extracts due to PP1 ( Figure 2—figure supplement 2 ) .", "( D ) In asynchronous S2 ( Drosophila ) cell extracts , anti-CDKS and anti-Endos have nearly identical dose-response curves to okadaic acid; these activities are slightly more sensitive to okadaic acid than is the phosphatase activity directed at Histone H3 substrate .", "In this panel , each symbol represents the average of four technical replicates; error bars are not shown for the anti-CDKS activity to aid readability , but their magnitude is consistent with those of the other assays .", "( E ) Evolutionary replicate of panels A–D performed with an extract made from asynchronous mouse embryo fibroblast ( MEF ) cells .", "( F ) Phosphatase activities against all three substrates are completely suppressed by calyculin A; reported IC50 values for this drug with respect to PP1 , PP2A , PP4 , PP5 , and PP6 are all similar ( Swingle et al . , 2007 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 00510 . 7554/eLife . 01695 . 006Figure 2—figure supplement 2 . Anti-Endos is mostly fostriecin-sensitive . In all parts of this figure , red circles depict anti-Endos , blue squares are anti-CDKS , green triangles are activity against Histone H3 , and purple stars are anti-H1v1 . 0 .", "Each symbol represents a single assay .", "( A ) The fostriecin sensitivities of anti-Endos in M phase ( CSF extracts ) and interphase Xenopus egg extracts are similar .", "A proportion of anti-Endos in these concentrated egg extracts is more fostriecin-resistant than is the anti-CDKS in the same extracts; the exact proportion is difficult to estimate because the maximal amount of fostriecin that could be added was insufficient even to inhibit anti-CDKS completely .", "( B–F )", "In extracts of Xenopus eggs diluted 1:4 in phosphatase buffer ( B ) , of Drosophila S2 cells ( C and D are biological replicates ) , or of mouse MEF cells ( F ) , anti-Endos activity is more resistant to fostriecin than is anti-CDKS , but is less resistant than are the phosphatase activities against Histone H1v1 . 0 or Histone H3 .", "In panel C , two technical replicates of the anti-Endos assay are shown .", "These data suggest that PP1-like enzymes account for most of the activity against Histone H1v1 . 0 and about half of that against Histone H3 .", "From the amount of anti-Endos activity remaining at fostriecin concentrations of 100 μM sufficient to inhibit anti-CDKS completely , we estimate that 70–90% of anti-Endos is fostriecin-sensitive ( depending on the extract ) and is therefore likely to be PP2A , PP4 , or PP6 .", "The minor fostriecin-resistant component is labile and is lost from certain extracts such as D and E ( see also Figure 2C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 00610 . 7554/eLife . 01695 . 007Figure 2—figure supplement 3 . Anti-Endos is more tautomycetin/tautomycin-resistant than anti-CDKS .", "( A ) As expected from the literature ( Mitsuhashi et al . , 2001 ) , purified PP1 ( orange stars ) is more sensitive to tautomycetin than is an equal molar amount of purified PP2A A-C dimer ( pink diamonds ) measured with myelin basic protein phosphorylated with PKA kinase as substrate .", "( B–F )", "The IC50 for tautomycetin ( B–E ) or tautomycin ( F ) is consistently ∼10-fold higher for anti-Endos than for anti-CDKS when measured in the same extract .", "The activity against CDK-phosphorylated Histone H1v1 . 0 is more sensitive than either to tautomycetin , consistent with the argument that the major phosphatase targeting this substrate is PP1 .", "Each data point represents a single assay; C and D are biological replicates using different extracts of S2 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 00710 . 7554/eLife . 01695 . 008Figure 2—figure supplement 4 . Anti-Endos is resistant to phosphomimetic Endos . Although anti-CDKS activity is strongly inhibited by phosphomimetic Endos ( S68D ) , anti-Endos activity is insensitive to addition of this molecule .", "The dephosphorylations of Histone H1v1 . 0 or Histone H3 substrates are also resistant to Endos S68D , as would be expected of substrates that are targeted by PP1 or any phosphatases other than PP2A-B55 .", "Each data point represents a single assay; panel A is a biological replicate of the results shown in Figure 2E . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 00810 . 7554/eLife . 01695 . 009Figure 2—figure supplement 5 . The specific activity of anti-Endos does not increase upon substrate dilution . The y-axis shows the phosphatase activity on the indicated substrates , normalized to the original volume of undiluted extract .", "Panel A is a biological replicate of the experiment shown in Figure 2F , n = 1; in panel B , the data points each represent the average of three technical replicates with error bars shown .", "The reason that the dilution effect is less pronounced for the extract in part A likely reflects the fact that the original concentration of this extract ( prior to dilution ) was about fourfold lower than that in panel A . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 00910 . 7554/eLife . 01695 . 010Figure 2—figure supplement 6 . Estimating relative levels of Twins ( B55 ) and Endos proteins in Drosophila S2 cells . Sequential dilutions of purified recombinant proteins and of total S2 cell extracts were compared on Western blots using antibodies against these two proteins .", "Total protein amounts in extracts were determined by nanodrop spectrophotometry . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 010 We next characterized the sensitivity of the anti-Endos phosphatase in concentrated extracts ( from Xenopus eggs and human , fly , or mouse tissue culture cells ) to common phosphatase inhibitors .", "The properties of anti-Endos studied in all of these extracts were nearly interchangeable .", "All of the activity in all extracts tested was suppressed by relatively low doses of okadaic acid or calyculin A ( Figure 2B , Figure 2—figure supplement 1 ) , but was completely resistant to the calcineurin ( PP2B ) inhibitor cyclosporin A ( data not shown ) .", "These results indicate that the enzyme ( s ) targeting the Gwl site in Endos belong to the PPP family of phospho-serine/threonine protein phosphatases , which include PP1 , PP2A , PP4 , PP5 , and PP6 ( Swingle et al . , 2007 ) .", "PP1 and PP5 are ∼10 , 000-fold more resistant to the inhibitor fostriecin than the PP2A/PP4/PP6 group of enzymes ( Swingle et al . , 2007 ) .", "In all extracts examined , the majority of anti-Endos activity was sensitive to the same doses of fostriecin that inhibit PP2A-B55’s anti-CDKS activity ( Figure 2C , Figure 2—figure supplement 2 ) .", "Anti-Endos and anti-CDKS activities were both considerably more sensitive to fostriecin than were the dephosphorylations of two other substrates , CDK-phosphorylated histone H1v1 . 0 , which is substantially targeted by PP1-like enzymes ( Paulson et al . , 1994; Qian et al . , 2011 ) , and histone H3 , which is apparently a substrate for both a fostriecin-sensitive and a fostriecin-resistant phosphatase .", "The predominant anti-Endos activity in many cell types ( ∼70–90% of the total depending on the experiment ) is thus due to PP2A , PP4 , or PP6 .", "Because the major anti-Endos activity displays closely similar sensitivities to okadaic acid or fostriecin when comparing M phase and interphase frog egg extracts , it appears that the same phosphatase is responsible for this activity during both cell cycle stages ( Figure 2—figure supplements 1C and 2A ) .", "A minor fraction of anti-Endos is nonetheless fostriecin-resistant; this part of the activity is labile and is seen in some extract preparations ( Figure 2—figure supplement 2A–C , F ) but not others ( Figure 2C , Figure 2—figure supplement 2D , E ) .", "This secondary activity , possibly due to some form of PP1 , is likely responsible for the modest okadaic acid resistance of anti-Endos ( relative to anti-CDKS ) seen in concentrated Xenopus extracts ( Figure 2—figure supplement 1C ) , where the proportion of fostriecin-resistant activity is highest ( Figure 2—figure supplement 2A , B ) .", "In the ‘Discussion’ , we argue that this minor component of anti-Endos is unlikely to be of great physiological importance; in the remainder of the ‘Results’ , we thus focus on the predominant fostriecin-sensitive enzyme .", "In spite of the fact that anti-Endos and anti-CDKS are both associated with the same highly related subfamily of PPP phosphatases including PP2A , PP4 , and PP6 , further experiments revealed clear divergences in the behavior of these two phosphatase activities .", "First , we characterized the response of anti-Endos and anti-CDKS activities to tautomycetin and its relative tautomycin .", "These drugs have been reported to be much more effective as inhibitors of PP1 than PP2A ( Gupta et al . , 1997; Mitsuhashi et al . , 2001; Kelker et al . , 2009 ) .", "This conclusion was verified in our own system using purified enzymes ( Figure 2—figure supplement 3A ) and by the tautomycetin sensitivity of phosphatase activity in extracts against the PP1-specific substrate Histone H1v1 . 0 ( Figure 2D ) .", "In contrast , the anti-Endos activity in all extracts tested is 6- to 10-fold more resistant to tautomycetin than is anti-CDKS ( Figure 2D , Figure 2—figure supplement 3B–F ) .", "This result is surprising because we had not anticipated the existence of a PPP family phosphatase more tautomycetin-resistant than PP2A-B55 .", "The tautomycetin/tautomycin resistance of anti-Endos is due to the major , fostriecin-sensitive component , because extracts lacking the labile fostriecin-resistant enzyme are still resistant to tautomycetin ( the extracts used in Figure 2C and Figure 2D are identical ) .", "Second , we found that in contrast to anti-CDKS , anti-Endos is relatively unaffected by the presence of constitutively activated Endos .", "Endosulfine thiophosphorylated by Gwl acts in vitro as a specific inhibitor of PP2A-B55’s anti-CDKS activity; this effect is also observed using Endos bearing a phosphomimetic mutation of the Gwl target site ( Gharbi-Ayachi et al . , 2010; Mochida et al . , 2010; Kim et al . , 2012 ) .", "However , the anti-Endos activity in extracts is substantially resistant to inhibition by phosphomimetic Drosophila Endos ( S68D ) ( Figure 2E , Figure 2—figure supplement 4 ) .", "Finally , it has previously been reported that the dilution of cellular extracts leads to large increases in the specific activity of protein phosphatases , possibly caused by the decreased association , due to mass action , of the phosphatase with weak inhibitors in the extracts ( Cohen , 1989; Mochida et al . , 2009 ) .", "We have verified this dilution effect for anti-CDKS and anti-H1v1 . 0 , but in contrast , anti-Endos activity is very much less affected by extract dilution ( Figure 2F , Figure 2—figure supplement 5 ) .", "The data to this point suggest that the major anti-Endos enzyme is some form of PP2A , PP4 , or PP6 .", "As we anticipated , anti-Endos is clearly differentiated from PP2A-B55 ( anti-CDKS ) in terms of its constitutive activity during M phase .", "Anti-Endos and anti-CDKS also diverge strongly in terms of their relative sensitivities to tautomycetin , phosphomimetic Endos , and extract dilution .", "To pinpoint which of the candidate phosphatases targets the Gwl phosphosite on pEndos , we examined anti-Endos activity in extracts of cultured cells depleted for phosphatase subunits ( Figure 3 ) .", "Treatment of Drosophila S2 cells with dsRNAs for the single PP2A catalytic subunit gene in the fly genome ( called mts ) caused the loss of 70–75% of the anti-Endos activity ( Figure 3A ) .", "Success of the RNAi was verified by Western blot ( Figure 3B ) , and by the almost complete loss of anti-CDKS activity in the dsRNA-treated cells ( Figure 3A ) .", "Depletion of the A ( structural ) subunit of PP2A also caused substantial loss of anti-Endos activity ( Figure 3 ) ; as expected from previous studies ( Silverstein et al . , 2002 ) , RNAi for fly PP2A-A destabilizes PP2A-C , and vice versa .", "In contrast , RNAi for PP1-87B , whose product accounts for more than 80% of the PP1 activity in Drosophila larvae ( Dombradi et al . , 1990 ) , only slightly decreased anti-Endos ( Figure 3 ) .", "dsRNAs against coding sequences for all other PPP family catalytic subunits ( PP4 , PP5 , and PP6 ) had no obvious effects on anti-Endos levels ( data not shown ) . 10 . 7554/eLife . 01695 . 011Figure 3 . Depletion of PP2A by RNAi disrupts a major component of anti-Endos activity .", "( A ) Phosphatase assays for anti-Endos , anti-CDKS , and anti-Histone H3 activities in mock-treated control Drosophila S2 tissue culture cells ( blue ) , and S2 cells treated with dsRNAs for the PP2A-A subunit ( red; the gene is PP2A-29B ) , the PP2A-C subunit ( green; the gene is mts ) , and the major PP1-C subunit ( yellow; the gene is PP1-87B , which accounts for more than 80% of PP1-related activity against generic substrates [Dombradi et al . , 1990] ) .", "Each column represents a separate biological replicate ( n = 4 for the controls and n = 2 for each of the three RNAi treatments ) ; values were normalized for total protein concentrations in the extracts .", "One of the four replicates of the control assay was arbitrarily chosen as the 100% reference .", "The data indicate that a form of PP2A is responsible for about 90% of anti-CDKS , ∼70 to 80% of anti-Endos , and ∼60% of anti-H3; the residual activities are likely due mostly to forms of PP1 , although PP5 may also contribute in the case of anti-H3 ( Figure 4 ) .", "( B ) Western blots showing effectiveness of the RNAi treatments .", "As reported elsewhere ( Kamibayashi et al . , 1992 ) , the stabilities of the PP2A-A and PP2A-C subunits are mutually interdependent . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 01110 . 7554/eLife . 01695 . 012Figure 3—figure supplement 1 . Neither PP4 nor PP5 is a major contributor to anti-Endos .", "( A and B )", "Treatment of HeLa cells with siRNA to the PP4 catalytic subunit strongly reduces the amount of PP4 but does not obviously affect anti-Endos levels .", "C1 represents control cells that are not treated with siRNA; C2 cells were treated with a control-scrambled siRNA .", "( C and D )", "No obvious changes in anti-Endos levels are seen in the mutant mouse MEF cells homozygous for a null mutation in the gene encoding the PP5 catalytic subunit ( Yong et al . , 2007 ) relative to control MEF cells .", "PP5 may be a minor contributor to Histone H3 dephosphorylation .", "In panels A and C , LC are background bands used as loading controls for the Western blots .", "In panels B and D , the number of technical replicates is indicated in each bar . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 012 Analogous experiments to deplete phosphatase catalytic subunits from HeLa cells using siRNAs were clearly successful only in the case of PP4 , where no effects on either anti-Endos or anti-CDKS activities were observed ( Figure 3—figure supplement 1A , B ) .", "Mouse MEF cells homozygous for a knockout allele of the PP5 gene ( Yong et al . , 2007 ) also displayed the same levels of anti-Endos and anti-CDKS activities as control cells ( Figure 3—figure supplement 1C , D ) .", "These knockdown/mutation experiments argue strongly that the predominant anti-Endos phosphatase is some form of PP2A .", "Most PP2A activity in vivo is ascribed to heterotrimeric enzymes containing the C and A subunits plus one of several kinds of regulatory B subunits ( Janssens et al . , 2008 ) .", "To distinguish which form of PP2A is anti-Endos , we took advantage of the fact that the Drosophila genome has a near-minimal set of genes encoding PP2A regulatory subunits ( e . g . , one B55-type subunit as opposed to four in mammals; and two B56-class [B′] subunits vs five in mammals ) .", "Null or strongly hypomorphic mutations are available for almost all of these fly genes ( Uemura et al . , 1993; Mayer-Jaekel et al . , 1994; Chen et al . , 2002; Hannus et al . , 2002; Viquez et al . , 2006 ) ; animals homozygous for these mutations usually survive until third instar larval or pupal stages because maternal contributions are sufficient for earlier stages of development ( Gatti and Goldberg , 1991 ) .", "Extracts prepared from mutant third instar larvae were assayed for anti-Endos and anti-CDKS activities , as well as phosphatase activity against Histone H1v1 . 0 as an additional control .", "The results were striking and unanticipated: Relative to wild-type , larvae homozygous for mutations in twins ( the gene encoding the sole B55-type subunit in flies ) were not only deficient in anti-CDKS as expected , but they also lacked anti-Endos; the same was true for brains isolated from these larvae ( Figure 4A ) .", "Activities against Histone H1v1 . 0 were virtually unaffected in twins mutants .", "On Western blots , the twins larvae had no detectable Twins ( B55 ) protein; moreover , gross levels of PP2A-A and PP2A-C were not diminished by the absence of Twins ( Figure 4B ) .", "None of these phosphatase activities were altered in larvae carrying mutations in genes for any other tested PP2A regulatory subunit , for the PPP4R3 regulatory subunit of PP4 ( flfl ) , or for any of the PP1 catalytic subunits ( with the possible exception of a decrease in Histone H1v1 . 0 dephosphorylation in PP1-87B larvae; Figure 4A ) . 10 . 7554/eLife . 01695 . 013Figure 4 . Drosophila larvae lacking B55 are deficient in anti-Endos .", "( A ) Extracts were prepared from larvae with null or strong loss-of-function alleles of the indicated genes , and assayed for anti-Endos , anti-CDKS , and anti-Histone H1v1 . 0 activities .", "Values were normalized for the total protein concentrations in the extracts; one replicate of the control assay ( on a wild-type larva ) was arbitrarily chosen as the 100% reference .", "Details about the genotypes involved are given in ‘Materials and methods’ .", "The number of biological replicates for each genotype is presented inside the corresponding bar , with standard deviations shown .", "Levels of anti-Endos are much lower in twins ( encoding the sole B55 subunit in Drosophila ) larvae than in wild-type ( WT ) controls ( p<10−20; Student’s two-tailed t test ) ; this is also the case as expected for anti-CDKS ( p<1010 ) .", "As a control , phosphatase activity against Histone H1v1 . 10 is relatively unaffected in twins mutant larvae .", "Anti-Endos and Anti-CDKS activities were also severely compromised in extracts made from brains isolated from twins mutant animals .", "No consistent effects were observed in extracts made from larvae mutant for genes encoding the other phosphatase subunits indicated , with two exceptions .", "First , extracts from larvae mutant for PP1-96A displayed only about half the level of phosphatase activities measured with all three substrates .", "This is probably a systematic error caused by the ebony mutation in these animals , producing a dark pigment that caused an overestimation of the amount of total protein concentration .", "Second , the lower level of activity against Histone H1v1 . 0 in animals mutant for PP1-87B ( the predominant PP1 catalytic subunit in Drosophila; Dombradi et al . , 1990 ) is likely caused by the targeting of this substrate by the PP1-87B phosphatase .", "( B ) Western blots of extracts from larvae mutant for genes encoding B55 ( twins ) and B56 ( widerborst [wdb] and well-rounded [wrd] ) regulatory subunits of PP2A .", "The blot at the right verifies that in twins mutants , the B55 protein is missing , while the PP2A-A and PP2A-C subunits are present in normal amounts . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 013 As just seen , depletion/mutation of any of the three components of the PP2A-B55 heterotrimer removes the large majority of the phosphatase activity directed against the Gwl site in pEndos .", "These findings were very surprising , given that PP2A-B55’s action against CDK-phosphorylated substrates differs in many properties from anti-Endos , and the sequence motifs phosphorylated by Gwl and CDKs are very different .", "We were thus concerned that the effects of PP2A-B55 depletion on pEndos dephosphorylation could be indirect: that is , PP2A-B55 might not be anti-Endos , but its loss might instead disrupt another phosphatase that does play such a role .", "To address whether anti-Endos is indeed a PP2A-B55 heterotrimer , we fractionated asynchronous HeLa cell extracts by Mono-Q chromatography ( Figure 5 ) .", "Assays of the fractions revealed that anti-Endos and anti-CDKS activities co-purified .", "The peak activities towards both substrates coincided with the fractions containing the three components of PP2A-B55 ( the A , B55 , and C subunits ) , although the PP1 catalytic subunit was also found in these same fractions .", "PP5 and PP6 elute from the Mono-Q column at much lower salt concentrations , while PP4 and the B56 and B‴ ( striatin ) regulatory subunits of PP2A elute at higher salt than the peak anti-Endos/anti-CDKS ( Figure 5 ) .", "The anti-Endos activity in the peak fractions has the same characteristics as in concentrated extracts: it is sensitive to okadaic acid and fostriecin , but insensitive to tautomycetin and phosphomimetic Drosophila Endos ( data not shown ) .", "The fractionation results are consistent with the idea that anti-Endos is indeed the PP2A-B55 heterotrimer and further exclude most other PPP enzymes as candidates . 10 . 7554/eLife . 01695 . 014Figure 5 . Mono-Q chromatography of HeLa extracts . Total extract from asynchronous HeLa cells ( high speed supernatant; HS ) was applied to the column; FT is the flow-through .", "Proteins were eluted with linear gradient from 150 mM ( Fraction 1 ) to 500 mM ( Fraction 75 ) of NaCl .", "Individual fractions were assayed ( n = 1 assay per data point ) for anti-Endos ( black circles ) and anti-CDKS ( black squares ) , and were also examined for the indicated phosphatase subunits by Western blot . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 014 We next purified recombinant PP2A-B55 heterotrimer from lysates of human embryonic kidney ( HEK ) cells transfected with constructs expressing FLAG-tagged B55α or B55δ isoforms , or the B56β isoform as a control ( as described in Adams and Wadzinski ( 2007 ) ) .", "On silver stained SDS-PAGE gels , the FLAG immune complexes showed predominant bands at the positions of the A , FLAG-B55 or FLAG-B56 , and C subunits of PP2A ( Figure 6—figure supplement 1 ) .", "Some variable contaminating bands were also seen in control immune complexes isolated from lysates of cells transfected with empty vectors .", "Mass spectrometry identified these proteins as keratins or immunoglobulin chains ( from the antibody on the FLAG affinity beads ) unlikely to contribute to phosphatase activities .", "Heterotrimers containing B55α and B55δ isoforms efficiently dephosphorylated pEndos and pCDKS substrates ( Figure 6A ) .", "Pilot experiments allowed us to choose concentrations of the enzymes and time of reactions in which subsequent investigations could be performed in the linear range for these variables ( Figure 6—figure supplement 2 ) .", "Neither control PP2A-B56β heterotrimers nor empty vector preparations made in parallel displayed any activity against either substrate .", "Neither the anti-Endos nor anti-CDKS functions of the PP2A-B55 preparations could be attributed to heterotrimer dissociation into A–C dimers , because purified dimer does not target either substrate even when in 10-fold excess relative to the heterotrimeric holoenzymes ( Figure 6A ) .", "The anti-Endos and anti-CDKS activities of the PP2A-B55 heterotrimer were similarly sensitive to fostriecin , as expected if the catalytic subunit is the same ( Figure 6B ) .", "However , the anti-Endos activity of PP2A-B55 was much less sensitive to either tautomycetin or phosphomimetic Endos than was the anti-CDKS activity of the same preparations ( Figure 6C , D ) , as was also true for unfractionated extracts ( review Figure 2D , E ) and the peak Mono-Q fractions ( data not shown ) . 10 . 7554/eLife . 01695 . 015Figure 6 . Anti-Endos activity of purified PP2A-B55 . ( A ) PP2A-B55 heterotrimers efficiently dephosphorylate Gwl-phosphorylated Endos .", "Each column represents an individual experiment ( a biological replicate ) in which the indicated enzyme preparations shown in Figure 6—figure supplement 1 ( normalized for the amount of PP2A-C subunit where possible , or for the volume of transfected cells in the case of the vector-only control preparation ) were assayed for anti-Endos activity , anti-CDKS activity , or generic activity against myelin basic protein ( MyBP ) phosphorylated with PKA .", "The y-axis indicates the percentage of radioactivity in the input substrate that was freed by enzyme treatment .", "The control preparation had no activity against any of these substrates , whereas PP2A-B55α and PP2A-B55δ heterotrimers showed similar strong levels of activity against all three substrates .", "PP2A-B56β heterotrimers and PP2A-A/C heterodimers , both of which dephosphorylated the generic MyBP substrate , failed to dephosphorylate either Gwl-phosphorylated pEndos or the pCDKS substrate .", "( B–D )", "Dose-response curves to phosphatase inhibitors ( fostriecin , tautomycetin , and phosphomimetic Endos S68D as indicated ) for the anti-Endos ( red circles ) and anti-CDKS ( blue squares ) activities of purified PP2A-B55α heterotrimers .", "Each point represents a single assay ( n = 1 ) .", "The anti-Endos activity of purified PP2A-B55α heterotrimers has the same characteristics as the predominant anti-Endos activity in whole cell extracts .", "In D , the fostriecin used has lost potency during the ∼6 months of storage after the experiments shown in Figure 2 were performed; fostriecin is well known to be somewhat labile in this time frame ( Weiser et al . , 2003 ) .", "It is nonetheless clear that both the anti-Endos and anti-CDKS functions of PP2A-B555α display similar dose responses to this drug .", "( E ) Technical replicates ( n = 3 ) of untreated purified PP2A-B55 and enzyme treated with the maximal doses of the inhibitors shown in panels B–D ( 20 μM fostriecin , 10 μM tautomycetin , and 20 μM Endos S68D ) were performed to assess reproducibility; symbols indicate average values and bars representing one standard deviation are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 01510 . 7554/eLife . 01695 . 016Figure 6—figure supplement 1 . Preparations of PP2A heterodimer and heterotrimers . In the first four lanes , HEK cells were transfected with the indicated constructs , and FLAG-tagged components were affinity purified as described in ‘Materials and methods’ .", "Final preparations were fractionated by SDS-PAGE and analyzed by silver staining ( top panel ) and by Western blot ( bottom panels ) .", "The major visible components in the silver staining are the A and C subunits of PP2A , the FLAG-tagged regulatory subunit whose expression construct was originally transfected ( B55α , B55δ , or B56β ) , and bovine serum albumin ( BSA ) added to stabilize the purified enzymes .", "An additional band of variable intensity located between the B55 and C bands , which is also seen in the vector alone control lane , was identified by mass spectrometry as keratin; yet another weak band seen in some preparations that migrates just faster than B55α/δ appears to be immunoglobulin heavy chain from the anti-FLAG column .", "At the right is a sample of commercially obtained A–C heterodimer ( Millipore 14-111 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 01610 . 7554/eLife . 01695 . 017Figure 6—figure supplement 2 . Characterization of phosphatase assays using purified PP2A-B55 . Panels A and B show the rate at which phosphate is released from labeled substrates as a function of the concentration of PP2A-B55 .", "The relationships are approximately linear , although the rate of the anti-Endos reaction slows slightly at higher enzyme concentrations; for this reason , the concentration of PP2A-B55 was held to 0 . 5 nM or below in most experiments .", "Panels C and D show time courses of the same reactions; the concentration of PP2A-B55 was 0 . 5 nM .", "As expected , the reactions slow down as more than 20% of either substrate is depleted and as the reaction time becomes long enough for the PP2A-B55 to lose activity due to instability or degradation .", "For these reasons , kinetic constants were measured in experiments with incubations sufficiently short to ensure that the maximal conversion of substrate stayed below this level and to minimize loss of enzyme function .", "In all panels , data points represent the average of three technical replicates with error bars shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 017 To understand the different properties of PP2A-B55 with respect to pEndos and CDK-phosphorylated substrates , we performed kinetic analyses of phosphatase activities using purified components and analyzed the data using the Michaelis–Menten reaction scheme allowing for tight-binding ( ‘Materials and methods’ ) .", "The results are summarized in Table 1; the data on which these results were based are presented in Figure 7 .", "Measurements of nanomolar or subnanomolar Km values for the dephosphorylation of pEndos by traditional plots of initial reaction rates as a function of the substrate concentration are technically challenging because of the very low concentrations of enzyme and substrate involved; these issues could lead to overestimates of the Km .", "We therefore supplemented these studies ( Figure 7A–C ) with a more accurate alternative approach based upon competition between pEndos and okadaic acid for access to the enzyme’s active site ( ‘Materials and methods’; Figure 7D , E ) . 10 . 7554/eLife . 01695 . 018Table 1 . Kinetic parameters of dephosphorylation by PP2A-B55DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 018SubstrateKm ( μM ) kcat ( sec−1 ) n*Method†Representative experimentCDKS71–9921–252v vs [S]Figure 7AFzy Ser50>100‡>15‡1v vs [S]Figure 7ApEndos0 . 0009–0 . 00170 . 021–0 . 0352v vs [S]Figure 7B , C0 . 018–0 . 066§16§0 . 0004–0 . 00110 . 005–0 . 0373OA competitionFigure 7D , E*Number of independent experiments; each point in each experiment included 3–4 measurements .", "†v vs [S] experiments measured the initial rate of reaction as a function of substrate concentration; OA competition experiments involved measurements of pEndos dephosphorylation as a function of okadaic acid competition .", "‡Because sufficiently high concentrations of the Fzy Ser50 substrate were not obtained , non-linear regression analysis was inherently inaccurate in determining kinetic parameters .", "The values given are conservative lower limits .", "§The specific activities of multiple independent preparations of purified PP2A-B55 were measured at high pEndos substrate concentrations ( 5 μM ) approximately 1000-fold in excess of the Km . 10 . 7554/eLife . 01695 . 019Figure 7 . Determinations of the kinetic parameters shown in Table 1 . ( A ) Determination of kinetic parameters for the dephosphorylation of CDKS and Fzy-Ser50 substrates .", "One representative experiment of each is shown .", "Initial rates of dephosphosphorylation were determined with increasing substrate concentrations; the concentration of purified PP2A-B55 was 1 nM .", "The amount of 32P phosphate released was in all samples less than 20% of the input .", "For pCDKS substrate , n = 4; for pFzy-Ser50 substrate , n = 3 ( technical replicates ) .", "The data were analyzed by non-linear regression analysis ( using Prism 6 software ) to determine Km and kcat values incorporated into the data shown in Table 1 .", "Because we could not obtain sufficiently concentrated preparations of the Fzy-Ser50 substrate , the uncertainty in these parameters is very high , but the graphs illustrate that the behaviors of these two substrates are nonetheless much more similar to each other than either is to pEndos .", "( B and C )", "Determination of kinetic parameters for the dephosphorylation of pEndos from the initial reaction velocity as a function of substrate concentration .", "One representative experiment is shown .", "Initial rates of dephosphosphorylation were determined with increasing substrate concentrations .", "The concentration of purified PP2A-B55 was 0 . 5 nM; n = 4 technical replicates at each substrate concentration point .", "The amount of 32P phosphate released was in all samples less than 20% of the input .", "The data were analyzed by non-linear regression analysis as detailed in the ‘Materials and methods’ to determine Km and kcat values shown in Table 1 .", "B and C graph the same data at different ranges of pEndos concentration .", "Note that in B , the rate of pEndos dephosphorylation remains constant near Vmax over a 20-fold range from 50 to 1100 nM , indicating that pEndos does not inhibit its own dephosphorylation .", "( D and E )", "Determination of kinetic parameters for the dephosphorylation of pEndos from competition with okadaic acid .", "Note the logarithmic scale on the y-axes .", "( D ) Initial rates of pEndos dephosphorylation were determined in the presence of increasing concentrations of okadaic acid; one representative experiment is shown .", "The amount of 32P phosphate released was in all samples less than 20% of the input .", "Two independent experiments ( technical replicates ) are indicated with pink squares and green stars .", "The concentration of pEndos in this reaction was 50 nM , the concentration of purified PP2A-B55 was 0 . 25 nM , and the concentration of okadaic acid varied as shown .", "The data were fit by non-linear regression analysis to the mathematical model described in ‘Materials and methods’; calibration curves based on this model are shown in ( E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 019 The data summarized in Table 1 indicate that the kcat for the dephosphorylation of pEndos by PP2A-B55 is two to three orders of magnitude slower than that for the reaction of the same enzyme with pCDKS substrate , while the Km values for the two substrates differ by more than four and perhaps even five orders of magnitude .", "Table 1 presents the results as a range of values representing multiple experiments with several independent preparations of PP2A-B55 and either pEndos or pCDKS .", "We believe the actual kcat for the pEndos reaction is likely to lie in the higher end of its range , because some measurements were taken with older preparations of enzyme that had lost some activity .", "We further anticipate that the actual Km for this same reaction is in the lower end of its range , because we obtained lower values using hexahistidine-tagged pEndos than with a pEndos substrate containing a bulkier tag ( maltose binding protein ) that might have slightly impaired the interaction .", "( To appreciate the difficulties inherent in these measurements , it is informative to consider that , of more than 6200 enzymatic reactions currently cataloged in the BRENDA enzyme database ( http://www . brenda-enzymes . org; Schomburg et al . , 2013 ) , only three have Km values less than 10 nM and none has a subnanomolar Km .", ") In any event , the differences between the reactions using pEndos or pCDKS substrates are so pronounced that any variations within the ranges shown in Table 1 are ultimately inconsequential to the models presented below .", "These unusual kinetic properties explain the apparent paradox that a single enzyme , PP2A-B55 , exhibits both anti-CDKS and anti-Endos activities that differ markedly in their responses to inhibition by both tautomycetin/tautomycin and an Endos phosphomimetic protein and to the dilution of extracts .", "In each case , the discrepancy is explained by careful quantitative attention to the relationship between the Ki of the inhibitor and the Km of the target: roughly speaking , an inhibitor will be effective only if its Ki is less than the Km for the substrate .", "Thus , as was seen in Figure 6C , tautomycetin , which has an IC50 ( which provides a very rough estimate of Ki ) of 62 nM ( Mitsuhashi et al . , 2001 ) , effectively inhibits PP2A-B55 dephosphorylation of pCDKS ( Km ∼ 90 μM , Table 1 ) , but only weakly inhibits PP2A-B55 dephosphorylation of pEndos ( Km ∼ 1 nM , Table 1 ) .", "The same consideration explains the difference in inhibition by the Drosophila S68 Endos phosphomimetic protein , whose IC50 ( approximately 500 nM from Figure 6D ) is again intermediate between that for dephosphorylation of pEndos and pCDKS .", "Although the identity of the inhibitor ( s ) present in extracts that presumably underlie the dilution effects seen in Figure 2F are unknown , the different sensitivities of anti-Endos and anti-CDK to extract dilution can be rationalized if the inhibitors’ Ki values are intermediate between the Km’s of pEndos and pCDKS .", "Two findings clearly indicate that pEndos interacts with the active site of PP2A-B55 .", "First , pEndos is a substrate that is dephosphorylated by this enzyme at a measurable rate; and second , pEndos competes with okadaic acid , a drug known to bind only to PP2A’s active site ( Xing et al . , 2006 ) .", "Our results provide no indication that pEndos inhibits PP2A-B55 by binding to a second , allosteric site separate from the active site at which it is dephosphorylated .", "Such allosteric down-regulation would cause the rate of pEndos dephosphorylation to decrease when the substrate is added at very high concentrations , but this is not the case ( Figure 7B ) .", "Furthermore , the different responses of purified PP2A-B55’s anti-Endos and anti-CDKS activities to the addition of phosphomimetic Drosophila Endos ( S68D in Figure 6D ) also argue strongly against inhibition at an allosteric site .", "If pEndos inhibited PP2A-B55 by binding to a site other than the active site , phosphomimetic Endos would have been expected to inhibit the dephosphorylations of pEndos and pCDKS at identical concentrations , and this is clearly not the case .", "Figure 8A provides yet another demonstration that the ability of pEndos to inhibit PP2A-B55 is mostly dependent on interactions at the enzyme’s active site .", "We assayed the response of PP2A-B55’s anti-CDKS activity to increasing concentrations of either unphosphorylated Endos or Endos previously thiophosphorylated by Gwl kinase; the incorporated thiophosphate cannot be removed by this enzyme ( Morgan et al . , 1976 , and data not shown ) .", "The IC50 of thiophosphorylated Endos determined in this way was 197 ± 14 pM; by comparison , the IC50 of nonphosphorylated Endos was measured to be 566 ± 65 nM .", "Thus , the ( thio ) phosphorylation of Endos increases its binding affinity for PP2A-B55 about 3000-fold .", "Although these data do not preclude the existence of interactions outside of the active site and involving regions of Endos distant from the Gwl-phosphorylated site , it is clear that most of the binding is dictated by insertion of the phosphorylated residue into the active site , where it can then be dephosphorylated .", "Indicative of the strong affinity between activated Endos and PP2A-B55 , the IC50 of thiophosphorylated Endos is only about twice that of okadaic acid measured in similar assays ( 107 ± 10 pM Figure 8A ) , in close accordance with previous determinations ( Cohen et al . , 1989; Kam et al . , 1993; Walsh et al . , 1997 ) . 10 . 7554/eLife . 01695 . 020Figure 8 . Dose-response curves for PP2A-B55 inhibitors .", "( A ) The ability of PP2A-B55 ( at 0 . 125 nM ) to dephosphorylate radioactive CDKS substrate ( at 5 μM ) was measured in the presence of varying amounts of the following inhibitors: okadaic acid ( green ) , thiophosphorylated Endos ( black ) , non-radioactive pEndos ( during a 30-min incubation [pink] and a 120-min incubation [red] ) , and unphospohorylated Endos ( blue ) .", "Thiophosphorylation by Gwl kinase makes Endos into a much stronger inhibitor of PP2A-B55 than unphosphorylated Endos; the affinity of thiophosphorylated Endos is only about twofold lower than that of okadaic acid .", "Non-radioactive pEndos is a less efficient inhibitor of PP2A-B55 than is thiophosphorylated Endos because the enzyme dephosphorylates pEndos during the course of incubation; the longer pEndos is exposed to the enzyme , the higher is the concentration of pEndos required to achieve the same degree of inhibition .", "For all points shown , n = 3 technical replicates .", "( B ) Direct demonstration of the automatic reset mechanism that allows PP2A-B55 reactivation upon anaphase onset .", "PP2A-B55 at 0 . 25 nM was mixed together on ice with buffer in the absence of Endos ( control ) or in the presence of 16 nM unlabeled Gwl-phosphorylated Endos or Gwl-thiophosphorylated Endos and incubated on ice for 5 min; radioactive pCDKS substrate was then added to a final concentration of 0 . 47 μM; and the reaction was then transferred to 30°C at t = 0 and aliquots assayed for the release of 32P from the pCDKS substrate .", "Each point represents the average of three technical replicates ( n = 3 ) with standard deviations shown .", "The dephosphorylation of pCDKS is suppressed until the majority of pEndos is inactivated by PP2A-B55-mediated dephosphorylation .", "( The gradual decrease in the slopes of the control and pEndos-added curves is probably due to instability and/or degradation of the PP2A-B55 during the extended time-course . )", "The control and cold pEndos data were fitted to curves calculated as described in ‘Materials and methods’; the best fit gave Km = 0 . 47 ± 0 . 14 nM and kcat = 0 . 03 s−1 .", "The stronger inhibition caused by the thiophosphorylated pEndos ( green triangles ) is expected because its Kd ( ∼0 . 12 nM ) is lower than the Km of pEndos . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 020 These results suggest that Gwl-phosphorylated pEndos inhibits the dephosphorylation of CDK-phosphorylated substrates through competition for PP2A-B55’s active site ( Figure 9 ) .", "We call our model for this mechanism ‘inhibition by unfair competition’ to reflect the large disparities between the kinetic parameters for pEndos in comparison with other substrates .", "pEndos binds rapidly to all available PP2A-B55 , while the release of Endos from the enzyme ( either through dissociation or the removal of dephosphorylated Endos product ) is very slow .", "Sequestration by pEndos would thus severely compromise the ability of PP2A-B55 to target alternative substrates with much higher Km values ( in the 1 μΜ range or above ) . 10 . 7554/eLife . 01695 . 021Figure 9 . Inhibition by unfair competition . In this model , pEndos and CDK-phosphorylated substrates compete for the active site of PP2A-B55 .", "During M phase , pEndos is in stoichiometric excess of PP2A-B55 , so due to the disparities in the kinetic constants of these substrates , almost all of the enzyme will accumulate as a tight-binding complex with pEndos .", "Although PP2A-B55 can slowly dephosphorylate pEndos , the inhibitor is replenished by action of Gwl until this kinase is inactivated at anaphase onset ( not shown ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 021 One prediction of the inhibition by unfair competition hypothesis is that PP2A-B55 should be able to auto-reactivate at the conclusion of M phase .", "By dephosphorylating pEndos inhibitor that can no longer be replenished once Gwl is inactivated , PP2A-B55 is now freed to recognize its normal , CDK-phosphorylated substrates .", "We tested this idea by setting up an in vitro analog of this aspect of M phase exit .", "We added varying amounts of non-radioactive pEndos to PP2A-B55 , and monitored the enzyme’s ability to dephosphorylate radioactive pCDKS added at the same time .", "The results , shown in Figure 8A , show clearly that pEndos indeed inhibits PP2A-B55-mediated pCDKS dephosphorylation .", "Much higher concentrations of pEndos than thiophosphorylated Endos are needed to achieve the same degree of inhibition , which is to be expected if pEndos is being inactivated during the incubation .", "As the time of incubation increases , PP2A-B55 consumes more pEndos , so even higher concentrations of pEndos are required for the same amount of inhibition .", "Comparing the effects of pEndos inhibition after 30- and 120-min incubations demonstrates that PP2A-B55 cannot recognize the pCDKS substrate until the majority of the pEndos in the same tube has been dephosphorylated .", "We estimate that the kcat for the dephosphorylation of pEndos in these reactions is between 0 . 03 and 0 . 12 s−1 , consistent with the range previously determined in Table 1 .", "Figure 8B provides straightforward evidence for the automatic reset of PP2A-B55 activity that we postulate occurs after the enzyme has succeeded in inactivating pEndos .", "At the beginning of this experiment , purified PP2A-B55 was mixed on ice with either buffer , nonradioactive pEndos , or nonradioactive thiophosphorylated Endos; then radioactive pCDKS substrate was added and the samples were incubated at 30°C starting at t = 0 .", "The time courses of pCDKS dephosphorylation , assayed by 32P release , were then measured .", "A clear delay in pCDKS dephosphorylation of roughly 35 min is visible in the sample containing pEndos , after which the rate of the reaction approaches the initial rate observed in the absence of Endos inhibitors .", "( This delay is longer than would occur in cells because the ratio of pEndos to the enzyme is much higher than the physiological condition . Also , even stronger in vivo inhibition is predicted because the physiological pEndos concentration is many times higher than the concentration used here , which was limited by experimental constraints . )", "The best-fit values to these data , Km = 0 . 47 ± 0 . 14 nM and kcat = 0 . 03 s−1 , are consistent with those determined by the okadaic acid competition experiments ( Table 1; ‘Materials and methods’ for the calculations underlying the theoretical curves ) ." ], [ "Three independent lines of evidence indicate that the major activity contributing to the dephosphorylation of Gwl-phosphorylated Endos is a phosphatase that includes the three subunits of the PP2A-B55 heterotrimer .", "( 1 ) Inhibitor specificities: all of the anti-Endos activity ( whether in M phase or interphase ) is sensitive to okadaic acid and calyculin A ( Figure 2B , Figure 2—figure supplement 1 ) , and the majority is highly sensitive to fostriecin ( Figure 2C , Figure 2—figure supplement 2 ) , suggesting that the catalytic subunit of the anti-Endos phosphatase is PP2A or its less abundant relatives PP4 or PP6 .", "Furthermore , the facts that tautomycetin and the S68D Drosophila phosphomimetic Endos protein are such weak inhibitors of anti-Endos ( Figure 2 , Figure 6 ) can be most easily explained if the anti-Endos phosphatase has a very low Km for pEndos—below that for either inhibitor—as is the case for PP2A-B55 .", "( 2 ) Ablation of phosphatase activities: depletion of PP2A-A or PP2A-C from S2 tissue culture cells removes most anti-Endos from extracts ( Figure 3 ) , while depletion of PP4 from HeLa cells does not affect this activity ( Figure 3—figure supplement 1 ) .", "Drosophila larvae mutant for twins , which encodes the sole B55-type regulatory subunit of PP2A in flies , exhibit very little anti-Endos , while mutations in genes for other PPP family catalytic and regulatory subunits do not impede pEndos dephosphorylation in larval extracts ( Figure 4 ) .", "( 3 ) Biochemical purification of the activity: anti-Endos copurifies with the anti-CDKS activity previously ascribed to PP2A-B55 heterotrimer ( Mochida and Hunt , 2007; Castilho et al . , 2009 ) , while on the same column it resolves away from other PPP phosphatase subunits ( Figure 5 ) .", "Furthermore , highly purified PP2A-B55 heterotrimer displays robust anti-Endos activity whose properties match that of the predominant anti-Endos activity in extracts ( Figure 6 ) .", "Because most of our experiments in Figure 2 were performed with extracts prepared from unsynchronous cells ( the large majority of which are in interphase ) , it is conceivable that a phosphatase other than PP2A-B55 , that is activated only for a very short period following anaphase onset , is the enzyme responsible for dephosphorylating pEndos during M phase exit .", "We believe that this possibility is extremely remote for several reasons .", "( i ) As discussed below , the inhibition by unfair competition mechanism we propose is sufficiently fast to account for the observed rapidity of M phase exit .", "( ii ) The characteristics of the M phase and interphase anti-Endos activities measured in Xenopus egg extracts are very similar ( Figure 2—figure supplements 1C and 2A ) ; PP2A-B55’s ability to dephosphorylate pEndos is therefore constitutive with respect to the cell cycle ( see also Figure 2A ) .", "( iii ) Because of its extremely low Km , pEndos is so tightly bound to PP2A-B55 during M phase that no other hypothetical phosphatase would be able to inactivate it in a reasonable time frame; the theoretical simulation in Figure 10 below illustrates this point .", "We thus see no reason to postulate the existence of such a transiently activated phosphatase , and we think this possibility is incompatible with the observed relationship between PP2A-B55 and pEndos . 10 . 7554/eLife . 01695 . 022Figure 10 . Theoretical time-course of pEndos dephosphorylation and PP2A/B55 activation at the end of M-phase . These calculations make three assumptions: First , consistent with published data ( Gharbi-Ayachi et al . , 2010; Mochida et al . , 2010 ) , Endos was completely phosphorylated during M phase .", "Second , because the Km of PP2A-B55 for pEndos ( ∼1 nM , Table 1 ) is much smaller than the pEndos concentration ( 1 μM ) , essentially all the PP2A-B55 was bound to pEndos during M phase .", "Third , the Gwl phosphorylation of Endos stopped at the end of M-phase ( t = 0 ) ( Castilho et al . , 2009; Mochida et al . , 2009; Gharbi-Ayachi et al . , 2010; Mochida et al . , 2010 ) .", "The curves show the fraction of Endos that remains phosphorylated ( purple ) and the fraction of PP2A-B55 that is released from pEndos sequestration ( orange ) as a function of time t in sec . ( A ) Scenario: PP2A-B55 is the only enzyme that can dephosphorylate pEndos .", "Parameter values estimated from the experimental data were used: Total PP2A/B55 concentration , 250 nM; initial total pEndos concentration , 1 μM; Km , 1 nM; and kcat , 0 . 05 s−1 .", "( B ) Scenario: pEndos is a target of both PP2A-B55 and equal amounts of PPX , a second phosphatase with parameters Km = 85 μM and kcat = 22 . 5 s−1 ( based on the dephosphorylation of pCDKS by PP2A-B55 in Table 1 ) .", "( C and D )", "Scenario: pEndos is only an inhibitor and not a substrate of PP2A-B55 , and only PPX dephosphorylates pEndos .", "In this case , the Kd = 0 . 12 nM of thiophosphorylated Endos was used; this value was calculated from the IC50 for thiophosphorylated Endos from the dose-response curve in Figure 7 according to the equation described in Sasaki et al . ( 1994 ) and Takai et al . ( 1995 ) .", "The time frame in D is an extension of that in C , showing that the time required for desequestration of 50% of PP2A-B55 activity would be ∼6200 s under this last scenario . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 02210 . 7554/eLife . 01695 . 023Figure 10—figure supplement 1 . PP2A-B55 sequesters pEndos from the action of other phosphatases . The graph models the scenario that cells contain an additional phosphatase PPX , present at the same concentration as PP2A-B55 ( 250 nM ) , which targets pEndos .", "The y-axis shows the fraction of the total initial rate of pEndos dephosphorylation contributed by the indicated enzyme ( red for PPX; blue for PP2A-B55 ) .", "The kinetic parameters used for PP2A-B55 and PPX were identical to those in Figure 9 .", "When the concentration of pEndos is below the intracellular concentration of PP2A-B55 , the contribution of PPX to pEndos dephosphorylation is negligible . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 023 Some extracts contain a secondary activity ( ∼10 to 30% of the total ) that also targets the Gwl site in Endos .", "We have not characterized this minor activity in detail , but some evidence suggests that it may be a form of PP1: it is relatively resistant to fostriecin ( Figure 2—figure supplement 2 ) , and RNAi depletion of PP1-87B , the most abundant form of the PP1 catalytic subunit , removes ∼20 to 25% of anti-Endos activity from Drosophila S2 cell extracts ( Figure 3 ) .", "Regardless of its exact composition , we believe this secondary activity is not very important to the ultimate goal of regulating PP2A-B55 function .", "Figure 10—figure supplement 1 models the scenario that another phosphatase ( called PPX in the figure , but presumptively PP1 ) exists that can target pEndos .", "We chose the Km and kcat values for this putative reaction to match those for the dephosphorylation of pCDKS by PP2A-B55 ( from Table 1 ) , and the concentration of PPX to be the same as that of PP2A-B55 ( about 100–250 nM by our estimation , depending on cell type ) , but in fact the basic conclusions would be essentially unaltered by 10-fold changes in any of these assumptions .", "At high pEndos concentrations above the intracellular concentration of PP2A-B55 , PPX could dephosphorylate the excess pEndos that was not bound to PP2A-B55 .", "However , this dephosphorylation would have little regulatory consequence: as soon as the pEndos concentration is reduced to the intracellular concentration of PP2A-B55 , the remaining pEndos would be protected from dephosphorylation by PPX due to the very tight ( nanomolar or subnanomolar ) binding of pEndos to PP2A-B55 .", "The consequences of this conclusion to the dynamics of the M phase-to-interphase transition will be further explored later in the ‘Discussion’ .", "Since the original submission of this manuscript , three other papers have been published whose results bear on our identification of PP2A-B55 as the major anti-Endos phosphatase .", "The results from two of these studies support this conclusion .", "First , S Mochida measured the IC50 of pEndos and thiophosphorylated Endos and obtained values very close to those shown in our Figure 8A ( Mochida , 2013 ) .", "Moreover , he demonstrated that Endos bound by PP2A-B55 is in close proximity to both the B55 and C ( catalytic ) subunits of the enzyme , consistent with a location at the active site .", "An extremely tight association of phosphorylated Endos with the PP2A-B55 active site is an essential feature of our inhibition by unfair competition model .", "Second , during the course of an investigation that will be discussed further below , the laboratory of F A Barr provisionally identified PP2A-B55 as the phosphatase responsible for dephosphorylating pEndos in a mammalian cell system ( Cundell et al . , 2013 ) .", "A third recent publication ( Hegarat et al . , 2014 ) came to a conclusion incompatible with that presented here: these authors maintain that the anti-Endos enzyme is Fcp1 , a phosphatase known to target phosphorylations of the C-terminal domain ( CTD ) of RNA polymerase II .", "We believe their report is incorrect for several reasons .", "( i ) We show here strong evidence that PP2A-B55 is responsible for the anti-Endos activity .", "Furthermore , Figure 10C , D demonstrates that the very tight binding of pEndos to the PP2A-B55 active site blocks its binding to other phosphatases .", "No other normal-binding phosphatase could contribute to pEndos inactivation rapidly enough to account for the timing of M phase exit .", "( ii ) Among other issues , this new publication is internally inconsistent .", "The authors show that the anti-Endos activity they ascribe to Fcp1 is okadaic acid sensitive ( supplemental Figure S3 in reference Hegarat et al . , 2014 ) , yet the literature contains many reports that Fcp1 is completely resistant to okadaic acid ( e . g . , Palancade et al . , 2001; Washington et al . , 2002; Kong et al . , 2005; Visconti et al . , 2012 ) , a fact that we verify in our Figure 11E . 10 . 7554/eLife . 01695 . 024Figure 11 . Fcp1 is not the anti-Endos phosphatase .", "( A and B ) .", "Depletion of Fcp1 does not decrease anti-Endos activity .", "( A ) Control extracts ( made from S2 cells treated with a mock double-stranded RNA ) or Fcp1 RNAi extracts ( made from S2 cells treated with double-stranded RNA corresponding to the Drosophila Fcp1 gene [CG12252] were assayed for phosphatase activities using pCDKS , pEndos , and pCTD ( the C-terminal domain of RNA polymerase II phosphorylated in vitro by mitogen-activated kinase 2 [MAPK2] ) substrates .", "No significant differences were observed in terms of anti-CDKS or anti-Endos activities .", "Fcp1 depletion slightly decreases activity against the CTD substrate , consistent with the fact that Fcp1 is only one out of several known phosphatases known to target the CTD ( Hsin and Manley , 2012 ) .", "Each bar represents the average of three technical replicates with the standard deviation shown .", "( B ) Western blot of the control and Fcp1 RNAi extracts assayed in part A , showing that more than 90% of the Fcp1 was removed by the treatment with Fcp1 double-stranded RNA .", "The loading control shows the signal revealed by antibody against the RNA polymerase II elongation factor TFIIS .", "( C and D ) .", "Purified S . pombe Fcp1 enzyme has minimal anti-Endos activity .", "Activities of purified Fcp1 against labeled pEndos , pCDKS , and pCTD .", "Each point represents a single assay .", "Note that the concentrations of Fcp1 employed in these experiments are in the micromolar range , as opposed to the subnanomolar-to-nanomolar concentrations of purified PP2A-B55 used in other figures .", "Consistent with previous reports for this enzyme ( Hausmann and Shuman , 2002 ) , the specific activity of Fcp1 against the CTD substrate is low ( but much higher than that against pEndos ) .", "( E ) The activities of purified wild-type Fcp1 against all three substrates are , as expected , resistant to high concentrations of okadaic acid ( 10 μM ) .", "A preparation of kinase-dead ( KD ) S . pombe Fcp1 with the D172N mutation ( Suh et al . , 2005 ) made in parallel has no activity against any of these three substrates . DOI: http://dx . doi . org/10 . 7554/eLife . 01695 . 024 We have also obtained results that more directly exclude Fcp1 as a major anti-Endos phosphatase .", "( iii ) In mono-Q chromatography , Fcp1 does not co-fractionate with the pEndos-dephosphorylating activity .", "Figure 5 shows that fractions with peak anti-Endos activity ( such as fractions from 37 to 39 ) have no detectable Fcp1 protein .", "( iv ) RNAi depletion of more than 90% of the Fcp1 in Drosophila S2 cells has no obvious effect on the ability of extracts made from these cells to dephosphorylate pEndos ( Figure 11A , B ) .", "( v ) Purified S . pombe Fcp1 enzyme has detectable anti-Endos activity , but this is very low .", "At a physiological concentration of the pEndos ( 1 μM ) , each molecule of Fcp1 has less than 1/1 , 000th the efficiency of a molecule of PP2A-B55 in dephosphorylating this substrate ( Figure 11C , D ) .", "Moreover , cells contain more than 10 times more PP2A-B55 than Fcp1 molecules ( based on values in the Pax-DB database of protein abundances determined by mass spectrometry; see www . pax-db . org and reference Wang et al . , 2012 ) .", "By this estimate , less than 0 . 01% of the total anti-Endos activity in cells can be ascribed to Fcp1 .", "It should be cautioned that our measurements of Fcp1 enzyme kinetics were made using the heterologous S . pombe enzyme , but the structure of Fcp1 is well conserved in evolution .", "In Figure 9 , we propose a straightforward model for the relationship between pEndos and PP2A-B55 .", "In essence , pEndos competes with a large class of CDK-catalyzed mitotic phosphosites ( of which pCDKS and pSer50 Fizzy are examples ) for access to the phosphatase active site .", "pEndos can successfully compete for the site because its affinity for PP2A-B55 is much higher than those of the competing substrates; that is , the Km for pEndos is extremely low .", "However , PP2A-B55 dephosphorylates pEndos at a much slower rate ( low kcat ) than the CDK phosphosites , so the tight interaction of PP2A-B55 and pEndos would be prolonged .", "Thus , pEndos would soon sequester the phosphatase from competing substrates , protecting such substrates against premature dephosphorylation .", "pEndos in this way permits M phase to begin and to be maintained as long as Gwl kinase is active .", "We call this mechanism ‘inhibition by unfair competition’ .", "In its essence , the model shown in Figure 9 clarifies how PP2A-B55 can be ‘off’ during M phase for CDK-phosphorylated substrates , but ‘on’ at the same time for the Gwl-phosphorylated pEndos substrate .", "In the context of the cell , the inhibition by unfair competition mechanism requires that pEndos be present in molar excess over PP2A-B55 in mitosis to account for the near-total absence of anti-CDKS activity observed during M phase ( Burgess et al . , 2010; Mochida and Hunt , 2007; Mochida et al . , 2009; Figure 2A ) .", "Considerable evidence exists that this requirement is met in many cell types .", "From our own quantitation on Western blots , we estimate the intracellular concentration of the PP2A B55 subunit to be between 100 and 250 nM and that of Endos to be between 500 nM and 1 μΜ , depending on the cell type .", "( One example of this analysis is seen in Figure 2—figure supplement 6 ) .", "The 5:1 ratio of Endos to PP2A-B55 observed in that figure is roughly consistent with estimates from other laboratories ( Cundell et al . , 2013 ) and with estimates from mass spectrometry studies of whole cell extracts , after accounting for all Endos and PP2A-B55 family members ( Brunner et al . , 2007; Beck et al . , 2011; Kolker et al . , 2012; Wang et al . , 2012 ) .", "For example , the Pax-DB database integrating the results of many mass spectrometry experiments on a variety of tissue types estimates that the abundance of Endos in Drosophila cells is 295 ppm while that of Twins ( B55 ) is 123 ppm; for human cells , the integrated datasets yield values of 83 . 2 ppm for Endos-family proteins ( ENSA and ARPP-19 ) and 23 . 1 ppm for B55-family proteins ( www . pax-db . org; Wang et al . , 2012 ) .", "Although we ourselves have not determined the fraction of the total Endos protein that is phosphorylated during M phase , other investigators have found that this proportion is roughly 50% in extracts of synchronized mammalian tissue culture cells ( perhaps some of which may not have been in M phase ) ( Cundell et al . , 2013 ) , and approaches 100% in frog egg M phase extracts ( Mochida et al . , 2010 ) .", "Sufficient ‘headroom’ thus exists to conclude that the molar concentration of pEndos during M phase in fact exceeds that of the PP2A-B55 phosphatase .", "A major virtue of the inhibition by unfair competition model is that it explains not only how pEndos inhibits PP2A-B55 dephosphorylation of pCDKS-class substrates , but also how pEndos can itself become inactivated: the system has an automatic reset that is intrinsic to the mechanism that inactivates PP2A-B55 phosphatase in the first place .", "When Gwl is inactivated at anaphase onset , the pEndos dephosphorylated by PP2A-B55 can no longer be replaced .", "PP2A-B55 is now free to work on CDK-phosphorylated substrates and thus to promote the M phase-to-interphase transition .", "The experiments presented in Figure 8 , in which we added non-radioactive pEndos and radioactive pCDKS to the same tube of PP2A-B55 , provide a direct in vitro test of the proposed mechanism by mimicking the events that occur during M phase exit .", "The results show that PP2A-B55 targets pCDKS only after the enzyme has dephosphorylated almost all of the pEndos .", "M phase exit is surprisingly rapid given the large number of phosphorylations which must be reversed; in Xenopus cycling extracts , for example , the M phase-to-interphase transition is completed within less than 5 min ( Zhao et al . , 2008; Mochida et al . , 2009 ) .", "Even though the turnover rate of pEndos dephosphorylation by PP2A-B55 is quite slow ( ∼0 . 05 s−1 ) , the inhibition by unfair competition mechanism is nevertheless compatible with the rapidity of M phase exit .", "The reason is that Endos is present in cells only in at most a fivefold stoichiometric excess with respect to the phosphatase , as was just discussed .", "Each molecule of PP2A-B55 thus needs to dephosphorylate only a few molecules of pEndos to effect the M phase-to-interphase transition .", "To explore this idea quantitatively , we modeled the dynamics of a simplified system consisting solely of Endos and PP2A-B55 at estimated physiological conditions ( Figure 10A ) .", "Because the Km of PP2A-B55 for pEndos is four-to-five orders of magnitude smaller than the Km for other substrates , typified by pCDKS ( Table 1 ) , and the pEndos concentration during M phase is in excess of the phosphatase concentration , we suggest that the approximation of ignoring the binding of PP2A-B55 to other substrates is appropriate , and that this simplified model should capture the essence of the regulatory process .", "The calculation begins at the end of M-phase ( t = 0 ) with the inactivation of Gwl .", "Almost all the PP2A-B55 is sequestered by pEndos until PP2A-B55-catalyzed dephosphorylation causes the pEndos concentration to decrease from 1 μM to ∼250 nM , the intracellular PP2A-B55 concentration .", "Beyond this point , PP2A-B55 is rapidly released from sequestration; half is available to act on other substrates within 74 s ( Figure 10A ) .", "Note that this calculation essentially recapitulates the actual experiments shown in Figure 8 , but here the inhibition of PP22A-B55 is stronger and the time to release is faster because physiological , not laboratory , conditions are being modeled .", "We previously showed in Figure 2—figure supplement 2 that cells likely harbor a pEndos-targeting phosphatase other than PP2A-B55; because this secondary activity is fostriecin-resistant , we speculate that it may be a form of PP1 .", "Figure 10B shows how the time courses of pEndos dephosphorylation and PP2A-B55 desequestration are modified if this second phosphatase ( again called PPX ) is present at the same concentration as PP2A-B55 and acts on pEndos with kinetic parameters matching the activity of PP2A-B55 on pCDKS .", "In this case , the lag in PP2A-B55 desequestration shown in Figure 10B is shortened , because PPX can dephosphorylate the pEndos that is not bound to PP2A/B55 .", "However , as illustrated in Figure 10—figure supplement 1 , the presence of PPX makes very little difference once the pEndos concentration decreases to the PP2A-B55 concentration of 250 nM , since almost all the remaining pEndos is bound to PP2A-B55 .", "In this scenario , half of the PPA-B55 is released from pEndos by 38 s , producing a modest acceleration in M phase exit .", "We regard the situation shown in Figure 10B as a reasonable description of the actual events in the cell , as it accounts for contributions from both PP2A-B55 and other phosphatases in pEndos inactivation .", "Further dynamic modeling surprisingly revealed a key insight: cells would be unable to exit M phase in a timely manner unless pEndos was in fact dephosphorylated by PP2A-B55 as demanded by unfair competition .", "Figure 10C , D assumes that pEndos is only an inhibitor and not a substrate of PP2A-B55; thus pEndos binds and sequesters PP2A-B55 , but is only dephosphorylated by PPX ( whose concentrations and properties are identical to those in Figure 10B ) .", "In this scenario , PP2A-B55 would protect the bound pEndos from dephosphorylation , so many hours—not just a few minutes—would be required for PP2A-B55 desequestration and M phase exit .", "In other words , the dephosphorylation of pEndos by PP2A-B55 is an absolute requirement for rapid completion of the M phase-to-interphase transition .", "Our identification of PP2A-B55 as the anti-Endos phosphatase poses a dilemma in terms of M phase exit: the inhibition by unfair competition model describes events that must occur downstream of the inactivation of Gwl kinase , but what then can inactivate Gwl upon anaphase onset ?", "According to our model , the rate-limiting Gwl-inactivating phosphatase cannot be PP2A-B55 , because the system would be futile .", "Gwl inactivation could not proceed until pEndos was depleted , but pEndos cannot be inactivated as long as Gwl is active .", "An argument can be made that PP2A-B55 should be the Gwl-inactivating phosphatase because Gwl is activated by CDKs ( Blake-Hodek et al . , 2012 ) , and PP2A-B55 targets at least a subset of CDK phosphosites ( Mochida and Hunt , 2007; Castilho et al . , 2009; Mochida et al . , 2009 ) .", "Indeed , one group has recently reported some evidence in favor of this idea ( Hegarat et al . , 2014 ) .", "However , it should be emphasized that PP2A-B55 does not act on all , or even perhaps the majority , of CDK phosphosites , as recently most forcefully demonstrated by Cundell et al . ( 2013 ) .", "The obvious resolution of this dilemma is therefore that a phosphatase other than PP2A-B55 inactivates Gwl during M phase exit .", "We have obtained preliminary evidence in support of this idea in two ways ( data not shown ) .", "First , in our hands purified PP2A-B55 in vitro is very inefficient in inactivating Gwl .", "Second , when active Gwl is added to interphase extracts from a variety of cell types , it becomes rapidly inactivated and dephosphorylated in a process that is insensitive to okadaic acid ( and cannot therefore be controlled by PP2A-B55 ) .", "Some evidence in favor of the existence of an okadaic acid-resistant anti-Gwl phosphatase has also recently been obtained by another group ( Hegarat et al . , 2014 ) ; the identity of this Gwl-inactivating enzyme is currently unknown .", "F A Barr et al . have recently proposed an elegant model in which the events of anaphase onset are ordered in time , with later events such as cytokinesis being controlled by the Gwl-Endos pathway ( Cundell et al . , 2013 ) .", "Our conclusions are in essence consistent with their hypothesis , and the automatic reset mechanism we propose provides an explanation for part of the delay they observe between CDK1-Cyclin B inactivation and the activation of PP2A-B55 , and thus for the dephosphorylation of late substrates involved in cytokinesis such as PRC1 .", "That is , the major determining factors for this delay would be the rate of Endos dephosphorylation by PP2A-B55 described here coupled with the time required to inactivate Gwl through mechanisms we do not yet understand .", "The virtues of inhibition by unfair competition are sufficiently compelling that one might ask whether other phosphatases are controlled in the same fashion .", "We believe this supposition is likely , based on findings published 30 years ago concerning inhibition of PP1 by a polypeptide called Inhibitor-1 that is , like Endosulfine , small and heat-stable ( Foulkes et al . , 1983 ) .", "Inhibition occurs only after Inhibitor-1 is phosphorylated at a conserved site by the cAMP-dependent protein kinase ( PKA ) , an enzyme whose structure and activation mechanism are closely related to those of its fellow AGC kinase family member , Gwl ( Blake-Hodek et al . , 2012 ) .", "The investigators found that purified PP1 could dephosphorylate PKA-phosphorylated Inhibitor-1 , and the values of Km and kcat were both considerably lower for this reaction than for dephosphorylation of the more normal substrate phosphorylase a ( Foulkes et al . , 1983 ) .", "In short , just as we report here for pEndos and PP2A-B55 , they concluded that pInhibitor-1 acts both as an inhibitor and a substrate for PP1 .", "These observations have long since been disregarded because PP1’s activity in inactivating pInhibitor-1 was very slow , and other phosphatases ( PP2A and calcineurin ) had substantial pInhibitor-1 dephosphorylating activity ( Ingebritsen et al . , 1983; Cohen , 1989 ) .", "In light of the arguments summarized in Figure 10 that a phosphatase will effectively sequester a tightly binding inhibitor from other phosphatases , we believe the biological significance of PP1’s activity in dephosphorylating pInhibitor-1 should be re-evaluated .", "Of interest , recent studies in Xenopus egg extracts have indicated that pInhibitor-1 dephosphorylation at the end of M phase is in fact dependent upon activity of PP1 , although this work could not distinguish whether the effect was direct or indirect ( Wu et al . , 2009 ) .", "The inhibition by unfair competition mechanism may well prove to have been utilized by AGC kinases other than Gwl to control phosphatases other than PP2A-B55 ." ], [ "Extracts from third instar larvae of the following genotypes were assayed for phosphatase activities: PP2A B55 regulatory subunit ( twins ) : twsP/tws196 ( Uemura et al . , 1993; Kim et al . , 2012 ) .", "PP2A B56 regulatory subunit type 1 ( widerborst ) : wdb12–1/wdb12–1 ( Kotadia et al . , 2008 ) .", "PP2A B56 regulatory subunit type 2 ( well-rounded ) : wrdKG01108/wrdKG01108 ( Rollmann et al . , 2007 ) .", "PP2A B‴ ( striatin ) regulatory subunit ( connector of kinase to AP-1 ) : cka05836/ckaS1883 ( Perrimon et al . , 1996; Spradling et al . , 1999 ) .", "PP4 regulatory subunit PP4R3 ( falafel ) : flflN42/flfl795 ( Sousa-Nunes et al . , 2009 ) .", "PP1 catalytic subunit ( protein phosphatase 1 at 87B ) : PP1-87B1/PP1-87B87Bg-3 ( Gausz et al . , 1981; Reuter et al . , 1987 ) .", "PP1 catalytic subunit ( protein phosphatase 1α at 96A ) : PP1-96A2/PP1-96A2 ( Kirchner et al . , 2007a ) .", "PP1 catalytic subunit ( flapwing; also known as PP1β-9C ) : flw6/flwGO172 ( Raghavan et al . , 2000; Bennett et al . , 2003; Kirchner et al . , 2007b ) .", "Stocks with these mutant alleles were obtained from the Drosophila Stock Center ( Bloomington , IN ) and rebalanced over chromosomes containing Tubby ( Tb ) to facilitate the identification of homozygous or trans-heterozygous mutant third instar larvae .", "For the genes on the third chromosome ( tws , wdb , flfl , PP1-87B , PP1α-96A ) the balancers used either were TM6B , Tb1 AntpHu or TM6C , Tb1 Sb1 .", "For the cka gene on the second chromosome , the balancer was CyO-TbA , while for the flw gene on the X chromosome , the balancer was FM7A-TbA ( Lattao et al . , 2011 ) .", "The wrdKG01108 mutation is homozygous viable .", "For certain genes , trans-heterozygous combinations of mutant alleles were employed as indicated by the genotypes above to obtain mutant third instar larvae; this was necessary because chromosomes bearing the single mutations have over time accumulated second-site lesions causing earlier lethality .", "HeLa CCL-2 and HEK293 CRL-1573 cells were obtained from the American Type Culture Collection ( Manassas , VA ) .", "PP5 +/+ , +/− and −/− mouse MEF cells ( Yong et al . , 2007 ) were the kind gift of Dr Wenian Shou , ( Indiana University-Purdue University , Indianapolis , IN ) .", "All mammalian cell lines were grown in Dulbecco’s Modified Eagle Medium +10% Fetal Bovine Serum ( Invitrogen , Carlsbad , CA ) .", "Drosophila S2 cells were grown in Insect-Xpress with L-Glutamine ( Lonza BioWhittaker , Walkersville , MD; catalog number 12-730Q ) .", "Both mammalian and insect cells were grown in the presence of 100 units/ml of penicillin and 100 μg/ml of streptomycin and a 1:100 dilution of the antimycotic Fungizone ( Gibco , Gaithersburg , MD ) .", "Substrates for phosphatase reactions were purified as previously described ( Castilho et al . , 2009 ) from E . coli transformed with recombinant plasmids constructed from the vector pMAL-C2X ( New England Biolabs , Ipswich , MA ) and expressing maltose binding protein fusions with the following peptides/proteins: full-length Xenopus Endosulfine ( Endos; Castilho et al . , 2009; Kim et al . , 2012 ) ; the region from Xenopus PP1γ surrounding the phosphosite Thr311 ( we term this substrate CDKS; Mochida and Hunt , 2007; Castilho et al . , 2009; Mochida et al . , 2009 ) ; the region surrounding the Ser50 phosphosite of Xenopus Fizzy ( Fzy; Mochida and Hunt , 2007; Castilho et al . , 2009; Mochida et al . , 2009 ) ; amino acids 1–27 of Drosophila Histone H3; and full-length H . sapiens histone H1 . 0 ( purified Histone H1 . 0 protein obtained from New England Biolabs yielded identical results ) .", "In a few experiments included in Table 1 , Xenopus Endos was cloned into the vector pQE30 ( Qiagen , Valencia , CA ) so as to express hexahistidine-tagged protein .", "Bovine myelin basic protein ( MyBP ) was from Millipore ( Temecula , CA ) .", "Substrates for phosphatase assays of the RNA polymerase II C-terminal domain ( CTD ) were purified as GST-CTD fusions as previously described ( Suh et al . , 2005 ) .", "The purified proteins were 32P-phosphorylated in vitro by Gwl kinase ( Endos ) , by CDK1-Cyclin A ( CDKS , Fzy Ser50 , and Histone H1 . 0 ) , by Protein Kinase A ( New England Biolabs; Histone H3 and MyBP ) , or by mitogen activated kinase 2 ( MAPK2; New England Biolabs; RNA polymerase II CTD ) using methods previously described ( Suh et al . , 2005; Castilho et al . , 2009 ) .", "Proteins were purified away from the kinase and other reaction components , and desalted and concentrated using Amicon Ultracel 10K centrifugal filter columns ( Millipore ) and substrate buffer ( 20 mM Tris , pH 7 . 5; 150 mM NaCl ) .", "The incorporation of phosphate was determined to be between 0 . 5 and 1 . 0 per mole of protein based on measurements of protein concentration and specific activity .", "For some studies , the Endos fusion protein was thiophosphorylated by Gwl in the presence of 1 mM ATP-[γ]-35S instead of ATP .", "Dephosphorylation of 35S-thio-phosphorylation by purified PP2A-B55 was not detectable .", "Xenopus CSF and interphase extracts were prepared as described ( Castilho et al . , 2009 ) .", "HeLa cells were extracted in ice-cold phosphatase buffer ( 50 mM Tris , pH 7 . 5 , 150 mM NaCl , 1 mM EDTA , 0 . 1 mM EGTA , 0 . 25% NP-40 ) according to Singh et al . ( 2010 ) , or M-PER mammalian cell lysis buffer ( Thermo Scientific , Rockford , IL ) with the same results .", "A 1:100 dilution of EDTA-free Proteoblock protease inhibitor ( Thermo Scientific ) was added to both extraction buffers .", "Drosophila whole larvae or larval brains were homogenized with a pestle in phosphatase buffer as above , to which was added a 1:100 dilution of phenylmethanesulfonyl fluoride ( PMSF; Thermo Scientific ) , a 1:50 dilution of Halt protease inhibitor cocktail ( Thermo Scientific ) , and a 1:100 dilution of a 100 mg/ml stock solution of soybean trypsin inhibitor ( AMRESCO , Solon , OH; catalog number M191 ) .", "Extracts prepared in phosphate buffer ( see the section on cell-free extracts above ) or purified enzymes appropriately diluted in PP2A reaction buffer ( see below ) were used for each phosphatase assay .", "A no-extract control was always included to verify that the substrate did not itself contain phosphatase activity .", "Unless otherwise noted , radiolabeled substrates were added to a final concentration of 5 μM in the reactions .", "Phosphatase assays were stopped with trichloracetic acid ( TCA ) after a time at which <20% of the substrate was dephosphorylated .", "The supernatant was mixed with ammonium molybdate and extracted with heptane-isobutyl alcohol according to Mochida and Hunt ( Mochida and Hunt , 2007 ) and the 32P measured in a scintillation counter .", "In certain experiments , the following phosphatase inhibitors were used after being dissolved in dimethyl sulfoxide ( DMSO ) : fostriecin , tautomycin , and tautomycetin ( all from Santa Cruz Biochemicals , Dallas , TX ) , and okadaic acid ( LC Labs , Boston , MA ) .", "The phosphomimetic Drosophila S68D Endos protein has been previously described ( Kim et al . , 2012 ) .", "Inhibitors were pre-incubated on ice with extracts for 10 min prior to addition of the substrate .", "siRNA oligonucleotides ( Thermo Scientific ) for PP1 , PP2A ( α and β isoforms ) , PP4 , PP5 , and PP6 catalytic subunits were transfected into HeLa cells using Dharmafect following the manufacturer’s protocols ( Dharmacon , now Thermo Scientific ) and harvested after 72 hr .", "The cells were lysed in phosphatase buffer and the lysates used to determine activity against 32P-labeled substrates in a phosphatase assay as described above , and for knockdown efficiency by Western blotting using the antibodies listed below .", "The technique used to effect RNAi in Drosophila S2 tissue culture cells has been described previously ( Williams et al . , 2003 ) .", "dsRNA was produced from cDNA clones for the given phosphatase subunit by PCR amplification using primers containing a T7 RNA polymerase promoter site at the 5′ end .", "The primer pair used for the catalytic subunit of PP2A ( microtubule star ) was: 5′ TAATACGACTCACTATAGGGAGACCTACGCAGCTTACATTTACACATA 3′ and 5′ TAATACGACTCACTATAGGGAGATAGGTTCGATTGGATTGTATCATTT 3′ .", "For the A subunit of PP2A ( PP2A-29B ) : 5′ TAATACGACTCACTATAGGGAGACAGAGTTTGCCATGTACTTGATTC 3′ and 5′ TAATACGACTCACTATAGGGAGAGGAATCAAATCGGACTTCAGATACT 3′ .", "For the major catalytic subunit of PP1 ( PP1-87B ) : 5′ TAATACGACTCACTATAGGGAGAACCACGAGCAGTCTTTTTCTATCTA 3′ and 5′ TAATACGACTCACTATAGGGAGAGTG GCTTTTAATCATGGTATTTGTC 3′ .", "RNAi depletion of Fcp1 from S2 tissue culture cells has been previously described ( Fuda et al . , 2012 ) .", "In all cases , S2 cell pellets were lysed and analyzed as just described for HeLa cells .", "Fractionation of HeLa cell lysates by Mono-Q chromatography was performed using modifications of published protocols ( Che et al . , 1998; Guo et al . , 2002 ) .", "HeLa cells were lysed in buffer containing 50 mM Tris-HCl , pH 7 . 4; 100 mM NaCl; 1 mM EDTA; 0 . 5 mM EGTA; 0 . 25% NP-40; and 10% glycerol .", "Cell lysate was then diluted to 20 ml with Buffer A ( 25 mM Tris-HCl , pH 7 . 4; 150 mM NaCl; 1 mM DTT; and 10% glycerol ) .", "Diluted lysate was loaded onto a 5-ml HiTrap Q HP column ( GE Healthcare , Uppsala , Sweden ) equilibrated with Buffer A at a flow rate of 0 . 5 ml/min .", "The column was then washed for with Buffer A for 16 min ( 0 . 5 ml/min ) followed by elution with a linear gradient to 500 mM NaCl over 30 min , and the column was then subjected to an additional 30 min wash at 500 mM NaCl .", "0 . 5 ml fractions were collected every minute .", "The fractions were split into 100 μl aliquots , flash frozen in liquid nitrogen , and then stored at −80°C for later analysis .", "pcDNA5 ( vector alone ) , pcDNA5/FLAG-B55α , pcDNA5/FLAG-B55δ , and pcDNA5/FLAG-B56β plasmid DNAs were transfected into HEK293 CRL-1573 cells using Lipofectamine 2000 ( Invitrogen ) using the manufacturer’s protocols and harvested after 72 hr .", "PP2A trimers were isolated exactly according to Adams and Wadzinski ( 2007 ) .", "Protein concentrations were measured in comparison to bovine serum albumin ( BSA ) standards on silver stained gels .", "Western blotting confirmed the identity of the protein bands in PP2A preparations .", "Phosphatase assays with the purified PP2A enzymes were carried out as described above .", "Enzymes were diluted to the appropriate concentration in PP2A reaction buffer ( 20 mM Tris , pH 7 . 5; 1 mg/ml bovine serum albumin; 0 . 1% β-mercaptoethanol ) prior to incubation with the substrate being tested .", "Recombinant Schizosaccharomyces pombe Fcp1 ( wildtype and kinase-dead D172N mutant ) proteins were purified after expression in E . coli cells as previously described ( Hausmann and Shuman , 2002; Kimura et al . , 2002; Suh et al . , 2005 ) .", "Samples were prepared by mixing equal volumes of the fraction with 2X SDS loading dye , followed by boiling for 2 min .", "Samples were then resolved on 10% SDS-polyacrylamide gels .", "Proteins were transferred to Immobilon-P membranes ( Millipore ) as previously described ( Castilho et al . , 2009 ) .", "The following primary antibodies were used to probe Western blots ( catalog numbers in parentheses ) : from Abgent ( San Diego , CA ) : PP6-C ( #AP8477b ) .", "From Abnova ( Taiwan ) : PP4-C ( #PAB14146 ) ; and PP5-C ( #H00005536-B01 ) .", "From Cell Signaling Technology ( Beverly , MA ) : PP2A-A ( #2309 ) ; PP1α ( #2582 ) ; Twins ( PP2A B55 subunit , from clone 100C1; #2290 ) ; and FlagTag ( DYKDDDDK; #2368 ) .", "From Santa Cruz Biotechnology: PP2A-Cα , from clone N-25 ( #sc-130237 ) ; PP1-C , from clone E−9 ( #sc-7482 ) ; and Fcp1 ( from clone H300; #sc32867 ) .", "From Stratagene ( La Jolla , CA ) : PP2A B′ ( B56 ) pan ( #B13009-51 ) .", "From Thermo Scientific: α-tubulin ( #MA5-14992 ) ; and striatin ( PP2A B‴; #PA1-46460 ) .", "From Upstate/Millipore ( Temecula , CA ) : PP2A-C subunit , from clone 1D6 ( #05-421 ) ; and PP2A B subunit , from clone 2G9 ( #05-592 ) .", "Antibodies against the two B56 regulatory subunits of PP2A in Drosophila ( Widerborst [Wdb] and Well-rounded [Wrd] ) were the kind gifts of Dr Timothy Megraw ( University of Texas Southwestern Medical Center , Dallas , TX ) and have been previously described ( Kotadia et al . , 2008 ) .", "Antibodies recognizing Drosophila Fcp1 ( made in rabbits ) and TFIIS ( made in guinea pigs ) were described in Fuda et al . ( 2012 ) .", "The secondary antibodies used were: goat anti-rabbit IgG ( H+L ) -HRP conjugate ( catalog #170-6515; Bio-rad , Hercules , CA ) at a 1:5000 dilution; goat anti-mouse IgG ( H+L ) -HRP conjugate ( catalog #170-6516; Bio-rad ) at a 1:3000 dilution; and donkey anti-guinea pig IgG ( H+L ) -HRP conjugate ( catalog #706-035-148; Jackson ImmunoResearch Laboratories , West Grove , PA ) at a 1:5000 dilution .", "Chemiluminescent signals were visualized by ECL Western Blotting Substrate ( Thermo Scientific ) according to the manufacturer’s instructions .", "Varying concentrations , [N]T , of 32P-labeled pEndos ( see Figure 7B , C ) were incubated with [P]T = 0 . 5 nM PP2A-B55 , and the initial velocities , v , of 32P release were measured in ( duplicate or quadruplicate ) samples in two independent experiments .", "Technical limitations precluded the use of a PP2A-B55 concentration that was much less than the Km , so the data was analyzed using the Michaelis–Menten reaction scheme with equations that accounted for tight binding .", "That is , we did not assume that [N] ≈ [N]T or that [P] ≈ [P]T .", "( Unscripted variables are unbound concentrations and the T subscript signifies total concentrations . )", "Combining the mass conservation and quasi-steady state equations for the concentration of the transient pEndos/PP2A-B55 complex , [NP] , gives:[NP]= ( [N]T−[NP] ) ( [P]T−[NP] ) /Kmv=kcat [NP] .", "This quadratic velocity equation has the solution according to Morrison ( 1969 ) :v=kcat { ( [P]T+[N]T+Km ) − ( [P]T+[N]T+Km ) 2−4[P]T [N]T } The parameters Km and kcat were determined from the data using nonlinear regression assuming equal fractional errors ( Mathematica Version 9 ) , and the weighted means and standard deviations were determined from two independent experiments .", "The results are displayed in Table 1 .", "The measurements described above gave relatively large errors in Km because of the difficulty of making measurements at the very low PP2A-B55 and pEndos concentrations imposed by the very low Km .", "To improve accuracy we used a modification of the approach used in Sasaki et al . ( 1994 ) and Takai et al . ( 1995 ) to measure the Km by a supplementary method: competing the PP2A-B55-catalyzed dephosphorylation of a fixed amount of pEndos with varying concentrations of okadaic acid ( Figure 7D , E ) .", "This approach was feasible because the dissociation constant of okadaic acid with respect to PP2A has been carefully determined ( Takai et al . , 1992; Sasaki et al . , 1994; Takai et al . , 1995 ) and because the affinity of pEndos for PP2A-B55 is in the same order of magnitude .", "By using an initial pEndos concentration ( [N]T ( 0 ) = 50 nM or 1 μM , depending on the experiment ) that was much greater than the PP2A-B55 concentration ( [P]T = 0 . 25 nM or 1 nM ) and by limiting the incubation time , we ensured that only a small fraction of pEndos was dephosphorylated , facilitating a more accurate determination of the Km .", "Moreover , this ensured that [P]T/{[N]T ( 0 ) + Km} < 0 . 005 << 1 , so the total quasi-steady state approximation that was used or mathematical analysis was good ( Borghans et al . , 1996 ) .", "Figure 8B: parallel aliquots containing [P]T ( 0 ) = 0 . 25 nM PP2A-B55 , [pC]T ( 0 ) = 0 . 47 μM 32P-phosphorylated pCDKS , and either no pEndos , [N]T ( 0 ) = 16 nM non-radioactive pEndos , or 16 nM thio-pEndos were incubated for the indicated times and the amount of 32P released was determined by scintillation counting after removing the pCDKS by TCA precipitation .", "Because the pCDKS concentration was much less than its Km , 71–99 μM ( Table 1 ) , it bound a negligible fraction of PP2A-B55; therefore , the analysis would have been identical to that of Figure 10A except that PP2A-B55 activity decreased continuously , even in the no pEndos control , presumably due to degradation or instability .", "This decrease was modeled assuming a constant degradation rate , kdeg , so d[P]T/dt = −kdeg [P]T and [P]T ( t ) =e−kdegt[P]T ( 0 ) .", "The fraction of pCDKS dephosphorylated was small , so we approximated [pC]T ( t ) = [pC]T ( 0 ) ≡ [pC]T .", "Therefore , the amount of 32P released from pCDKS in the no-pEndos control was:d [P32] ( t ) dt= ( kcatpCDKS/KmpCDKS ) [pC]T [P]T ( t ) [P32 ( t ) ]= ( kcatpCDKS/KmpCDKS ) [pC]T [P]T ( 0 ) ( 1−e−kdegt ) /kdeg , where , the second line was computed by integrating the first line .", "An excellent fit was obtained using the values determined by nonlinear regression: kcatpCDKSKmpCDKS = 0 . 52 ± 0 . 01/ ( μM sec ) and kdeg = 0 . 020 ± 0 . 001/min .", "The analysis in the presence of pEndos used the quasi-steady-state equations used for Figure 10A except that they were modified by the replacement [P]T → [P]T ( t ) to account for the instability of PP2A-B55 .", "[P]T ( t ) was computed using the value of kdeg as described except that the onset of degradation was postponed for 25 min to accord with the data , which shows that the PP2A-B55 activity ( determined by the slope of the curve ) after release from pEndos inhibition ( e . g . , at t ∼ 50 min ) is greater than that in the no-pEndos control .", "( This might have been due to stabilization of PP2A-B55 by pEndos binding but , in any case , does not significantly affect the analysis . )", "These equations along with the equation above determining d [P32]dt and the value of kcatpCDKSKmpCDKS determined in the no-pEndos experiment were numerically integrated to determine [P32] ( t ) as a function of Km and kcat These values , Km = 0 . 47 ± 0 . 14 nM and kcat = 0 . 03 ± 0 . 0005/sec , were determined by nonlinear regression to the data .", "Figure 10A: the values for pEndos PP2A-B55 binding of kon = 0 . 057/ ( sec nM ) and koff = 0 . 0068/sec were determined from the experimentally measured Kd ( for thiophosphorylated Endos ) and kcat .", "Since ε=kcat[P]T/{kon ( [N]T ( 0 ) +[P]T+Km ) 2}=5 . 6×10−7≪1 , the total quasi-steady state approximation is expected to be good except for a transient during the small time interval 0 ≤ t ≤ tc , where 1/{ ( kon ( [N]T ( 0 ) + Km ) } ≈ 0 . 02 s ( Borghans et al . , 1996 ) .", "Therefore , we used the total quasi-steady state equations:[N] ( t ) =[N]T ( t ) 1+[P] ( t ) /Km[P] ( t ) =[P]T1+[N] ( t ) /Kmd[N]T ( t ) dt=−kcat[P] ( t ) [N] ( t ) /Km .", "These were numerically integrated using Mathematica Version 9 with [P]T = 250 nM , kcatX = 0 . 05/sec , and KmP= 1 . 0 nM with the boundary condition [N]T ( 0 ) = 1 μM .", "To be certain that the quasi-steady state assumption was valid over the entire time course ( i . e . , including the period when [N] ( t ) < [P] ( t ) ) , we recomputed the plots using the explicit mass-action equations for d[N]/dt , d[P]/dt , and d[NP]/dt ( where NP denotes the PP2A-B55/pEndos transient complex ) .", "These plots were visually indistinguishable from those computed using the quasi-steady state assumption except , as expected , for a brief transient during 0 ≤ t ≤ tc .", "Figure 10B: We assume that the kinetic constants for the additional phosphatase PPTX are KmX = 85 μM and kcatX= 22 . 5 s−1 , which are based on the values for PP2A dephosphorylation of CDKS ( Table 1 ) .", "Since KmX is so large , it is certain that ε≪1 ( see definition above ) so the total quasi-steady state assumption can again be used .", "In this case , the equations are:[N] ( t ) =[N]T ( t ) 1+[P] ( t ) /KmP+[X] ( t ) /KmX[P] ( t ) =[P]T1+[N] ( t ) /KmP[X] ( t ) =[X]T1+[N] ( t ) /KmXd[N]T ( t ) dt=−{kcatP [P] ( t ) /KmP+kcatX [X] ( t ) /KmX} [N] ( t ) , where , [X] and [X]T are the concentrations of unbound and bound phosphatase X , and superscripts P and X on the kinetic parameters kcat and Km identify the relevant enzymes .", "These equations were numerically integrated .", "Figure 10C and D: When PP2A/B55 is assumed to bind , but not dephosphorylate , pEndos with dissociation constant KdP , the equations are the same with the substitutions KmP→KdP and kcatP→0 .", "These equations were numerically integrated using KdP= 0 . 12 nM , which was based on the dissociation constant of the pEndos thiosulfate phosphomimetic ( computed from the data shown in Figure 8 ) .", "Figure 10—figure supplement 1: the first three equations described above for Figure 10B were solved to determine [N] , [P] , and [X] as functions of [N]0 ( using the notation above ) .", "The amounts of dephosphorylating velocity coming from PP2A/B55 and PPX were then:vP=kcatP[P] [N]/KmPvX=kcatX[X] [N]/KmX and the fractional velocities were:fP=vPvP+vXfX=vXvP+vX ." ] ]
[ "During M phase , Endosulfine ( Endos ) family proteins are phosphorylated by Greatwall kinase ( Gwl ) , and the resultant pEndos inhibits the phosphatase PP2A-B55 , which would otherwise prematurely reverse many CDK-driven phosphorylations .", "We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit .", "The kinetic parameters for PP2A-B55’s action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates , suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition .", "As the name suggests , during M phase PP2A-B55’s attention is diverted to pEndos , which binds much more avidly and is dephosphorylated more slowly than other substrates .", "When Gwl is inactivated during the M phase-to-interphase transition , the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced , so the phosphatase can refocus its attention on CDK-phosphorylated substrates .", "This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos , and how pEndos controls PP2A-B55 ." ]
[ "The most dramatic stage of the cell division cycle is M phase , when the cell splits into two genetically identical daughter cells .", "If this process goes wrong , the cell might die , so cells employ a complicated regulatory process to ensure that M phase begins and ends at the right time .", "As with many biological processes , regulation of the cell cycle depends on the activation and inhibition of a range of enzymes .", "Enzymes act as biological catalysts , binding target molecules ( substrates ) to active sites so that chemical reactions can take place .", "However , the activity of the enzyme can be shut down if a different type of molecule , called a competitive inhibitor , binds to the active site .", "For M phase to proceed , an enzyme called M phase promoting factor adds phosphate groups to hundreds of target proteins .", "At the end of M phase , a different enzyme , called PP2A-B55 , removes these phosphate groups .", "Cells can enter M phase because an inhibitor called Endosulfine blocks the active site of the PP2A-B55 enzyme .", "However , the cells need to unblock the PP2A-B55 enzyme at the end of M phase .", "Williams et al . have now established the mechanism behind this unblocking of the PP2A-B55 enzyme .", "One basis for this mechanism is that Endosulfine works as an inhibitor only when it is phosphorylated ( contains a phosphate group ) .", "Throughout M phase , a plentiful supply of newly phosphorylated Endosulfine inhibitor molecules binds very tightly to the active sites of the PP2A-B55 enzyme molecules , blocking the enzyme’s more loosely binding substrates from accessing the active sites .", "The second basis for this mechanism is that PP2A-B55 can also slowly remove the phosphate groups from Endosulfine molecules bound at the active site .", "In other words , phosphorylated Endosulfine works as an inhibitor only because it is really a substrate with special properties .", "It binds very tightly to the active site , where it is destroyed very slowly .", "For this reason , Williams et al . have named the process inhibition by unfair competition .", "In the final stages of M phase , the cell cannot produce any more phosphorylated Endosulfine molecules , and the PP2A-B55 enzyme can then destroy all the existing inhibitors .", "Even though this reaction is relatively slow , it is still achieved within a couple of minutes .", "After its active site is no longer blocked , the PP2A-B55 enzyme is then free to remove the phosphate groups from the target proteins , and M phase can come to an end ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "chromosomes and gene expression", "evolutionary biology" ]
Evolution of extreme resistance to ionizing radiation via genetic adaptation of DNA repair
elife-01322-v1
[ [ "Ionizing radiation ( IR ) is encountered humans in the form of medical X-rays and tumor irradiation , and very rarely in the context of nuclear power plant malfunction .", "The study of organisms with extreme resistance to IR has received increasing attention as a potential source of mechanistic insights that might permit the modulation of IR resistance in cells .", "Deinococcus radiodurans , which can absorb IR doses in excess of 5 kGy without lethality ( over 1000 times the lethal dose for humans ) , has become a key model organism for understanding this phenotype ( Cox and Battista , 2005 , Blasius et al . , 2008 ) .", "Mainly through the formation of reactive oxygen species ( ROS ) , IR can lead to the damage of protein , DNA , and all other cellular macromolecules ( Sonntag Cv , 2005 ) .", "Ongoing research into the molecular basis of extreme IR resistance has suggested three potential classes of mechanisms .", "These are ( A ) a condensed nucleoid structure , which could potentially facilitate DNA repair processes ( Zimmerman and Battista , 2005 , Levin-Zaidman et al . , 2003 ) , ( B ) an enhanced capacity for amelioration of protein damage , which could protect DNA repair systems and make them more readily available following irradiation ( Daly , 2012 , Daly , 2009 ) , and ( C ) potential specialized pathways for DNA repair ( Zahradka et al . , 2006 , Cox and Battista , 2005 ) .", "The amelioration of protein oxidation has received extensive experimental support as a mechanism to account for much if not all of the observed IR resistance in D . radiodurans ( Daly et al . , 2004 , Slade and Radman , 2011 , Krisko and Radman , 2010 , Daly , 2012 , Daly , 2009 ) .", "Increases in cytosolic antioxidant capacity , particularly an increase in the cellular Mn/Fe ratio ( Daly et al . , 2004 ) , appear to make the major contributions .", "Increasing the Mn/Fe ratio limits the Fenton chemistry that produces ROS ( Krisko and Radman , 2010 , Daly , 2012 ) .", "The hypothesis that the condensed nucleoid of Deinococcus facilitates efficient DNA repair has been questioned , since the presence of condensed nucleoids does not correlate reliably with radiation resistance in bacterial species ( Zimmerman and Battista , 2005 ) .", "Repair of IR-induced DNA damage is clearly important to survival .", "However , the constellation of DNA repair functions in D . radiodurans is unremarkable by bacterial standards .", "D . radiodurans and other IR resistant species appear to have approximately the same toolbox of DNA repair pathways as non-resistant species .", "Thus , the argument has been advanced that specialized DNA repair plays little or no role in IR resistance ( Daly , 2012 , 2009 ) .", "Instead , antioxidants prevent protein oxidation and render the classical DNA repair systems more readily available to correct the effects of IR .", "Bacteria are being used in long-term laboratory experiments that have elucidated many aspects of evolutionary biology ( Barrick et al . , 2009 , Hindre et al . , 2012 , Elena and Lenski , 2003 ) .", "Directed bacterial evolution can be used as a tool to determine the molecular underpinnings of adaptation to a stress or a new environment .", "Does IR resistance always arise via enhanced protection of otherwise commonplace DNA repair proteins from oxidative inactivation ?", "Alternatively , can a special facility for DNA repair or some other mechanism make a significant contribution to this extremophile phenotype ?", "In an earlier study , we demonstrated that the naturally IR sensitive bacterium , E . coli , could acquire an extreme IR resistance phenotype by directed evolution ( Harris et al . , 2009 ) .", "In brief , we carried out 20 rounds of selection , with each round consisting of IR exposure sufficient to kill ∼99% of the population , followed by outgrowth .", "The experiment was carried out four times , generating four separately evolved populations ( IR-1-20 , IR-2-20 , IR-3-20 , and IR-4-20 ) .", "In the evolved populations , survival at 3000 Gy was typically increased by 3–4 orders of magnitude ( Harris et al . , 2009 ) .", "We reported genomic sequences from seven isolates of IR-1-20 , one from IR-2-20 , and one from IR-3-20 .", "Genetic alterations in the evolved strains were quite numerous ( within the range of 40–80 per isolate ) , suggesting a complex and somewhat variable pattern of genetic innovation .", "None of the isolates in that study had mutator phenotypes relative to the Founder strain , although it is possible that mutators appear and thrive in the various populations for periods during the many cycles of selection ( Harris et al . , 2009 ) .", "An approximately fivefold variation in levels of resistance to IR was evident in a screen of 62 separate isolates from population IR-1-20 ( Harris et al . , 2009 ) .", "It remained for us to determine which of those mutations underlay the increase in radiation resistance , and what each of them might contribute .", "If amelioration of protein oxidation uniquely explains high level IR resistance in bacteria , then the mutations that contribute substantially to the phenotype might follow a fairly predictable pattern .", "We now provide a greatly expanded analysis involving many additional evolved isolates .", "The results reveal multiple contributions to IR resistance as well as the detailed molecular basis of the phenotype in one isolate ." ], [ "In the present study , we augmented the earlier data with the complete genomic sequences of 13 new isolates from populations IR-2-20 , IR-3-20 , and IR-4-20 .", "In addition , we examined the complete genomic sequences of nine isolates derived from either 20 or 30 rounds of further evolution of CB1000 , an isolate derived from IR-1-20 .", "We note that IR resistance in this more highly evolved population ( IR-CB1000-30 ) was impossible to measure accurately .", "The cells in this population grew more slowly than the Founder , but growth continued during irradiation at 6 Gy/min ( John R Battista , unpublished data ) .", "The results from the previous ( Harris et al . , 2009 ) and new sequencing efforts are combined in Supplementary file 1A , B .", "The overall selection scheme is illustrated in Figure 1 .", "The results give us a much-enhanced view of mutational patterns that are likely to contribute to the acquired IR resistance . 10 . 7554/eLife . 01322 . 003Figure 1 . Directed evolution scheme for the evolved isolates described in this paper . Red arrows denote cycles of irradiation and outgrowth .", "Evolved populations have titles beginning with “IR” .", "Isolates were derived from each listed population , as indicated by blue arrows .", "Isolates from each population are listed under the respective blue arrows , and each isolate features a name beginning with CB .", "Isolates listed in green text are described for the first time in this study .", "The sequences of the remaining isolates were described previously ( Harris et al . , 2009 ) , and are listed here since the genomic data was utilized in the current analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 01322 . 003 A summary of mutation types detected in the various sequenced isolates is presented in Table 1 .", "In each case , the genomic sequence was compared to the 4 , 639 , 675-bp reference genome .", "In the isolates derived from the original four evolved populations , there were between 44 and 77 genetic alterations .", "The numbers of mutations jumped appreciably in the isolates derived from the further evolution of CB1000 , with 242–267 mutations present in these strains .", "Transition mutations dominated the mutational spectrum . 10 . 7554/eLife . 01322 . 004Table 1 . Summary of the mutational spectrum observed in strains derived from directed evolution of resistance to ionizing radiationDOI: http://dx . doi . org/10 . 7554/eLife . 01322 . 004IR-1-20IR-2-20IR-3-20IR-4-20IR-CB1000-20IR-CB1000-30TransitionsCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB1111111222222233333334111111111000000000000000000000011111111101111220000011000011102222300000234545034794602684790245701458 C → T192061015691213111211991414131413121214596469696360626362 G → A189111611111217171615131311171113131281510606578716057606060 T → C776461341181610991111413117697515140444747474747 A → G1516788141110667765614101091168424342545046505050Transversions G → T44111353423542554446421097865666 C → A1121156536334624213416325564554 A → C1121112112211742244333 T → G111111111121222411111 G → C111221111111211243333 C → G1231111112121122222 A → T1211312143112111221233333 T → A1111111111311121332333332Insertions 767bp IS12111111Deletions1 e141111111111111111111111111111111 93bp1Totals70634046495653665564565350516357636155535453243251249265250236246247244The mutational spectrum of the population is inferred based on the genetic alterations observed in the subset of strains sequenced .", "One straightforward pattern noted in the earlier study was continued .", "The only genetic change that is universal to all strains sequenced is deletion of the e14 prophage , a defective lambdoid prophage that is 15 . 4 kb in length .", "The deletion of occurs early , within the first three rounds of selection for IR resistance ( EA Wood and JR Battista , unpublished data ) , and it makes a significant contribution ( an approximately 10-fold increase in survival at 3000 Gy ) to the overall IR resistance phenotype ( Harris et al . , 2009 ) .", "Population IR-1-20 has a particularly complex population structure and is strikingly different from the IR-2-20 and IR-3-20 populations .", "Outside of the e14 deletion , there are no mutations shared by all seven of the IR-1-20 isolates .", "There are three groupings of mutations in subsets of the clonal isolates that reflect clonal interference ( Gerrish and Lenski , 1998 , Perron et al . , 2012 , Rozen et al . , 2002 ) in this population ( JR Battista , unpublished results ) .", "In contrast , isolates from the IR-2-20 and IR-3-20 populations each exhibit a number of mutations ( 23 in IR-2-20 and 30 in IR-3-20 ) that are present in all seven isolates from their respective populations , and are effectively fixed .", "The apparent fixation of a mutation in one of these populations may reflect its importance to the phenotype .", "Alternatively , this may reflect a genetic bottleneck at some stage in the evolution of these populations , and speaks to the close relationship of the isolates .", "To identify the mutations that are most likely to contribute to the IR resistance phenotype , we focused on genetic alterations that exhibited the following criteria: ( I ) the mutation is present in all or most isolates sequenced in at least one population , ( II ) the mutated gene was a prominent mutational target in at least one other sequenced population or mutations are found in genes in the same operon or pathway in other populations .", "Application of the second criterion requires data that was unavailable in our earlier study ( Harris et al . , 2009 ) , and provides a pattern implicating a particular mutation in the acquisition of an extreme IR resistance phenotype .", "This criterion also assumes that there is a significant level of phenotypic parallelism ( Hindre et al . , 2012 , Futuyama , 1986 ) between the independently evolved populations .", "Table 2 summarizes the results of applying these criteria , with nine altered genes or systems meeting these two criteria .", "The entire complement of mutations found in all isolates from the four evolved populations , both those sequenced for this study and those analyzed in the earlier study ( Harris et al . , 2009 ) , are provided in Supplementary file 1A . 10 . 7554/eLife . 01322 . 005Table 2 . Summary of prominent mutational patterns observed in multiple evolved populationsDOI: http://dx . doi . org/10 . 7554/eLife . 01322 . 005IR-1-20IR-2-20IR-3-20IR-4-20IR CB1000−+GenePositionRef . CCCCCCCCCCCCCCCCCCCCCCChangeMutationBBBBBBBBBBBBBBBBBBBBBB111111122222223333333400000000000000000000000111122000001100001110Allele0234545034794602684790TypeclpP456127AGG+Y75CNclpP/clpX456637GAAAAAAA-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 in red denote mutations that are present in CB2000 .", "The prominent genetic alterations present in the various evolved isolates are not identical .", "We thus set out to define the genetic basis of the observed IR resistance in one representative isolate .", "We focused on the isolated strain CB2000 from the IR-2-20 population for several reasons .", "First , CB2000 is one of the most radiation resistant of the isolated strains from the original evolved populations ( Harris et al . , 2009 ) .", "Second , it is particularly well characterized ( Harris et al . , 2009; and this study ) .", "Third , it was isolated from a population that lacks any sign of clonal interference , allowing us to focus on patterns present in a relatively simple population .", "Finally , the patterns of mutations we highlight below overlap patterns that are evident in other populations , making CB2000 a good barometer of mechanisms by which radiation resistance evolved in many of our strains .", "Of the 9 genes and/or systems reflected in Table 2 , seven are represented in CB2000 .", "The candidate CB2000 genes can be grouped into three functional categories: ( I ) DNA repair and replication ( recA , dnaB , yfjK ) , ( II ) oxidative damage suppression ( rsxB and gsiB ) , and ( III ) cell wall biogenesis ( wcaK and nanE ) .", "All of these represent prominent mutational patterns .", "To assess the importance of these seven CB2000 mutations quantitatively , we took three approaches:Mutations identified in CB2000 were moved into the radiosensitive Founder strain .", "This was done both individually and collectively with one or more of the other mutations .", "The e14 prophage was deleted ( Δe14 ) in our wild-type background , since this genetic alteration occurs very early in the evolution of IR resistance in all of our strains , and the effects of this deletion have already been characterized ( Harris et al . , 2009 ) .", "If any of the 7 mutations we identify above are contributing to the phenotype , they are doing so in a background that excludes the e14 prophage .", "In addition to the seven mutations identified in Table 2 , we selected the glpD mutation as an additional target for analysis since it fulfilled criterion I above but did not quite fit criterion II .", "This mutation was fixed in population IR-2-20 .", "Although this gene was not mutated in any other population , a mutation that might affect regulation of glpD was found in three isolates of IR-3-20 . The same eight total mutations ( including glpD ) were reverted back to the original Founder sequence in CB2000 both individually and in combination with other mutations .", "This allows us to determine if the mutations contribute to IR resistance in a genetic background that includes all or most of the other 69 mutations present in CB2000 . We deleted the nonessential genes carrying these mutations in the radiosensitive Founder strain ( in a genetic background deleted for e14 ) individually .", "We wished to determine if a definitive knockout of an individual gene could mimic any observed effects of the individual mutations observed in CB2000 .", "If the answer was yes , we reasoned that the mutation we observed in CB2000 was likely to be a loss of function mutation .", "All of the strains constructed for this effort were assayed for survival to 3000 Gy to measure the contribution of individual mutations and mutation combinations to the IR resistance phenotype .", "We examined the baseline metabolic profiles of the Founder strain and several of the IR resistant isolates .", "The strains were not subjected to irradiation prior to analysis .", "The results measure the state of the cells when they first encounter irradiation .", "We used RNA-Seq ( Marioni et al . , 2008 ) to directly sequence and map RNAs that are expressed in the Founder , CB1000 , and CB2000 , with the comparison reported in Supplementary file 1C .", "Overall , there were few genes with different expression profiles when comparing the strains with a 1 . 5-fold cutoff enforced .", "The only commonality in expression patterns between the evolved strains was the >1 . 5-fold decrease in the transcript abundance compared to Founder of the following genes: fruBKA ( fructose metabolism ) , sdaC ( serine transport ) and proK ( proline tRNA ) .", "Transcription of the entire fimbrial operon is increased in CB1000 , possibly due to phase variation of the fimS region , but in CB2000 , only the fimC gene exhibits an increase in transcription of more than 1 . 5-fold .", "In both evolved strains icd ( b4519 ) transcript levels are increased , likely due to the excision of the e14 prophage , which reconstitutes the icd gene with a different 3′ end of the gene containing 2 base substitutions .", "In general , changes are minimal .", "There are few genes that display a newly constitutive level of expression that is strikingly higher than in the parent Founder strain , in spite of the existence of mutations in a number of genes encoding global regulators .", "Using NMR , we investigated metabolite concentrations in the total soluble fraction of the cytosol from Founder , two evolved radioresistant strains , CB1000 and CB2000 , and in CB1013 ( an isolate from the IR-1-20 population that has a distinctively different mutational profile relative to CB1000 ) ( Figure 4A ) .", "There are no significant changes in metabolites between any of the strains measured , with the possible exception of small apparent decreases in the levels of acetate and succinate in CB2000 as compared to Founder .", "Unlike D . radiodurans , there is no evidence of accumulation of intermediate metabolites that could act as antioxidants .", "The measured metabolites included glutathione , a molecule that plays a particularly important role in cellular redox chemistry . 10 . 7554/eLife . 01322 . 008Figure 4 . ( A ) , Measurements of metabolites from three representative evolved E . coli strains as compared to Founder . Metabolites from whole cell pellets collected during logarithmic growth in LB were identified using a two-dimensional 1H-13C Heteronuclear Single Quantum Coherence ( HSQC ) experiment .", "Each metabolite is expressed as a ratio of the amount measured in the evolved strain ( CB1000 , CB1013 , or CB2000; black , gray , and white bars , respectively ) relative to the Founder .", "( B ) Ratios of manganese to iron are plotted for all isolates for which genomic sequences were obtained .", "The average increase in Mn/Fe ratio in strains derived from the further evolution of CB1000 is 1 . 4-fold . DOI: http://dx . doi . org/10 . 7554/eLife . 01322 . 00810 . 7554/eLife . 01322 . 009Figure 4—figure supplement 1 . Relative levels of metals are unchanged in evolved E . coli . Cells sampled during mid-logarithmic growth were subjected to trace metal analysis as described in ‘Materials and methods’ .", "The amount of each metal was plotted as a ratio normalized to the amount of potassium present in each sample , K , which was chosen arbitrarily to correct for variation in metal extraction . DOI: http://dx . doi . org/10 . 7554/eLife . 01322 . 009 We also used trace metal analysis to measure total metal content of all strains for which we obtained genomic sequences .", "Mn/Fe ratios are reported in Figure 4B , and complete listings of metal ion measurements are provided in Figure 4—figure supplement 1 .", "In spite of the demonstrable increases in radiation resistance exhibited by all of our isolates , we did not see a significant change in the Mn/Fe ratio ( nor significant increases in the concentration of either metal ) in most of our directly evolved highly radioresistant strains of E . coli .", "There is a minor elevation in manganese in the one evolved isolate from population IR-4-20 ( CB4000 ) and in some strains derived from the further evolution of CB1000 .", "There is no universal change in metal content in the evolved strains that mirror the apparent adaptation seen in D . radiodurans and other IR-resistant bacteria ." ], [ "To the three potential mechanisms described in the introduction of this article—amelioration of protein oxidation , novel DNA repair systems , and nucleoid condensation—we now document a fourth .", "IR resistance can be increased—dramatically—by functional enhancement and/or adaptation of existing DNA repair enzymes .", "Although classical DNA repair systems may be shared widely in different IR resistant and IR sensitive bacterial species , those DNA repair systems are biologically malleable .", "Classical DNA repair pathways can adapt to facilitate more efficient repair when cells are exposed to high levels of IR .", "In our evolved isolates , adaptations to DNA repair , involving genetic alterations in well-studied enzymes that are present in most bacterial species , represent a substantial and sometimes the dominant adaptation that contributes to the IR resistance phenotype .", "In one well-characterized isolate , three mutations—all in DNA repair functions—largely account for the increase in IR resistance .", "The metabolic profile of our cells indicates that the evolved strains possess little or no unusual gene expression , metabolite concentration , or metal ion adaptations when they first encounter irradiation .", "We acknowledge that irradiation may lead to an alteration of the profile of genes induced in the IR resistant isolates , and those isolates may thus adapt to the challenge of irradiation more rapidly .", "In CB2000 , changes in the rsxB gene might bring about some significant and beneficial changes in the IR response .", "However , in the case of CB2000 , it is clear that changes to DNA repair genes—not regulatory genes—play the major role in the observed IR resistance .", "The complement of DNA repair systems present tend to be quite similar from one bacterial species to another , but they are not identical .", "The differences , some subtle and some significant , inevitably reflect the lifestyle and environment that applies to each species .", "Whereas Deinococcus radiodurans has a fairly standard set of DNA repair functions ( Daly , MJ , 2012 ) , there are important distinctions in both the relevant gene catalogue ( Cox and Battista , 2005 , Blasius et al . , 2008 ) and the mechanisms used to recover from exposure to high levels of ionizing radiation ( Slade et al . , 2009 ) .", "In the present study , we directly demonstrate that genetic innovation involving the cellular DNA repair systems can directly contribute , and contribute substantially , to the acquisition of extreme resistance to ionizing radiation .", "Nevertheless , we emphasize that there are other effects clearly evident in the data .", "Evolution of a complex extremophile phenotype , such as extreme resistance to ionizing radiation , has no single molecular explanation .", "There are multiple paths; multiple mutations affecting multiple cellular systems make significant contributions .", "The mutational patterns suggest that improvements in the amelioration of protein oxidation , as first pointed out by Daly et al . ( Daly et al . , 2004 , Slade and Radman , 2011 , Krisko and Radman , 2010 , Daly , 2012 , 2009 ) , may also play a role in some of the evolved populations .", "Although that role appears to be relatively minor in CB2000 , it may well predominate in one or more of the other populations .", "Given the genes affected , any mechanisms contributing to the amelioration of protein oxidation in these isolates may be somewhat different than those described to date for D . radiodurans .", "We note that the mutations to DNA repair genes might , in principle , simply render the protein products of those genes less vulnerable to oxidation .", "This does not appear to be the case for the changes we observe in the recA gene .", "We have characterized the proteins encoded by the recA gene variants described in this report ( JR Piechura and MM Cox , unpublished data ) , particularly the RecA D276A and D276N mutant proteins .", "In brief , the altered RecA proteins nucleate filament formation more rapidly and extend those filaments more slowly than the wild-type protein , leading to larger numbers of shorter filaments .", "Those same proteins function better as shorter filaments , and are less sensitive to inhibition by ADP .", "In general , the proteins exhibit functional alterations that are easily rationalized in the context of a recombinational system that must simultaneously deal with large numbers of double strand breaks in cells where metabolic processes might be compromised .", "Contributions to the extreme IR resistance phenotype by additional biochemical processes appear likely , and remain to be explored ." ], [ "All strains used in this study are E . coli K-12 derivatives and are listed in Supplementary file 1D .", "Genetic manipulations were performed by site directed mutagenesis ( Stratagene ) and as previously described ( Datsenko and Wanner , 2000 ) .", "Radioresistant populations IR-1-20 , IR-2-20 , IR-3-20 , and IR-4-20 were generated in a directed evolution experiment described previously ( Harris et al . , 2009 ) .", "To further evolve the CB1000 isolate , 1 ml of mid logarithmic phase liquid culture grown in LB was placed into two 1 . 5 ml plastic tubes .", "One was archived at −80°C , and the other was exposed to IR ( 60Co source from a Shepherd model 484 irradiator; 19 Gy/min ) .", "After irradiation , appropriate dilutions were plated to estimate survival .", "The balance of the irradiated culture was used to inoculate fresh LB broth .", "Survivors were grown to stationary phase ( 12–18 hr ) and the protocol was repeated .", "The administered radiation dose was adjusted to allow approximately 1% survival after 1 day at 37°C , with the dose increasing as radio-resistance increased .", "Survivors at the end of 20 and 30 rounds of irradiation constitute a population of cells , designated IR-CB1000-20 and IR-CB1000-30 , respectively .", "Single colony isolates from both populations were isolated and designated with the prefix ‘CB’ .", "All archived populations and strains were stored at −80°C with 15% vol/vol DMSO added as cryoprotectant .", "New evolved isolates described in this study , were sequenced at the Joint Genome Institute , Walnut Creek , CA , as detailed previously ( Harris et al . , 2009 ) .", "We used Illumina-based next generation sequencing that typically generates very low error levels in bacterial genome sequencing .", "SNP calls required their presence in at least three reads .", "Typically , the SNPs were based on 50–150 reads .", "To estimate error levels , the sequence alignments for one dataset ( CB2004 ) were examined manually .", "Since this is a haploid genome , loci which contained multiple alleles ( a mixture or reference and ‘variant’ alleles ) are indications of alignment errors or sequence specific errors ( Nakamura et al . , 2011 ) and were not called as SNPs No false positives were identified in the manual examination and we estimate their appearance at rates of <2% .", "If reads can be aligned to multiple locations in the genome , their exact placement is ambiguous and assigned a map quality score of zero ( MQ = 0 ) .", "It is not possible to call SNPs in regions that contain only MQ = 0 reads and false negative calls are potentially present .", "Approximately , 100 , 000 bp of the genome was covered by only MQ = 0 reads , and thus the potential for false negatives extends over about 2 . 2% of the genome .", "Cells from a fresh single colony of each strain were cultured in Luria–Bertani ( LB ) broth ( Miller , 1992 ) at 37°C with aeration .", "After growth overnight , cultures were diluted 1:1000 into 10 ml fresh LB broth in 125 ml flasks and grown at 37°C with shaking until an optical density ( OD600 ) of ∼0 . 2 was reached .", "Each culture was incubated on ice for 5 min before a 1 ml sample was transferred to an eppendorf tube and irradiated in a Mark I 137Cs irradiator ( from JL Shepherd and Associates , San Fernando , CA , USA ) for a time corresponding to 3 kGy ( ∼7 Gy/min ) .", "Irradiated samples as well as the non-irradiated control samples for each culture were diluted appropriately , and plated on LB 1 . 5% agar medium to determine the total number of colony forming units ( CFUs ) .", "Percent survival was calculated by dividing the titer of the surviving population by the titer of the non-irradiated control sample .", "Initial cell densities ranged from 2 to 6 × 107 CFU/ml ( average 4 × 107 CFU/ml ) .", "For each strain , 3–5 biological replicates were carried out .", "Cells from a fresh single colony of each strain were cultured in LB broth ( Miller , 1992 ) at 37°C with aeration .", "After growth overnight , competition cultures were started by inoculating 10 ml fresh LB broth with 35 μl of competition Ara+ and Ara−strains in 125 ml flasks and grown at 37°C with shaking until an optical density ( OD600 ) of ∼0 . 2 was reached .", "Each competition culture was incubated on ice for 5 min before a 1 ml sample was transferred to an eppendorf tube and irradiated in a Mark I 137Cs irradiator ( from JL Shepherd and Associates , San Fernando , CA ) for a time corresponding to 2 kGy and 3 kGy ( ∼7 Gy/min ) .", "Irradiated samples were diluted appropriately , and plated on TA plates to determine the total number of surviving red and white colony forming units .", "A non-irradiated control sample for each competition culture was diluted and plated to determine the titer of each culture and the percent Ara+ vs Ara−cells before irradiation .", "For each competition , three biological replicates were carried out .", "Percent survival was calculated by dividing the titer of the surviving population by the titer of the non-irradiated control sample , for both Ara+ and Ara−strains .", "Selection rate , r , also referred to as log ( advantage ) , was calculated as log ( N1 ( IR ) ) / ( N1 ( No-IR ) ) − log ( N2 ( IR ) ) / ( N2 ( No-IR ) ) , where N1 ( No-IR ) and N2 ( No-IR ) represent the initial densities of the two competing strains before IR treatment , and N1 ( IR ) and N2 ( IR ) represent the corresponding densities after IR exposure .", "Normally , in a direct competition experiment , plates with fewer than 20 colonies of either competitor are usually excluded to reduce the effect of outliers caused by low counts ( Breed and Dotterrer , 1916 ) .", "However , because the differences in fitness after IR treatment is so great between CB2000 Ara−and CB2000 wtRecA wtDnaB wtYfjK and between Founder Δe14 Ara−and Founder Δe14 RecA D276N DnaB P80H YfjK A151D , it was virtually impossible to retrieve at least 20 colonies of the sensitive strains in a range that we could also use to calculate the density of the resistant strains .", "The selection rates in Figure 3E are approximate , because there were less than 20 colonies counted on plates for the sensitive strains .", "However , the trend that we show in Figure 2 is strongly conserved .", "By reverting the three mutations in DNA metabolism genes , CB2000 loses virtually all of its IR resistance .", "We report in Figure 3E that when treated with 2000 Gy , CB2000 Ara− has at least a two log fitness advantage over CB2000 wtRecA wtDnaB wtYfjK and inversely , Founder Δe14 Ara- RecA D276N DnaB P80H YfjK P80H has at least a two log advantage over Founder Δe14 .", "Because their sensitivities to IR were so similar , we did not have a problem with retrieving more than 20 colonies for each strain in the competition of Founder Δe14 and CB2000 wtRecA wtDnaB wtYfjK .", "Rather , CB2000 wtRecA wtDnaB wtYfjK had less than a half log advantage ( less than threefold ) over Founder Δe14 , again illustrating the importance of mutations in these three genes for extreme radiation resistance .", "This method is reviewed in Croucher and Thomson ( 2010 ) .", "Sample preparation: samples were prepared as described in ( Durfee et al . , 2008 ) with modification as described here .", "Cell growth: Overnight cultures from single-cell inoculates grown in LB were used to inoculate 20 ml of LB in a 125 ml flask with appropriate antibiotic to an initial OD600 of 0 . 02 .", "Cultures were grown at 37°C with aeration until an OD600 of ∼0 . 2 was reached .", "Aliquots of 15 ml of each culture were mixed with 30 ml of RNAprotect bacterial reagent ( Qiagen ) , inverted to mix , and incubated at room temperature for 5 min .", "Cells were centrifuged at 4000×g for 20 min at 4° and the cell pellets were stored at −80°C .", "Total RNA was isolated using the MasterPure RNA purification kit according to the manufacturer’s specifications ( Epicentre , Madison , WI , USA ) .", "Nucleic acid pellets were treated with 0 . 05 U/µl DNase I for 45 min at 37°C and then repurified with MasterPure .", "10 μg of total RNA was enriched for mRNA by targeted removal of rRNA using the MICROBExpress bacterial mRNA enrichment kit ( Ambion ) .", "The resulting enriched mRNA was isopropanol precipitated , and the pelleted mRNA resuspended in TE .", "The enriched mRNA concentration was quantified by A260 measurement on a NanoDrop 1000 instrument .", "10 µg of purified total RNA was reverse transcribed using the Superscript II double-stranded cDNA kit ( Invitrogen ) followed by RNase digestion and cDNA purification by phenol chloroform extraction and precipitation .", "cDNA samples were submitted to JGI for library preparation and sequencing using the Illumina Genome Analyzer IIx to generate single-ended 36 bp reads .", "Libraries were prepared for sequencing according to the manufacturer’s instructions .", "Analysis was performed using the CLC-Bio Genomics Workbench version 3 . 7 .", "There were two biological replicates for each of the three samples ( Founder , CB1000 , CB2000 ) .", "Read ends were trimmed to remove low quality and ambiguous bases and all reads less than 20 nt were discarded .", "Trimmed reads were mapped to the annotated CDSs of the reference genome ( E . coli K-12 MG1655 m56 reference genome , RefSeq Accession Number NC_000913 . 2 ) with two mismatches allowed and 10 bases of each read were allowed to map beyond ORF boundaries .", "Expression was calculated independently for each duplicate sample .", "Any read that could be mapped to more than four locations was discarded .", "Genes encoding rRNA and tRNA transcripts were masked by removing their annotations from the reference genome prior to mapping so that did not affect normalization expression estimates of protein-coding genes .", "Expression values were reported in RPKM ( Mortazavi et al . , 2008 ) .", "Cell pellets were submitted for analysis in acid-cleaned polypropylene vials and treated at 40°C with ultrapure HNO3 and subsequently diluted to volume for analysis with 2% HNO3 .", "Samples were analyzed in the Trace Element Clean Laboratory at the Wisconsin State Laboratory of Hygiene , Madison , WI , using high-resolution inductively coupled plasma mass spectrometry .", "Each sample was measured twice .", "The metal content is reported as μg per pellet of bacteria submitted .", "Overnight cultures of Founder , CB1000 , CB2000 , and CB1013 were diluted into 1L M9 media containing glucose in 6-L flasks to an initial OD600 of 0 . 05 and grown at 37°C with aeration .", "When an OD600 of ∼0 . 80 was achieved , cultures were chilled on ice for 30 min before being centrifuged at 8000 rpm for 10 min at 4°C in a JLA 8 . 1 rotor .", "The supernatant was discarded , and cell pellets were washed in 25 ml of 1X M9 salts and transferred to a JA-20 tube before centrifuged 20 min at 5000×g at 4°C .", "The supernatant was discarded and 16 ml of boiling water with 250 μM MES ( 2- ( N-morpholino ) ethanesulfonic acid ) was added to each pellet , vortexed briefly to loosen the pellet , and placed in boiling water for 7 . 5 min .", "Tubes were briefly vortexed again , and then centrifuged in a JA-20 rotor at 7000×g for 20 min at 4°C to clear cell debris .", "The supernatant was poured off into a clean 50-ml sterile polypropylene tube and frozen .", "Supernatants were transferred to microfilters ( Sartorius Stedim Vivaspin 20 , 3000 MWCO ) .", "The low MW fraction was frozen and lyophilized .", "Dried metabolites were dissolved in 800 μl D2O containing 300 μl DSS and 300 μl NaN3 and titrated to pH 7 . 40 ( ±0 . 01 ) with DCl/NaOD as needed .", "Samples were transferred to 5-mm NMR tubes ( Wilmad Lab Glass , Vineland , NJ , USA ) .", "Spectroscopy was performed at the Nuclear Magnetic Resonance Facility at Madison ( NMRFAM ) in Madison , WI on a 600 MHz Varian Spectrometer with a cryoprobe and VNMRJ software .", "Two-dimensional 1H-13C HSQC spectra were acquired using 4 transits , 32 steady state transits , 0 . 3 s acquisition time , and 512 increments .", "Analysis was performed using rNMR , an open source software package developed at NMRFAM ( Lewis et al . , 2009 ) .", "To quantify signals , standard compounds of the observed metabolites were prepared at 2 , 5 , and 10 mM .", "The resonances from these compounds were linearly regressed in order to measure concentration as a function of intensity .", "The samples were normalized to 5 mM MES and their resultant peak intensities used to obtain concentrations of the measured metabolites using the regression slopes ." ] ]
[ "By directed evolution in the laboratory , we previously generated populations of Escherichia coli that exhibit a complex new phenotype , extreme resistance to ionizing radiation ( IR ) .", "The molecular basis of this extremophile phenotype , involving strain isolates with a 3-4 order of magnitude increase in IR resistance at 3000 Gy , is now addressed .", "Of 69 mutations identified in one of our most highly adapted isolates , functional experiments demonstrate that the IR resistance phenotype is almost entirely accounted for by only three of these nucleotide changes , in the DNA metabolism genes recA , dnaB , and yfjK .", "Four additional genetic changes make small but measurable contributions .", "Whereas multiple contributions to IR resistance are evident in this study , our results highlight a particular adaptation mechanism not adequately considered in studies to date: Genetic innovations involving pre-existing DNA repair functions can play a predominant role in the acquisition of an IR resistance phenotype ." ]
[ "X-rays and other forms of ionizing radiation can damage DNA and proteins inside cells .", "The radiation interacts with aqueous solutions to produce reactive forms of oxygen , which then cause the damage .", "A range of mechanisms exist to moderate and/or repair this damage , with certain species being able to tolerate extraordinary levels of radiation .", "The bacterium D . radiodurans , for example , can survive radiation levels that are over 1000 times higher than the levels that can kill human cells .", "The molecular basis of high-level resistance to ionizing radiation is not well understood , and several mechanisms have been proposed .", "Recent work has focused on passive mechanisms that are based on changes in cellular levels of certain small molecules that prevent damage by reactive forms of oxygen molecules .", "Now , based on experiments on E . coli , Byrne et al . demonstrate that active mechanisms , involving adaptations in the cellular DNA repair systems , can bring about dramatic increases in radiation resistance .", "The experiments were performed on populations of E . coli cells that had been subjected to an evolutionary selection for extremely high resistance to ionizing radiation .", "This involved exposing the E . coli cells to ionizing radiation that killed most of the population , and then growing up the survivors .", "Many repetitions of this process led to a population of cells with a resistance that was comparable to that of the bacterium D . radiodurans .", "The same evolution experiment was carried out four times , generating four separate populations of bacteria that were resistant to ionizing radiation .", "Byrne et al . sequenced the genomes of the E . coli after 20 , 40 or 50 rounds of the selection process , and compared mutations found in the four separate evolved populations .", "This showed that nine genes were particularly prone to mutations .", "Together , these genes had roles in repairing and copying DNA sequences , in decreasing damage caused by reactive forms of oxygen , and in manufacturing the molecular wall that shields cells .", "To assess the importance of the mutations in the nine genes , Byrne et al . took Founder cells from the initial population of E . coli cells–which were not resistant to ionizing radiation–and introduced the very same mutations , one at a time .", "Then the mutations that had the largest positive effects on resistance to ionizing radiation were combined .", "Introducing particular mutations into three DNA repair genes resulted in the highest aggregate levels of resistance .", "Finally , evolved E . coli cells that were already resistant were made more sensitive to radiation by repairing the same individual mutations .", "Again , the biggest change was observed with the DNA repair genes .", "Indeed , repairing the mutations in just the three DNA repair genes completely removed the radiation resistance .", "The next step is to determine how the properties of the mutated proteins change , and how those changes lead to radiation resistance .", "Also , there are clues in the work that suggest the presence of additional ways for cells to become radiation resistant , and these remain to be explored ." ]
2014
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "stem cells and regenerative medicine", "tools and resources", "genetics and genomics" ]
Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6
elife-27873-v3
[ [ "The current gold standard method for studying human hematopoietic stem and progenitor cell ( HSPC ) gene function has been either overexpression or RNAi-mediated knockdown of genes using lentiviral vectors ( Doulatov et al . , 2012; Chan et al . , 2015 ) .", "While these methods have provided great insights into HSPC biology , they come with several confounders , such as random integration of the vector into the host genome , unregulated transgene expression , and incomplete gene knockdown ( Woods et al . , 2006; Naldini , 2015 ) .", "More recently , programmable nucleases such as zinc finger nucleases ( ZFNs ) , transcription activator-like effector nucleases ( TALENs ) , and CRISPR/Cas9 have been utilized to disrupt genes by the introduction of site-specific DNA double strand breaks ( DSBs ) that are corrected through non-homologous end-joining ( NHEJ ) ( Hendel et al . , 2015; Holt et al . , 2010; Saydaminova et al . , 2015; Mandal et al . , 2014; Schumann et al . , 2015; Kim et al . , 2014; Lin et al . , 2014 ) .", "This error-prone system creates a heterogeneous mixture of cells with various genotypes of SNPs and small insertions or deletions ( INDELs ) ; moreover , not all of the genetic changes from INDELs cause functional gene disruption as they may preserve the open reading frame and may not change amino acids essential for protein functions ( Shi et al . , 2015; Hultquist et al . , 2016 ) .", "In a prior study , defined gene deletions were created in HSPCs using a dual sgRNA approach , however , more than half of the alleles were not modified leading to residual gene expression ( Mandal et al . , 2014 ) .", "Another limitation of this prior study is that successfully modified cells were not distinguishable from unmodified wild type ( WT ) cells , and therefore could not be tracked or isolated as an enriched population .", "Although the versatility of the CRISPR/Cas9 system allows for simultaneous manipulation at multiple genetic loci in a single cell , multiplexing of NHEJ-based gene editing has mainly been performed in immortalized human cancer cell lines and mouse cells ( Hultquist et al . , 2016; Cong et al . , 2013; Heckl et al . , 2014; Platt et al . , 2014; Brown et al . , 2016 ) .", "Finally , these interesting multiplexed proof-of-concept studies , only used NHEJ-mediated editing and did not harness the power of homologous recombination ( HR ) to create more sophisticated alterations to the genome at multiple alleles and/or loci .", "Here , we report an HR-mediated genome engineering method in human HSPCs and T cells that overcomes these limitations and enables the generation and enrichment of HSPC or T cell populations with complete gene knockout or gene replacement at multiple genetic loci .", "This method has the power to reveal functional gene networks during hematopoiesis and immune system disease pathogenesis and could be combined with the concepts of synthetic biology to create novel stem cell based therapeutics ." ], [ "We and others have previously shown that HR in human HSPCs can be efficiently induced by site-specific nucleases in combination with homologous donor DNA delivered as single-stranded oligonucleotides ( ssODNs ) , integration-defective lentiviral vectors ( ÍDLVs ) , or by recombinant adeno-associated virus serotype 6 ( rAAV6 ) vectors ( Dever et al . , 2016; DeWitt et al . , 2016; De Ravin et al . , 2017; Wang et al . , 2015; Hoban et al . , 2016 ) .", "We previously showed targeted integration in the beta-globin gene ( HBB ) by combining delivery of Cas9 protein pre-complexed with chemically modified sgRNAs ( RNP ) and delivery of an AAV6 donor .", "After successful on-target integration of a reporter transgene , FACS-based sorting of transgene reporterhigh-expressing HSPCs was used to purify an HSPC population with >90% targeted integration that displayed long-term repopulation capacity in NSG mice ( Dever et al . , 2016 ) .", "To extend this method beyond the HBB locus for therapeutic genome editing approaches of hemoglobinopathies , we tested six additional loci for their potential to be modified through HR by CRISPR/Cas9 in combination with AAV6-derived donor delivery .", "These genes are associated with hematopoiesis , hematopoietic malignancies , or safe harbor sites and include: interleukin-2 receptor gamma chain ( IL2RG ) , chemokine ( C-C motif ) receptor 5 ( CCR5 ) , runt-related transcription factor one isoform c ( RUNX1c ) , additional sex combs like 1 ( ASXL1 ) , stromal antigen 2 ( STAG2 ) , and adeno-associated virus integration site 1 ( AAVS1 ) ( Tebas et al . , 2014; Genovese et al . , 2014; Patel et al . , 2012; Mazumdar et al . , 2015; Kotin et al . , 1992 ) .", "Following electroporation with Cas9 RNP , containing a chemically-modified sgRNA targeting a single site in the selected locus , and transduction with an rAAV6 donor vector carrying homology arms for the targeted site and an expression cassette encoding a fluorescent reporter gene ( Figure 1—figure supplement 1a ) , we observed at early time points ( day", "4 ) a cell population with increased fluorescence intensity detectable by flow cytometry ( reporterhigh cells ) compared to cells receiving only the rAAV6 donor without electroporation of Cas9 RNP ( reporterlow ) ( Figure 1a and Supplementary file 1a ) .", "For cells targeted at either CCR5 or IL2RG , reporterhigh , reporterlow , and reporterneg populations were sorted at day four post-electroporation and cultured up to 22 days .", "Reporterhigh populations remained 99 . 2 ± 0 . 7% reporter positive ( Figure 1b ) while sorted reporterlow and reporterneg populations were 29 . 3 ± 5 . 4% and 0 . 6 ± 0 . 2% reporter positive , respectively .", "Dividing the reporterlow cells into three sub fractions based on fluorescence intensity revealed that GFP intensity at day four post-electroporation positively correlated with the propensity for maintaining GFP expression at day 20 ( Figure 1—figure supplement 1b–c ) .", "In addition , single reporterhigh cells were plated in methylcellulose to assess integration events at the clonal level .", "Targeted HSPCs formed a mix of myeloid ( CFU-M/GM ) and erythroid colonies ( BFU-E , CFU-E ) indicating that they retained HSPC function .", "‘In-Out PCR’ ( one donor-specific primer and one locus-specific primer outside of the respective homology arms ) on genomic DNA ( gDNA ) from single cell-derived methylcellulose colonies confirmed that 99% , 92% , and 100% of reporterhigh HSPCs targeted at CCR5 ( 338 clones analyzed ) , IL2RG ( 117 clones analyzed ) , and RUNX1 ( 36 clones analyzed ) , respectively , had at least a monoallelic targeted integration ( Figure 1c and Figure 1—figure supplement 2 ) .", "Analyses of clones with only mono-allelic integration showed gene-specific differences in the modification of the non-integrated alleles ranging from 38% INDELs for IL2RG to 89% INDELs for CCR5% and 88% INDELs for RUNX1 , among which the majority was gene-disrupting ( Figure 1—figure supplement 2 and Supplementary file 1b ) .", "Collectively , these data indicate that the observed log-fold transgene expression shift following rAAV6 and RNP delivery is due to HR at the intended locus and that reporter expression can be used to enrich gene-targeted HSPCs .", "To evaluate the applicability of this technology in a biologically relevant setting we decided to modify the cohesin complex member , STAG2 , in primary CD34+ HSPCs .", "The cohesin complex has previously been shown to play an essential part in maintaining normal erythroid differentiation potential of hematopoietic stem and progenitor cells ( Mazumdar et al . , 2015; Viny et al . , 2015; Mullenders et al . , 2015 ) .", "Since the STAG2 gene is located on the human X chromosome , single-allele integration of a fluorescent reporter in male cells would be sufficient to fully knock out the gene .", "As expected , Cas9 RNP combined with rAAV6 donor transduction resulted in the generation of a reporterhigh population that could be sorted for subsequent differentiation experiments .", "Single cell methylcellulose assays of reporterhigh cells revealed an almost complete loss in the capacity to form erythroid colonies compared to cells that had only been exposed to rAAV6 and not Cas9 RNP , and also compared to cells with targeted integration at the AAVS1 locus ( Figure 1d ) .", "These proof-of-concept studies provide evidence that gene-specific enrichment of reporterhigh cells can be used to study HSPC gene function .", "To determine if this method could be used to enrich HSPCs with biallelic gene disruption , necessary for complete functional gene knockout , we targeted the ASXL1 gene and simultaneously provided GFP and BFP-encoding rAAV6 donors .", "Four days after electroporation and transduction , 10 . 4% of cells were double positive for GFPhigh and BFPhigh compared to 0 . 2% for the AAV only sample ( Figure 2a ) .", "Similarly , double-positive populations were apparent when targeting three other genes ( RUNX1 , HBB , and CCR5 ) with two rAAV6 donors with various color combinations ( Figure 2—figure supplement 1 and Supplementary file 1c ) .", "Double-positive cells sorted at day four after electroporation remained 94% double-positive for more than two weeks in culture ( Figure 2b ) .", "‘In-out PCR’ on gDNA from single cell-derived methylcellulose clones confirmed on-target integration of one transgene into one allele and the other transgene into the second allele ( Figure 2c ) .", "We next tested if the biallelic targeting approach could be extended to another blood cell type and therefore targeted primary human T cells for biallelic HR at CCR5 .", "After electroporation with CCR5-targeting Cas9 RNP followed by transduction with GFP and mCherry CCR5 rAAV6 donors , a GFPhigh/mCherryhigh double-positive population was observed , indicative of biallelic integration at the CCR5 gene ( Figure 2d ) .", "No significant toxicity was associated with biallelic targeting in T cells ( Figure 2—figure supplement 2 ) .", "Overall , these results demonstrate the utility of using rAAV6 , Cas9 RNP , and FACS to enrich for primary human HSPCs and T cells that have undergone biallelic homologous recombination , which may have applications for studying hematological and immunological diseases or generating HSPC or T cell therapeutics that require gene modifications or gene knockout at both alleles .", "The vast majority of hematopoietic functions and immune diseases are governed by complex , polygenic networks ( Seita and Weissman , 2010 ) .", "To potentially study gene-gene interactions and/or generate cell therapeutics with HR modifications at two separate genes , we tested whether our methodology could facilitate simultaneous di-genic ( two different genes ) HR in HSPCs .", "We therefore co-delivered HBB-tdTomato and IL2RG-GFP rAAV6 donors with Cas9 RNP targeting both genes .", "This strategy produced 10 . 2% double positive GFPhigh/tdTomatohigh HSPCs compared to 0 . 1% for the AAV only control sample ( Figure 3a ) .", "We also generated double reporterhigh positive populations when testing other combinations of di-genic HR ( IL2RG/CCR5 , RUNX1/ASXL1 , and HBB/CCR5 ) ( Figure 3—figure supplement 1 and Supplementary file 1c ) .", "Again , double reporterhigh positive cells sorted at day four post-electroporation remained 94% double positive for 15 days in culture ( Figure 3b ) .", "‘In-Out PCR’ on double positive methylcellulose myeloid and erythroid clones showed on-target integration at both loci in 88% of clones ( 57 clones analyzed ) ( Figure 3c and d ) .", "Since the combination of two sgRNAs has previously been used to create and study oncogenic translocations ( Maddalo et al . , 2014 ) , and multiplexed TALEN-mediated gene editing in primary human T cells led to translocation frequencies between the two targeted genes of 0 . 01–1% with monocentric translocations occurring most frequently ( Poirot et al . , 2015 ) , we assessed if our di-genic targeting scheme would enrich for translocations after purification of dual-reporter positive cells .", "Therefore , we analyzed one of the monocentric translocations between HBB and AAVS1 ( Figure 3—figure supplement 2a ) .", "We targeted HBB and AAVS1 with a GFP and BFP reporter , respectively , and sorted the four different populations ( double negative , single positives ( each gene ) , and double positive ) seven days after targeting ( Figure 3e , left panel ) .", "INDEL rates at HBB and AAVS1 were comparable among all four sorted populations , with a small enrichment of INDELs in the three populations positive for the reporter ( Figure 3e , middle panel ) .", "Droplet digital PCR ( ddPCR ) quantification of the translocation showed frequencies ranging from 0 . 14–0 . 28% , and importantly , no evidence of enrichment of the translocation was observed in the population sorted for di-genic targeting ( Figure 3e , right panel and Figure 3—figure supplement 2c ) .", "Cloning and sequencing of PCR products spanning the translocation showed a wide variety of translocation junctions derived from different DNA end-processing products ( Figure 3—figure supplement 2b ) .", "To confirm that HSPCs with long-term and multi-lineage engraftment potential were targeted , we again targeted HBB and AAVS1 with a GFP and BFP reporter , respectively , and transplanted the four different sorted populations into immune-compromised NSG mice ( Figure 3f ) .", "12 weeks after transplantation , human multi-lineage engraftment was evident in the bone marrow of the transplanted mice of all four groups ( Figure 3g and Figure 3—figure supplement 3 ) .", "Collectively , these data show that human HSPCs that have undergone di-genic HR are not enriched for translocations , and maintain their multi-lineage colony forming capacity and long-term engraftment potential .", "We next tested if we could combine the di-genic and biallelic targeting approach to simultaneously target both alleles of ASXL1 ( GFP and mCherry ) as well as both alleles of RUNX1c ( BFP and E2-Crimson ) ( tetra-allelic ) ( for schematic see Figure 4—figure supplement 1a ) .", "Delivery of Cas9 RNPs targeting both genes followed by transduction of four rAAV6 donors gave rise to 1 . 1% GFPhigh/mCherryhigh/BFPhigh/E2Crimsonhigh quadruple-positive cells ( Figure 4a and Figure 4—figure supplement 1b–c ) .", "A similar quadruple-positive population was evident when targeting all four combined alleles of HBB and RUNX1c ( Figure 4—figure supplement 1e–h and Supplementary file 1e ) .", "Mixed , myeloid , and erythroid colonies were formed at frequency and ratio comparable to AAV only controls ( Figure 4b ) .", "Genotyping of colonies revealed on-target integration at both alleles at both loci in 78% of clones ( 73 clones analyzed ) ( Figure 4c ) .", "Flow-cytometric analysis of individual colonies confirmed expression of all four reporters ( BFP/GFP/mCherry/E2Crimson ) at high levels ( Figure 4—figure supplement 1d ) .", "The total number of genetic changes in this enriched population , which could be used for synthetic biology purposes is six: two endogenous genes inactivated ( both alleles of each gene ) plus the addition of four different transgenes ( represented in our experiment by four genes encoding different fluorescent proteins ) .", "Thus , this methodology could be used for studying interaction of genes that need both copies disrupted to lose function , such as tumor suppressor genes .", "Multi-genic HR in HSPCs would allow for the characterization of functional gene networks during human hematopoiesis ( Bystrykh et al . , 2005 ) .", "To validate that our methodology could multiplex HR in HSPCs in more than two genes simultaneously , we electroporated HSPCs with RNPs targeting HBB , CCR5 , and IL2RG , and then transduced them with gene-specific rAAV6 donors ( HBB-tdTomato , CCR5-tNGFR , IL2RG-GFP ) ( for schematic see Figure 4—figure supplement 2a ) .", "At day four post-electroporation , 4 . 1% of HSPCs were triple-positive ( Figure 4d and Figure 4—figure supplement 2b ) .", "‘In-Out PCR’ on gDNA from myeloid and erythroid colonies derived from this population showed that 78% ( 27 clones analyzed ) had an integration event at all 3 loci , indicating at least mono-allelic integrations at each targeted locus ( Figure 4e ) .", "Further analyses showed that 85% of these clones with tri-genic integrations were modified on all alleles either by biallelic integration or INDELs on the non-integrated allele that were mostly disruptive ( Supplementary file 1d ) .", "These data confirm that the methodology can efficiently enrich for HSPCs with multiplexed HR .", "Targeting at another combination of three genes ( RUNX1/HBB/ASXL1 ) showed 2 . 9% triple-positive cells ( Figure 4—figure supplement 2c–e ) , and collectively , tri-genic targeting experiments yielded an average of 4 . 5% triple-positive cells , with the highest frequency of 14% ( N = 5 ) ( Supplementary file 1e ) .", "To test if multiplexing HR caused cellular senescence or more cell death than mono or di-genic targeting in HSPCs , we evaluated cell death and apoptosis rates at day three post-targeting and proliferation for up to 10 days post-targeting ( corresponding to 7 days post-sorting ) .", "We observed similar proliferation rates comparing modified and unmodified cells ( data not shown ) and only a minor , non-statistically significant decrease in cell viability ( p=0 . 333 ) when targeting three genes compared to one ( Figure 4—figure supplement 3 ) .", "Finally , we targeted HSPCs for tetra-genic HR ( HBB , CCR5 , ASXL1 , RUNX1 ) and found after four days in culture that 1% of cells were reporterhigh positive for all four reporters ( Figure 4f ) .", "Targeting the same four genes with other combinations of reporter genes gave 0 . 41% and 0 . 78% tetra-genic targeting frequencies in the total cell population ( Supplementary file 1e ) .", "Strikingly , 41–71% of HSPCs with tri-genic HR had undergone tetra-genic HR , suggesting that HR events at different genes may not be independent of each other , in contrast to recent findings for multiplexed NHEJ ( Hultquist et al . , 2016 ) .", "Because rAAV vectors can be captured at DSBs via NHEJ ( Miller et al . , 2004 ) , we performed experiments that aimed to detect the frequency of capture events by including a non-homologous rAAV donor in targeting experiments .", "We found that 89–98% of reporterhigh cells were derived from on-target homologous recombination , confirming a relatively low rate of AAV capture ( Figure 4—figure supplement 4 ) ." ], [ "Table 1 summarizes the HR multiplex experiments ( seven total genes targeted ) and shows that by using Cas9 RNP , rAAV6 , and flow cytometry-based sorting , we can reproducibly generate HSPC populations that have undergone HR events at multiple loci .", "For synthetic biology purposes , the tetra-genic targeting method , for example , can generate an enriched population of cells with eight genetic modifications: the knockout of at least a single allele of four different genes while introducing four different transgenes ( in this proof-of-concept we used three fluorescent protein reporter genes and one biologically inert cell surface marker ( tNGFR ) that has been previously used in human clinical trials to track genetically modified hematopoietic stem cells over the course of decades ) .", "Our approach to studying gene function in human HSPCs has several advantages over lentiviral-based approaches because it enables: ( 1 ) multigenic targeted integration ( at least four genes ) , ( 2 ) enrichment of highly pure edited populations , ( 3 ) the ability to trace cells with a specific genotype , ( 4 ) enrichment of a population with biallelic targeting of at least two genes , and ( 5 ) fluorescent protein-based hematopoietic cell lineage tracing .", "Our methodology has the potential to advance the biological understanding of gene functions in canonical HSC processes , including self-renewal , differentiation , and engraftment , all of which are critical aspects of fundamental stem cell biology and may augment the efficacy of stem cell based therapeutics .", "By knocking in four different transgenes into four different genes , the method generates four gene disruptions and four gene additions .", "However , the use of multiple sgRNAs also increases the chances for off-target effects and chromosomal translocations .", "By looking for monocentric translocations between two genes ( HBB and AAVS1 ) , we observed low levels of translocation events similar to previously published studies ( Poirot et al . , 2015 ) .", "Such effects are likely sgRNA and target gene-specific and need to be assessed on a case-by-case basis .", "The observed tetra-genic targeting efficiencies at >0 . 5% are high enough to be experimentally useful , and though some applications may be restricted by HSPC source and starting cell numbers , our targeting methodology may be combined with recent advances in HSPC expansion protocols ( Fares et al . , 2014; Cutler et al . , 2013; de Lima et al . , 2012; Popat et al . , 2015 ) or with transplantation into a humanized bone marrow ossicle xenotransplantation model , which supports higher engraftment levels compared to a standard NSG model ( Reinisch et al . , 2016 ) .", "By using reporters as transgenes , one can both enrich and track the modified cells , and by using a transgene cassette in which a potentially biologically active transgene is linked through a 2A peptide or IRES to a reporter gene , one can enrich and track cells that could have up to four different new potentially bioactive genes expressed .", "Additionally , we and others have recently demonstrated the feasibility of knocking in a cDNA immediately after the start codon of the gene , thereby maintaining endogenous regulatory control over gene expression ( Dever et al . , 2016; Hubbard et al . , 2016; Voit et al . , 2014 ) .", "This provides a genetic engineering toolbox where different types of alleles ( WT , knockout , mutant cDNA forms ) are fluorescently tagged and can be enriched or tracked in a population with mixed allele combinations .", "One potential caveat is the requirement for reporter gene expression and the fact that cells must be cultured for 2–3 days until reporter gene expression is detectable and cells can be sorted .", "Even though we have not detected any obvious negative impact in this or previous studies ( Dever et al . , 2016; Bak and Porteus , 2017 ) , future studies may further investigate and optimize ex vivo culturing conditions , as well as promoter and reporter choice for minimal impact on biology and repopulation potential of edited HSPCs .", "Our methodology could be used for the characterization of gene interactions during blood and immune system disease pathogenesis .", "For example , functional knockouts can be created at one gene ( e . g . reporter knock-in into tumor suppressor gene ) , while introducing disease-causing polymorphisms at another gene ( cDNA expression cassette knock-in into proto-oncogene ) ( see Figure 4—figure supplement 5 for schematic ) .", "For example , Zhao et al . , showed that the loss of p53 cooperates with the KrasG12D mutation to promote acute myeloid leukemia ( AML ) in mouse HSPCs using a retroviral methodology ( Zhao et al . , 2010 ) .", "Our system could be used to address whether these findings can be translated to human HSPCs by achieving site specific HR that would simultaneously knock out a tumor suppressor ( e . g . TP53 ) and drive mutant KRAS under endogenous regulatory conditions , instead of using strong constitutive exogenous viral promoters with little control over proviral copy number and heterogeneity of transgene expression .", "However , in cDNA knock-in experiments , proper expression should always be validated since elements in the adjacent reporter expression cassette or the lack of UTRs and introns could influence cDNA expression ( Sweeney et al . , 2017 ) .", "We also show biallelic integration in primary human T cells at CCR5 , which could be therapeutically applicable for engineering HIV-resistance , where biallelic knockout of CCR5 could be combined with expression of different HIV restriction factors ( Voit et al . , 2013 ) .", "Additionally , this approach could be useful to extend recently published studies showing high potency of chimeric antigen receptors ( CARs ) that were site-specifically integrated into the TRAC gene using CRISPR and AAV6 in primary human T cells ( Eyquem et al . , 2017 ) .", "Multiplexed gene editing may be used to knock-in different CARs or co-stimulatory ligands into genes that are desirable to knock-out in CAR T cell therapy .", "We anticipate in the future that multiplexed HR mediated cell engineering will facilitate even more sophisticated uses of synthetic biology-based stem cell therapeutics than the examples we have given .", "Our methodology should also be widely applicable to other cell types of the hematopoietic system besides HSPCs and T cells , and even to cells of non-hematopoietic origin .", "In conclusion , we anticipate that this method will be applicable to studying human hematopoiesis and immune system disease pathogenesis through multiplexed , site-specific genome engineering by HR , which has the potential to lead to new discoveries in human hematopoietic stem cell biology ." ], [ "AAV vector plasmids were cloned in the pAAV-MCS plasmid ( Agilent Technologies , Santa Clara , CA ) containing ITRs from AAV serotype 2 ( AAV2 ) .", "CCR5 , IL2RG , HBB , RUNX1 , ASXL1 , and CXCL12 vectors contained an SFFV promoter , a reporter gene such as tNGFR , MaxGFP ( or Citrine ) , BFP , mCherry , tdTomato or E2Crimson and BGH polyA .", "MaxGFP and Citrine are referred to as GFP throughout .", "For translocation and NSG transplantation experiments , a UbC promoter ( approx . 1200 bp ) was used in the HBB donor instead of an SFFV promoter .", "For the T cell experiments , donors carried an EF1α promoter ( approx . 1200 bp ) .", "The homology arms for IL2RG , ASXL1 , and CCR5 were 800 bp , whereas left and right homology arms for HBB were 540 bp and 420 bp , respectively .", "The homology arms for RUNX1 , STAG2 , and AAVS1 were 400 bp .", "CCR5 donors used in T cell experiments expressed Citrine or mCherry from the PGK promoter and contained 400 bp homology arms .", "rAAV6 vectors were produced as described with a few modifications ( Khan et al . , 2011 ) .", "Briefly , 293FT cells ( Life Technologies , Carlsbad , CA , USA ) were seeded at 13 × 106 cells per dish in ten 15 cm dishes one day before transfection .", "Each 15 cm dish was transfected using standard PEI transfection with 6 μg ITR-containing plasmid and 22 μg pDGM6 ( gift from David Russell , University of Washington , Seattle , WA , USA ) , which contains the AAV6 cap genes , AAV2 rep genes , and adenovirus five helper genes .", "Cells were incubated for 72 hr until rAAV6 was harvested from cells by three freeze-thaw cycles followed by a 45 min incubation with TurboNuclease ( Abnova , Heidelberg , Germany ) or Benzonase ( Thermo Fisher ) at 250 U/mL .", "AAV vectors were purified on an iodixanol density gradient by ultracentrifugation at 48 , 000 rpm for 2 . 25 hr at 18°C .", "AAV vectors were extracted at the 58–40% iodixanol interface and dialyzed three times in PBS with 5% sorbitol in the last dialysis using a 10K MWCO Slide-A-Lyzer G2 Dialysis Cassette ( Thermo Fisher Scientific , Santa Clara , CA , USA ) .", "Vectors were added pluronic acid to a final concentration of 0 . 001% , aliquoted , and then stored at −80°C until further use .", "rAAV6 vectors were titered using quantitative PCR to measure number of vector genomes as described before ( Aurnhammer et al . , 2012 ) .", "Frozen CD34+ HSPCs derived from mobilized peripheral blood or cord blood were purchased from AllCells ( Alameda , CA , USA ) and thawed according to manufacturer’s instructions .", "Fresh CD34+ HSPCs from cord blood were acquired from donors under informed consent via the Binns Program for Cord Blood Research at Stanford University and used without freezing .", "Fresh CD34+ HSPCs from bone marrow were obtained from Stanford BMT Cell-Therapy Facility after informed consent .", "CD34+ cells were isolated using a human CD34 MicroBead Kit ( Miltenyi Biotec , San Diego , CA , USA ) .", "Generally , CB-derived HSPCs perform better in HR experiments .", "CD34+ HSPCs were cultured in stem cell retention media consisting of StemSpan SFEM II ( Stemcell Technologies , Vancouver , Canada ) supplemented with SCF ( 100 ng/ml ) , TPO ( 100 ng/ml ) , Flt3-Ligand ( 100 ng/ml ) , IL-6 ( 100 ng/ml ) , UM171 ( Stemcell Technologies ) ( 35 nM ) and StemRegenin1 ( 0 . 75 mM ) .", "Mycoplasma contamination testing was not performed .", "Cells were cultured at 37°C , 5% CO2 , and 5% O2 .", "Primary human CD3+ T cells were isolated from buffy coats obtained from the Stanford School of Medicine Blood Center using a human T Cell Isolation Kit ( Miltenyi ) according to manufacturer’s instructions .", "Cells were cultured in X-VIVO 15 ( Lonza , Walkersville , MD , USA ) containing 5% human serum ( Sigma-Aldrich , St . Louis , MO , USA ) , 100 IU/ml human rIL-2 ( Peprotech , Rocky Hill , NJ , USA ) and 10 ng/ml human rIL-7 ( BD Biosciences , San Jose , CA , USA ) .", "T cells were activated directly after isolation with immobilized anti-CD3 antibody ( clone: OKT3 , Tonbo Biosciences , San Diego , CA , USA ) and soluble anti-CD28 antibody ( clone: CD28 . 2 , Tonbo Biosciences ) for 72 hr .", "Mycoplasma contamination testing was not performed .", "T cells were cultured at 37°C , 5% CO2 , and ambient oxygen levels .", "All synthetic sgRNAs were purchased from TriLink BioTechnologies ( San Diego , CA , USA ) .", "sgRNAs were chemically modified with three terminal nucleotides at both the 5′ and 3′ ends containing 2′ O-Methyl 3′ phosphorothioate and HPLC-purified .", "The genomic sgRNA target sequences with PAM in bold ) were: HBB: 5’-CTTGCCCCACAGGGCAGTAACGG-3’ , CCR5: 5’-GCAGCATAGTGAGCCCAGAAGGG-3’ , IL2RG: 5’-TGGTAATGATGGCTTCAACATGG-3’ , RUNX1c: 5’-TACCCACAGTGCTTCATGAGAGG-3’ ASXL1: 5’-ACAGATTCTGCAGGTCATAGAGG-3’ , STAG2: 5’-AGTCCCACATGCTATCCACAAGG-3’ , AAVS1: 5’-GGGGCCACTAGGGACAGGATTGG-3’ .", "Cas9 protein was purchased from Life Technologies and Integrated DNA Technologies .", "Cas9 RNP was made by incubating protein with sgRNA at a molar ratio of 1:2 . 5 at 25°C for 10 min immediately prior to electroporation into CD34+ HSPCs or T cells .", "CD34+ HSPCs were electroporated 1–2 days after thawing or isolation .", "T cells were electroporated three days following activation .", "Both CD34+ HSPCs and T cells were electroporated using the Lonza Nucleofector 2b ( program U-014 ) or 4D ( program EO-100 ) ( we have not detected any device-specific differences in electroporation efficiencies ) and the Human T Cell Nucleofection Kit ( VPA-1002 , Lonza ) with the following conditions: 5 × 106 cells/ml , 150–300 µg/ml Cas9 protein complexed with sgRNA at 1:2 . 5 molar ratio .", "Following electroporation , cells were incubated for 15 min at 37°C after which they were added rAAV6 donor vectors ( generally at an MOI ( vector genomes/cell ) of 50 , 000–100 , 000 for each gene ) .", "A mock-electroporated control was included in most experiments where cells were handled the same and was electroporated in the same electroporation buffer , but without Cas9 RNP .", "For experiments targeting multiple loci , electroporation volume and cell numbers were kept the same as stated above , and 150–300 µg/ml Cas9 RNP and MOIs of 50 , 000–100 , 000 were used for each targeted locus , but with no more than a total of 60 ug Cas9 per electroporation and 200 , 000 vector genomes/cell .", "All AAV vectors were added simultaneously and directly to the cell culture after which the cells were transferred to the incubator without further manipulation .", "AAV volume was kept less than 20% of the total culturing volume and medium was either supplemented or replaced with fresh medium after overnight culture .", "Reporterhigh expression was measured by flow cytometric analyses after 3–4 days post-electroporation and transduction using gates for multiplexed targeted integration set so that ‘AAV only’ samples ( no nuclease ) were less than 1% since previous data ( not presented ) have shown that after ~14 days in culture the frequency of reporter+ cells ( from persistent episomal expression , random integration , and/or non-nuclease mediated HR ) is generally less than 1% .", "The truncated NGFR receptor ( tNGFR ) where the cytoplasmic intracellular signaling domain is removed and is signaling incompetent , solely served the purpose of a reporter for targeted CD34+ HSPCs in indicated experiments ( Bonini et al . , 2003 ) .", "Targeted integration of a tNGFR expression cassette was measured by flow cytometry of cells stained with APC-conjugated anti-human CD271 ( NGFR ) antibody ( clone: ME20 . 4 , BioLegend , San Diego , CA ) .", "For enriching of reporterhigh populations , cells were sorted on a FACS Aria II SORP using DAPI , PI ( both Thermo Fisher , 1 µg/ml ) or LIVE/DEAD Fixable Cell Stain Kit ( Life Technologies ) to discriminate live and dead cells according to manufacturer's instructions .", "Single reporterhigh cells were either single-cell sorted into 96-well plates ( Corning ) pre-filled with 100 µl of methylcellulose and water in the outer wells or plated at 500 cells per 6 cm dish with methylcellulose ( Methocult , StemCell Technologies ) .", "After 14 days , colonies were counted and scored as BFU-E , CFU-M , CFU-GM and CFU-GEMM according to the manual for ‘Human Colony-forming Unit ( CFU ) Assays Using MethoCult’ from StemCell Technologies and prior expertise ( Majeti et al . , 2007 ) .", "For DNA extraction from 96-well plates , PBS was added to wells with colonies , and the contents were mixed and transferred to a U-bottomed 96-well plate .", "From 6 cm dishes , colonies were picked and transferred to PBS .", "Cells were pelleted by centrifugation at 300xg for 5 min followed by a wash with PBS .", "Finally , cells were resuspended in 25 µl QuickExtract DNA Extraction Solution ( Epicentre , Madison , WI , USA ) and transferred to PCR plates , which were incubated at 65°C for 10 min followed by 100°C for 2 min .", "For CCR5 , a 3-primer PCR was set up with a forward primer binding in the left homology arm , a forward primer binding in the insert , and a reverse primer binding in CCR5 outside the right homology arm CCR5_inside_LHA: 5’-GCACAGGGTGGAACAAGATGG-3’ , CCR5_insert: 5’-AAGGGGGAGGATTGGGAAGAC-3’ , CCR5_outside_RHA: 5’-TCAAGAATCAGCAATTCTCTGAGGC-3’ .", "For all other genes , gene-specific integration was detected by ‘In-Out’ PCR using a primer that binds outside the homology arm ( HA ) and a primer specific for the transgene cassette ( insert ) .", "HBB_outside_LHA: GAAGATATGCTTAGAACCGAGG , HBB_insert: ACCGCAGATATCCTGTTTGG IL2RG_insert: 5’-GTACCAGCACGCCTTCAAGACC-3’ , IL2RG_outside_RHA: 5’-CAGATATCCAGAGCCTAGCCTCATC-3’ , RUNX1_outside_RHA: 5’- GAAGGGCATTGCTCAGAAAA-3’ , RUNX1_insert: 5’- AAGGGGGAGGATTGGGAAGAC-3’ , ASXL1_outside_RHA: 5’- AAGGGGGAGGATTGGGAAGAC-3’ , ASXL1_insert: 5’- CCTCCCAAGCTGGAACTACA-3’ .", "For detecting IL2RG non-integrated ( non_int ) alleles the following primers were used: IL2RG_non_int_fw: 5’-TCACACAGCACATATTTGCCACACCCTCTG-3′ , IL2RG_non_int_rv: 5′-TGCCCACATGATTGTAATGGCCAGTGG-3’ .", "For detecting dual integration of GFP and tdTomato into two HBB alleles , a primer in HBB outside the right homology arm was used together with either a GFP or tdTomato-specific primer: HBB_outside_RHA: 5’-GATCCTGAGACTTCCACACTGATGC-3’ , GFP: 5’-GTACCAGCACGCCTTCAAGACC-3’ , tdTomato: 5’-CGGCATGGACGAGCTGTACAAG-3’ .", "Clones with di-genic GFP ( HBB ) /mCherry ( CCR5 ) and tri-genic GFP ( IL2RG ) /tdTomato ( HBB ) /tNGFR ( CCR5 ) integrations were screened for integrations using the same primers as above .", "All integrated PCR bands were subjected to Sanger sequencing to confirm perfect HR at the intended locus .", "For flow-cytometric analysis of colonies generated from cells with quadruple-allelic HR , individual colonies were picked and directly resuspended in FACS buffer containing LIVE/DEAD staining solution ( LIVE/DEAD Fixable Near-IR Dead Cell Stain , Thermo ) .", "After 30 min incubation ( 4°C , dark ) cells were washed in FACS buffer and subjected to analysis .", "Dead cells were excluded from analysis based on APC-Cy7 positivity .", "6 to 8 week-old NOD scid gamma ( NSG ) mice were used ( Jackson laboratory , Bar Harbor , ME USA ) .", "The experimental protocol was approved by Stanford University’s Administrative Panel on Lab Animal Care ( IACUC 25065 ) .", "Four days after electroporation/transduction , different populations of live ( DAPI-negative ) targeted cells were sorted .", "Mock-treated cells were also sorted to control for the effect of the sorting procedure .", "Directly after sorting , cells were transplanted into one femur of sub-lethally irradiated mice ( 200 rad , 24 hr before transplant ) .", "Mice were randomly assigned to each experimental group and analyzed in a blinded fashion .", "12 weeks after transplantation , mice were sacrificed , mouse bone marrow ( BM ) was harvested from the transplanted femur by flushing .", "Non-specific antibody binding was blocked ( 10% vol/vol , TruStain FcX , BioLegend ) and cells were stained ( 30 min , 4°C , dark ) with monoclonal anti-human HLA-ABC APC-Cy7 ( W6/32 , BioLegend ) , anti-mouse CD45 . 1 PE-Cy7 ( A20 , eBioScience , San Diego , CA , USA ) , CD19 APC ( HIB19 , BD511 Biosciences ) , CD33 PE ( WM53 , BD Biosciences ) , and anti-mouse mTer119 PE-Cy5 ( TER-119 , BD Biosciences ) antibodies , and Propidium Iodide to detect dead cells .", "Human engraftment was defined as HLA-ABC+ cells .", "Genomic DNA was extracted from sorted populations using QuickExtract DNA Extraction Solution .", "For ddPCR quantification of translocations , ddPCR droplets were generated on a QX200 Droplet Generator ( Bio-Rad ) according to manufacturer’s protocol .", "Briefly , PCR reactions were set up in a 25 µL total volume per reaction with the ddPCR Supermix for Probes ( No dUTP ) ( Bio-Rad ) .", "A HEX reference assay detecting copy number input of the TERT gene was used to normalize for genomic DNA input ( Bio-Rad: saCP1000100 ) .", "A custom assay designed to detect the translocations between HBB and AAVS1 consisted of: Forward primer: 5’-TCAGGGCAGAGCCATCTATTGC-3’ , Reverse primer: 5’-CCAGATAAGGAATCTGCCTAACAGG-3' , 5'−6FAM/ZEN/3'-IBFQ-labeled Probe ( IDT ) : 5’-CTTCTGACACAACTGTGTTCACTAGCAACC-3’ .", "The translocation assay was used at a final concentration of 900 nM for each of the primers and a final concentration of 250 nM for the probe .", "20 µL of the PCR reaction was used for droplet generation , and 40 µL of the droplets was used in the following PCR conditions: 95° - 10 min , 50 cycles of 94° - 30 s , 57°C – 30 s , and 72° - 2 min , finalize with 98° - 10 min and 4°C until droplet analysis .", "Droplets were analyzed on a QX200 Droplet Reader ( Bio-Rad ) detecting FAM and HEX positive droplets .", "Control samples with non-template control ( H2O ) or genomic DNA from mock-electroporated samples were included in the entire process .", "Translocation frequencies were calculated as the translocation copy number per µL divided by the TERT copy number per µL .", "For sequencing of translocations , PCR products were generated using Phusion polymerase ( Fisher Scientific ) with the forward and reverse primers listed above for the translocation ddPCR assay .", "PCR amplicons were gel-purified and cloned into the pMiniT 2 . 0 plasmid using the NEB PCR Cloning Kit ( NEB ) according to manufacturer’s recommendations .", "Ligated plasmid reactions were transformed into XL-1 Blue competent cells , plated on ampicillin-containing agar plates , and single colonies were sequenced by MCLAB ( South San Francisco , CA , USA ) using rolling circle amplification followed by sequencing using the following primer: 5’-ACCTGCCAACCAAAGCGAGAAC-3’ .", "Modified cells were FACS-sorted into individual wells of a 96-well U bottom plate and expanded in HSPC retention media ( see above ) at a density of <100 , 000 cells per mL .", "To check viability and proliferation after multiplexed HR , cells from a single well were recovered and a known number of absolute counting beads ( CountBright beads , Invitrogen ) was added .", "Cells were stained with Ghost Dye Red 780 ( Tonbo Biosciences ) for 30 min at 4°C in the dark and analyzed on a FACS-Aria II without further manipulation to reduce potential cells loss .", "Viable cells were determined as GhostDye Red 780 negative and exact cell counts were assessed through concomitant acquisition of 10 , 000 beads .", "Cell counts were calculated based on ratio of beads to cells within the suspension ." ] ]
[ "Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system .", "Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells .", "We describe a method to multiplex homologous recombination ( HR ) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins ( RNPs ) .", "In addition , the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR .", "We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks , but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics ." ]
[ "Our DNA contains thousands of sections called genes that encode the information needed to make all the cells in the human body .", "To understand what the genes do and how they contribute to diseases , it is crucial for researchers to be able to switch individual genes on or off or make precise changes to the ‘letters’ in their code .", "Since most genes act in complicated networks it would be very useful to be able to edit several genes at the same time , especially when studying cancer and other diseases that are caused by defects in multiple genes .", "CRISPR/Cas9 is a relatively new technique that allows the code of individual genes to be precisely edited .", "To edit a gene , CRISPR/Cas9 first breaks the DNA at the site of interest and this break is subsequently repaired using new DNA templates that introduce the desired change in the code .", "In this way , the letters of the code can be changed with the same precision that one edits the letters and words of a document .", "This technique has been successfully used to edit the code of single genes , but it is much more difficult to use it to edit several genes at the same time .", "To import new DNA repair templates into human and other mammalian cells , researchers have used harmless virus-like particles called rAAV vectors .", "Researchers load the DNA templates into rAAV vectors , which are able to enter the cells and carry the templates to the DNA of the cells .", "Bak , Dever , Reinisch et al . combined CRISPR/Cas9 with rAAV template delivery to precisely edit several genes in human cells , including blood stem cells .", "In this new system , CRISPR/Cas9 directs the insertion of new pieces of DNA carried by rAAV6 vectors into specific genes .", "The system developed by Bak , Dever , Reinisch et al . allows several genes to be precisely edited at the same time .", "Furthermore , the system includes fluorescent markers that enable successfully edited cells to be identified and tracked .", "In the future , this technique could be used to study how genes work together to control various characteristics , and how cancer and other diseases develop ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Decreased microRNA levels lead to deleterious increases in neuronal M2 muscarinic receptors in Spinal Muscular Atrophy models
elife-20752-v1
[ [ "Spinal Muscular Atrophy ( SMA ) is an autosomal recessive neurodegenerative disease and the leading genetic cause of infant death in the US ( Cusin et al . , 2003; Pearn , 1978 ) .", "SMA is caused by homozygous deletion or mutation of the SMN1 ( Survival Motor Neuron 1 ) gene , resulting in reduced Survival of Motor Neuron ( SMN ) protein levels ( Lefebvre et al . , 1995 ) .", "SMN expression is ubiquitous , but particularly essential for motor neuron survival ( Lefebvre et al . , 1997 ) .", "Disease severity , as well as spinal cord α-MN dysfunction and degeneration , correlates with the extent of SMN loss ( Lefebvre et al . , 1997 ) .", "Understanding why SMN loss impairs function should offer insight into SMA and may reveal therapeutic targets .", "SMN is conserved across species ( Miguel-Aliaga et al . , 1999 ) .", "Studies of various SMA models suggest a role for SMN in several cellular processes including snRNP assembly ( Golembe et al . , 2005; Yong et al . , 2002 ) , messenger RNA ( mRNA ) transport ( Fallini et al . , 2011 ) , and local translation ( Dimitriadi et al . , 2010; Kye et al . , 2014 ) .", "SMN function , however , has not been linked definitively to MN degeneration or synaptic transmission defects caused by SMN loss .", "microRNAs ( miRNAs ) are non-coding RNAs that often repress protein translation , by a mechanism that requires miRNA binding to the 3’UTR of mRNA targets .", "Disruption of the miRNA pathway in spinal MNs leads to severe degeneration ( Haramati et al . , 2010 ) .", "SMN loss alters levels and/or activity of specific miRNAs ( Haramati et al . , 2010; Kye et al . , 2014; Valsecchi et al . , 2015; Wang et al . , 2014 ) , but the cellular mechanisms leading to altered miRNA expression and/or function are unknown .", "The RNA helicase Gemin3 associates with both SMN and RNA-induced silencing complex components ( Charroux et al . , 1999; Höck et al . , 2007; Hutvágner and Zamore , 2002; Meister et al . , 2005; Mourelatos et al . , 2002; Murashov et al . , 2007 ) .", "Gemin3 and SMN levels decrease concomitantly , suggestive of a functional link ( Feng et al . , 2005; Helmken et al . , 2003 ) .", "We took advantage of the C . elegans SMA model to examine the connection between SMN , Gemin3 , and miRNA function .", "SMN1 , Gemin3 , and multiple miRNA pathway components are conserved in C . elegans ( Grishok et al . , 2001; Miguel-Aliaga et al . , 1999; Minasaki et al . , 2009 ) .", "Loss-of-function ( lf ) mutations in smn-1 , the C . elegans ortholog of SMN1 , cause behavioral and morphological abnormalities , premature death , and sterility ( Briese et al . , 2009; Sleigh et al . , 2011 ) .", "smn-1 ( lf ) animals also have neuromuscular junction ( NMJ ) defects , suggesting a functional role for SMN-1 in MNs ( Briese et al . , 2009 ) .", "MNs in smn-1 ( lf ) animals do not die , likely because of their short lifespan .", "However , smn-1 ( lf ) neuromuscular defects may correspond to the early stages of SMA pathogenesis , characterized by NMJ dysfunction prior to MN degeneration ( Miguel-Aliaga et al . , 1999; Yoshida et al . , 2015 ) .", "We find that the C . elegans Gemin3 ortholog , MEL-46 , is perturbed by SMN-1 loss , impacting miR-2 suppression of the M2 muscarinic receptor ortholog , GAR-2 ( Lee et al . , 2000 ) .", "Across species in SMA mouse models , we find decreased levels of miR-128 , a potential miR-2 ortholog , and increased expression of the GAR-2 ortholog , m2R .", "Notably , m2R inhibition ameliorates axon outgrowth defects in MNs from a SMA mouse model , consistent with our results in C . elegans ." ], [ "The C . elegans Gemin3 ortholog is MEL-46 .", "Homozygous loss of smn-1 or mel-46 results in lethality ( Briese et al . , 2009; Miguel-Aliaga et al . , 1999 ) , but maternal loading of smn-1 or mel-46 mRNA and protein allows many homozygous , loss of function animals to survive into the last larval stage , called L4 ( Miguel-Aliaga et al . , 1999; Minasaki et al . , 2009 ) .", "Loss of smn-1 results in neuromuscular defects including decreased pharyngeal pumping rates , followed by overtly altered locomotion and subsequent death ( Briese et al . , 2009 ) .", "Like smn-1 loss in L4 stage animals , we found that mel-46 ( tm1739 ) homozygous loss of function animals had severely decreased pharyngeal pumping rates .", "Pharyngeal pumping was restored to normal rates in mel-46 ( tm1739 ) animals using a previously described , broadly expressed mel-46 rescue array ( also referred to as [mel-46 ( + ) #1] ) , which utilizes the mel-46 promoter ( Figure 1A ) ( Minasaki et al . , 2009 ) .", "mel-46 partial loss of function alleles , yt5 and ok3760 , also caused pumping defects as did global mel-46 RNA interference ( RNAi ) or cholinergic neuron-specific mel-46 ( RNAi ) ( Figure 1—figure supplement 1A–E ) .", "We conclude that MEL-46 is necessary for normal neuromuscular function . 10 . 7554/eLife . 20752 . 003Figure 1 . Decreased MEL-46 function in C . elegans results in defective NMJ signaling .", "( a ) mel-46 ( tm1739 ) animals had reduced pharyngeal pumping rates versus wild type ( N2 ) control animals .", "Defects were fully rescued by global expression of MEL-46 behind its own promoter ( [mel-46 ( + ) #1] ) .", "Mean ± SEM; Mann-Whitney U-test , two-tailed .", "( b ) mel-46 ( tm1739 ) animals paralyzed more slowly when exposed to aldicarb , an acetylcholinesterase inhibitor .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mel-46 ( tm1739 ) , and mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] early larval stage L4 animals .", "Reintroduction of mel-46 restored normal aldicarb sensitivity .", "Log-rank test .", "( c ) Cholinergic neuron-specific mel-46 ( RNAi ) causes resistance to aldicarb .", "Time course for paralysis on 1 . 5 mM aldicarb for empty ( RNAi ) , smn-1 ( RNAi ) , mel-46 ( RNAi ) , and goa-1 ( RNAi ) young adult animals .", "Animals sensitive to RNAi in only cholinergic neurons ( XE1581 ) were fed bacteria expressing double-stranded RNA ( dsRNA ) against mel-46 , smn-1 , or goa-1 ( positive control ) .", "Control animals were fed bacteria expressing an empty vector control: empty ( RNAi ) .", "Data set previously published without mel-46 ( RNAi ) ( Dimitriadi et al . , 2016 ) .", "Log-rank test .", "( d ) mel-46 ( tm1739 ) animals had reduced RFP::SNB-1 ( synaptobrevin ) .", "Percent change from wild type ( N2 ) control for RFP::SNB-1 in the dorsal cord of mel-46 ( tm1739 ) and mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] animals for ‘punctaanalyzer’ parameters: puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "Asterisks denote significance compared to wild type; shading indicates significant change for mel-46 ( tm1739 ) versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] .", "Mann-Whitney U-test , two-tailed .", "Expression of mel-46 rescued RFP::SNB-1 puncta width defects in mel-46 ( tm1739 ) animals ( wild type versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] p=0 . 82 ) ; mel-46 ( tm1739 ) versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] p=0 . 03 ) , rescued SNB-1 puncta intensity defects ( wild type versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] p=0 . 85; mel-46 ( tm1739 ) versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] p=0 . 005 ) and partially ameliorated SNB-1 puncta linear density defects ( wild type versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] p=0 . 0004 ) ; mel-46 ( tm1739 ) versus mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] p=0 . 0001 ) .", "( h–j )", "Representative images of RFP::SNB-1 expressed in the dorsal cord of cholinergic DA MNs for wild type , mel-46 ( tm1739 ) , and mel-46 ( tm1739 ) ;[mel-46 ( + ) #1] animals .", "These images were taken as part of data collection .", "Scale bar , 5 μm .", "For statistical analyses in all figures: *p≤0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "A helpful summary of C . elegans phenotypes reported throughout this article for selected loss of function alleles can be found in Supplementary file 1 .", "Additional information on C . elegans strains used in Figures 1–6 is provided in Supplementary file 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 00310 . 7554/eLife . 20752 . 004Figure 1—source data 1 . Raw Data for Figure 1—figure supplement 2 . Raw data and statistical analysis that correspond to RFP::SNB-1 localization analysis in Figure 1—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 00410 . 7554/eLife . 20752 . 005Figure 1—figure supplement 1 . MEL-46 ( Gemin3 ) is necessary for proper NMJ function .", "( a ) Schematic representation of the predicted mel-46 gene .", "Large arrow indicates the direction of translation .", "Also shown are the positions of the yt5 G to A transition , the tm1739 deletion and the ok3760 complex substitution , for which the inserted sequence is indicated ( Minasaki et al . , 2009 ) .", "( b ) mel-46 ( yt5 ) animals had reduced pharyngeal pumping rates versus wild type ( N2 ) control .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( c ) mel-46 ( ok3760 ) animals had reduced pharyngeal pumping rates versus wild type ( N2 ) control .", "Mean ± SEM; t-test , two tailed .", "This data was collected alongside data in Figure 1A .", "( d ) Animals sensitive to RNAi in all tissues ( KP3948 ) fed bacteria expressing double-stranded RNA ( dsRNA ) against mel-46 had reduced pharyngeal pumping rates versus control animals fed bacteria expressing an empty vector control .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( e ) Animals sensitive to RNAi in only cholinergic neurons ( XE1581 ) fed bacteria expressing double-stranded RNA ( dsRNA ) against mel-46 had reduced pharyngeal pumping rates versus control animals fed bacteria expressing an empty vector control .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( f ) mel-46 ( yt5 ) animals were resistant to the acetylcholinesterase inhibitor , aldicarb .", "Broad expression of MEL-46 with [mel-46 ( + ) #3] , which uses the mel-46 promoter , did not significantly restore mel-46 ( yt5 ) aldicarb resistance .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mel-46 ( yt5 ) , and mel-46 ( yt5 ) ;[mel-46 ( + ) #3] early larval stage L4 animals .", "Log-rank test .", "( g ) mel-46 ( ok3760 ) animals were resistant to the acetylcholinesterase inhibitor , aldicarb .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) and mel-46 ( ok3760 ) young adult animals .", "Log-rank test .", "This data was collected alongside data in Figure 1B .", "( h ) GABA neuron-specific mel-46 RNAi or smn-1 RNAi results in aldicarb hypersensitivity .", "Time course for paralysis on 1 mM aldicarb for empty ( RNAi ) , smn-1 ( RNAi ) , mel-46 ( RNAi ) , and unc-25 ( RNAi ) animals .", "Animals sensitive to RNAi in only GABAergic neurons ( XE1375 ) were fed bacteria expressing double-stranded RNA ( dsRNA ) against mel-46 , smn-1 , or unc-25 ( positive control ) .", "Control animals were fed bacteria expressing an empty vector control: empty ( RNAi ) .", "Log-rank test .", "For all statistical analyses in figure supplements: *p≤0 . 05 , **p<0 . 01 , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 00510 . 7554/eLife . 20752 . 006Figure 1—figure supplement 1—source data 1 . Raw Data for Figure 1—figure supplement 1 . Raw data and statistical analysis that correspond to pumping and aldicarb resistance assays in Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 00610 . 7554/eLife . 20752 . 007Figure 1—figure supplement 2 . MEL-46 ( Gemin3 ) loss causes increased APT-4 ( AP2 α-adaptin ) linear density .", "( a ) mel-46 ( tm1739 ) animals had increased APT-4 ( AP2 α-adaptin ) linear density .", "Percent change from wild type control for APT-4 in the dorsal nerve cord of mel-46 ( tm1739 ) animals for ‘punctaanalyzer’ parameters: puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "Mann-Whitney U-test , two-tailed .", "( b–c )", "Representative images of APT-4::GFP in the dorsal nerve cord of cholinergic DA MNs of wild type and mel-46 ( tm1739 ) animals .", "These images were taken as part of data collection .", "Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 00710 . 7554/eLife . 20752 . 008Figure 1—figure supplement 2—source data 1 . Raw Data for Figure 1—figure supplement 2 . Raw data and statistical analysis that correspond to RFP::SNB-1 localization analysis in Figure 1—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 008 SMN-1 is required for normal NMJ function in C . elegans cholinergic MNs ( Dimitriadi et al . , 2016 ) .", "Aldicarb is an acetylcholinesterase inhibitor that leads to acetylcholine accumulation in the NMJ and consequently , paralysis ( Mahoney et al . , 2006 ) .", "The time course of aldicarb-induced paralysis was slowed by decreased SMN-1 activity ( Dimitriadi et al . , 2016 ) .", "We tested if a decrease in MEL-46 function causes similar resistance to aldicarb and found that mel-46 loss of function resulted in aldicarb resistance across multiple alleles ( Figure 1B; Figure 1—figure supplement 1F and G ) , reminiscent of smn-1 loss .", "Reintroduction of mel-46 using the [mel-46 ( + ) #1] rescue array restored aldicarb sensitivity in mel-46 ( tm1739 ) animals .", "Tissue-specific knock-down of mel-46 in cholinergic neurons resulted in aldicarb resistance , thus confirming that MEL-46 function is required in cholinergic neurons , as is SMN-1 ( Figure 1C ) ( Dimitriadi et al . , 2016 ) .", "We also showed that knock-down of mel-46 or smn-1 in inhibitory GABAergic neurons resulted in aldicarb hypersensitivity ( Figure 1—figure supplement 1H ) .", "Our findings , taken together with previous work , suggest that MEL-46 and SMN-1 are required in both cholinergic and GABAergic neurons for normal NMJ function .", "smn-1 loss causes changes in presynaptic protein localization ( Dimitriadi et al . , 2016 ) .", "Do similar changes occur in mel-46 ( tm1739 ) animals ?", "We evaluated localization of presynaptic proteins SNB-1 ( synaptobrevin ) and APT-4 ( AP2 α-adaptin ) in cholinergic dorsal A-type ( DA ) MNs of mel-46 ( tm1739 ) animals ( Ch'ng et al . , 2008; Sieburth et al . , 2005 ) .", "SNB-1 is a v-SNARE protein required for SV exocytosis , while APT-4 associates with clathrin-coated endocytic vesicles ( Kamikura and Cooper , 2006; Nonet et al . , 1998 ) .", "In the dorsal cord , cholinergic DA MNs do not have presynaptic inputs; they form en passant presynaptic connections in a punctate pattern ( Ch'ng et al . , 2008; White et al . , 1976 ) .", "Three parameters were measured to evaluate fluorescently labeled SNB-1 and APT-4 localization to presumptive synapses: puncta width ( μm ) , intensity ( AU ) , and linear density ( puncta/μm ) , as previously described ( Kim et al . , 2008 ) .", "Loss of smn-1 or mel-46 resulted in similar SNB-1 synaptic localization defects: decreased SNB-1 puncta width , intensity and linear density ( Figure 1D–G ) ( Dimitriadi et al . , 2016 ) .", "Loss of smn-1 leads to decreased APT-4 puncta width and intensity , but increased linear density ( Dimitriadi et al . , 2016 ) .", "mel-46 ( tm1739 ) animals also had increased APT-4 linear density , but no changes in puncta width or intensity compared to controls ( Figure 1—figure supplement 2A–C ) .", "Therefore , decreased mel-46 causes synaptic protein defects that overlap partially with defects observed when SMN-1 levels decrease .", "Given the similarities between SMN-1 and MEL-46 loss in aldicarb resistance , decreased pharyngeal pumping rates , and defective synaptic protein localization , we decided to explore whether SMN-1 and MEL-46 act in common pathways required for NMJ function .", "MEL-46 might act together with or downstream of SMN-1 in pathways necessary for NMJ function .", "To test these and other possibilities , we generated integrated multicopy transgenic lines expressing GFP-tagged MEL-46 expressed under control of the unc-17 cholinergic-specific promoter ( Figure 2A ) .", "MEL-46::GFP was found in both the cell bodies and processes of neurons .", "No obvious changes were seen in cytoplasmic MEL-46::GFP , leading us to evaluate localization of MEL-46::GFP in MN dorsal cord processes in smn-1 ( ok355 ) animals .", "Because ok355 deletion in smn-1 leads to a complete loss of function , smn-1 ( ok355 ) animals were maintained over an hT2 balancer and sterile smn-1 ( ok355 ) homozygous progeny carry some maternally-loaded SMN-1 protein ( Briese et al . , 2009 ) .", "We found that MEL-46::GFP localizes to small granular structures in dorsal cord processes in control ( smn-1 ( + ) ) and smn-1 ( ok355 ) animals .", "Our finding is consistent with previous work showing that Gemin3 localizes to granular structures in mammalian neurites; Gemin3 co-localizes with SMN in 50–60% of these granules , along with multiple mRNAs ( Todd et al . , 2010a , 2010b; Zhang et al . , 2006 ) .", "In smn-1 ( ok355 ) animals , we found that the density of MEL-46::GFP-positive granular structures was doubled compared to smn-1 ( + ) controls ( Figure 2B–D ) .", "Furthermore , the mean intensity of MEL-46::GFP fluorescence and the maximum fluorescence for each sample were decreased in smn-1 ( ok355 ) animals ( Figure 2B–D; Figure 2—figure supplement 1A ) .", "These results suggest that decreased SMN-1 leads to MEL-46 mislocalization in cholinergic MN processes and diminished MEL-46 levels in granules .", "Our findings suggest that SMN-1 impairs MEL-46 function , which could contribute to smn-1 ( ok355 ) synaptic defects ( Dimitriadi et al . , 2016 ) .", "To test this hypothesis , we increased mel-46 gene dosage in smn-1 ( ok355 ) animals using the [mel-46 ( + ) #1] rescue array and showed that this ameliorated smn-1 ( ok355 ) aldicarb resistance defects ( Figure 2E ) .", "We also showed that increasing mel-46 specifically in cholinergic neurons , using the cholinergic-specific unc-17 ( ACh ) promoter in an integrated array , referred to as [ACh::mel-46::GFP] , rescued smn-1 ( ok355 ) aldicarb resistance ( Figure 2—figure supplement 1B and C ) .", "The aldicarb resistance observed with broad expression of mel-46 in control animals ( Figure 2E ) was not observed when we overexpressed mel-46 in cholinergic neurons only .", "Under this condition we observed mild hypersensitivity in one integrated line ( referred to as [ACh::mel-46::GFP#1] ) ( Figure 2—figure supplement 1C ) and no difference from control animals in a second line ( referred to as [ACh::mel-46::GFP#2] ) ( Figure 2—figure supplement 1B ) .", "It is possible that high levels of MEL-46 in cholinergic neurons cause aldicarb hypersensitivity , whereas broad overexpression of MEL-46 may impact NMJ function independent of cholinergic neurons .", "Taken together , our results suggest that loss of SMN-1 negatively impacts MEL-46 function , resulting in perturbed NMJ signaling .", "Our finding is consistent with observations in humans that reduced human SMN levels result in Gemin3 downregulation ( Feng et al . , 2005; Helmken et al . , 2003 ) , 10 . 7554/eLife . 20752 . 009Figure 2 . MEL-46 localization and levels are perturbed in smn-1 ( lf ) animals .", "( a ) Illustration: mel-46 was tagged with GFP at the C-terminus and expression was driven by the cholinergic ( ACh ) unc-17 promoter .", "Two lines were generated by UV integration .", "( b ) smn-1 ( ok355 ) animals exhibited mislocalization and reduction of MEL-46::GFP in dorsal cord processes of cholinergic neurons .", "MEL-46::GFP localizes to granular punctate structures in dorsal cord processes .", "Percent change from smn-1 ( + ) control for MEL-46::GFP in the dorsal cord of smn-1 ( ok355 ) animals for ImageJ parameters: puncta density ( puncta/area ) , puncta intensity ( AU ) , and puncta size ( pixels/puncta ) .", "The ImageJ analysis was used instead of the ‘punctaanalyzer’ program since MEL-46::GFP had a scattered non-linear pattern in smn-1 ( ok355 ) animals; a linear pattern is necessary for accurate ‘punctaanalyzer’ analysis .", "Asterisks denote significance compared to wild type .", "Mann-Whitney U-test , two-tailed .", "( c–d )", "Representative images of MEL-46::GFP in dorsal cord cholinergic DA MN processes for control smn-1 ( + ) and smn-1 ( ok355 ) animals .", "These images were taken as part of data collection .", "Scale bar , 5 μm .", "( e ) Increasing expression of mel-46 rescued smn-1 ( ok355 ) aldicarb response defects .", "Time course for paralysis on 1 mM aldicarb for smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[mel-46 ( + ) #1] , and smn-1 ( + ) ;[mel-46 ( + ) #1] early larval stage L4 animals .", "smn-1 ( + ) ;[mel-46 ( + ) #1] animals were resistant to paralysis by aldicarb .", "Log-rank test .", "( f ) Increasing mel-46 rescued smn-1 ( ok355 ) RFP::SNB-1 synaptic localization defects .", "Percent change from smn-1 ( + ) control for RFP::SNB-1 in the dorsal cord of smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[mel-46 ( + ) #1] , and smn-1 ( + ) ;[mel-46 ( + ) #1] animals for ‘punctaanalyzer’ parameters: puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "Asterisks denote significance compared to smn-1 ( + ) control; shading indicates significant difference from smn-1 ( ok355 ) ;[mel-46 ( + ) #1] .", "Mann-Whitney U-test , two-tailed .", "Expression of mel-46 restored RFP::SNB-1 puncta width defects ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;[mel-46 ( + ) #1] p=0 . 05; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;[mel-46 ( + ) #1] p=0 . 001 ) , rescued SNB-1 puncta intensity defects ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;[mel-46 ( + ) #1] p=0 . 035; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;[mel-46 ( + ) #1] p=0 . 0004 ) , but did not rescue SNB-1 puncta linear density defects ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;[mel-46 ( + ) #1] p=0 . 036; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;[mel-46 ( + ) #1] p=0 . 19 ) .", "( G–J )", "Representative images of cholinergic DA MN RFP::SNB-1 in the dorsal nerve cord of smn-1 ( + ) , smn-1 ( ok355 ) and smn-1 ( ok355 ) ;[mel-46 ( + ) #1] .", "These images were taken as part of data collection .", "Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 00910 . 7554/eLife . 20752 . 010Figure 2—source data 1 . Raw Data for Figure 2 . Raw data and statistical analysis that correspond to MEL-46::GFP localization , aldicarb resistance assays , and RFP::SNB-1 localization analysis in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01010 . 7554/eLife . 20752 . 011Figure 2—figure supplement 1 . Expressing mel-46 restores neuronal defects in smn-1 ( lf ) animals .", "( a ) Decreased SMN-1 resulted in a 21% decrease in maximum MEL-46::GFP fluorescence in dorsal cord DA motor neuron processes .", "Histogram of maximum fluorescence ( AU ) for smn-1 ( + ) and smn-1 ( ok355 ) animals .", "t-test .", "p<0 . 01 .", "( b ) Increasing cholinergic expression using the unc-17 ( ACh ) promoter of mel-46 partially rescued smn-1 ( ok355 ) aldicarb response defects .", "Time course for paralysis on 1 mM aldicarb for smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[ACh::mel-46::GFP #1] , and smn-1 ( + ) ;[ACh::mel-46::GFP #1] early larval stage L4 animals .", "Log-rank test .", "( c ) Increasing cholinergic expression of mel-46 resulted in rescue of smn-1 ( ok355 ) aldicarb response defects .", "Time course for paralysis on 1 mM aldicarb for smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[ACh::mel-46::GFP #1] , and smn-1 ( + ) ;[ACh::mel-46::GFP #1] early larval stage L4 animals .", "smn-1 ( + ) ;[ACh::mel-46::GFP #1] were hypersensitive to paralysis by aldicarb .", "Log-rank test .", "( c–d )", "The same MEL-46::GFP integrated lines were used to evaluate levels and localization in Figure 2B–D .", "Despite rescue smn-1 ( ok355 ) aldicarb resistance , these animals expressing cholinergic MEL-46::GFP still exhibit MEL-46 mislocalization likely leading to decreased function .", "This rescue may occur because enough MEL-46::GFP is expressed to overcome mislocalization-related functional deficits .", "( d ) smn-1 ( ok355 ) animals had reduced pharyngeal pumping rates versus smn-1 ( + ) control animals; defects were not rescued by increasing mel-46 using the [mel-46 ) + ) #1] array .", "smn-1 ( + ) ; [mel-46 ( + ) #1] animals had pharyngeal pumping rates indistinguishable from smn-1 ( + ) controls .", "Mean ± SEM; Mann-Whitney U-test , two-tailed .", "( e ) Broad expression of MEL-46 using the mel-46 promoter ameliorated the APT-4 ( AP2 α-adaptin ) linear density defect in smn-1 ( ok355 ) animals .", "Percent change from smn-1 ( + ) control for APT-4 in the dorsal cord of smn-1 ( ok355 ) and smn-1 ( ok355 ) ;[mel-46 ( + ) #2] animals for ‘punctaanalyzer’ parameters: puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "Asterisks denote significance versus smn-1 ( + ) control; shading indicates smn-1 ( ok355 ) is significantly different from smn-1 ( ok355 ) ;[mel-46 ( + ) #2] .", "Mann-Whitney U-test , two-tailed .", "Expression of MEL-46 did not rescue APT-4 puncta width ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;[mel-46 ( + ) #2] p=0 . 006; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;[mel-46 ( + ) #2] p=0 . 48 ) , did not rescue APT-4 total puncta intensity ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;[mel-46 ( + ) #2] p=0 . 002; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;[mel-46 ( + ) #2] p=0 . 27 ) , but ameliorated APT-4 puncta linear density ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;[mel-46 ( + ) #2] p=0 . 93; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;[mel-46 ( + ) #2] p=0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01110 . 7554/eLife . 20752 . 012Figure 2—figure Supplement 1—source data 1 . Raw Data for Figure 2—figure supplement 1 . Raw data and statistical analysis that correspond to MEL-46::GFP localization , APT-4::GFP localization analysis , aldicarb resistance and pumping assays in Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01210 . 7554/eLife . 20752 . 013Figure 2—figure supplement 2 . Expressing mel-46 does not increase SMN-1 levels .", "( a ) Illustration: smn-1 ( rt280 ) was generated by CRISPR-mediated insertion of GFP upstream of smn-1 exon 1 .", "( b ) Increasing MEL-46 expression using the [mel-46 ( + ) #2] array led to decreased GFP::SMN-1 fluorescence .", "Quantification of mean smn-1 ( rt280 ) GFP fluorescence in wild type and [mel-46 ( + ) #2] backgrounds .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( c ) Representative images of smn-1 ( rt280 ) GFP expression in wild type and [mel-46 ( + ) #2] larval stage L4 animals .", "Scale bar , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01310 . 7554/eLife . 20752 . 014Figure 2—figure Supplement 2—source data 1 . Raw Data for Figure 2—figure supplement 2 . Raw data and statistical analysis that correspond to GFP::SMN-1 quantification in Figure 2—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 014 The interdependency we report , between SMN-1 and MEL-46 levels , may be specific to particular tissues and/or neural circuits .", "For example , pharyngeal pumping rate defects were not ameliorated in smn-1 ( ok355 ) animals by increased mel-46 levels ( [mel-46 ( + ) #1] rescue array , Figure 2—figure supplement 1D ) , suggesting a privileged relationship between SMN-1 and MEL-46 in cholinergic NMJ signaling .", "Increasing mel-46 did rescue smn-1 ( ok355 ) synaptic protein localization defects .", "Using the [mel-46 ( + ) #1] rescue array , we rescued smn-1 ( ok355 ) defective SNB-1 puncta width and intensity to normal levels , but did not ameliorate linear density defects ( Figure 2F–J ) .", "Notably , increased mel-46 in an smn-1 ( + ) control background did not increase SNB-1 levels , suggesting that mel-46-induced up-regulation of SNB-1 is specifically beneficial in a smn-1 ( ok355 ) background .", "Increasing mel-46 with a second broadly-expressed mel-46 rescue array line , [mel-46 ( + ) #2] , also restored APT-4 puncta linear density to normal levels ( Figure 2—figure supplement 1E ) , without rescuing other metrics .", "These results are consistent with the aldicarb/NMJ functional rescue studies presented here and suggest increasing mel-46 improves neuromuscular signaling in smn-1 ( ok355 ) animals by partially restoring levels and localization of synaptic proteins .", "Furthermore , these results suggest that mel-46 may act with or downstream of smn-1 in a pathway essential for NMJ function .", "Alternatively , we considered the possibility that increasing mel-46 stabilizes maternally-loaded SMN-1 protein and mRNA , a scenario which could also explain mel-46 rescue of smn-1 ( ok355 ) NMJ function .", "Since mammalian Gemin3 directly binds SMN ( Charroux et al . , 1999 ) , increasing MEL-46 ( Gemin3 ) might decrease the rate of SMN-1 loss .", "To test this possibility , we used CRISPR/Cas9-targeted genome editing to insert GFP coding sequences at the N-terminus of smn-1 on chromosome I , resulting in fluorescent SMN-1 protein ( Figure 2—figure supplement 2A ) ( Dickinson et al . , 2015 ) .", "The GFP-tagged protein was functional; animals were viable and fertile .", "We found that increasing mel-46 using the [mel-46 ( + ) #2] rescue array did not increase GFP::SMN-1 levels , but unexpectedly caused a modest overall decrease ( Figure 2—figure supplement 2B and C ) .", "It therefore seems unlikely that smn-1 ( ok355 ) rescue by increased mel-46 is due to stabilization of maternally-loaded SMN-1 .", "Using the same CRISPR-based method , we were unable to tag endogenous smn-1 on balancer chromosomes necessary for maintaining the smn-1 ( ok355 ) line .", "This approach would have allowed us to examine the effects of MEL-46 overexpression on the stability of maternally-loaded SMN-1 in smn-1 ( ok355 ) animals .", "Therefore , although we cannot rule out stability of maternally-loaded SMN-1 as a contributing factor , the large effect that decreased SMN-1 has on MEL-46 localization favors a mechanism in which MEL-46 overexpression rescues smn-1 ( ok355 ) defects , at least in part , by restoring MEL-46 functional deficits in this background .", "The pathways in MNs downstream of SMN and Gemin3 that are linked to SMA are unknown .", "Here , we consider a role for these two proteins in miRNA regulation .", "As mammalian Gemin3 co-localizes and co-purifies with RISC pathway components ( Höck et al . , 2007; Hutvágner and Zamore , 2002; Meister et al . , 2005; Mourelatos et al . , 2002; Murashov et al . , 2007 ) , we considered a role for miRNA regulation in NMJ function .", "miRNA miR-2 is enriched in neurons , expressed at all developmental stages , and predicted to regulate expression of many proteins involved in neuronal development and function ( Marco et al . , 2012; Martinez et al . , 2008 ) .", "We hypothesized that miR-2 is necessary for proper NMJ function and that it might be perturbed by loss of either SMN-1 or MEL-46 .", "To test this possibility , we first examined the aldicarb response of mir-2 ( lf ) animals .", "Two different deletion alleles , gk259 and n4108 , caused resistance to aldicarb paralysis compared to wild type animals ( Figure 3A; Figure 3—figure supplement 1A ) .", "This defect was partially rescued by expressing miR-2 under the control of a the unc-17 ( ACh ) cholinergic neuron-specific promoter ( referred to as ( [ACh::mir-2 ( + ) ] ) ( Figure 3A ) .", "Loss of miR-2 also resulted in a mild pharyngeal pumping defect ( Figure 3—figure supplement 1B ) .", "Taken together , we conclude that miR-2 is required in cholinergic neurons for proper NMJ signaling . 10 . 7554/eLife . 20752 . 015Figure 3 . miR-2 is required in cholinergic neurons for proper NMJ function .", "( a ) mir-2 ( gk259 ) animals were resistant to paralysis by aldicarb .", "Expression of miR-2 behind the unc-17 ( ACh ) cholinergic promoter partially restored mir-2 ( gk259 ) sensitivity to aldicarb compared to transgenesis controls expressing GFP behind the same promoter .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mir-2 ( gk259 ) , mir-2 ( gk259 ) ;[ACh::mir-2 ( + ) ] and mir-2 ( gk259 ) ;[ACh::GFP] young adult animals .", "Log-rank test .", "( b ) mir-2 ( gk259 ) animals had increased RFP::SNB-1 ( synaptobrevin ) linear density .", "Percent change from wild type ( N2 ) control for RFP::SNB-1 in the dorsal nerve cord of mir-2 ( gk259 ) animals for ‘punctaanalyzer’ parameters: puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "t-test , two-tailed .", "( c–d )", "Representative images of cholinergic DA MN RFP::SNB-1 in the dorsal cord of wild type and mir-2 ( gk259 ) animals .", "These images were taken as part of data collection .", "Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01510 . 7554/eLife . 20752 . 016Figure 3—source data 1 . Raw Data for Figure 3 . Raw data and statistical analysis that correspond to aldicarb resistance assays and RFP::SNB-1 localization analysis in Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01610 . 7554/eLife . 20752 . 017Figure 3—figure supplement 1 . miR-2 is required for NMJ function .", "( a ) mir-2 ( n4108 ) animals were resistant to the acetylcholinesterase inhibitor , aldicarb .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) and mir-2 ( n4108 ) young adult animals .", "Log-rank test .", "( b ) mir-2 ( gk259 ) animals had reduced pharyngeal pumping rates versus wild type control .", "Mean ± SEM; t-test , two-tailed .", "( c ) mir-2 ( gk259 ) SYD-2 ( α-liprin ) levels were indistinguishable from wild type ( N2 ) .", "Percent change from wild type control for SYD-2 in the dorsal nerve cord of mir-2 ( gk259 ) animals for ‘punctaanalyzer’ parameters: average puncta width ( μm ) , total intensity ( AU ) , and linear density ( number/μm ) .", "t-test , two-tailed .", "( d ) mir-2 ( lf ) had reduced expression of ITSN-1 ( DAP160/Intersectin ) .", "Percent change from wild type ( N2 ) control for ITSN-1 in the dorsal nerve cord of mir-2 ( gk259 ) and mir-2 ( n4108 ) animals as above .", "t-test , two-tailed .", "( e–f )", "Representative images of ITSN-1::GFP expressed in the dorsal nerve cord of cholinergic DA motor neurons for wild type and mir-2 ( n4108 ) animals .", "These images were taken as part of data collection .", "Scale bar , 5 μm .", "( g ) mir-2 ( gk259 ) and mir-2 ( n4108 ) animals had reduced APT-4 ( AP2 α-adaptin ) levels .", "Percent change from wild type ( N2 ) control for the APT-4 in mir-2 ( gk259 ) animals as above .", "t-test , two-tailed .", "( h–i )", "Representative images of cholinergic DA motor neuron APT-4::GFP in the dorsal cord of wild type ( N2 ) and mir-2 ( gk259 ) animals .", "These images were taken as part of data collection . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01710 . 7554/eLife . 20752 . 018Figure 3—figure Supplement 1—source data 1 . Raw Data for Figure 3—figure supplement 1 . Raw data and statistical analysis that correspond to SYD-2::GFP localization analysis , ITSN-1::GFP localization analysis , APT-4::GFP analysis , aldicarb resistance and pumping assays in Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 018 As a first step towards evaluating how miR-2 loss impacts cholinergic MN presynaptic function , we examined the effects of miR-2 loss on localization of presynaptic proteins in DA MNs .", "Four fluorescently-tagged presynaptic proteins were examined: SNB-1 ( synaptobrevin ) , SYD-2 ( α-liprin ) , ITSN-1 ( DAP160/Intersectin ) , and APT-4 ( AP2 α-adaptin ) .", "Analysis of tagged SNB-1 in mir-2 ( gk259 ) animals revealed increased SNB-1 puncta linear density , but no change in puncta width or intensity compared to wild type animals ( Figure 3B–D ) .", "Additionally , mir-2 ( gk259 ) animals were indistinguishable from wild type control animals with respect to SYD-2 synaptic localization for all metrics analyzed ( Figure 3—figure supplement 1C ) , suggesting that pre-synaptic active zones are unchanged in number and size; thus , synaptic changes are likely not the result of altered active zone number or size ( Zhen and Jin , 1999 ) .", "ITSN-1 puncta width and intensity , but not linear density , were decreased in mir-2 ( gk259 ) and mir-2 ( n4108 ) , animals compared to wild type controls ( Figure 3—figure supplement 1D–F ) .", "Finally , both mir-2 ( gk259 ) and mir-2 ( n4108 ) had decreased APT-4 puncta width , intensity , and linear density ( Figure 3—figure supplement 1G–I ) .", "ITSN-1 , similar to APT-4 , is involved in vesicle recycling at the NMJ ( Wang et al . , 2008 ) .", "Together with results from aldicarb resistance studies , our results suggest that loss of miR-2 results in synaptic dysfunction at the NMJ , consistent with decreased cholinergic synaptic release ( Ch'ng et al . , 2008; Sieburth et al . , 2005 ) .", "Additionally , we observed considerable overlap between synaptic protein localization defects resulting from miR-2 loss with those of smn-1 ( ok355 ) animals ( Dimitriadi et al . , 2016 ) , a finding consistent with miR-2 and SMN-1 acting in partially redundant pathways at the NMJ .", "To address mechanistically how miR-2 loss impacts NMJ function , we searched for mRNA targets of miR-2 .", "Canonically , miRNA loss results in overexpression of direct mRNA targets ( Elbashir et al . , 2001 ) .", "Since miR-2 loss leads to aldicarb resistance , loss of the target ( s ) is expected to cause hypersensitivity to aldicarb .", "Following a literature search for genes whose loss of function results in hypersensitivity , we selected the following genes with putative miR-2 3’UTR binding sites for study: gar-2 , dbl-1 , sek-1 , and vab-2 ( Jan et al . , 2011; Lewis et al . , 2005; Paraskevopoulou et al . , 2013; Reczko et al . , 2012; Vashlishan et al . , 2008 ) .", "Loss of a bona fide target gene is predicted to suppress aldicarb resistance caused by miR-2 loss .", "Therefore , we crossed a deletion allele for each gene into the mir-2 ( gk259 ) background .", "Loss of any of these four genes suppressed mir-2 ( gk259 ) to some extent , but gar-2 ( ok520 ) , which contains a large deletion removing gar-2 exons 6 and 7 , resulted in the most complete suppression , thus suggesting that GAR-2 acts downstream of miR-2 ( Figure 4A; Figure 4—figure supplement 1A–C ) .", "GAR-2 is a G protein-coupled acetylcholine receptor orthologous to the mammalian M2 muscarinic receptor ( m2R ) ( Lee et al . , 2000 ) . 10 . 7554/eLife . 20752 . 019Figure 4 . miR-2 binds the gar-2 3’UTR and represses GAR-2 translation .", "( a ) Loss of m2R ortholog , GAR-2 , suppressed aldicarb response defects of animals lacking mir-2 ( gk259 ) .", "gar-2 ( ok520 ) animals were hypersensitive to paralysis by aldicarb .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mir-2 ( gk259 ) , gar-2 ( ok520 ) and mir-2 ( gk259 ) ;gar-2 ( ok520 ) young adult animals .", "Log-rank test .", "( b ) Schematic representation of changes made to the endogenous gar-2 3’UTR using CRISPR .", "For the wild type control ( gar-2 UTRwtC ) , the miR-2 binding site remained intact , however , a C>T PAM site change was made .", "For the experimental condition ( gar-2 UTRscrC ) , the miR-2 binding site was scrambled in addition to the C>T PAM site alteration .", "( c ) gar-2 ( rt318 ) , referred to as gar-2 UTRscrC , animals were resistant to the acetylcholinesterase inhibitor , aldicarb , compared to gar-2 ( rt317 ) , referred to as gar-2 UTRwtC .", "Time course for paralysis on 1 mM aldicarb for young adult animals .", "Log-rank test .", "( d ) Scrambling the predicted endogenous gar-2 3’UTR miR-2 binding site increased gar-2 messenger RNA levels .", "Quantification of gar-2 mRNA levels in young adult gar-2 UTRwtC and gar-2 UTRscrC animals .", "t-test , two-tailed ( n = 4 for gar-2 UTRwtC and gar-2 UTRscrC ) .", "( e ) Reporter constructs used to assess miR-2 regulation of gar-2 3’UTR in cholinergic neurons: rtIs56 ( unc-17p-ACh::GFP::gar-2 3’UTRwt ) and rtIs57 or rtIs58 ( unc-17p-ACh::GFP::gar-2 3’UTRscr ) .", "unc-17p-ACh::GFP::gar-2 3’UTRwt construct contains the unc-17 promoter expressing NLS::GFP upstream of the gar-2 3’UTR , which has a predicted miR-2 binding site .", "Red text indicates intact seed region .", "unc-17p-ACh::GFP::gar-2 3’UTRscr is the same construct with the predicted miR-2 binding site scrambled identically to the sequence in gar-2 UTRscrC animals .", "( f ) Representative images of unc-17p-ACh::GFP::gar-2 3’UTRwt expression in cholinergic neurons of wild type ( N2 ) and mir-2 ( gk259 ) larval stage L4 animals .", "Scale bar , 50 μm .", "( g ) Representative images of unc-17p-ACh::GFP::gar-2 3’UTRscr ( rtIs57 ) expression in cholinergic neurons of wild type ( N2 ) and mir-2 ( gk259 ) larval stage L4 animals .", "( h ) Ratio representation of mean GFP fluorescence for wild type and mir-2 ( gk259 ) animals .", "t-test , two-tailed .", "Ratio was calculated by dividing the mean GFP fluorescence of unc-17p-ACh::GFP::gar-2 3’UTRwt for each genotype by the corresponding mean GFP fluorescence of unc-17p-ACh::GFP::gar-2 3’UTRscr for that genotype .", "UTRwt represents mean fluorescence for each genotype expressing the unc-17p-ACh::GFP::gar-2 3’UTRwt reporter , while UTRscr represents mean fluorescence for each genotype expressing the unc-17p-ACh::GFP::gar-2 3’UTRscr control reporter .", "Error bars represent the cumulative SEM for each genotype across transgenes .", "( see Figure 4—figure supplement 2D ) .", "( i ) gar-2 transcript levels did not increase in a mir-2 loss of function background .", "Quantification of gar-2 mRNA levels in young adult mir-2 ( gk259 ) animals compared to wild type ( N2 ) controls .", "t-test , two-tailed ( n = 4 for mir-2 ( gk259 ) and N2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 01910 . 7554/eLife . 20752 . 020Figure 4—source data 1 . Raw Data for Figure 4 . Raw data and statistical analysis that correspond to aldicarb resistance assays , qPCR quantification , and GFP reporter analysis in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02010 . 7554/eLife . 20752 . 021Figure 4—figure supplement 1 . Loss of predicted miR-2 mRNA targets suppresses mir-2 ( lf ) aldicarb resistance .", "( a ) Loss of DBL-1 suppressed mir-2 ( gk259 ) aldicarb resistance .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mir-2 ( n4108 ) , mir-2 ( n4108 ) ;dbl-1 ( nk3 ) , and dbl-1 ( nk3 ) young adult animals .", "Log-rank test .", "( b ) Loss of SEK-1 suppressed mir-2 ( gk259 ) aldicarb resistance .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mir-2 ( n4108 ) , mir-2 ( n4108 ) ;sek-1 ( km4 ) , and sek-1 ( km4 ) young adult animals .", "Log-rank test .", "( c ) Loss of VAB-2 suppressed mir-2 ( gk259 ) aldicarb resistance .", "Time course for paralysis on 1 mM aldicarb for wild type ( N2 ) , mir-2 ( n4108 ) , mir-2 ( n4108 ) ;vab-2 ( ju1 ) , and vab-2 ( ju1 ) young adult animals .", "Log-rank test . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02110 . 7554/eLife . 20752 . 022Figure 4—figure Supplement 1—source data 1 . Raw Data for Figure 4—figure supplement 1 . Raw data and statistical analysis that correspond to aldicarb resistance assays in Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02210 . 7554/eLife . 20752 . 023Figure 4—figure supplement 2 . miR-2 inhibits translation by binding the gar-2 3’UTR .", "( a ) Loss of miR-2 results in increased expression of unc-17p-ACh::GFP::gar-2 3’UTRwt .", "unc-17p-ACh::GFP::gar-2 3’UTRwt mean GFP fluorescence in wild type ( N2 ) and mir-2 ( gk259 ) backgrounds .", "Mean ± SEM; t-test , two-tailed .", "( b–c )", "Expression of unc-17p-ACh::GFP::gar-2 3’UTRscr was indistinguishable in mir-2 ( gk259 ) versus wild type ( N2 ) animals for two independent integrated lines .", "unc-17p-ACh::GFP::gar-2 3’UTRscr mean GFP fluorescence in wild type ( N2 ) and mir-2 ( gk259 ) backgrounds .", "Mean ± SEM; t-test , two-tailed .", "( d ) Formula used to calculate the ratio and standard error of the mean ( SEM ) shown in Figure 4 .", "UTRwt represents mean fluorescence for each genotype expressing the unc-17p-ACh::GFP::gar-2 3’UTRwt reporter , while UTRscr represents mean fluorescence for each genotype expressing the unc-17p-ACh::GFP::gar-2 3’UTRscr control reporter .", "For SEM , s represents the standard deviation of the population and n represents the number of animals analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02310 . 7554/eLife . 20752 . 024Figure 4—figure Supplement 2—source data 1 . Raw Data for Figure 4—figure supplement 2 . Raw data and statistical analysis that correspond to GFP reporter analysis in Figure 4—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 024 To determine if miR-2 regulates GAR-2 expression directly , we examined the consequences of perturbing the putative miR-2 binding site in the gar-2 3’UTR .", "Using CRISPR/Cas9-targeted genome editing , we scrambled the 18 base pair gar-2 3’UTR region corresponding to the endogenous miR-2 binding site ( Figure 4B ) .", "Compared to control animals ( gar-2 UTRwtC ) carrying the disrupted PAM site mutation ( C>T ) , we found that animals with the scrambled miR-2 binding site ( gar-2 UTRscrC ) were resistant to aldicarb , similar to miR-2 loss ( Figure 4C ) .", "To evaluate whether disruption of the miR-2 binding site influenced gar-2 transcript levels , we compared gar-2 mRNA levels in gar-2 UTRscrC animals and gar-2 UTRwtC controls and found a 40% increase in gar-2 transcript in gar-2 UTRscrC ( Figure 4D ) .", "Disruption of the 3’UTR site likely inhibits binding of other miR-2 family members , possibly contributing to the effect we observe ( Ibáñez-Ventoso et al . , 2008 ) .", "To test the effects of miR-2 loss on GAR-2 function in cholinergic neurons , we undertook in vivo GFP reporter analysis of GAR-2 expression .", "We generated a construct encoding GFP with a gar-2 3’UTR , whose expression is driven under the control of a cholinergic-specific promoter ( referred to as unc-17p-ACh::GFP::gar-2 3’UTRwt ) .", "A second control version of the construct contained the same scrambled UTR sequence as used in gar-2 UTRscrC animals ( referred to as unc-17p-ACh::GFP::gar-2 3’UTRscr ) ( Figure 4E ) .", "Transgenic lines were created by multicopy insertion for each construct .", "Increased GFP levels were observed in mir-2 ( gk259 ) animals expressing the intact 3’UTR construct as compared to control animals ( Figure 4F; Figure 4—figure supplement 2A ) .", "Whereas , loss of the mir-2 gene did not affect GFP levels in animals expressing the scrambled 3’UTR construct compared to control ( Figure 4G; Figure 4—figure supplement 2B and C ) .", "To quantify impact of miR-2 on expression of these GFP reporters in various genetic backgrounds , we compared relative changes in GFP levels of transgenic lines using a ratiometric strategy; we determined GFP expression for lines carrying unc-17p-ACh::GFP::gar-2 3’UTRwt and compared these values to lines carrying unc-17p-ACh::GFP::gar-2 3’UTRscr in the same genetic background ( Figure 4—figure supplement 2D ) .", "By this method , we observed a ~13% increase in relative GFP expression associated with loss of miR-2 ( Figure 4H ) , indicating that miR-2 directly suppresses translation of gar-2 mRNA by binding the gar-2 3’UTR at this 3’UTR site .", "We also assessed the effect of miR-2 loss on gar-2 transcript levels and found no significant difference in gar-2 mRNA levels between wild type animals and mir-2 ( gk259 ) loss of function animals ( Figure 4I ) .", "These results , combined with our reporter data , suggest that miR-2 binds and inhibits gar-2 mRNA translation , but does not reduce transcript levels .", "Previous studies have reported that miRNAs can influence protein synthesis of targets without destabilizing mRNA levels ( Cloonan , 2015; Selbach et al . , 2008 ) .", "Next , we determined if smn-1 loss altered miR-2 regulation of the gar-2 3’UTR .", "Both GFP reporter transgenes were crossed into the smn-1 ( ok355 ) background .", "Using the same ratiometric strategy from Figure 4H , we observed a small increase in relative GFP reporter expression ( ~5% ) in smn-1 ( ok355 ) animals compared to smn-1 ( + ) controls ( Figure 5A; Figure 5—figure supplement 1A and B ) .", "This finding suggested that smn-1 ( ok355 ) animals may have decreased miR-2 function and , as a consequence , an increase in GAR-2 translation .", "We showed above that increased mel-46 levels ameliorates smn-1 ( ok355 ) NMJ defects ( Figure 2; Figure 2—figure supplement 1 ) , therefore we assessed the impact of increased mel-46 on miR-2 activity using the [mel-46 ( + ) #2] rescue array .", "In control smn-1 ( + ) animals , increasing mel-46 did not alter relative expression of the miR-2 GFP reporter .", "However , in smn-1 ( ok355 ) animals , increasing mel-46 caused decreased relative reporter expression by ~15% , compared to smn-1 ( ok355 ) animals lacking the mel-46 rescue array ( Figure 5A; Figure 5—figure supplement 1A and B ) .", "These data suggest that MEL-46 overexpression decreases GAR-2 levels in smn-1 ( ok355 ) by increasing miR-2 activity . 10 . 7554/eLife . 20752 . 025Figure 5 . smn-1 loss of function abrogated miR-2 repression of GAR-2 expression .", "( a ) Loss of smn-1 caused a relative increase in unc-17p-ACh::GFP::gar-2 3’UTRwt expression .", "Expressing mel-46 using the broadly expressed [mel-46 ( + ) #2] array decreased relative unc-17p-ACh::GFP::gar-2 3’UTRwt expression in smn-1 ( ok355 ) animals .", "Ratio representation of mean GFP fluorescence for smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[mel-46 ( + ) #2] , and smn-1 ( + ) ;[mel-46 ( + ) #2] animals .", "Mann-Whitney U-test , two-tailed .", "Ratio calculation was completed in the same manner as Figure 4H ( see also Figure 4—figure supplement 2D )", "( b ) miR-2 levels were decreased in neurons when either SMN-1 or MEL-46 were decreased .", "Quantification of mature miR-2 for empty ( RNAi ) , smn-1 ( RNAi ) , and mel-46 ( RNAi ) young adult animals relative to housekeeping miRNA miR-60 .", "t-test , two-tailed ( n = 6 for each condition ) .", "( c ) gar-2 transcript levels did not change when SMN-1 or MEL-46 levels decreased in neurons .", "Quantification of gar-2 mRNA for empty ( RNAi ) , smn-1 ( RNAi ) , and mel-46 ( RNAi ) young adult animals .", "t-test , two-tailed ( n = 3 for each condition ) .", "( c–d )", "Animals sensitive to RNAi in only neurons ( TU3401 ) were fed bacteria expressing double-stranded RNA ( dsRNA ) against mel-46 or smn-1 .", "Control animals were fed bacteria expressing an empty vector control: empty ( RNAi ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02510 . 7554/eLife . 20752 . 026Figure 5—source data 1 . Raw data for Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02610 . 7554/eLife . 20752 . 027Figure 5—figure supplement 1 . Increasing MEL-46 ( Gemin3 ) ameliorates smn-1 ( lf ) defective miR-2 activity .", "( a ) Expression of mean unc-17p-ACh::GFP::gar-2 3’UTRscr ( rtIs57 ) fluorescence was decreased in smn-1 ( ok355 ) compared to smn-1 ( + ) control animals .", "Increasing MEL-46 levels using the [mel-46 ( + ) #2] array did not alter expression versus smn-1 ( + ) controls .", "Mean unc-17p-ACh::GFP::gar-2 3’UTRwt fluorescence in smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[mel-46 ( + ) #2] , and smn-1 ( + ) ;[mel-46 ( + ) #2] animals .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( b ) Expression of mean unc-17p-ACh::GFP::gar-2 3’UTRwt fluorescence was indistinguishable between smn-1 ( + ) and smn-1 ( ok355 ) animals; increasing MEL-46 using the [mel-46 ( + ) #2] array reduced expression versus control .", "Mean unc-17p-ACh::GFP::gar-2 3’UTRwt fluorescence in smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;[mel-46 ( + ) #2] , and smn-1 ( + ) ;[mel-46 ( + ) #2] animals .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( c ) miR-2 levels were decreased in neurons when either SMN-1 or MEL-46 were decreased .", "Quantification of mature miR-2 for empty ( RNAi ) , smn-1 ( RNAi ) , and mel-46 ( RNAi ) young adult animals relative to housekeeping gene act-1 .", "t-test , two-tailed ( n = 6 for each condition ) .", "( d ) miR-2 levels were decreased in neurons when either SMN-1 or MEL-46 were decreased .", "Quantification of mature miR-2 for empty ( RNAi ) , smn-1 ( RNAi ) , and mel-46 ( RNAi ) young adult animals relative to housekeeping rRNA 18s .", "t-test , two-tailed ( n = 6 for each condition ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02710 . 7554/eLife . 20752 . 028Figure 5—figure Supplement 1—source data 1 . Raw Data for Figure 5—figure supplement 1 . Raw data and statistical analysis that correspond to GFP reporter analysis and qPCR quantification in Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 028 Increased GAR-2 translation in animals lacking SMN-1 might be due to decreased mature miR-2 levels .", "To test this possibility , we used quantitative RT-PCR studies .", "After neuron-specific RNAi knock-down of either SMN-1 or MEL-46 , we found decreases in mature miR-2 levels ( Figure 5B; Figure 5—figure supplement 1C and D ) , but no change in gar-2 transcript levels ( Figure 5C ) .", "This result is consistent with our finding that gar-2 transcript levels were unchanged in miR-2 complete loss conditions ( Figure 4I ) , despite alterations in GFP reporter expression ( Figure 4H ) .", "These results suggest neuronal miR-2 levels are decreased when MEL-46 or SMN-1 levels decrease .", "Collectively , our data above are consistent with a model where diminished SMN-1 leads to decreased miR-2 levels and activity , resulting in increased GAR-2 expression .", "Since M2 receptors inhibit synaptic release at cholinergic NMJs across species ( Dittman and Kaplan , 2008; Parnas et al . , 2005; Slutsky et al . , 2003 ) , overexpression of these receptors in smn-1 ( ok355 ) MNs might contribute to the NMJ defects previously observed in these animals ( Dimitriadi et al . , 2016; Sleigh et al . , 2011 ) .", "Furthermore , increased MEL-46/Gemin3 might have an ameliorative effect in animals lacking SMN-1 by stimulating miR-2 activity , thus decreasing GAR-2 levels and disinhibiting cholinergic release .", "If increased GAR-2 levels exacerbate NMJ dysfunction in animals lacking SMN-1 , then decreasing GAR-2 function should ameliorate the NMJ defects caused by smn-1 loss of function .", "Loss of GAR-2 did not improve pharyngeal pumping rates in smn-1 ( ok355 ) animals ( Figure 6—figure supplement 1A ) , similar to our results in animals with increased mel-46 levels ( Figure 2—figure supplement 1D ) .", "However , similar to increasing mel-46 , gar-2 ( ok520 ) restored normal response to aldicarb in both smn-1 ( ok355 ) and smn-1 ( rt248 ) animals ( Figure 6A; Figure 6—figure supplement 1B ) , consistent with improved NMJ function .", "The rt248 smn-1 allele causes a frameshift and loss of SMN-1 function similar to ok355 ( Dimitriadi et al . , 2016 ) .", "Additionally , gar-2 ( ok520 ) restored normal response to aldicarb in mel-46 ( tm1739 ) animals ( Figure 6B ) .", "Our results indicate that decreasing GAR-2 likely improves presynaptic function in animals with decreased SMN-1 or MEL-46 .", "Consistent with this conclusion , gar-2 ( ok520 ) also rescued numerous presynaptic protein localization defects caused by SMN-1 loss; SNB-1 puncta width , intensity and linear density defects were rescued in both smn-1 ( ok355 ) and smn-1 ( rt248 ) backgrounds ( Figure 6C–6G; Figure 6—figure supplement 1D–H ) .", "GAR-2 loss in smn-1 ( + ) control animals resulted in increased SNB-1 puncta width and intensity , but did not alter SNB-1 puncta linear density . 10 . 7554/eLife . 20752 . 029Figure 6 . Loss of gar-2 ameliorated smn-1 ( lf ) NMJ defects .", "( a ) Loss of gar-2 rescued smn-1 ( ok355 ) aldicarb response defect .", "Time course for paralysis on 1 mM aldicarb for smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) early larval stage L4 animals .", "Log-rank test .", "( b ) Loss of gar-2 rescued mel-46 ( tm1739 ) aldicarb response defect .", "Time course for paralysis on 1 mM aldicarb for mel-46 ( + ) , mel-46 ( tm1739 ) , mel-46 ( tm1739 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) early larval stage L4 animals .", "Log-rank test .", "For these experiments , mel-46 ( tm1739 ) was maintained over the nT1 balancer and therefore , control mel-46 ( + ) animals were obtained as +/+ animals from +/nT1 heterozygous mothers .", "( c ) Loss of gar-2 rescued smn-1 ( ok355 ) RFP::SNB-1 synaptic localization defects .", "gar-2 loss in the smn-1 ( + ) background resulted in increased RFP::SNB-1 puncta width and intensity .", "Percent change from smn-1 ( + ) control for RFP::SNB-1 in the dorsal nerve cord of smn-1 ( ok355 ) , smn-1 ( ok355 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) animals for ‘punctaanalyzer’ parameters: puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "Asterisks denote significance compared to smn-1 ( + ) control; shading indicates significant difference from smn-1 ( ok355 ) ;gar-2 ( ok520 ) .", "Mann-Whitney U-test , two-tailed .", "Loss of gar-2 rescued RFP::SNB-1 puncta width defects ( smn-1 ( + ) control animals versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 76; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 0001 ) , restored SNB-1 puncta intensity ( smn-1 ( + ) control animals versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=1 . 00; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 0001 ) and rescued SNB-1 puncta linear density defects ( smn-1 ( + ) control animals versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 08; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 02 ) .", "( d–g )", "Representative images of cholinergic DA MN RFP::SNB-1 in the dorsal nerve cord of smn-1 ( + ) , smn-1 ( ok355 ) , smn-1 ( ok355 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) .", "These images were taken as part of data collection .", "Scale bar , 5 μm .", "Figures D and G are also shown in Figure 6—figure supplement 1 since this control data was collected alongside both ok355 and rt248 RFP::SNB-1 data . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 02910 . 7554/eLife . 20752 . 030Figure 6—source data 1 . Raw Data for Figure 6 . Raw data and statistical analysis that correspond to RFP::SNB-1 localization analysis and aldicarb resistance assays in Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 03010 . 7554/eLife . 20752 . 031Figure 6—figure supplement 1 . Decreasing GAR-2 ( m2R ) levels rescues NMJ defects in smn-1 ( lf ) and mel-46 ( lf ) animals .", "( a ) smn-1 ( ok355 ) animals had reduced pharyngeal pumping rates versus smn-1 ( + ) control animals; defects were not rescued by loss of GAR-2 .", "Mean ± SEM; Mann-Whitney U-test , two tailed .", "( b ) Loss of GAR-2 ameliorated smn-1 ( rt248 ) aldicarb response .", "smn-1 ( + ) ;gar-2 ( ok520 ) animals were hypersensitive to aldicarb .", "Time course for paralysis on 1 mM aldicarb in smn-1 ( + ) , smn-1 ( rt248 ) , smn-1 ( rt248 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) early larval stage L4 animals .", "Log-rank test .", "( c ) Loss of GAR-2 did not rescue smn-1 ( ok355 ) APT-4 ( AP2 α-adaptin ) defects .", "Loss of GAR-2 in the smn-1 ( + ) background resulted in decreased APT-4 puncta width and intensity .", "Percent change from smn-1 ( + ) control for APT-4 in the dorsal cord of smn-1 ( ok355 ) , smn-1 ( ok355 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) animals for ‘punctaanalyzer’ parameters: average puncta width ( μm ) , intensity ( AU ) , and linear density ( number/μm ) .", "Asterisks denote significance compared to smn-1 ( + ) control .", "Mann-Whitney U-test , two-tailed .", "GAR-2 loss did not rescue APT-4 puncta width ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 12; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 48 ) , did not rescue APT-4 puncta intensity ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 08; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 54 ) and did not rescue APT-4 puncta linear density ( smn-1 ( + ) control versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 66; smn-1 ( ok355 ) versus smn-1 ( ok355 ) ;gar-2 ( ok520 ) p=0 . 07 ) .", "( d ) Loss of GAR-2 ameliorated smn-1 ( rt248 ) SNB-1 ( synaptobrevin ) defects .", "Percent change from smn-1 ( + ) control for SNB-1 in the dorsal nerve cord of smn-1 ( rt248 ) , and smn-1 ( rt248 ) ;gar-2 ( ok520 ) animals for all ‘punctaanalyzer’ parameters: average puncta width ( μm ) , total intensity ( AU ) , and linear density ( number/μm ) .", "smn-1 ( + ) ;gar-2 ( ok520 ) percent change was collected alongside this data and is shown in Figure 5B .", "Asterisks denote significance compared to smn-1 ( + ) control; shading indicates significant difference from smn-1 ( rt248 ) ;gar-2 ( ok520 ) .", "Mann-Whitney U-test , two-tailed .", "Loss of GAR-2 rescued SNB-1 puncta width ( smn-1 ( + ) control versus smn-1 ( rt248 ) ;gar-2 ( ok520 ) p=0 . 83; smn-1 ( rt248 ) versus smn-1 ( rt248 ) ;gar-2 ( ok520 ) p=0 . 004 ) , restored SNB-1 total puncta intensity ( smn-1 ( + ) control versus smn-1 ( rt248 ) ;gar-2 ( ok520 ) p=0 . 91; smn-1 ( rt248 ) versus smn-1 ( rt248 ) ;gar-2 ( ok520 ) p=0 . 004 ) and rescued SNB-1 puncta linear density ( smn-1 ( + ) control versus smn-1 ( rt248 ) ;gar-2 ( ok520 ) p=0 . 97; smn-1 ( rt248 ) versus smn-1 ( rt248 ) ;gar-2 ( ok520 ) p=0 . 002 ) .", "( Martinez et al . )", "Representative images of SNB-1::RFP expressed in the dorsal nerve cord of cholinergic DA motor neurons for smn-1 ( + ) , smn-1 ( rt248 ) , smn-1 ( rt248 ) ;gar-2 ( ok520 ) , and smn-1 ( + ) ;gar-2 ( ok520 ) .", "These images were taken as part of data collection .", "Scale bar , 5 μm .", "Figures E and H were taken from Figure 5 since this data was collected alongside both ok355 and rt248 SNB-1::RFP data . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 03110 . 7554/eLife . 20752 . 032Figure 6—figure Supplement 1—source data 1 . Raw Data for Figure 6—figure supplement 1 . Raw data and statistical analysis that correspond to APT-4::GFP localization analysis , RFP::SNB-1 localization analysis , aldicarb resistance and pumping assays in Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 032 Research in vertebrates has demonstrated m2R internalization normally occurs in response to chronic m2R stimulation , either by pharmacological agonist application or acetylcholinesterase inhibition ( Clancy et al . , 2007 ) .", "Since endocytosis is defective in animals lacking SMN-1 , perturbed endocytosis , in combination with miRNA misregulation , could contribute to GAR-2 accumulation at the membrane leading to decreased SNB-1 in smn-1 ( ok355 ) motor neurons ( Dimitriadi et al . , 2016 ) .", "Loss of GAR-2 did not rescue smn-1 ( ok355 ) APT-4 puncta defects ( Figure 6—figure supplement 1C ) , but decreased APT-4 puncta width and intensity in smn-1 ( + ) control animals .", "As loss of GAR-2 did not restore APT-4 synaptic defects , we conclude that there are additional pathways affected by smn-1 loss , beyond GAR-2 misregulation .", "Nevertheless , these results suggest that C . elegans GAR-2 levels are increased by smn-1 loss , which might contribute to NMJ defects in smn-1 ( lf ) animals .", "The closest human ortholog of GAR-2 is the M2 muscarinic receptor ( m2R ) , encoded by the CHRM2 gene .", "GAR-2 and m2R are functionally conserved , as activation of these presynaptic receptors by acetylcholine in different species results in hyperpolarization and decreased NMJ acetylcholine release across species ( Dittman and Kaplan , 2008; Dudel , 2007; Parnas et al . , 2005; Slutsky et al . , 2003 ) .", "Previous research suggests decreased SMN function across species might impact miRNA activity across species , which could increase m2R levels consistent with our work in C . elegans .", "The CHRM2 mRNA is a predicted target of miR-128 in mice and humans ( Figure 7A ) ( Jan et al . , 2011; Lewis et al . , 2005; Paraskevopoulou et al . , 2013 ) .", "Based on results from C . elegans , we predicted that vertebrate SMN loss might disrupt miR-128 activity , also leading to increased m2R .", "m2R protein levels were examined in MNs isolated from E13 . 5 SMA mice ( Smn-/-;SMN2tg/0 ) .", "A ~50% increase in m2R levels was observed , compared to wild type control MNs ( Figure 7B and C ) .", "miR-128 levels in SMA mouse MNs were decreased compared to wild type ( Figure 7D ) .", "Combined , these results suggest that diminished SMN protein causes decreased levels of mature miR-128 , thus disinhibiting m2R expression in MNs across species . 10 . 7554/eLife . 20752 . 033Figure 7 . Increased m2R muscarinic receptor levels in SMA mouse model MNs contribute to axon outgrowth defects .", "( a ) Alignment of predicted miR-2 or miR-128 binding sites for C . elegans , mouse and human gar-2 or CHRM2 3’UTRs .", "CHRM2 encodes the mR2 muscarinic receptor ( Paraskevopoulou et al . , 2013; Reczko et al . , 2012 ) .", "Predicted nucleotide pairing shown by vertical lines .", "Red text indicates predicted miRNA seed region .", "A black line indicates potential seed region conservation .", "( b ) Representative image for two E13 . 5 wild type and two Smn-/-;SMN2tg/0 DIV10 spinal MN immunoblots probed for m2R and control β-Actin .", "( c ) Quantification of immunoblot , t-test , two-tailed , p=0 . 017 ( n = 14 for WT and n = 13 for SMA ) .", "( d ) Quantification of miR-128 levels in DIV10 spinal MNs from E13 . 5 wild type and Smn-/-;SMN2tg/0 animals .", "t-test , two-tailed ( n = 24 from 12 mice for WT; n = 20 from 10 mice for SMA ) .", "( e ) Longest axon length for E13 . 5 wild type and Smn-/-;SMN2tg/0 DIV5 spinal MNs treated with 0 nm , 50 nm , and 500 nm methoctramine .", "t-test , two-tailed .", "( n = 103 for WT 0 nM , n = 131 for WT 50 nM , n = 98 for WT 500 nM , n = 102 for SMA 0 nM , n = 67 for SMA 50 nM and n = 53 for SMA 500 nM )", "( f ) Proposed model: SMN acts via MEL-46 to influence microRNAs that play important roles in NMJ function and MN survival .", "SMN loss affects other pathways as well .", "C . elegans genes shown on left side; human genes on right side . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 03310 . 7554/eLife . 20752 . 034Figure 7—figure supplement 1 . m2R inhibition by methoctramine increases axon length in SMA mouse model MNs .", "( a ) Total axon length for E13 . 5 wild type and Smn-/-;SMN2tg/0 DIV5 spinal MNs treated with 0 nm , 50 nm , and 500 nm methoctramine .", "‘Total axon length’ is a measurement of all axon branches .", "t-test , two-tailed .", "n = 103 for WT 0 nM , n = 131 for WT 50 nM , n = 98 for WT 500 nM , n = 102 for SMA 0 nM , n = 67 for SMA 50 nM and n = 53 for SMA 500 nM .", "Neurons are from at least three biological samples . DOI: http://dx . doi . org/10 . 7554/eLife . 20752 . 034 Decreased SMN levels results in axon outgrowth defects in MNs derived from a SMA mouse model ( Rossoll et al . , 2003 ) and increased m2R might contribute to this functional defect .", "To test this , we examined the impact of m2R pharmacological inhibition on axon length for DIV5 MNs from E13 . 5 wild type ( FVB ) and SMA mice ( Smn-/-;SMN2tg/0 ) ( Figure 7E ) .", "Wild type and SMA MNs were cultured in the presence of 50 nm or 500 nm methoctramine , an m2R antagonist .", "In wild type MNs , methoctramine decreased mean longest axon length .", "Conversely , methoctramine treatment in SMA MNs increased both mean longest axon length and total axon length ( Figure 7E; Figure 7—figure supplement 1A ) .", "We conclude that m2R inhibition rescues MN axon outgrowth defects in a SMA mouse model , consistent with a deleterious impact of increased m2R activity in SMA model MNs ." ], [ "The most direct molecular connection between SMN and the miRNA pathway is Gemin3 .", "Results presented here indicate that decreases in SMN-1 ( SMN ) or MEL-46 ( Gemin3 ) result in similar neuromuscular defects as well as decreased miR-2 levels in neurons .", "This analysis further suggests that decreases in C . elegans SMN-1 result in increased GAR-2 ( m2R ) through miR-2 misregulation and that increasing MEL-46 ( Gemin3 ) reduces GAR-2 protein levels in smn-1 ( ok355 ) animals via modulation of miR-2 .", "Several lines of evidence support this conclusion , including miR-2 GFP reporter expression results , NMJ aldicarb sensitivity , synaptic protein localization changes , and qPCR measurements of mature miR-2 and gar-2 mRNA levels ( Figures 1–5 ) .", "There is a paucity of research addressing the functional importance of Gemin3 , in either the Gemin or the RISC complexes .", "Biochemical and co-localization studies support a role for Gemin3 in spliceosome assembly , mRNA transport , and miRNA function ( Charroux et al . , 1999; Dostie et al . , 2003; Feng et al . , 2005; Mourelatos et al . , 2002; Zhang et al . , 2006 ) .", "It is possible that decreased and/or mislocalized Gemin3 impairs RISC function , leading to the diminished miR-2 activity and levels reported in Figure 5 .", "However , since Gemin3 likely functions in numerous pathways , it may influence miR-2 through other , more indirect pathways .", "In Drosophila , Gemin3 is necessary for motor function , but the molecular mechanisms underlying this defect are unclear ( Cauchi et al . , 2008 ) .", "Results presented here in C . elegans further support a requirement for Gemin3 in motor function and additionally , show that Gemin3 is capable of modifying miRNA levels and activity .", "miR-2 belongs to the invertebrate K box family ( motif: CUGUGAUA ) of miRNAs ( Ibáñez-Ventoso et al . , 2008 ) .", "It was suggested previously that miR-2 does not have well-conserved mammalian orthologs ( Marco et al . , 2012 ) , but another study suggested that human miR-128 is a member of this miRNA family ( Ibáñez-Ventoso et al . , 2008 ) .", "These two bioinformatics studies differ in their definition of the miRNA binding site , also known as the seed region .", "Our alignment of miR-2/miR-128 miRNAs and gar-2/CHRM2 mRNAs suggests that the CUGUG seed region may be a conserved motif for miRNA binding ( Figure 7A ) .", "Both miR-2 and miR-128 are enriched in the nervous system and share conserved mRNA targets ( Marco et al . , 2012; Martinez et al . , 2008 ) .", "More studies will be needed to confirm that miR-2 and miR-128 are orthologs .", "Regardless , the results presented here , in conjunction with previous research , suggest that overall miRNA misregulation contributes to neuronal defects when SMN levels decrease ( Haramati et al . , 2010; Kye et al . , 2014; Valsecchi et al . , 2015; Wang et al . , 2014; Wertz et al . , 2016 ) .", "Altered function of miR-2/miR-128 or GAR-2/m2R is not sufficient to explain all of the dysfunction observed in models of SMN deficiency .", "In C . elegans , miR-2 loss does not cause overt defects and GAR-2 loss does not restore viability , fecundity , or normal development to animals lacking SMN-1 or MEL-46 .", "This suggests miR-2 loss does not contribute to defects outside the NMJ caused by SMN-1 loss .", "And , at the NMJ , synaptic protein perturbations are more severe in animals with diminished SMN-1 or MEL-46 , compared to those lacking either miR-2 or GAR-2 , which is consistent with a broader range of defects caused by decreased SMN-1 levels .", "In mice , complete miR-128 knock-out results in decreased motor activity and premature death ( Tan et al . , 2013 ) , but it is currently unknown how miR-128 loss might specifically impact cholinergic MN function .", "We found that m2R levels were increased 50% overall in SMN-deficient mouse MNs compared to wild type by Western blot analysis ( Figure 7B and C ) .", "In C . elegans , relative GAR-2 translation was increased by only 5% globally when SMN-1 levels dropped , based on GFP reporter expression in cholinergic neurons ( Figure 5A ) .", "These two assays are not directly comparable , since the C . elegans experiment only investigated the contribution of miR-2 perturbation in regards to increased GAR-2 translation .", "Certainly , additional pathways are affected by SMN loss , beyond miRNAs , contributing to the greatly increased m2R levels in SMN-deficient MNs .", "These pathways may include mRNA transport metabolism , endocytosis , as well as spliceosome and ribonucleoprotein assembly ( Dimitriadi et al . , 2016; Fallini et al . , 2011; Hosseinibarkooie et al . , 2016; Pellizzoni et al . , 2002; Yong et al . , 2002 ) .", "Extensive analysis would be required to understand how defects in these pathways contribute to increased m2R in SMN-deficient MNs .", "m2R is expressed in α-MNs , with little to no expression in smaller gamma MNs ( Welton et al . , 1999 ) .", "This expression profile correlates with the pattern of neurodegeneration in an SMA mouse model: selective loss of α-MNs , while gamma MNs remain unaffected ( Powis and Gillingwater , 2016 ) .", "Within α-MNs , m2R is distributed along the membrane and concentrates at postsynaptic connections with C-boutons ( Deardorff et al . , 2014 ) .", "α-MNs in SMNΔ7 mice have increased C-bouton sites ( Tarabal et al . , 2014 ) , which could be an additional cause or consequence of increased m2R levels in α-MNs .", "Taken together , this evidence suggests that increased m2R is consistent with multiple features of SMA pathology .", "We also consider three additional previously defined m2R pathways as possible contributors to MN functional defects in SMN-deficient α-MNs: GIRK channels , Ca2+ channels , and SK channels .", "Classically , m2R receptor activation leads to GIRK-channel-dependent efflux of K+ cations , resulting in neuronal hyperpolarization and decreased SV release ( Sun et al . , 2013 ) .", "Therefore , increased m2R levels are consistent with the synaptic defects observed in SMA models across species ( Dimitriadi et al . , 2016; Kong et al . , 2009 ) .", "m2R activation of GIRK channels is conserved in C . elegans ( Lee et al . , 2000 ) , suggesting GAR-2 loss may rescue NMJ defects in animals lacking SMN-1 by reducing GIRK channel activation .", "Interestingly , sustained GIRK activation has been previously linked to neurodegeneration ( Coulson et al . , 2008 ) .", "m2R inhibits N-type Ca2+ ( Cav2 . 2 ) and P/Q-type Ca2+ ( Cav2 . 1 ) channels resulting in decreased SV release at the NMJ ( Slutsky et al . , 2003; Yan and Surmeier , 1996 ) .", "And , Cav2 . 1-deficient mice exhibit NMJ degeneration and decreased active zone proteins ( Fox et al . , 2007; Nishimune et al . , 2004 ) .", "Additionally , in a mouse model of SMA , distal axons and growth cones had reduced Ca2+ transients resulting from defective Cav2 . 2 excitability and accumulation ( Jablonka et al . , 2007 ) .", "Increased m2R is consistent with decreased Ca2+ channel activity observed in SMN-deficient animals .", "Moreover , Cav2 . 2 channels activate SK channels in α-MNs , suggesting increased m2R may lead to decreased SK channel currents ( Goldberg and Wilson , 2005 ) .", "The drug riluzole , which ameliorated motor defects in smn-1 ( ok355 ) animals , may act via SK channels ( Dimitriadi et al . , 2013 ) .", "Increased m2R levels may result in excessive inhibition of SK channels , contributing to defective synaptic transmission in SMA models across species; this connection may offer additional mechanistic insight into the ameliorative effects of riluzole .", "Previous reports suggest that decreases in Ca2+ transients hinder axon outgrowth ( Hutchins and Kalil , 2008 ) .", "SMN loss also decreases these currents ( Jablonka et al . , 2007 ) , consistent with defective axon outgrowth in SMN-deficient cultured neurons .", "Here , we show that inhibition of m2R by methoctramine ameliorates axon outgrowth defects in SMA mouse model MNs .", "As we find m2Rs are overexpressed in SMA MNs , methoctramine rescue of axon outgrowth may be the result of restored Ca2+ channel function .", "Taken together , these data suggest that increased m2R expression contributes to axon outgrowth defects in SMA MNs and that m2R inhibition promotes axon outgrowth in SMA MNs .", "We connect SMN functionally and mechanistically to the miRNA pathway .", "As an exemplar of this connection in two species , we demonstrate that decreased SMN levels lead to downregulation of specific miRNAs and consequent increased expression of M2 muscarinic receptors .", "Increased m2R activity is deleterious and consistent with a subset of the NMJ defects seen in SMA models , across species .", "We suggest future studies might address the possible benefits of m2R inhibition in SMA models , as a combinatorial approach with other therapies ." ], [ "Strains listed in Supplementary file 2A were maintained under standard conditions at 25°C ( Brenner , 1974 ) ; we provide complete genotypes with unique strain identifiers , consistent with the rigorous standards of the C . elegans community .", "Abbreviated names are sometimes used for arrays , integrated lines or alleles in Figures 1–5; additional information about abbreviations can be found in Supplementary file 2C .", "For experiments with smn-1 ( ok355 ) and smn-1 ( rt248 ) , animals assayed were first generation progeny of hermaphrodites heterozygous for the hT2 balancer .", "To maintain a common genetic background , control smn-1 ( + ) animals were also derived from +/hT2 parents .", "Similarly , for APT-4::GFP synaptic localization ( Figure 2—figure supplement 1E ) and aldicarb response studies ( Figure 6B ) , mel-46 ( tm1739 ) animals were first generation progeny of parents heterozygous for the nT1 balancer .", "Control mel-46 ( + ) animals were derived from +/nT1 animals .", "For all other assays involving mel-46 ( tm1739 ) , animals were first generation progeny of parents carrying the ytEx211[mel-46 ( + ) ] rescue array; animals tested did not carry the array unless specified .", "For these experiments , N2 animals served as wild type controls .", "We attempted to generate smn-1 ( ok355 ) ;mel-46 ( tm1739 ) double mutant animals , but generation of heterozygous double mutant animals was not possible , using either balancer chromosomes or the ytEx211[mel-46 ( + ) ] rescue array .", "The pHA#756 ( unc-17p::mir-2::unc-54 3’UTR ) plasmid was generated by excising a 867 bp fragment from pHA#755 ( aex-3p::mir-2::unc-54 3’UTR ) using NheI and SpeI .", "This fragment , containing the genomic mir-2 pre-miRNA sequence along with unc-54 3’UTR sequence , was subcloned into pPD95 . 77 ( pPD95 . 77 was a gift from Andrew Fire; Addgene , Cambridge , Massachusetts plasmid 1495 ) between NheI and SpeI sites , resulting in removal of the GFP sequence .", "Additionally , a 4466 bp fragment corresponding to the unc-17 promoter was inserted between pPD95 . 77 SphI and AscI sites .", "Information for all amplification primers can be found in Supplementary file 2B .", "pHA#757 ( unc-17p::GFP::unc-54 3’UTR ) was generated by inserting the unc-17 promoter fragment between pPD95 . 77 SphI and AscI sites , without altering the GFP sequence .", "Plasmid pHA#758 ( NLS::GFP::gar-2 3’UTRwt ) contains a 269 bp fragment corresponding to the gar-2 3’UTR that was subcloned into pPD95 . 67 ( pPD95 . 67 was a gift from Andrew Fire; Addgene plasmid 1490 ) as a EcoRI and SpeI product .", "pHA#759 ( unc-17p::NLS::GFP::gar-2 3’UTRwt ) was generated by excising a 1286 bp fragment containing the NLS::GFP sequence and gar-2 3’UTR from pHA#758 using MscI and SpeI and ligating this fragment into pHA#756 , thus removing the genomic mir-2 pre-miRNA and unc-54 3’UTR sequences .", "pHA#760 was generated by ligating the gar-2 3’UTR fragment into pBluescript KS+ ( Stratagene , La Jolla , California ) using EcoRI and SpeI .", "To construct pHA#761 , the last 85 bp of the gar-2 3’UTR were removed from pHA#760 using NcoI and SpeI .", "This fragment was replaced with an identical 85 bp sequence , but with 19 bp scrambled at the predicted miR-2 binding site sequence ( gar-2 3’UTRscr ) .", "Primers were annealed to produce this 85 bp sequence ( Supplementary file 2C ) .", "Plasmid pHA#762 ( NLS::GFP::gar-2 3’UTRscr ) was generated by subcloning the 269 bp gar-2 3’UTRscr fragment from pHA#761 into pPD95 . 67 with EcoRI and SpeI .", "pHA#763 ( unc-17p::NLS::GFP::gar-2 3’UTRscr ) was produced by subcloning the 1286 bp fragment containing NLS::GFP and gar-2 3’UTRscr sequences from pHA#762 into pHA#756 using MscI and SpeI , while removing the genomic mir-2 pre-miRNA and unc-54 3’UTR sequences .", "pHA#790 ( unc-122p::mel-46::unc-54 3’UTR ) was created by amplifying the MEL-46 coding region from the pRM8 plasmid ( Minasaki et al . , 2009 ) and inserting this fragment into the pHA#729 EcoRI site ( Dimitriadi et al . , 2016 ) .", "Using SphI and MscI restriction enzymes , the unc-122 promoter was then excised and replaced with the 4466 bp unc-17 promoter fragment excised from pHA#763 with the same enzymes , thus generating pHA#791 ( unc-17::mel-46::unc-54 3’UTR ) .", "To create pHA#792 ( unc-17p::mel-46::GFP::unc-54 3’UTR ) , a 906 bp GFP sequence was amplified from pHA#763 and subcloned by Gibson assembly into pHA#791 just before the MEL-46 stop codon ( TGA ) .", "The small guide RNA ( sgRNA ) plasmids targeting the smn-1 gene ( pHA#764 and pHA#765 ) and the sgRNA plasmid targeting the gar-2 3’UTR ( pHA#793 ) for CRISPR/Cas9-mediated genome editing were produced by amplification of PU6::klp-12 ( Friedland et al . , 2013 ) .", "Plasmid pHA#766 contains a GFP insertion template and self-excising cassette flanked by smn-1 arms of homology that were subcloned by Gibson assembly into pDD282 following a protocol from ( Dickinson et al . , 2015 ) .", "Integrated arrays rtIs64 and rtIs65 [unc-17p::mel-46::GFP::unc-54 3’UTR] were created by UV irradiation of rtEx871 , which were generated by standard injection of pHA#792 at 50 ng/μl , alongside 5 ng/μl myo-3p::mCherry ( pCFJ104 - myo-3p::mCherry::unc-54utr was a gift from Erik Jorgensen ( Frøkjaer-Jensen et al . , 2008 ) , and 75 ng/μl pBluescript KS+ .", "To generate rtEx855[pRM8 ( mel-46 ( + ) ; myo-2p::RFP] , wild type animals were injected with 133 ng/μl PRM8 plasmid ( Minasaki et al . , 2009 ) , 5 ng/μl myo-3p::mCherry; Addgene plasmid 19328 ) and 75 ng/μl pBluescript KS+ .", "Animals injected with rtEx855[pRM8 ( mel-46 ( + ) ; myo-3p-RFP ) ] were crossed into a mel-46 ( tm1739 ) background to assure rescue of viability before further experiments were undertaken .", "Notably , expression of either rtEx855 or ytEx211 in smn-1 ( lf ) animals did not rescue lethality or adult survival , further emphasizing a privileged relationship between SMN-1 and MEL-46 in cholinergic NMJ signaling .", "Lines for rtEx853[unc-17p::mir-2; myo-2p::mCherry] and rtEx854[unc-17p::GFP; myo-2p::mCherry] were produced by injecting mir-2 ( gk259 ) animals with pHA#756 or pHA#757 , respectively , at 40 ng/μl alongside 2 . 5 ng/μl myo-2p::mCherry ( pCFJ90 - myo-2p::mCherry::unc-54utr was a gift from Erik Jorgensen ( Frøkjaer-Jensen et al . , 2008 ) ; Addgene plasmid 19327 ) and 77 . 5 ng/μl pBluescript KS+ .", "rtIs56[unc-17p::GFP::gar-2 3’UTRwt; myo-2p::mCherry] was integrated by UV irradiation into the genome and is derived from extrachromosomal array rtEx856 , containing pHA#759 , which was injected into wild type animals at 20 ng/μl with 2 . 5 ng/μl myo-2p::mCherry and 77 . 5 ng/μl pBluescript KS+ .", "Integrated arrays rtIs57 and rtIs58 [unc-17p::GFP::gar-2 3’UTRscr; myo-2p::mCherry] are two separate lines generated by UV irradiation of extrachromosomal array rtEx857 , containing pHA#763 , which was injected into wild type animals at 20 ng/μl with 2 . 5 ng/μl myo-2p::mCherry and 77 . 5 ng/μl pBluescript KS+ .", "gar-2 ( rt317 ) and gar-2 ( rt318 ) alleles were generated by injecting pha-1 ( e2123 ) animals with the pHA#793 sgRNA plasmid targeting the gar-2 3’UTR at 25 ng/μl with either 50 ng/μl of a mutant single-strand oligo DNA ( ssODN ) repair template ( rt318 ) or a control ssODN repair template ( rt317 ) , alongside the injection cocktail as described in Ward ( 2015 ) .", "Progeny from this injection were screened as described ( Ward , 2015 ) .", "Information on ssODN template sequences can be found in Supplementary file 2C .", "To generate smn-1 ( rt280 ) , which contains a GFP N-terminal insertion , wild type animals were injected with both pHA#764 and pHA#765 sgRNA plasmids targeting smn-1 at 50 ng/μl alongside 20 ng/μl of the GFP template plasmid pHA#766 and the standard injection cocktail described in Dickinson et al . ( 2015 ) .", "Progeny from this injection were screened as described ( Dickinson et al . , 2015 ) .", "Consistent with Miguel-Aliaga et al . , we noticed that the tagged-SMN protein was expressed in all blastomeres throughout embryonic development with redistribution from the nucleus to the cytoplasm during mitotic stages ( data not shown ) .", "The presence of GFP::SMN during such early stages indicates that GFP::SMN is maternally transmitted during germline development ( Miguel-Aliaga et al . , 1999 ) .", "RNAi studies involved animals from an RNAi-enhanced background ( KP3948 ) ( Kennedy et al . , 2004 ) , neuron-specific RNAi-sensitized background ( TU3401 ) ( Calixto et al . , 2010 ) , cholinergic neuron-specific RNAi-sensitized background ( XE1581 ) , or GABA neuron-specific RNAi-sensitized background ( XE1375 ) ( Firnhaber and Hammarlund , 2013 ) .", "Aldicarb response , pumping rates , and RNA quantification were evaluated in animals that had been reared for at least two generations on HT115 bacteria containing control vector L4440 , C41G7 . 1/smn-1 ( RNAi ) , C26C6 . 2/goa-1 ( RNAi ) , T06A10 . 1/mel-46 ( RNAi ) , or Y37D8A . 23/unc-25 ( RNAi ) ( Kamath and Ahringer , 2003 ) .", "Primer sequences used to generate the PCR products specific to each gene of interest can be found on the Kim Lab Stanford University website ( http://cmgm . stanford . edu/~kimlab/primers . 12-22-99 . html ) .", "Aldicarb resistance assay: 1 mM aldicarb assays were completed in at least three independent trials blinded to genotype ( n ≥ 30 animals/genotype ) as described in previous work ( Mahoney et al . , 2006; Sato et al . , 2009 ) .", "Paralysis induced by aldicarb was scored as inability to move or pump in response to prodding with a platinum wire .", "Experiments involving smn-1 ( ok355 ) , smn-1 ( rt248 ) or mel-46 ( tm1739 ) animals were completed at the early L4 stage .", "All other aldicarb experiments were done with young adult animals .", "Pharyngeal pumping: Assays were performed blinded to genotype as previously described ( Dimitriadi et al . , 2010 ) .", "Pumping events were scored as grinder movement in any axis .", "Average pumping rates ( ± Standard Error of the Mean ( SEM ) ) were pooled from at least two independent trials ( n > 20 animals/genotype ) .", "Experiments involving smn-1 ( ok355 ) , smn-1 ( rt248 ) or mel-46 ( tm1739 ) animals were completed at day three post-hatching ( animals were kept at 25°C for two days and then 20°C for one day ) .", "Pumping experiments involving all other genotypes were done with young adult animals .", "Animals were mounted on 2% agar pads and immobilized using 30 mg/mL BDM ( Sigma ) in M9 buffer .", "Dorsal cord protein localization: Images were obtained as Z-stacks of the dorsal cord above the posterior gonad reflex ( 100x objective , Zeiss ( Jena , Germany ) AxioImager ApoTome and Axiovision software v4 . 8 ) .", "For MEL-46::GFP analysis , a set area was defined for each image along the dorsal cord ( 25 µm x 5 µm ) .", "Using ImageJ ( RRID:SCR_003070 ) , a uniform threshold was used to eliminate background .", "The number ( density ) , mean fluorescence ( intensity ) and area ( size ) for MEL-46::GFP granular structures were calculated using the ImageJ ‘particle analyzer’ program .", "For synaptic protein localization , mean puncta width ( meanfixedwidth ) , intensity ( meanfixedvolume ) and linear density ( fixedwidthlineardensity ) were quantified with an in-house developed program called ‘Punctaanalyser’ using MatLab software ( v6 . 5; Mathworks , Inc . , Natick , MA , USA; RRID:SCR_001622 ) ( Kim et al . , 2008 ) .", "At least three independent trials ( n > 17 animals/genotype ) were performed .", "For data sets involving smn-1 ( ok355 ) , smn-1 ( rt248 ) , or mel-46 ( tm1739 ) animals , all genotypes were examined at the early L4 stage , while other data sets were collected with young adult stage animals .", "GFP Fluorescence Quantification: GFP images of L4 animals were acquired ( 10x objective , Zeiss V20 stereoscope and Axiovision software v4 . 8 ) .", "Mean GFP fluorescence was quantified using ImageJ ( RRID:SCR_003070 ) .", "A threshold was set to eliminate background fluorescence .", "For each data set , thresholds were kept constant .", "Average fluorescence values ( ±SEM ) were combined from at least three independent trials for n > 25 animals/genotype; however certain backgrounds containing rtEx855[mel-46 ( + ) ] had a lower n ( reported in legends ) as these animals went sterile and/or did not throw many progeny carrying the mel-46 array .", "Ratios in Figure 4H and Figure 5A were calculated as average mean fluorescence for each genotype in the rtIs56 background and divided by their respective average mean fluorescence in the control rtIs57 background .", "Ratio SEM was calculated by summing the SEM for each population ( see Figure 4—figure supplement 2D ) .", "All representative images shown were analyzed as part of data collection .", "For each RNA sample , animals were synchronized by collecting eggs for 6 hr from gravid adults on large seeded NGM plates .", "After two days at 25°C , young adult progeny were washed off , rinsed and flash frozen .", "Total RNA was extracted after a 15 min Trizol ( Thermo Fisher , Waltham , Massachusetts ) incubation .", "1 ng total RNA was used for reverse transcription with either the miScript II RT kit ( Qiagen #218160 ) for miRNA or the SuperScript III First-Strand Synthesis Supermix kit ( Invitrogen #11752050 ) for mRNA .", "Methodology followed manufacturer’s instructions .", "miRNA levels were determined in a 10 µl reaction using miScript SYBR Green PCR kit ( #218073 , Qiagen , Venlo , Netherlands ) and 300 nM of mature miR-2 primer/probe .", "miR-60 was used to normalize miR-2 expression as it is not expressed in the nervous system where SMN-1 or MEL-46 were knocked-down .", "Forward primer sequences for miR-2 and miR-60 were , respectively: 5’-TATCACAGCCAGCTTTGATGTGC-3’ and 5’-TATTTATGCACATTTTCTAGTTCA-3’ .", "A universal reverse probe was provided by Qiagen .", "Primer sequences for act-1: 5’-acgccaacactgttctttcc-3’ and 5’-gatgatcttgatcttcatggttga-3’ ( Ly et al . , 2015 ) .", "Primer sequences for 18S rRNA: 5’-TTGCTGCGGTTAAAAAGCTC’3’ and 5’-CCAACCTCAAACCAGCAAAT-3’ ( Essers et al . , 2015 ) .", "The stability of miR-60 , 18S rRNA , and act-1 housekeeping RNAs were evaluated using the ‘model-based approach to estimation of expression variation’ ( Andersen et al . , 2004 ) .", "mRNA levels were determined in a 10 µl reaction using Power SYBR Green PCR Master Mix ( Thermo Fisher Scientific # 4368706 ) , and 300 nM of each primer .", "PGK-1 was used to normalize gar-2 expression , as the mammalian orthologue has been used previously as a housekeeping gene for experiments involving SMN ( Abera et al . , 2016; Simard et al . , 2007 ) .", "Primer sequences for gar-2: 5’-CCTGAACTCTCATTGCCCTTTATTGATGC-3’ and 5’-CTAGCAGTCCCTTGCATTGAAAC-3’ .", "Primer sequences for pgk-1: 5’-GGCCCTCGACAACCCAGCTCGTC-3’ and 5’-CGGCGGAGGAATGGCCTATACC-3 .", "All reactions were performed in triplicate .", "Melting curve analysis and electrophoresis in agarose gel of every PCR product was conducted after each qRT-PCR to control amplification specificity .", "Gene expression level was calculated as the fold change of relative DNA amount of a target gene in a target sample and a reference sample normalized to a reference gene using the comparative ΔΔCT method as previously described ( Kurrasch et al . , 2004 ) .", "E13 . 5 mouse MNs were isolated from WT ( FVB/NJ; RRID:IMSR_JAX:001800 ) and SMA mice ( FVB , Smn-/-;SMN2tg/0; generated by crossing lines RRID:IMSR_JAX:005058 and RRID:IMSR_JAX:005024 ) ( Riessland et al . , 2010 ) as described ( Wiese et al . , 2001 ) .", "Isolated mouse MNs were differentiated 10 days in NB/B27 media supplemented with growth factors to promote survival; brain derived neurotrophic factor ( BDNF ) 10 ng/ml , ciliary neurotrophic factor ( CNTF ) 10 ng/ml and glial-derived neurotrophic factor ( GDNF ) 50 ng/ml .", "Fifty percent of medium was replaced every three days .", "To reduce the amount of glia and fibroblasts in culture , 1 µM cytosine arabinoside ( AraC ) was added at day 3 .", "After 10 days in vitro culture , total RNA was extracted from MNs using the mirVana total RNA isolation kit ( Thermo Scientific ) .", "Nanodrop was used to measure RNA amount .", "Using 100 ng of total RNA , miR-128 expression levels were determined by real time PCR with mature miR-128a primer/probe ( TaqMan MicroRNA Assays , #4427975 , Thermo Scientific ) .", "Actin-beta was used to normalize miR-128a expression .", "Primer sequences for actin-beta: 5'-agccatgtacgtagccatcc-3' and reverse 5'-ctctcagctgtggtggtgaa-3' .", "Methodology followed manufacturer’s instruction ( Kye et al . , 2014 ) .", "Proteins were extracted from motor neurons , after 10 days in vitro culture , using RIPA buffer and protease inhibitor cocktail ( Smith et al . , 2014 ) .", "Expression of m2R and β-actin was measured using Western blot .", "Antibodies against m2R ( ABCAM , ab109226; RRID:AB_10858602; 1:1000 ) and β-actin ( Santa Cruz , sc-47778; RRID:AB_626632; 1:1000 ) were used to detect proteins .", "Methoctramine ( Sigma , M105 ) was treated 48 hr from DIV3 to DIV5 in various concentrations .", "After 5 days of in vitro culture , neuronal morphology was visualized with Tau ( Santa Cruz , A-10 ) staining .", "Axon length was analyzed with ImageJ ( RRID:SCR_003070 ) .", "Log-rank test , two-tailed Mann-Whitney U-test , or t-test were used for C . elegans statistical analysis .", "The Mann Whitney U-test was chosen over t-test for experiments where homogeneity could not be assured ( i . e . RNAi; extrachromosomal arrays; or potential maternal loading from a heterozygous parent ) .", "t-test was used to determine significance for spinal motor neuron Western blot quantification and qPCR quantification ." ] ]
[ "Spinal Muscular Atrophy ( SMA ) is caused by diminished Survival of Motor Neuron ( SMN ) protein , leading to neuromuscular junction ( NMJ ) dysfunction and spinal motor neuron ( MN ) loss .", "Here , we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs .", "Loss of the C . elegans SMN ortholog , SMN-1 , causes NMJ defects .", "We found that increased levels of the C . elegans Gemin3 ortholog , MEL-46 , ameliorates these defects .", "Increased MEL-46 levels also restored perturbed microRNA ( miR-2 ) function in smn-1 ( lf ) animals .", "We determined that miR-2 regulates expression of the C . elegans M2 muscarinic receptor ( m2R ) ortholog , GAR-2 .", "GAR-2 loss ameliorated smn-1 ( lf ) and mel-46 ( lf ) synaptic defects .", "In an SMA mouse model , m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects .", "Collectively , these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation , followed by increased m2R expression , and neuronal dysfunction in SMA ." ]
[ "Spinal muscular atrophy is a genetic disease that causes muscles to gradually weaken .", "In people with the disease , the nerve cells that control the movement of muscles – called motor neurons – deteriorate over time , hindering the person’s mobility and shortening their life expectancy .", "Spinal muscular atrophy is usually caused by genetic faults affecting a protein called SMN ( which is short for “Survival of motor neuron” ) and recent research suggested that disrupting this protein alters the function of short pieces of genetic material called microRNAs .", "However , the precise role that microRNAs play in the disease and their connection to the SMN protein was not clear .", "MicroRNAs interfere with the production of proteins by disrupting molecules called messenger RNAs , which are temporary strings of genetic code that carry the instructions for making protein .", "By disrupting messenger RNAs , microRNAs can delay or halt the production of specific proteins .", "This is an important part of the normal behavior of a cell , but disturbing the activity of microRNAs can lead to an unwanted rise or fall in crucial proteins .", "O’Hern et al . made use of engineered nematode worms and mice that share genetic features with spinal muscular atrophy patients , including disruption of the gene responsible for producing the SMN protein .", "These animal models of the disease were used to examine the relationship between decreased SMN levels and microRNAs in motor neurons .", "The experiments showed that reduced SMN activity affects a specific microRNA , which in turn causes motor neurons to produce more of a protein called m2R .", "This protein is a receptor for a molecule , called acetylcholine , which motor neurons use to send signals to muscle cells .", "Increased m2R may be detrimental to motor neurons .", "As such , O’Hern et al . decreased m2R protein activity to determine whether this could reverse the defects in motor neurons that arise in the animal models of the disease .", "Indeed , blocking this receptor rescued some of the defects seen in the animal models , supporting the link to spinal muscular atrophy .", "Several treatments that block m2R are already available to treat other conditions .", "As such , the next step is to determine whether these existing treatments are able to protect mice models of spinal muscular atrophy against muscle deterioration or increase their lifespan .", "If successful , this could open new avenues for the development of treatments in people ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "biochemistry and chemical biology", "cell biology" ]
Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection
elife-21687-v3
[ [ "DNA end resection – the nucleolytic digestion of the 5’ strands of a DSB – is essential for the initiation of homologous recombination ( HR ) or related recombination-based mechanisms ( reviewed in [Cejka , 2015; Symington , 2014; Symington and Gautier , 2011] ) .", "At the same time , resection interferes with ligation-based repair ( non-homologous end-joining , NHEJ ) and thus is the critical step for repair pathway choice .", "In mitotically dividing cells , recombination-based repair critically depends on the presence of a sister-chromatid .", "DSB repair pathway choice and accordingly DNA end resection are therefore highly regulated during the cell cycle: in G1 phase , little resection occurs and NHEJ is therefore favoured .", "Conversely , in S , G2 and M phase , resection is up-regulated and HR becomes more prevalent ( Cejka , 2015; Ira et al . , 2004; Symington , 2014; Symington and Gautier , 2011 ) .", "The nucleases that mediate resection can be subdivided into resection initiation ( by Mre11-Rad50-Xrs2 and Sae2 in budding yeast ) and long-range resection ( by Exo1 or Dna2 with Sgs1-Top3-Rmi1 in budding yeast ) pathways ( Cannavo and Cejka , 2014; Cejka et al . , 2010; Mimitou and Symington , 2008; Niu et al . , 2010; Zhu et al . , 2008 ) .", "So far , Sae2 and Dna2 were shown to be cell cycle - controlled by cyclin-dependent kinase ( CDK ) phosphorylation in yeast ( Chen et al . , 2011; Huertas et al . , 2008 ) and EXO1 in human cells ( Tomimatsu et al . , 2014 ) .", "Notably , however , a bypass of this control is not sufficient to allow efficient end resection to occur in G1 , suggesting that other factors may be involved in the cell cycle control of DNA end resection .", "Resection is also influenced by the surrounding chromatin .", "Nucleosomes themselves can be inhibitory to resection enzymes ( Adkins et al . , 2013 ) .", "Additionally , nucleosome-associated proteins such as budding yeast Rad9 or its functional ortholog in humans , 53BP1 , can inhibit resection ( Bunting et al . , 2010; Lazzaro et al . , 2008; Trovesi et al . , 2011 ) .", "Consistent with a barrier function of chromatin and/or Rad9 , nucleosome remodellers are recruited to DSBs and promote resection , although the mechanism is poorly understood ( Bennett and Peterson , 2015; Bennett et al . , 2013; Chai et al . , 2005; Morrison et al . , 2004; van Attikum et al . , 2007 , 2004 ) .", "The Swr1-like family remodeller Fun30 ( SMARCAD1 in humans ) was found to be a critical regulator of resection ( Chen et al . , 2012; Costelloe et al . , 2012; Eapen et al . , 2012 ) .", "Fun30 localizes to chromatin surrounding DSBs and fun30△ mutant cells show a pronounced defect in long-range resection ( Chen et al . , 2012; Costelloe et al . , 2012; Eapen et al . , 2012 ) .", "Importantly , also SMARCAD1 promotes DNA end resection in human cells , suggesting evolutionary conservation ( Costelloe et al . , 2012 ) .", "Fun30 itself is a substrate for CDK phosphorylation ( Chen et al . , 2012 , 2016; Ubersax et al . , 2003 ) , but it has remained unclear by which mechanism Fun30 function is regulated during the cell cycle , how Fun30 is targeted to DNA lesions and if this regulation imposes a bottleneck in the regulation of DNA end resection .", "Here , we show that CDK phosphorylation enables Fun30 to form a complex with the phospho-protein-binding scaffold protein Dpb11 and the DNA damage sensor 9-1-1 .", "Formation of this complex is required for proper localization of Fun30 and for efficient long-range resection in M phase cells .", "Notably , when we bypass the CDK requirement by directly fusing Fun30 to a subunit of the 9-1-1 complex , we observe long-range resection even in G1–arrested cells .", "This suggests that the cell cycle regulation of long-range resection can be bypassed solely by artificially targeting Fun30 to DSBs .", "Finally , we show that also human SMARCAD1 binds to TOPBP1 ( human ortholog of Dpb11 ) in a CDK phosphorylation-dependent manner that involves conserved interaction surfaces , suggesting that the formation of a Fun30-Dpb11 complex is a conserved mechanism of cell cycle regulation that could control DNA end resection and repair pathway choice throughout eukaryotes ." ], [ "We identified Fun30 in a two-hybrid screen for interactors of the scaffold protein Dpb11 .", "Dpb11 is a critical regulator of genome stability in budding yeast and as such is found in several distinct protein complexes ( Gritenaite et al . , 2014; Ohouo et al . , 2010 , 2013; Pfander and Diffley , 2011; Puddu et al . , 2008; Tanaka et al . , 2007; Zegerman and Diffley , 2007 ) .", "Crucial for the formation of these complexes are the two tandem BRCT domains of Dpb11 , which are phospho-protein binding modules ( Leung and Glover , 2011 ) specific for discrete sets of phosphorylation-dependent interactors .", "In case of Fun30 , the interaction is mediated by BRCT1+2 , but not BRCT3+4 ( Figure 1A , Figure 1—figure supplement 1 ) .", "Using Dpb11 expressed from the strong GPD promoter , we also observed an interaction between Fun303FLAG and Dpb11 in co-immunoprecipitation ( Co-IP ) experiments ( Figure 1B ) .", "All Dpb11 complexes characterized so far are cell cycle-regulated ( Gritenaite et al . , 2014; Ohouo et al . , 2013; Pfander and Diffley , 2011; Tanaka et al . , 2007; Zegerman and Diffley , 2007 ) .", "Thus , we tested the interaction between Dpb11 and Fun30 from cells at different cell cycle stages .", "We observed that Fun30 interacted with Dpb11 only during late S to M phase , but not in G1 ( Figure 1B–C , Figure 1—figure supplement 2 ) and this interaction was not influenced by DNA damage ( Figure 1D ) . 10 . 7554/eLife . 21687 . 003Figure 1 . Fun30 and Dpb11 interact in a cell cycle- and CDK phosphorylation-dependent manner and this targets Fun30 to DSBs .", "( a ) Two-hybrid assay with GAL4-AD and -BD constructs as indicated reveals a physical interaction between the N-terminal region of Fun30 ( aa 1–188 ) and the BRCT1+2 domain of Dpb11 .", "Rad9 and Ddc1 represent known interactors of BRCT1+2 and BRCT3+4 , respectively .", "( b–e )", "Characterization of the Fun30-Dpb11 interaction by Fun303FLAG Co-IP experiments .", "Dpb11 was expressed from the strong , constitutive GPD promoter .", "( b ) Fun303FLAG specifically binds Dpb11 in cells arrested in M but not G1 phase .", "( c ) Fun303FLAG purified from cells synchronously progressing through the cell cycle binds Dpb11 only at 45’ and 90’ time points corresponding to late S and M phase ( Figure 1—figure supplement 2 for FACS analysis and western analysis of cell cycle progression ) .", "( d ) No enhancement of the Fun30-Dpb11 interaction by CPT or phleomycin treatment as measured by Fun303FLAG Co-IP .", "For DNA damage treatment , 50 µM CPT or 50 µg/ml phleomycin were added to asynchronously dividing yeast cells .", "DNA damage checkpoint activation was measured by Rad53 phosphorylation in IP extracts ( lowest blot panel ) .", "( e ) CDK inhibition using the cdc28-as1 allele and 1-NMPP1 treatment diminishes the Fun303FLAG-Dpb11 interaction in M phase arrested cells .", "( f ) Purified Fun30 interacts with a BRCT1+2 fragment of Dpb11 in the presence of CDK phosphorylation .", "Purified Fun303FLAG or the positive control MBPRad9 ( Pfander and Diffley , 2011 ) were incubated with a model CDK and ATP before binding to bead-bound GSTDpb11 BRCT1+2 .", "( g ) Mapping analysis of the two-hybrid interaction between Fun30 and Dpb11 reveals a binding site close to the N-terminus of Fun30 .", "( h–i )", "Putative CDK sites on Fun30 ( S20 and S28 ) are required for Dpb11 binding .", "( h ) Two-hybrid assay as in", "( a ) but in five-fold serial dilution and with WT , S20A , S28A and SS20 , 28AA variants of Gal4-AD-Fun301-188 .", "( i ) Co-IP as in", "( b ) but with mutant variants of Fun303FLAG growing asynchronously .", "( j ) Efficient Fun30 localization to damaged chromatin requires the Dpb11-Fun30 interaction .", "ChIP of Fun303FLAG to chromatin locations 3 , 8 , 10 and 15 kb distant of a non-repairable DSB induced at the MAT locus in M phase-arrested cells .", "fun30 mutants were expressed from the endogenous promoter as only copy of FUN30 .", "The FUN30-DPB11 fusion contains fun30-SSAA and dpb11△N mutations .", "WT , fun30-SSAA and FUN30-DPB11 fusion cells were crosslinked at indicated timepoints after DSB induction .", "Plotted values represent means from two independent experiments , error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 00310 . 7554/eLife . 21687 . 004Figure 1—figure supplement 1 . Expression control of two-hybrid constructs used in Figure 1A . Two-hybrid constructs are detected with anti-Gal4-AD ( AD-Fun30 1–188 , AD-Ddc1 , AD-Rad9 ) and with anti-Gal4-BD ( for BD-Dpb11 constructs ) antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 00410 . 7554/eLife . 21687 . 005Figure 1—figure supplement 2 . Control of the cell cycle states of the experiment in Figure 1C . Left panel: DNA content measurements with FACS .", "Right panel: Western Blot analysis using S/M-phase ( hyperphosphorylated Sld2 ) or M-phase ( Clb2 ) markers .", "Asterisk indicates a cross reactive band . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 00510 . 7554/eLife . 21687 . 006Figure 1—figure supplement 3 . Expression control of two-hybrid constructs used in Figure 1G . Two-hybrid constructs are detected with anti-Gal4-AD ( AD-Fun30 constructs ) and with anti-lexA-BD ( for BD-Dpb11 BRCT1 +2 construct ) antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 00610 . 7554/eLife . 21687 . 007Figure 1—figure supplement 4 . Expression control of two-hybrid constructs used in Figure 1H . Two-hybrid constructs are detected with anti-Gal4-AD ( AD-Fun30 1–188 and mutant derivatives ) and with anti-Gal4-BD ( for BD-Dpb11 BRCT1 +2 constructs ) antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 00710 . 7554/eLife . 21687 . 008Figure 1—figure supplement 5 . Efficient Fun30 localization to damaged chromatin requires the Dpb11-Fun30 interaction . ChIP of Fun303FLAG to chromatin in proximity of a non-repairable DSB induced at the MAT locus in M phase arrested cells .", "Same experiment as in Figure 1J , using WT , fun30-SSAA and FUN30-DPB11 fusion cells , but here Fun30 ChIP is shown at additional loci ( 1 . 1 , 3 , 8 , 10 , 15 and 20 kb distance from break ) .", "Cells were crosslinked at distinct time points after DSB induction .", "Plotted values represent error bars from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 008 Since Fun30 is phosphorylated by CDK ( Chen et al . , 2012 , 2016; Ubersax et al . , 2003 ) and Dpb11 was shown to bind several CDK targets ( Gritenaite et al . , 2014; Pfander and Diffley , 2011; Tanaka et al . , 2007; Zegerman and Diffley , 2007 ) , we tested if CDK phosphorylation mediates the Fun30-Dpb11 interaction .", "Indeed , upon CDK inhibition ( using the cdc28-as1 allele and 1-NMPP1 inhibition ) Dpb11 binding to Fun30 was strongly reduced ( Figure 1E ) .", "Accordingly , purified Fun303FLAG was able to interact with GSTDpb11-BRCT1+2 in vitro but only after pre-phosphorylation by CDK ( Figure 1F ) , suggesting that the Fun30-Dpb11 interaction as well as its regulation by CDK phosphorylation are direct .", "Therefore , we sought to identify the CDK phosphorylation sites on Fun30 , which are relevant for Dpb11 binding .", "Interaction mapping using truncated constructs placed the Dpb11 interaction site close to the N-terminus of Fun30 ( Figure 1G , Figure 1—figure supplement 3 ) .", "Within this region , we identified S20 as well as S28 as critical residues for the Fun30-Dpb11 interaction by two-hybrid and Co-IP binding assays using non-phosphorylatable versions of Fun30 ( Figure 1H–I , Figure 1—figure supplement 4 ) .", "This suggests that phosphorylation of both residues may create a composite binding surface for Dpb11 BRCT1+2 , perhaps similar to the Dpb11-binding surfaces on Rad9 and Sld3 ( Pfander and Diffley , 2011; Tanaka et al . , 2007; Zegerman and Diffley , 2007; Zegerman et al . , 2010 ) .", "It seemed likely that Dpb11 is involved in targeting Fun30 to DNA lesions .", "We therefore tested recruitment of WT Fun303FLAG or the corresponding fun30-SSAA variant to a site-specific , non-repairable DSB using chromatin immunoprecipitation ( ChIP ) .", "Indeed , we observed that Fun30-SSAA binding to regions distal of the DSB was reduced compared to WT ( 8–20 kb , Figure 1J , Figure 1—figure supplement 5 ) , while both versions bound similarly to the immediate vicinity of the DSB ( 1–3 kb , Figure 1J , Figure 1—figure supplement 5 ) .", "This result thus confirms recent observations showing a DSB recruitment defect of fun30-S20A and fun30-S28A mutants ( Chen et al . , 2016 ) .", "Importantly , we could expand these data by generating an experimental tool , which restores the Fun30-Dpb11 interaction in a phosphorylation-independent manner .", "Since conventional phospho-mimetic mutations failed to promote binding ( data not shown ) , we generated a covalent fusion of the Fun30-SSAA protein directly to Dpb11△N lacking BRCT1+2 ( FUN30-AA-DPB11-276-C expressed as the only copy of Fun30 from the endogenous promoter , referred to as FUN30-DPB11 fusion in the following ) .", "Importantly , the fusion protein localized efficiently to damaged chromatin and thus restored the defect of the fun30-SSAA mutation ( Figure 1J , Figure 1—figure supplement 5 ) .", "This finding suggests that the interaction with Dpb11 is indeed involved in targeting Fun30 to DSBs and that the covalent fusion is sufficient to bypass the CDK regulation of Fun30 .", "Notably , DSB recruitment of the Fun30-Dpb11 fusion protein was stronger than Fun30 consistent with the replacement of a transient , PTM-dependent interaction by a covalent interaction .", "Therefore , we reason that the FUN30-DPB11 fusion deregulates Fun30 in two ways: first , it uncouples Fun30 from its cell cycle regulation .", "Second , it leads to enhanced DSB localization to DSBs , thus potentially enhancing Fun30 activity at damaged chromatin .", "We also note that the apparently normal recruitment of Fun30-SSAA to the immediate vicinity of the DSB could be explained by an additional CDK phosphorylation-independent , but resection-dependent recruitment mechanism , such as via binding to RPA ( Chen et al . , 2012 ) .", "Our data thus suggest the existence of two Fun30–targeting mechanisms: one that is Dpb11-dependent and recruits Fun30 to sites of ongoing resection , and a second that is Dpb11-independent and tethers Fun30 to DNA that has already been resected .", "We utilized our system of abolishing and constitutively forcing Fun30 binding to Dpb11 in order to investigate the biological function of the Dpb11-dependent Fun30 targeting mechanism and its role in regulating DNA end resection .", "We measured resection at an HO-induced , non-repairable DSB in M phase-arrested cells using the combined read-out of", "( a ) the accumulation of the ssDNA-binding protein RPA ( Figure 2A , upper panel ) around the DSB by ChIP and", "( b ) the specific DNA loss ( occurring due to ssDNA formation , Figure 2A , lower panel ) .", "Indeed , compared to WT cells , fun30△ mutants showed a pronounced defect in long-range resection , visible by a reduced spreading of both RPA-ChIP and DNA loss , to regions greater than 10 kb away from the break ( Figure 2A , Figure 2—figure supplement 1 ) , confirming previous observations ( Chen et al . , 2011 , 2012; Costelloe et al . , 2012; Eapen et al . , 2012 ) .", "Notably , the same defect in long-range resection was also observed in the Dpb11-binding deficient fun30-SSAA mutant and was fully restored by the FUN30-DPB11 fusion ( Figure 2A , Figure 2—figure supplement 1 ) .", "To corroborate these findings , we also analysed resection-dependent DSB repair in a single-strand annealing ( SSA ) assay , where cellular survival upon an HO-induced DSB in the absence of Rad51 critically depends on the efficient resection of 25 kb of DNA ( Figure 2B , [Vaze et al . , 2002] ) .", "In fact , Dpb11-binding deficient fun30 mutants were deficient in SSA-mediated survival and this defect was completely rescued by covalent fusion of Fun30-SSAA to Dpb11 ( Figure 2B ) .", "Thus , the CDK-regulated interaction between Fun30 and Dpb11 is required for efficient long-range resection as well as subsequent resection-coupled repair . 10 . 7554/eLife . 21687 . 009Figure 2 . The Fun30-Dpb11 complex is required for efficient long-range resection .", "( a ) Long-range resection of a DSB is dependent on the Fun30-Dpb11 interaction .", "A non-repairable DSB at MAT was induced in M phase-arrested WT , fun30△ , fun30-SSAA and FUN30-DPB11 fusion strains and DNA end resection measured at indicated times .", "Upper panel: fold enrichment of a given locus in an RPA ChIP relative to undamaged control loci .", "Lower panel: DNA loss relative to control loci located in non-damaged chromatin .", "( b ) Single-strand annealing ( SSA ) is dependent on the Fun30-Dpb11 interaction .", "FUN30 mutants as indicated were combined with the rad51△ deletion , a DSB at the leu2::HO cutsite was induced by plating cells on galactose .", "Cells need to resect 25 kb up to the homologous his4::leu2 locus in order to survive by SSA . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 00910 . 7554/eLife . 21687 . 010Figure 2—figure supplement 1 . The Fun30-Dpb11 interaction is required for efficient long-range resection . Long-range resection of a site-specific DSB is partially deficient in CDK-phosphorylation site mutants that are deficient in the Fun30-Dpb11 interaction .", "A non-repairable DSB at MAT was induced in M-phase arrested WT , fun30-S20A3FLAG , fun30-S28A3FLAG , fun30-SSAA3FLAG and fun30∆ cells .", "DNA end resection was measured at indicated time points by RPA ChIP .", "Plotted is the fold enrichment of a given locus relative to three undamaged control loci . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 010 Fun30 participates in chromatin organization in the absence of DNA damage ( Neves-Costa et al . , 2009 ) and previous studies could therefore not rule out the possibility that the DNA end resection defect of the fun30△ mutant is a consequence of general changes in chromatin organization ( Chen et al . , 2012; Costelloe et al . , 2012; Eapen et al . , 2012 ) .", "However , we found that the fun30-SSAA mutant ( unlike the fun30△ mutant ) did not display any defect in silencing at telomeres or at the silent mating type locus and thus differs from the fun30△ mutant ( Figure 3 ) .", "The fun30-SSAA mutant thus separates Fun30 functions and the associated resection phenotype of this mutant therefore provides strong support for a direct role of Fun30 and the Fun30-Dpb11 complex during DNA end resection . 10 . 7554/eLife . 21687 . 011Figure 3 . The Fun30-Dpb11 interaction is not involved in Fun30-dependent gene silencing at telomeric heterochromatin and a silent mating type locus . The silencing defect of the fun30∆ mutant is not recapitulated by the fun30-SSAA mutant .", "Two silencing tester strains were used: the first ( upper panels ) had URA3 integrated in telomeric heterochromatin at the end of the left arm of chromosome VII , the second ( lower panels ) had URA3 integrated at the HML silent mating type locus .", "A silencing defect leads to enhanced growth on –Ura medium and less growth on medium supplemented with 5-FOA ( e . g . fun30∆ ) .", "Shown is a spotting in 5-fold serial dilutions on non-selective medium , medium lacking uracil or containing 5-FOA . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 011 Mutants with DNA end resection defects such as exo1△ sgs1△ , sae2△ or fun30△ are hypersensitive towards the Top1 inhibitor camptothecin ( CPT ) ( Chen et al . , 2012; Costelloe et al . , 2012; Eapen et al . , 2012; Neves-Costa et al . , 2009 ) ( Figure 4A–C ) , most likely because of repair defects of replication-borne DSBs at CPT-induced Top1 stall sites .", "Indeed , also the fun30-SSAA mutant showed hyper-sensitivity to CPT albeit not as strong as the fun30 deletion .", "Importantly , the CPT sensitivity of fun30-SSAA was rescued by expressing the covalent FUN30-DPB11 fusion ( Figure 4A , Figure 4—figure supplement 1 ) , emphasizing again the importance of the Fun30-Dpb11 interaction for DSB repair . 10 . 7554/eLife . 21687 . 012Figure 4 . The Fun30-Dpb11 interaction is required for the response towards CPT , as is functional long- and short-range resection .", "( a ) The Fun30-Dpb11 interaction is required for the response towards CPT .", "WT , fun30△ , fun30-SSAA and FUN30-DPB11 fusion were spotted in five-fold serial dilutions on plates containing indicated amounts of CPT and incubated at 37°C for two days .", "( b ) A double mutant of exo1∆ and sgs1∆ is hyper-sensitive to low doses of CPT .", "Spotting in 5-fold serial dilutions was incubated for two days at 30°C .", "( c ) The fun30∆/fun30-SSAA mutants enhance the CPT hyper-sensitivity of sae2∆ mutants .", "Cells were spotted in 5-fold serial dilutions and incubated for two days at 30°C .", "( d ) A rad9∆ deletion rescues CPT hyper-sensitivity of fun30∆ and fun30-SSAA mutant alleles . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 01210 . 7554/eLife . 21687 . 013Figure 4—figure supplement 1 . Mutants of Fun30 show no discernable phenotype upon chronic exposure to HU , MMS or phleomycin . Spotting in 5-fold serial dilutions on medium containing indicated dosages of DNA damaging agents .", "Plates were incubated two days at 30°C . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 01310 . 7554/eLife . 21687 . 014Figure 4—figure supplement 2 . The catalytic activity of Fun30 is required for the suppression of the CPT phenotype in the context of the FUN30-DPB11 fusion . Spotting of strains with indicated genotypes in 5-fold serial dilutions on CPT containing medium .", "The K603R mutation is located in the Walker A motif of Fun30 .", "Plates were incubated for two days at 37°C . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 014 Genetic evidence suggests that Fun30 may promote DNA end resection by antagonizing the resection inhibitor Rad9 ( Chen et al . , 2012 ) .", "Similar to what has been described for the fun30△ mutant , we observed that the CPT-hypersensitivity of the fun30-SSAA was suppressed by an additional rad9 deletion ( Figure 4D ) , suggesting that the Fun30-Dpb11 complex antagonizes Rad9 .", "Interestingly , Rad9 also binds to Dpb11 , and Fun30 and Rad9 share the same interaction site on Dpb11 ( Pfander and Diffley , 2011 ) .", "While it is currently unknown whether Dpb11-associated Rad9 ( in contrast to nucleosome-associated Rad9 ) contributes to the inhibition of DNA end resection , the overlapping binding site raised the possibility that Fun30 may interfere with Rad9 function via competition .", "Therefore , in order to exclude that the FUN30-DPB11 fusion rescues resection simply by blocking the Rad9-Dpb11 interaction , we inactivated the ATPase activity of Fun30 by a Walker A motif mutation ( K603R ) in the context of the FUN30-DPB11 fusion and found the K603R mutant fusion did not restore WT resistance to CPT ( Figure 4—figure supplement 2 ) .", "Therefore , competition does not explain the effects of the FUN30-DPB11 fusion and the catalytic activity of Fun30 is required for the resection-promoting function of the Fun30-Dpb11 complex .", "Overall , these data thus suggest that cell cycle-regulated targeting of Fun30 by Dpb11 is required for efficient DNA end resection .", "In several organisms , recruitment of Dpb11 and its orthologs to DSBs has been shown to be facilitated by the 9-1-1 complex ( Delacroix et al . , 2007; Du et al . , 2006; Furuya et al . , 2004; Pfander and Diffley , 2011; Puddu et al . , 2008 ) , a signalling platform ( Parrilla-Castellar et al . , 2004 ) which is loaded at DNA damage sites .", "Given that 9-1-1 interacts with BRCT3+4 of Dpb11 ( Wang and Elledge , 2002 ) , we tested whether Dpb11 could simultaneously bind to Fun30 and 9-1-1 .", "Indeed , Fun303FLAG co-precipitated the 9-1-1 subunits Mec3 and Ddc1 and this binding was absent in cells arrested in G1 or in the respective Dpb11 interaction-deficient mutants ( ddc1-T602A or fun30-SSAA; Figure 5A–B , Figure 5—figure supplement 1 ) .", "We thus conclude that Fun30 , Dpb11 and 9-1-1 can form a ternary complex , which is regulated by the cell cycle stage ( model in Figure 5—figure supplement 2 ) .", "Moreover , we observed a reduction of the Fun30 binding in the proximity of a DSB by ChIP , when we interfered either with the 9-1-1-Dpb11 interaction ( ddc1-T602A mutant ) or with the Fun30-Dpb11 interaction ( SLD3-DPB11∆N mutant strain , which expresses as only copy of DPB11 a truncated version of Dpb11 lacking the Fun30 binding site , Zegerman and Diffley , 2007 ) , further supporting a role of 9-1-1 in targeting Fun30 to DSBs ( Figure 5C ) . 10 . 7554/eLife . 21687 . 015Figure 5 . The 9-1-1 complex forms a ternary complex with Fun30-Dpb11 .", "( a ) Fun30 , Dpb11 and 9-1-1 form a ternary complex .", "The 9-1-1 subunit Mec3 interacts with Fun303FLAG when purified from M phase cells , where also Dpb11 binds to Fun30 .", "( b ) The ddc1-T602A mutation abolishes binding of Mec3 to Fun30-Dpb11 in Fun303FLAG Co-IPs , but leaves the Fun30-Dpb11 interaction intact .", "( c ) Mutants disrupting the interaction between 9-1-1 and Dpb11 ( ddc1-T602A ) or Fun30 and Dpb11 ( SLD3-dpb11∆N , lacks Fun30 binding site , only copy of Dpb11 ) impair efficient localization of Fun30 to DSBs in Fun303FLAG ChIPs of M phase-arrested cells .", "Experiment performed as in Figure 1J , plotted values represent means of two independent experiments , error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 01510 . 7554/eLife . 21687 . 016Figure 5—figure supplement 1 . The interaction between Fun30 and 9-1-1 depends on mutual interactions with Dpb11 , suggesting that Dpb11 forms a molecular bridge in the Fun30-Dpb11-9-1-1 complex . The fun30-SSAA mutation abolishes binding of Dpb11 and also Ddc19myc in Fun303FLAG Co-IPs .", "Cells were either left untreated or treated with 50 µg/ml phleomycin , which induced the DNA damage checkpoint ( Rad53 activation ) , but did not influence Dpb11 or Ddc1 binding . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 01610 . 7554/eLife . 21687 . 017Figure 5—figure supplement 2 . Model of the Fun30-Dpb11-9-1-1 association and its regulation . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 017 Given that Dpb11 seems to function as an adaptor between Fun30 and 9-1-1 , we also generated a covalent fusion of Fun30-SSAA and the 9-1-1 subunit Ddc1 ( referred to as DDC1-FUN30 fusion ) .", "Also this fusion rescued the CPT phenotype of the fun30-SSAA mutant in a manner that depended on the catalytic activity of Fun30 ( Figure 6A , Figure 6—figure supplement 1 ) .", "The DDC1-FUN30 fusion targeted Fun30 even more efficiently to damaged chromatin than the FUN30-DPB11 fusion and , notably , the corresponding strain was able to survive at very high CPT concentrations , where little growth could be detected even for WT cells , indicating that the DDC1-FUN30 fusion promotes hyper-resistance to CPT ( Figure 6A ) .", "It is thus possible to at least partially overcome the limits of cellular resistance to CPT by providing very efficient targeting of Fun30 to damaged chromatin and uncoupling it from cell cycle control . 10 . 7554/eLife . 21687 . 018Figure 6 . A covalent fusion of Fun30 to the 9-1-1 subunit Ddc1 generates a bypass of the cell cycle regulation of long-range resection .", "( a ) The DDC1-FUN30 fusion confers cellular hyper-resistance to CPT .", "Spotting of indicated strains as in Figure 4A , but using CPT concentrations of up to 12 µg/ml .", "( b ) The DDC1-FUN30 fusion localizes efficiently to a DSB in G1-arrested cells .", "Fun303FLAG ChIPs from WT , fun30-SSAA , FUN30-DPB11 and DDC1-FUN30 cells as in Figure 1J , but from G1 or M phase-arrested cells .", "Additional Fun303FLAG ChIP data can be found in Figure 6—figure supplement 3 .", "( c ) The DDC1-FUN30 fusion enhances long-range resection in G1-arrested cells .", "Resection assay as in Figure 2A , but with G1 or M phase-arrested cells .", "Additional resection assay data can be found in Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 01810 . 7554/eLife . 21687 . 019Figure 6—figure supplement 1 . The DDC1-FUN30 fusion rescues the CPT sensitivity of the fun30△ mutant in a manner that depends on the Fun30 catalytic activity . WT , fun30∆ , DDC1-FUN30 fusion and DDC1-FUN30 ( K603R ) fusion mutants are spotted on CPT as in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 01910 . 7554/eLife . 21687 . 020Figure 6—figure supplement 2 . Flow cytometric analysis of DNA content for experiments shown in Figure 6B–C and Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02010 . 7554/eLife . 21687 . 021Figure 6—figure supplement 3 . The DDC1-FUN30 fusion protein efficiently localizes to DSBs and promotes hyper-resection in M phase as well as allowing long-range resection in G1 phase . Cells ( WT , fun30∆ and DDC1-FUN30 fusion ) were arrested in G1 or M phase prior to DSB induction .", "Fun30 localization was investigated by anti-FLAG ChIP after break induction ( upper panels ) .", "DNA end resection was investigated by the combined read-out of RPA ChIP and DNA loss ( lower panels ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 021 DNA end resection is up-regulated in S , G2 , M phases of the cell cycle , thus shifting the DSB repair pathway choice from NHEJ to recombination-dependent mechanisms ( Cejka , 2015; Symington and Gautier , 2011 ) .", "Previous efforts to bypass this regulation have focussed on nucleases ( Huertas et al . , 2008 ) .", "Thus , we tested if the CDK-regulation of Fun30 may contribute to the cell cycle regulation of DNA end resection or may even be a limiting factor for this regulation .", "We used the DDC1-FUN30 fusion , which in contrast to WT Fun30 efficiently localized to a DSB in G1 ( Figure 6B , Figure 6—figure supplement 3 ) .", "This indicates that the fusion may in principle allow Fun30 to act on damaged chromatin in G1 , consistent with 9-1-1 being loaded to damaged chromatin in G1 ( Barlow et al . , 2008; Janke et al . , 2010 ) .", "Indeed , the DDC1-FUN30 fusion protein promoted resection in G1 to a significantly larger reach compared to WT Fun30 , since RPA recruitment could be observed up to 25 kb distance from the DSB ( Figure 6C , Figure 6—figure supplement 3 ) .", "Notably , the spreading of resection under the DDC1-FUN30 conditions was even more pronounced than in WT cells arrested in M phase ( Figure 6C , Figure 6—figure supplement 3 ) .", "This effect is thus consistent with the very efficient targeting of Fun30 to DNA damage sites by the DDC1-FUN30 fusion .", "This hyperactivation of resection thus indicates that forced tethering of Fun30 to DSB sites is able to bypass the bottleneck that limits long-range resection in G1 .", "It needs to be pointed out that within the resected region the fold enrichment of RPA recruitment and the extent of DNA loss was not restored to similar levels as observed in M phase ( Figure 6C ) .", "Moreover , precise ligation of a cut plasmid by NHEJ did not appear to be influenced by the DDC1-FUN30 fusion ( Figure 7 ) .", "These data thus suggest that the overall cell cycle regulation of DNA end resection was not bypassed completely , presumably because other resection proteins and in particular resection initiation are additional targets of cell cycle regulation ( Albuquerque et al . , 2008; Chen et al . , 2011; Huertas et al . , 2008; Pfander and Diffley , 2011; Zhang et al . , 2009 ) .", "We therefore compared G1 resection in the DDC1-FUN30 strain to another mutant – sae2-S267E – that is thought to at least partially bypass the CDK regulation of resection initiation ( Cannavo and Cejka , 2014; Huertas , 2010 ) .", "Notably , the sae2-S267E mutant showed no increase in the reach of resection and only led to a slight increase in the fold enrichment of the RPA ChIP , both in WT and DDC1-FUN30 background ( Figure 8 ) .", "Overall , these data are thus consistent with a model , whereby sae2-S267E partially bypasses the cell cycle regulation of resection initiation , while the DDC1-FUN30 fusion bypasses the cell cycle regulation of long-range resection .", "This highlights that chromatin has a barrier function towards resection and that formation of the Fun30-Dpb11 complex is the limiting step that needs to be up-regulated during recombination-permissive cell cycle phases in order to overcome this barrier . 10 . 7554/eLife . 21687 . 022Figure 7 . The DDC1-FUN30 fusion does not significantly inhibit non-homologous end-joining ( NHEJ ) .", "Precise re-ligation of BamHI-cut pRS316 as measured by cell viability on SC-Ura plates and subsequent sequencing of single colonies was dependent on Ku70 but not significantly affected in DDC1-FUN30 of fun30∆ mutant cells .", "Plotted are values from three independent experiments representing the viability rate of cells on SC-Ura plates relative to the total cell number and the transformation efficiency of the mock-digested plasmid .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02210 . 7554/eLife . 21687 . 023Figure 8 . The DDC1-FUN30 fusion specifically enhances long-range resection in G1 , while the sae2-S267E phospho-mimicry leads to a small increase in resection initiation . The sae2-S267E mutant has little effect on the spreading of DNA end resection in G1 , but slightly stimulates the RPA fold enrichment in WT and the DDC1-FUN30 fusion mutant .", "This suggests that sae2-S267E in contrast to the DDC1-FUN30 fusion does not affect long-range resection .", "DNA end resection in the indicated strains was analysed by RPA ChIP as in Figure 5C but with G1 arrested cells . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 023 Fun30’s role in promoting DNA end resection is conserved to its human ortholog SMARCAD1 ( Costelloe et al . , 2012; Densham et al . , 2016 ) .", "Strikingly , in an independent screen we identified an N-terminal fragment of SMARCAD1 ( aa 55–274 ) as interactor of TOPBP1 ( using a TOPBP1 BRCT0-2 construct ) , the human ortholog of Dpb11 .", "Furthermore , we found this interaction to require the phospho-protein binding sites of TOPBP1 BRCT1+2 ( Figure 9A , Figure 9—figure supplement 1 ) .", "We verified the SMARCAD1-TOPBP1 interaction in a pulldown approach using purified GST-TOPBP1-BRCT0/1/2 fragments and in vitro phosphorylation of cell extracts with purified CDK .", "GFP-SMARCAD1-55-445 bound efficiently to GST-TOPBP1-BRCT0/1/2 , but only after addition of active CDK to the cell extract ( Figure 9B ) .", "This suggests that CDK phosphorylation promotes the interaction , similar to the regulation in yeast .", "We therefore queried for the TOPBP1 interaction site on SMARCAD1 using mutagenesis of CDK consensus motifs .", "Using this approach , we found that the T71A variant , but none of the other S/TP site mutants tested caused strongly reduced TOPBP1 binding in two-hybrid and Co-IP ( Figure 9C–D , Figure 9—figure supplement 2 ) .", "These data therefore suggest that SMARCAD1 interacts with TOPBP1 via the CDK-site T71 .", "To our surprise , we observed in two-hybrid experiments that human SMARCAD1 also interacted with yeast Dpb11 and in a manner that was dependent on the T71 phosphorylation site ( Figure 9E , Figure 9—figure supplement 3 ) , despite low sequence conservation .", "This raised the possibility that SMARCAD1 and FUN30 could also functionally complement each other .", "Expression of SMARCAD1 from the inducible GAL-promoter lead only to a slight suppression of the CPT sensitivity of a fun30△ strain ( data not shown ) , suggesting that there is an aspect of Fun30 function or regulation that is not recapitulated by SMARCAD1 .", "In contrast , when we generated a SMARCAD1-Fun30 chimera lacking the Dpb11-binding region of Fun30 but containing the TOPBP1-binding region of SMARCAD1 ( SMARCAD1-1-300-FUN30-30-C ) , this chimera was largely able to rescue the CPT sensitivity of the fun30△ mutant ( Figure 9F , Figure 9—figure supplement 5 ) .", "In contrast , the Fun30-30-C fragment alone was unable to provide a rescue and showed reduced protein stability as well ( without tag or GFP-tagged , figure Figure 9—figure supplements 4 , 6 ) .", "This experiment may thus indicate that the TOPBP1-binding region of SMARCAD1 could replace the Dpb11-binding region of Fun30 in vivo .", "Overall , we therefore conclude from the data in Figure 9 that the Fun30/SMARCAD1 interaction with Dpb11/TOPBP1 , its regulation by CDK phosphorylation and the corresponding interaction surfaces show a remarkable conservation over more than a billion years of eukaryotic evolution . 10 . 7554/eLife . 21687 . 024Figure 9 . Yeast Fun30 and human SMARCAD1 underlie a conserved regulation .", "( a ) SMARCAD1 and TOPBP1 interact and their interaction depends on functional phospho-binding pockets in BRCT1 and BRCT2 of TOPBP1 .", "lexA-BD TOPBP1 1–360 ( harbouring BRCT0/1/2 ) or lexA-BD TOPBP1 1–766 ( harbouring BRCT0-5 ) were tested as WT versions or as K155E , KK154 , 155AM ( affecting BRCT1 ) or K250E ( affecting BRCT2 ) mutant derivatives .", "Interaction was tested against the Gal4-AD SMARCAD1 55–274 .", "3AT was added to –His plates to suppress auto-activation and to increase the stringency of the two-hybrid .", "Two-hybrid interactions with the lexA-BD TOPBP1 1–360 construct were generally stronger compared to lexA-BD TOPBP1 1–766 , leading to milder effects of the K155E and K250E single-mutants , particularly at low 3AT concentrations .", "( b ) SMARCAD1 interacts with TOPBP1 after CDK phosphorylation .", "GFPSMARCAD1 ( 55-445 ) was bound to a GSTTOPBP1 BRCT0/1/2 construct after phosphorylation with CDK .", "This CDK-dependent interaction was seen with several N-terminal SMARCAD1 constructs , but not with FL , perhaps due to low expression .", "( c–d )", "Threonine 71 of SMARCAD1 , a putative CDK phosphorylation site , is required for TOPBP1 binding .", "( c ) Two-hybrid analysis of ADSMARCAD1 ( 1-220 ) and phospho-mutant derivatives to BDTOPBP1 BRCT0/1/2 .", "( d ) Co-IP as in", "( a ) , but additionally using a T71A variant of GFPSMARCAD1 ( 55-274 ) .", "( e ) Dpb11 can bind to human SMARCAD1 , and T71 is important for the interaction .", "Two-hybrid analysis as in", "( b ) , but using a BDDpb11 BRCT1+2 construct .", "( f ) A SMARCAD1-Fun30 chimera lacking the Dpb11-binding site of Fun30 , but containing the TOPBP1-binding site of SMARCAD1 restores sensitivity to CPT .", "The SMARCAD1-Fun30 chimera is expressed from the pGAL1-10 promoter and induced by galactose .", "Spotting on CPT medium as in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02410 . 7554/eLife . 21687 . 025Figure 9—figure supplement 1 . The interaction between SMARCAD1 and TOPBP1 depends on functional phospho-binding pockets in BRCT1 and 2 of TOPBP1 . Expression control for two-hybrid constructs in Figure 9A using anti-lexA-BD and anti-Gal4-AD antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02510 . 7554/eLife . 21687 . 026Figure 9—figure supplement 2 . Threonine 71 of SMARCAD1 , a putative CDK phosphorylation site , is required for TOPBP1 binding . Expression control for two-hybrid constructs in Figure 9C using anti-lexA-BD and anti-Gal4-AD antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02610 . 7554/eLife . 21687 . 027Figure 9—figure supplement 3 . Dpb11 can bind to human SMARCAD1 , and T71 is important for the interaction . Expression control for two-hybrid constructs in Figure 9E using anti-Gal4-BD and anti-Gal4-AD antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02710 . 7554/eLife . 21687 . 028Figure 9—figure supplement 4 . A SMARCAD-FUN30 chimera lacking the Dpb11 binding site of Fun30 but containing the putative TOPBP1 binding site of SMARCAD1 restores sensitivity to CPT , while expression of the Fun30 construct lacking the Dpb11 binding site does not . The SMARCAD1-FUN30 chimera , FUN30 30–C and GFP-FUN30 30–C constructs are expressed from the pGAL1-10 promoter and induced by galactose .", "Spotting on CPT medium as in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02810 . 7554/eLife . 21687 . 029Figure 9—figure supplement 5 . Expression control of the SMARCAD1-FUN30 chimera in Figure 9F . SMARCAD1-FUN30 3FLAG chimerais expressed from the GAL1-10 promoter by addition of galactose .", "Fun30 3FLAG expressed from the endogenous promoter serves as control to visualize expression levels of the chimera . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 02910 . 7554/eLife . 21687 . 030Figure 9—figure supplement 6 . Expression control of the SMARCAD1-FUN30 chimera , FUN30-30-C and GFP-FUN30 30-C in Figure 9—figure supplement 4 .", "SMARCAD1-FUN303FLAG chimera , FUN30-30-C and GFP-FUN30 30 C are expressed from the GAL1-10 promoter by addition of galactose , which however leads to a stronger expression of the chimera constructs than the truncated FUN30 fragment alone . DOI: http://dx . doi . org/10 . 7554/eLife . 21687 . 030" ], [ "Our study reveals that the function of Fun30/SMARCAD1 at DSBs is cell cycle-regulated via interaction with Dpb11/TOPBP1 .", "In budding yeast , this interaction seems to facilitate localization of Fun30 to damaged chromatin in a manner that depends on the 9-1-1 complex .", "Notably , other interactions may also contribute to Fun30 targeting or function , given that Fun30 was shown to interact with RPA and nucleases ( Chen et al . , 2012 ) and that SMARCAD1 was recently shown to interact with H2A-ubiquitin ( Densham et al . , 2016 ) .", "Importantly , however , our data suggests that the interaction with Dpb11 and 9-1-1 is essential for the resection function of Fun30 during the cell cycle .", "In contrast to the Fun30-Dpb11 complex , the other interactions are seemingly cell cycle-independent and future research will need to show whether they are at all critical for Fun30/SMARCAD1 function .", "Once recruited to a lesion , Fun30 will then promote the action of the long-range resection machinery by generating resection-permissive chromatin .", "It seems clear that Fun30 antagonizes the resection inhibitor Rad9 ( Chen et al . , 2012 ) , but different , non-exclusive mechanisms remain possible .", "For example , Fun30 could directly remove Rad9 from DNA damage sites or render Rad9-containing chromatin resection-permissive , but also could interfere with Rad9 recruitment by changing chromatin composition .", "We predict that our system of forced targeting Fun30 to damaged chromatin will be useful to discriminate between these possibilities in the future .", "It is furthermore possible that a direct competition for Dpb11 binding between Fun30 and Rad9 contributes to the functional antagonism , similar to what has been suggested for Slx4 and Rad9 ( Cussiol et al . , 2015; Dibitetto et al . , 2016; Ohouo et al . , 2013 ) .", "Dpb11 thus interacts with pro- and anti-resection factors , and the same is true for TOPBP1 ( Cescutti et al . , 2010; Moudry et al . , 2016 ) .", "It will thus be interesting to figure out in the future , how binding of potential antagonizing factors is balanced .", "Overall , our data suggest that at least two layers of cell cycle regulation of DNA end resection can be distinguished .", "First , nucleases and nuclease-associated factors are substrate for CDK phosphorylation ( Chen et al . , 2011; Huertas et al . , 2008 ) and this may directly activate these enzymes , as has for example been shown for the endonuclease activity of MRX-Sae2 ( Cannavo and Cejka , 2014 ) .", "Second , chromatin and nucleosome remodellers may be regulated in a way that generates resection-permissive chromatin at damage sites in cell cycle phases when resection is favoured .", "The Fun30-Dpb11 complex clearly falls in this second category , as does perhaps Rad9/53BP1 , the cell cycle regulation of which we are only beginning to understand ( Cescutti et al . , 2010; Pfander and Diffley , 2011 ) .", "Notably , deregulation of the second layer such as in the experiments with the DDC1-FUN30 fusion has so far been the most successful strategy to bypass the cell cycle regulation of DNA end resection ( Figure 6 ) .", "This emphasizes the importance of resection regulation by its chromatin substrate and suggests that chromatin ( more specifically the Fun30 target on chromatin ) is the factor that limits long-range resection in G1 phase cells .", "Experimentally manipulating DSB repair pathway choice is a key challenge for future research , because it may allow gene targeting in G1/post-mitotic cells , which are currently refractory to this type of approach , since HR is inefficient under these conditions ( Orthwein et al . , 2015 ) .", "Notwithstanding the overall complexity of DSB repair pathway choice , our results suggest that modification of the DSB-surrounding chromatin by Fun30/SMARCAD1 should be explored further – particularly in higher eukaryotes – as a tool to experimentally channel DSBs into the HR pathway independently of cell cycle stage ." ], [ "All yeast strains used in this study derive from W303 MATa ( strains listed in Supplementary file 1A ) and were constructed using standard methods ( Janke et al . , 2004 ) .", "Cells were grown in YP glucose or YP raffinose media at 30°C .", "For sensitivity spottings on camptothecin , plates were incubated at 37°C for 2 days .", "The inhibitor-sensitive CDK allele cdc28-as1 ( Bishop et al . , 2000 ) was inhibited by supplementing 1NM-PP1 ( final concentration 1 . 5 µM ) to the medium .", "Cell cycle synchronization was performed using alpha-factor ( 5μg/ml ) or nocodazole ( 5μg/ml ) for 2–3 hr .", "HEK293-T cells were used in mammalian cell culture experiments .", "Cells were obtained from the cell services facility of CRUKs London Research Institute , authenticated using STR profiling ( Promega Mannheim , Germany ) and species determination .", "They were also tested negative for mycoplasma contamination .", "For molecular cloning , genes were amplified from yeast genomic DNA and inserted in plasmids using the In-Fusion HD cloning kit ( Clontech Saint-Germain-en-Laye , France ) .", "For site-directed mutagenesis , a PCR-based protocol with mutagenic oligonucleotides was used .", "All plasmids used in this study are listed in Supplementary file 1B .", "The yeast two-hybrid analyses of the protein-protein interactions were performed using either the Gal4-based plasmid system ( pGAD-C1 , pGBD-C1 [James et al . , 1996] ) in PJ69-7a cells , or the lexA-based plasmid system ( pBTM116 , Clontech Saint-Germain-en-Laye , France ) in L40 cells ( Invitrogen Schwerte , Germany ) .", "Transformants were spotted in serial ( 1:5 ) or single dilution either on SC-Leu-Trp plates ( control ) or on SC-Leu-Trp-His plates ( selection ) and grown at 30°C for 2–4 days .", "For a specific interaction between TOPBP1 1–360 and SMARCAD1 55–247 , spotting plates were supplemented with different concentrations of 3-Amino-1 , 2 , 4-triazole ( 3-AT ) ( 2 . 5–10 mM ) .", "To assess the phosphorylation-specific interaction between SMARCAD1 and Dpb11 , cells were additionally spotted on SC-Leu-Trp-His-Ade plates .", "All experiments ( Figure 1A , G and H; Figure 9A , C , E ) were performed in three technical repetitions per biological repetition ( spotting of the same yeast cultures on three separate selection plates ) and each interaction was observed in several ( 2-10 ) independent experiments ( a biological replicate corresponds to a fresh transformation of the Y2H expression vectors , raising of the transformed cells and spotting on selective plates ) .", "Yeast cells were freshly transformed with pUK1 ( pAG416 GPD-Dpb11 ) and grown to log-phase ( OD600 0 . 5 ) in SC-Ura medium + 2% glucose ( YPD ) at 30°C .", "Cells were cell cycle synchronized as describe above , the arrest was controlled by flow cytometry .", "To release cells from G1 ( Figure 1C , Figure 1—figure supplement 2 ) , BAR1+ cells were synchronized with 5 μg/ml alpha-factor , washed twice in pre-warmed SC-Ura medium and resuspended in pre-warmed SC-Ura medium supplemented with nocodazole .", "For preparation of extracts , 300 OD yeast cells were harvested , washed in ice-cold sorbitol buffer ( 1 M sorbitol , 25 mM Hepes pH 7 . 6 ) , and resuspended in 2 ml lysis buffer with protease and phosphatase inhibitors ( 100 mM Hepes , 200 mM KOAc , 0 . 1 % NP-40 , 10% glycerol , 2 mM β-mercaptoethanol , 100 nM ocadaic acid , 10 mM NaF , 20 mM β-glycerophosphate , 400 μM PMSF , 4 μM aprotinin , 4 mM benzamidin , 400 μM leupeptin , 300 μM pepstatin A ) and prepared for lysis using a Spex Sample Prep cryo mill .", "The extracts were cleared by centrifugation and incubated with anti‐FLAG agarose resin ( Sigma Munich , Germany ) for 30 min ( 4°C , rotation ) .", "After six washes with lysis buffer , Fun30-3FLAG was eluted twice with 0 . 5 mg/ml 3X FLAG peptide ( Sigma Munich , Germany ) .", "The elutions were pooled and proteins were precipitated with TCA prior to analysis on 4–12% NuPAGE gradient gels ( Invitrogen Schwerte , Germany ) and standard Western blotting .", "Yeast in vivo co-immunoprecipitation experiments were not performed in technical replicates .", "The number of biological replicates ( fresh transformation with the GPD-Dpb11 overexpressing plasmid , raising of the cells , lysis and IP ) was two or more , with the exception of Figures 1C and 5B , Figure 5—figure supplement 1 .", "In vitro experiments ( Figure 1F; Figure 9B , D ) as depicted were performed once , but confirmed in several different experimental setups ( Figure 9B and D ) .", "For in vitro pulldown of Fun30 with Dpb11 after in vitro CDK phosphorylation , GST-Dpb11-N or GST ( approx . 18 μg per reaction ) were immobilized on Sepharose beads for 1 hr at 4°C .", "The beads were washed twice in lysis buffer ( 200 mM KOAc , 100 mM HEPES KOH pH 7 . 6 , 10% glycerol , 0 . 1% NP-40 , 2 mM β-mercaptoethanol ) and resuspended in 100 μl lysis buffer per reaction .", "For phosphorylation of Fun30 and Rad9 ( control ) , 5 μg purified protein per reaction were dialyzed against lysis buffer ( 4°C ) and supplied with 4 mM ATP and 5 mM MgOAc .", "Buffer or CDK ( 2 . 5 μg per reaction ) were added and the reactions were incubated for 30 min at 24°C .", "Then , the pre-bound Dpb11/GST-beads were added to the phosphorylated proteins and incubated for 1 hr at 4°C .", "The beads were washed five times in lysis buffer and eluted by boiling in 2x Laemmli buffer .", "Proteins were separated by SDS-PAGE and analyzed with standard Western Blotting techniques .", "CDK-dependent pulldowns of SMARCAD1-TOPBP1 ( Figure 9B and D ) were carried out as described ( Boos et al . , 2011 ) for TRESLIN-TOPBP1 with modifications .", "HEK293T cells were transfected with pCS2-SMARCAD1-55-275 or pCS2-SMARCAD1-55-445 ( carrying an N-terminal GFP tag ) and native cell lysates were prepared by lysing the cell pellets in 5x lysis buffer ( 20 mM HEPES pH 7 . 5 , 250 mM KCl , 0 . 1% Triton X-100 , 5 mM β-mercaptoethanol , 5% glycerol and Complete EDTA-free Protease Inhibitor Cocktail ( Roche Mannheim , Germany ) ) .", "For CDK phosphorylation , the extract was supplemented with 5 mM ATP , 5 mM MgCl2 and approx .", "67 ng/μl cycA/CDK2 or buffer ( as control ) and incubated for 5 min at 25°C .", "200 μl of cell extract were incubated with approx .", "10 μg immobilized GST-TopBP1-BRCT0/1/2 for 2 hr at 4°C .", "The beads were washed with lysis buffer and WCE and bound material were analysed by SDS PAGE , Western Blotting and ponceau staining .", "For chromatin immunoprecipitation of Fun30 and RPA , cells were grown in YP-Raffinose to an OD of 0 . 5 and - as indicated for the individual experiments- cell cycle arrest was induced .", "A double-strand break was introduced by inducing the HO endonuclease from the galactose promoter by addition of galactose to the cultures ( 2% final ) .", "100 ml samples were crosslinked with formaldehyde ( final 1% ) for 16 min at indicated timepoints and the reaction was quenched with glycine .", "Cells were harvested by centrifugation , washed in ice-cold PBS and snap-frozen ( RPA ChIPs ) or directly processed ( Fun30-3FLAG ChIPs ) .", "For lysis , cell pellets were resuspended in 800 μl lysis buffer ( 50 mM HEPES KOH pH 7 . 5 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 1% Na-deoxycolate , 0 . 1% SDS ) and grinded with zirconia beads using a bead beating device .", "The chromatin was sonified to shear the DNA to a size of 200–500 bp .", "Subsequently the extracts were cleared by centrifugation , 1% was taken as input sample and 40% were incubated with either anti FLAG M2 magnetic beads ( Sigma Munich , Germany ) for 2 hr ( Fun30-3FLAG ChIPs ) or 1 . 5 hr with anti RFA antibody ( AS07-214 , Agrisera Vännäs , Sweden ) followed by 30 min with Dynabeads ProteinA ( Invitrogen Schwerte , Germany , for RPA ChIPs ) .", "The beads were washed 3x in lysis buffer , 2x in lysis buffer with 500 mM NaCl , 2x in wash buffer ( 10 mM Tris-Cl pH 8 . 0 , 0 . 25 M LiCl , 1 mM EDTA , 0 . 5% NP-40 , 0 . 5% Na-deoxycholate ) and 2x in TE pH 8 . 0 .", "DNA-protein complexes were eluted in 1% SDS , proteins were removed with Proteinase K ( 3 hr , 42°C ) and crosslinks were reversed ( 8 hr or overnight , 65°C ) .", "The DNA was subsequently purified using phenol-chloroform extraction and ethanol precipitation and quantified by quantitative PCR ( Roche LightCycler480 System , KAPA SYBR FAST 2x qpCR Master Mix , KAPA Biosystems London , UK ) at indicated positions with respect to the DNA double-strand break .", "As control , 2–3 control regions on other chromosomes were quantified and used for normalization .", "Chromatin Immunoprecipitation ( ChIP ) experiments were generally performed in technical replicates ( on the qPCR level , each sample was measured three times ) .", "Our experimental design ( excluding Figure 1J and figure Figure 1—figure supplement 5 ) includes further replicates , which can be considered as technical as well as biological replicates: we took samples at different timepoints after induction of the DNA break , which showed a consistent trend over the experiment .", "Additionally , we measured signals with 15–20 qPCR primer pairs over the damaged chromosome including three control regions on unaffected chromosomes .", "Therefore , we plot results from single experiments and timepoints and do not include error bars .", "Nonetheless , 2–6 repetitions ( independent cell growth and crosslinking ) were performed for mutants analysed in Figure 2A; Figure 6B–C; Figure 2—figure supplement 1 , Figure 8 with the exception of Figure 6—figure supplement 3 .", "The ChIP experiment in Figure 1J and figure Figure 1—figure supplement 5 was performed three times , error bars represent the standard deviation .", "Yeast growth assays ( DNA damage sensitivity spottings Figures 2B and 4A–D; Figure 6A , 9F; Figure 4—figure supplements 1 , 2; Figure 6—figure supplement 1; Figure 9—figure supplement 5 and URA3 silencing assay Figure 3 ) were performed in three technical repetitions per biological repetition ( spotting of the same yeast cultures on three separate selection plates ) , biological replicates refer to raising of the mutant strains and spotting on plates with indicated conditions .", "The genotypes were additionally confirmed by comparing several clones of each strain .", "Each experiment was spotted with three technical replicates .", "In order to assay for precise non-homologous end-joining , 40 OD of transformation-competent yeast cells were transformed with 500 ng BamHI-linearized or mock-digested pRS316 .", "Transformed cells were plated in a five-fold serial dilution on SC-Ura and SC-complete agar plates and grown for two days .", "Clones on plates containing 50–200 clones were counted to calculate the re-ligation rate ( ratio of clones from +BamHI –Ura by +BamHI +Ura divided by the equivalent ratio of mock digested plasmid transformations ) .", "Each sample was plated in triplicates and the experiment was independently repeated three times .", "Error bars represent standard deviations .", "50–75 clones from the BamHI-digest transformation on –Ura plates were sequenced to analyse the precise NHEJ event .", "We found that the majority of cells had precisely re-joined the BamHI overhangs , with a subset having added a single G-C basepair at the cutsite ( shown as rate in % ) .", "yku70∆ cells are NHEJ-deficient and showed very low NHEJ rates with exclusively precisely re-joined plasmid sequences , indicating that this number represents the background of uncut plasmid in the reactions ." ] ]
[ "DNA double strand breaks ( DSBs ) can be repaired by either recombination-based or direct ligation-based mechanisms .", "Pathway choice is made at the level of DNA end resection , a nucleolytic processing step , which primes DSBs for repair by recombination .", "Resection is thus under cell cycle control , but additionally regulated by chromatin and nucleosome remodellers .", "Here , we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller .", "Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1 , respectively .", "In yeast , this protein assembly additionally comprises the 9-1-1 damage sensor , is involved in localizing Fun30 to damaged chromatin , and thus is required for efficient long-range resection of DSBs .", "Notably , artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection , indicating that chromatin remodelling during resection is underlying DSB repair pathway choice ." ]
[ "DNA is continually exposed to chemicals and radiation that cause various forms of DNA damage .", "One of the most toxic forms of DNA damage is the double strand break , in which both strands of the double helix are broken .", "These breaks can be mended in two ways: by directly joining the broken ends together , or via a process called homologous recombination .", "In homologous recombination , a duplicate DNA molecule is used as a template to repair the broken DNA strands .", "These duplicates only form during particular phases of the cell division cycle , which limits when homologous recombination can take place .", "A cell can choose which pathway it uses to repair double strand breaks .", "However , the first step of homologous recombination – trimming the broken DNA ends in a process called resection – commits a cell to the homologous recombination repair pathway .", "Cell cycle kinases regulate the cell division cycle and control DNA end resection .", "This control takes two forms: on the one hand by regulating whether the enzymes that trim the DNA ends are active; and on the other hand by regulating the remodelling of the structure into which DNA is packaged , which is called chromatin .", "However , it is not known which of these two targets is the limiting factor that determines whether homologous recombination occurs .", "A protein called Fun30 that remodels chromatin had been found to be important for promoting resection in budding yeast .", "Bantele et al . now reveal how the activity of Fun30 is regulated by the cell cycle to limit extensive resection to certain cell cycle phases , where homologous recombination is wanted .", "During those stages , cell cycle kinases add phosphate groups to Fun30 .", "This enables Fun30 to engage in a protein complex that directs Fun30 to the site of a double strand break to facilitate the resection process .", "Bantele et al . also studied artificial versions of Fun30 that were directly fused to components of the protein complex , and so bypassed the controls that limit homologous recombination to particular phases of the cell cycle .", "These forms of Fun30 enabled resection to take place in phases of the cell cycle where it does not normally occur .", "This suggests that the remodelling of chromatin by Fun30 is a critical step at which resection is regulated by the cell cycle .", "Further experiments showed that the cell cycle regulation of human proteins that are equivalent to Fun30 and another protein in the resection complex is similar to that seen for the yeast proteins .", "In the future , knowing how these proteins are regulated during resection could help researchers to develop new gene editing methods based on homologous recombination that can be used in cells at any stage of the cell cycle ." ]
2017
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "structural biology and molecular biophysics", "neuroscience" ]
Functional synergy between the Munc13 C-terminal C1 and C2 domains
elife-13696-v2
[ [ "The release of neurotransmitters by Ca2+-evoked synaptic vesicle exocytosis is a key event for communication between neurons and involves several steps , including vesicle docking at presynaptic active zones , a priming reaction ( s ) that leaves the vesicles ready for release , and fast Ca2+-triggered fusion of the vesicle and plasma membranes ( Sudhof , 2013 ) .", "Studies of the complex machinery that controls release have shown that eight proteins are particularly important and have established defined roles for them ( Rizo and Xu , 2015; Jahn and Fasshauer , 2012; Brunger et al . , 2015; Sudhof and Rothman , 2009 ) :", "i ) the soluble N-ethylmaleimide-sensitive factor attachment protein receptors ( SNAREs ) syntaxin-1 , SNAP-25 and synaptobrevin bring the membranes together by forming a four-helix bundle called SNARE complex ( Sollner et al . , 1993; Poirier et al . , 1998; Sutton et al . , 1998 ) , which is critical for membrane fusion ( Hanson et al . , 1997 ) ;", "ii ) N-ethylmaleimide sensitive factor ( NSF ) and soluble NSF attachment proteins ( SNAPs; no relation to SNAP-25 ) disassemble the SNARE complex ( Sollner et al . , 1993 ) to recycle the SNAREs ( Mayer et al . , 1996; Banerjee et al . , 1996 ) ;", "iii ) Munc18-1 and Munc13s orchestrate SNARE complex assembly , which involves initial binding of Munc18-1 to a self-inhibited ‘closed’ conformation of syntaxin-1 ( Dulubova et al . , 1999; Misura et al . , 2000 ) and opening of syntaxin-1 by Munc13 ( Richmond et al . , 2001; Ma et al . , 2011; Yang et al . , 2015 ) ;", "iv ) and Synaptotagmin-1 ( Syt1 ) acts as the Ca2+ sensor that triggers fast release ( Fernandez-Chacon et al . , 2001 ) , likely via interactions with both membranes ( Arac et al . , 2006 ) and with the SNARE complex ( Brewer et al . , 2015; Zhou et al . , 2015 ) .", "Reconstitution experiments ( Weber et al . , 1998 ) have contributed to establishing some of these key concepts , providing a powerful tool to study the mechanism of synaptic vesicle fusion ( Brunger et al . , 2015 ) .", "It is now clear that synaptobrevin-liposomes can fuse with syntaxin-1-SNAP-25 liposomes under some conditions but not others , and that fusion is stimulated to different degrees by Syt1 , Munc18-1 or Munc13-4 ( Weber et al . , 1998; van den Bogaart et al . , 2010; Tucker et al . , 2004; Yu et al . , 2013; Kyoung et al . , 2011; Lee et al . , 2010; Boswell et al . , 2012; Parisotto et al . , 2012 ) , but such fusion is abolished by NSF and αSNAP because they disassemble syntaxin-1-SNAP-25 complexes ( Weber et al . , 2000; Ma et al . , 2013 ) .", "However , inclusion of Munc18-1 and a Munc13-1 fragment enable fusion at least in part because they protect against the disassembly activity of NSF-αSNAP while coordinating SNARE complex assembly ( Ma et al . , 2013 ) .", "These results explained the essential nature of Munc18-1 and Munc13s for neurotransmitter release ( Verhage et al . , 2000; Richmond et al . , 1999; Varoqueaux et al . , 2002; Aravamudan et al . , 1999 ) and correlated with earlier studies of the role of the HOPS tethering complex in yeast vacuolar fusion ( Xu et al . , 2010 ) .", "Despite these advances , fundamental questions remain about the mechanism of neurotransmitter release , in particular regarding the functions of Munc13s ( Rizo and Xu , 2015 ) .", "These large proteins of presynaptic active zones contain a variable N-terminal region that in some isoforms include a C2 domain ( the C2A domain ) , and a highly conserved C-terminal region that includes ( see Figure 1A for Munc13-1 ) :", "i ) a C1 domain involved in diacyglycerol ( DAG ) -phorbol ester-dependent augmentation of release ( Rhee et al . , 2002; Basu et al . , 2007 ) ; a C2B domain that regulates release probability and modifies short term plasticity through its Ca2+- and phosphatidylinositolphosphate-binding activities ( Shin et al . , 2010 ) ; a MUN domain that is key for the crucial function of Munc13 in release ( Basu et al . , 2005 ) and mediates the activity of Munc13 in opening syntaxin-1 ( Richmond et al . , 2001; Ma et al . , 2011; Yang et al . , 2015 ) ; and a C2C domain that is also important for release ( Madison et al . , 2005; Stevens et al . , 2005 ) and is not predicted to bind Ca2+ but may bind phospholipids because this is a common property of C2 domains ( Rizo and Sudhof , 1998 ) .", "The central function of Munc13s in neurotransmitter release was initially associated to an essential role in vesicle priming ( Augustin et al . , 1999 ) , but later studies that used stringent definitions of vesicle docking ( see discussion ) uncovered a critical role for Munc13s in docking that was attributed to their activity in mediating SNARE complex assembly ( Weimer et al . , 2006; Hammarlund et al . , 2007; Imig et al . , 2014 ) .", "However , it is unknown whether Munc13s participate in upstream interactions that might help bridging the two membranes to promote docking and priming .", "This possibility is attractive because the MUN domain is related to tethering factors involved in diverse forms of membrane traffic ( Pei et al . , 2009; Li et al . , 2011 ) and is flanked by domains with demonstrated or potential lipid-binding properties .", "Moreover , while there is evidence that Munc13s modulate release probability and have a role beyond docking [e . g . ( Rhee et al . , 2002; Hammarlund et al . , 2007; Shin et al . , 2010 ) ] , it is unclear whether they form part of the primed complex after SNARE complex assembly , influencing membrane fusion downstream of priming . 10 . 7554/eLife . 13696 . 003Figure 1 . Munc13-1 C1C2BMUN strongly stimulates lipid mixing between V- and T-liposomes .", "( A ) Domain diagram of Munc13-1 .", "CaMb = calmodulin-binding sequence .", "( B–C )", "Lipid mixing assays between V- and T-liposomes alone ( T+V ) or in the presence of different combinations of Munc13-1 C1C2BMUN , Syt1 C2AB fragment , Munc18-1 ( M18 ) , NSF-αSNAP ( NSF/SNAP ) and synaptobrevin cytoplasmic domain ( Syb-cd ) .", "T-liposomes contained 1% DAG and 1% PIP2 .", "( D–E )", "Analogous lipid mixing assays performed in the presence of C1C2BMUN ( D ) or C1C2BMUN plus Munc18-1 and NSF-αSNAP ( NSF/SNAP ) ( E ) with T-liposomes containing 1% DAG and 1% PIP2 , 1% PIP2 ( -DAG ) , 1% DAG ( -PIP2 ) or no DAG and PIP2 ( -DAG-PIP2 ) .", "Controls of T+V ( D ) or T+V in the presence of C1C2BMUN plus NSF-αSNAP ( NSF/SNAP ) ( E ) , both with T-liposomes containing 1% DAG and 1% PIP2 , are shown in orange .", "All experiments were started in the presence of 100 μM EGTA , and Ca2+ ( 600 μM ) was added after 300 s . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 00310 . 7554/eLife . 13696 . 004Figure 1—figure supplement 1 . Quantification of the lipid mixing experiments of Figure 1 . Panels ( A–D ) correspond to panels ( B–E ) of Figure 1 , respectively .", "Bars represent averages of the normalized NBD fluorescence observed after 500 s ( 200 s after Ca2+ addition ) in experiments performed at least in triplicate .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 004 To shed light into these questions , we have used a combination of reconstitution and dynamic light scattering ( DLS ) assays together with electrophysiological experiments .", "Our results show that both the C1-C2B region and the C2C domain of Munc13-1 play important functions in release , and suggest that these domains help bridging synaptic vesicles to the plasma membrane , facilitating the activity of the Munc13-1 MUN domain in promoting SNARE complex assembly .", "Moreover , our results indicate that the neuronal SNAREs , Munc18-1 , NSF , αSNAP and a Munc13-1 fragment including the C1 , C2B , MUN and C2C domains ( C1C2BMUNC2C ) are sufficient to generate a ‘primed’ state that is ready to trigger fast membrane fusion upon addition of Ca2+ , thus resembling the primed state of synaptic vesicles ." ], [ "In experiments that followed our recent reconstitution study ( Ma et al . , 2013 ) and were directed at analyzing how different factors affect the efficiency of membrane fusion , we first analyzed lipid mixing between synaptobrevin-liposomes ( V-liposomes ) and syntaxin-1-SNAP-25 liposomes ( T-liposomes ) by monitoring de-quenching of the fluorescence of NBD-labeled lipids incorporated in the synaptobrevin-liposomes ( Weber et al . , 1998 ) ( Figure 1 ) .", "These experiments were initiated in the absence of Ca2+ , and Ca2+ was added at 300 s to examine the Ca2+ -dependence of the results .", "The liposomes contained a synaptic-like lipid composition and , unless otherwise specified , the T-liposomes included DAG and PIP2 , which activate the Munc13-1 C1 and C2B domains ( Ma et al . , 2013 ) .", "We illustrate the reproducibility of some of the data in Supplementary Figures , showing the quantification of the data at 500 s .", "In the NBD de-quenching assays we observed that lipid mixing between V- and T-liposomes is strongly stimulated by a Munc13-1 fragment spanning its C1 , C2B and MUN domains ( C1C2BMUN ) ( Figure 1B; red data ) , which contrasts with the smaller stimulation observed earlier [Figure S8 of ( Ma et al . , 2013 ) ] .", "We note that we have been able to reproduce all other results from this previous study and we speculate that the sample of C1C2BMUN used for the experiments of Figure S8 of Ma et al . ( 2013 ) , which were performed at the end of that study , might have been partially inactivated during storage in the freezer , as we have reproduced the results shown in Figure 1B in more than 20 subsequent reconstitution experiments employing at least five different preparations of C1C2BMUN .", "The enhancement of lipid mixing induced by C1C2BMUN was independent of Ca2+ and was SNARE-dependent , as it was strongly impaired by addition of the cytoplasmic region of synaptobrevin ( Syb-cd ) ( Figure 1B and Figure 1—figure supplement 1A ) .", "The extent of lipid mixing between V- and T-liposomes in the presence of C1C2BMUN was comparable to that caused by a soluble fragment spanning the two C2 domains of Syt1 ( C2AB fragment ) in the presence of Ca2+; in contrast , Munc18-1 had only a small stimulatory effect on lipid mixing , and did not enhance the stimulation caused by C1C2BMUN ( Figure 1B and Figure 1—figure supplement 1A ) .", "As expected , addition of NSF-αSNAP abolished the strong stimulatory effect of C1C2BMUN but lipid mixing was highly efficient again when both C1C2BMUN and Munc18-1 were added in the presence of NSF-αSNAP ( Figure 1C and Figure 1—figure supplement 1B ) , consistent with the notion that C1C2BMUN and Munc18-1 mediate SNARE complex assembly in an NSF-αSNAP-resistant manner ( Ma et al . , 2013 ) .", "Note that these experiments did not include Syt1 C2AB and yet lipid mixing was Ca2+-dependent in the presence of C1C2BMUN , Munc18-1 and NSF-αSNAP .", "Moreover , removal of DAG , PIP2 or both from the T-liposomes caused increasingly stronger impairments of lipid mixing in these experiments but had much milder effects on the stimulation of lipid mixing caused by C1C2BMUN in the absence of NSF-αSNAP ( Figure 1D , E and Figure 1—figure supplement 1C , D ) .", "These data show that the effect of Munc13-1 C1C2BMUN alone on lipid mixing arises from a property that is largely independent of Ca2+ , DAG and PIP2 , whereas the lipid mixing observed in the more complete reconstitutions including C1C2BMUN , Munc18-1 and NSF-αSNAP is stimulated by Ca2+ , DAG and PIP2 , thus exhibiting properties that are more similar to those of neurotransmitter release .", "The ability of Syt1 C2AB to bind simultaneously to two membranes in a Ca2+-dependent manner ( Arac et al . , 2006 ) underlies at least in part its activity in stimulating SNARE-dependent lipid mixing ( Tucker et al . , 2004; Xue et al . , 2008 ) .", "Hence , we tested whether Munc13-1 C1C2BMUN is also able to bridge two membranes by monitoring the formation of liposome clusters by DLS .", "While C1C2BMUN did not cluster plain liposomes lacking phosphatidylserine ( PS ) , dramatic increases in particle size observed by DLS revealed efficient clustering of PS-containing vesicles caused by C1C2BMUN ( Figure 2A , B ) .", "Inclusion of synaptobrevin , DAG+PIP2 or syntaxin-1 in the PS-vesicles did not have major effects on the clustering induced by C1C2BMUN , as shown by the bar diagrams of Figures 2B–E and by the corresponding intensity autocorrelation curves ( Figure 2F ) .", "The two types of representations provide different views of the DLS data; below we use one or the other depending on the aspect that we want to emphasize .", "We note that there is some degree of variability among the particle sizes observed , which makes it difficult to draw firm conclusions from the small differences observed in Figures 2B–E .", "Hence , these data show that PS is the main determinant for the vesicle clustering activity of C1C2BMUN , although we cannot rule out that synaptobrevin , syntaxin-1 , DAG or PIP2 might affect clustering to a small degree .", "Similarly , Ca2+ did not have a major effect on clustering of PS-vesicles by C1C2BMUN , although it might increase clustering to a small extent ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 13696 . 005Figure 2 . Munc13-1 C1C2BMUN clusters PS-containing liposomes .", "( A–E )", "The particle size in samples containing phospholipid vesicles alone ( gray bars ) or after incubation with Munc13-1 C1C2BMUN for 5 min ( red bars ) in the absence of Ca2+ was measured by DLS .", "The liposomes had a standard lipid composition including no PS ( A ) , PS ( B ) , PS and synaptobrevin ( C ) , PS+DAG+PIP2 ( D ) or PS and syntaxin-1 ( E ) .", "( F ) Intensity autocorrelation curves corresponding to the experiments shown in ( A–E ) after incubation with Munc13-1 C1C2BMUN for 5 min . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 00510 . 7554/eLife . 13696 . 006Figure 2—figure supplement 1 . Ca2+ does not stimulate liposome clustering by C1C2BMUN strongly . The diagram shows intensity autocorrelation curves measured by DLS at 25°C for PS-containing vesicles alone or after 5 min incubation with C1C2BMUN in the presence of 100 μM EGTA or 500 μM Ca2+ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 006 These results correlate with the observation that the strong stimulatory activity of C1C2BMUN in lipid mixing between V- and T-liposomes does not require Ca2+ , DAG or PIP2 ( Figures 1B , D ) , indicating that this activity arises from its ability to bind simultaneously to two PS-containing membranes in a Ca2+-independent manner , thus favoring SNARE complex assembly .", "This activity might contribute to the role of Munc13s in synaptic vesicle docking and is distinct from the function of Munc13-1 in mediating the transition from the syntaxin-1-Munc18-1 complex to the SNARE complex ( Ma et al . , 2011 ) , but likely potentiates this function in the reconstitutions that include Munc18-1 and NSF-αSNAP by placing the MUN domain near the SNARE-Munc18-1 machinery .", "As expected , Syt1 C2AB could not stimulate lipid mixing between V- and T-liposomes in the presence of NSF-αSNAP because NSF-αSNAP disassemble the syntaxin-1-SNAP-25 t-SNARE complex ( Ma et al . , 2013 ) , but it was surprising that Syt1 C2AB did not have marked effects on the lipid mixing observed in the presence of Munc13-1 C1C2BMUN , Munc18-1 and NSF-αSNAP ( data not shown; see also below ) .", "This observation prompted us to investigate to what extent the lipid mixing observed in these experiments reflects real membrane fusion .", "For this purpose , we used an assay that simultaneously measures lipid mixing from de-quenching of the fluorescence of Marina Blue-labeled lipids and content mixing from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes ( Zucchi and Zick , 2011 ) .", "Addition of unlabeled streptavidin to the reaction ensures that the observed FRET arises only from content mixing .", "Using this approach , we again observed that efficient lipid mixing in the presence of NSF-αSNAP required C1C2BMUN and Munc18-1 , as well as Ca2+ , and that Syt1 C2AB did not markedly affect lipid mixing under these conditions ( Figure 3A , C ) .", "However , content mixing in the presence of Munc13-1 C1C2BMUN , Munc18-1 , NSF-αSNAP and Ca2+was inefficient in the absence of Syt1 C2AB and was strongly enhanced by Syt1 C2AB ( Figure 3B , D ) .", "The difference between lipid and content mixing in the absence of Syt1 C2AB emphasizes the fact that lipid mixing may not necessarily reflect true membrane fusion , as described in previous studies [e . g . ( Chan et al . , 2009; Zick and Wickner , 2014; Kyoung et al . , 2011; Diao et al . , 2012; Lai et al . , 2014 ) ] .", "Overall , our results show that Syt1 C2AB selectively enhances content mixing but not lipid mixing under our conditions .", "These findings indicate that Syt1 plays a role in membrane fusion , in agreement with results from single vesicle assays using full-length Syt1 ( Kyoung et al . , 2011; Diao et al . , 2012 ) . 10 . 7554/eLife . 13696 . 007Figure 3 . Syt1 is required for efficient content mixing but not lipid mixing in reconstitutions including Munc18-1 , Munc13-1 C1C2BMUN and NSF-αSNAP . Lipid mixing ( A , C ) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B , D ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "The assays were performed in the presence of different combinations of Munc13-1 C1C2BMUN , Munc18-1 ( M18 ) and NSF-αSNAP ( NSF/SNAP ) , and in the absence ( A , B ) or presence ( C , D ) of Syt1 C2AB fragment .", "Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 00710 . 7554/eLife . 13696 . 008Figure 3—figure supplement 1 . Assessment of leakiness in content mixing assays . Content mixing assays monitoring the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes were performed as in Figure 3D in the presence of Munc13-1 C1C2BMUN , Munc18-1 , NSF-αSNAP , and Syt1 C2AB fragment with ( red curve ) or without ( black curve ) 5 μM streptavidin . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 008 Experiments performed without streptavidin revealed a small amount of leakiness in reactions containing C1C2BMUN , Munc18-1 , NSF-αSNAP and Syt1 C2AB , but the leakiness occurred mostly in the beginning and likely arises because of the presence of a population of small , relatively unstable vesicles ( Figure 3—figure supplement 1 ) .", "Note that in these assays much of the Cy5 fluorescence increase caused by FRET from PhycoE ( reflecting content mixing ) should occur during the first round of fusion and that no further substantial increases are thus expected in subsequent rounds of fusion or upon detergent addition .", "Correspondingly , the maximum Cy5 fluorescence observed in our most efficient fusion reactions was similar to that observed upon detergent addition ( e . g . Figure 3D , red curve; see Materials and Methods ) .", "In contrast , the lipid mixing signal expressed as percentage of maximum Marina Blue fluorescence is much smaller in the same reactions ( e . g . Figure 3C , red curve ) because fluorescence de-quenching is expected to continue in successive rounds of fusion and to undergo a further , large increase upon detergent addition due to additional probe dilution .", "The Ca2+-dependent membrane fusion observed in the presence of C1C2BMUN , Munc18-1 , NSF-αSNAP and Syt1 C2AB is efficient but is much slower than that of neurotransmitter release , suggesting that our reconstitutions lack at least one key factor that contributes to the high speed of release in vivo .", "We hypothesized that the Munc13-1 C2C domain might be such a factor based on evidence suggesting that this domain plays an important role in release ( Stevens et al . , 2005; Madison et al . , 2005 ) .", "To test this hypothesis , we performed fusion assays between V- and T-liposomes , with or without Syt1 C2AB , in the presence of Munc18-1 , NSF-αSNAP and fragments of Munc13-1 that contained the MUN domain alone or together with the C1C2B region , the C2C domain , or both ( MUN , C1C2BMUN , MUNC2C and C1C2BMUNC2C , respectively ) .", "We found that the MUN and MUNC2C fragments did not support membrane fusion but C1C2BMUNC2C was much more efficient than C1C2BMUN in facilitating Ca2+-dependent fusion; in fact , Syt1 C2AB had no marked effect in the experiments performed with C1C2BMUNC2C ( Figure 4 and Figure 4—figure supplement 1 ) , likely because this fragment is already highly efficient in promoting fusion in the time scale of our measurements .", "Note that , in the absence of Ca2+ , C1C2BMUNC2C was also more active than C1C2BMUN in promoting lipid mixing , but did not stimulate content mixing . 10 . 7554/eLife . 13696 . 009Figure 4 . The Munc13-1 C2C domain strongly stimulates membrane fusion . Lipid mixing ( A , C ) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B , D ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "The assays were performed in the presence of Munc18-1 , NSF-αSNAP and distinct Munc13-1 fragments as indicated , without ( A , B ) or with ( C , D ) Syt1 C2AB fragment .", "Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s .", "( E ) Intensity autocorrelation curves measured by DLS for isolated V- or T-liposomes , or at different time points as indicated in a fusion reaction performed as in ( A , B ) with C1C2BMUNC2C and 8-fold dilution of all proteins and liposomes .", "Lipid and content mixing curves for this reaction , as well as particle size distributions corresponding to several of these intensity autocorrelation curves , are shown in Figure 4—figure supplement 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 00910 . 7554/eLife . 13696 . 010Figure 4—figure supplement 1 . Quantification of lipid and content mixing experiments of Figure 4 . Panels ( A–D ) correspond to panels ( A–D ) of Figure 4 , respectively .", "Bars represent averages of the normalized fluorescence observed after 500 s ( 200 s after Ca2+ addition ) in experiments performed at least in triplicate .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01010 . 7554/eLife . 13696 . 011Figure 4—figure supplement 2 . Dependence of lipid and content mixing on DAG and PIP2 . Lipid ( A ) and content ( B ) mixing assays were performed as in Figure 4 in the presence of Munc18-1 , NSF-αSNAP , Munc13-1 C1C2BMUNC2C and Syt1 C2AB fragment with T-liposomes that contained or lacked 1% DAG and/or 1% PIP2 . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01110 . 7554/eLife . 13696 . 012Figure 4—figure supplement 3 . Ca2+-dependence of membrane fusion . Content mixing assays were performed as in Figure 4 in the presence of Munc18-1 , NSF-αSNAP , Munc13-1 C1C2BMUNC2C and Syt1 C2AB fragment , starting in the presence of 100 μM EGTA and adding 100 μM Ca2+ ( black curve ) or 120 μM Ca2+ ( red curve ) after 300 s .", "Note that the extent of content mixing is comparable in both experiments to that observed when 600 μM Ca2+ was added at 300 s ( Figure 4D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01210 . 7554/eLife . 13696 . 013Figure 4—figure supplement 4 . Analysis of particle size during fusion assays between V- and T-liposomes in the presence of Munc18-1 , NSF-αSNAP and Munc13-1 C1C2BMUNC2C .", "( A , B )", "Lipid mixing ( A ) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "The assays were performed in the presence of Munc18-1 , NSF-αSNAP and distinct Munc13-1 fragments as in Figures 4A , B but with all protein and liposome concentrations divided by 2 , 4 or 8 ( C/2 , C/4 or C/8 , respectively ) .", "Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s .", "( C–F )", "Bar diagrams showing particle size distributions for several of the intensity autocorrelation curves shown in Figure 4E ( same color coding ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 013 These results show that , indeed , the Munc13-1 C2C domain plays a key role in stimulating liposome fusion , but the lack of activity of the MUNC2C fragment shows that the region spanning the C1 and C2B domains is also important for such stimulation .", "Since these two domains bind DAG and PIP2 , respectively , we tested the effects of removing DAG , PIP2 or both in the T-liposomes in the full reconstitutions including C1C2BMUNC2C and observed considerable impairments of fusion ( Figure 4—figure supplement 2 ) , similar to those observed in the lipid mixing assays of Figure 1E .", "We also attempted to examine the Ca2+-dependence of fusion in these full reconstitutions , which were normally performed with 100 μM EGTA to chelate any residual Ca2+ before addition of 600 μM Ca2+ ( to make the concentration of free Ca2+ 500 μM ) .", "However , experiments where we added 100 or 120 μM Ca2+ at 300 s ( Figure 4—figure supplement 3 ) yielded similar fusion efficiency to that observed when we added 600 μM Ca2+ ( Figure 4D ) .", "Since the EGTA present should chelate most of the added 100 μM Ca2+ , these results suggest that a small amount of residual Ca2+ ( likely in the 1 μM range or below ) is sufficient to trigger fusion in these experiments , but further research will be required to assess the Ca2+-dependence more accurately .", "Note that the sensitivity of the reaction to such low Ca2+ concentrations , compared to those that activate Syt1 ( Fernandez-Chacon et al . , 2001 ) , can be attributed to the C2B domain present in C1C2BMUNC2C ( Shin et al . , 2010 ) ( see below ) , and that residual Ca2+ might arise from the purified C1C2BMUNC2C fragment , which we did not treat with Ca2+ chelators to avoid removal of the Zn2+ ions bound to the C1 domain .", "We also compared the effects of the Munc13-1 C1C2BMUN and C1C2BMUNC2C fragments on liposome fusion in the absence of NSF-αSNAP .", "Interestingly , C1C2BMUNC2C alone stimulated fusion between V- and T-liposomes strongly but in a Ca2+ independent manner ( Figure 5A , B and Figure 5—figure supplement 1A , B ) , unlike the reactions that included Munc18-1 and NSF-αSNAP ( Figure 4 ) .", "The fusion efficiency caused by C1C2BMUNC2C alone was similar to that induced by Syt1 C2AB alone in the presence of Ca2+ and appeared to be somewhat increased by addition of Munc18-1 , even though Munc18-1 alone did not stimulate fusion ( Figure 5 and Figure 5—figure supplement 1 ) .", "Syt1 C2AB did not enhance fusion further in the reactions containing Munc18-1 and C1C2BMUNC2C , presumably because fusion is already highly efficient .", "However , Syt1 C2AB did enhance fusion in the presence of Munc18-1 and C1C2BMUN fragment , which is less efficient than C1C2BMUNC2C ( Figure 5 and Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 13696 . 014Figure 5 . Munc13-1 C1C2BMUNC2C can induce Ca2+-independent fusion of V- and T-liposomes in the absence of NSF-αSNAP . Lipid mixing ( A , C ) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B , D ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "The assays were performed in the presence of different combinations of Munc18-1 ( M18 ) , Syt1 C2AB fragment and Munc13-1 C1C2BMUN or C1C2BMUNC2C as indicated .", "Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01410 . 7554/eLife . 13696 . 015Figure 5—figure supplement 1 . Quantification of lipid and content mixing experiments of Figure 5 . Panels ( A–D ) correspond to panels ( A–D ) of Figure 5 , respectively .", "Bars represent averages of the normalized fluorescence observed after 500 s ( 200 s after Ca2+ addition ) in experiments performed at least in triplicate .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 015 Our results suggest that the Munc13-1 C2C domain contributes strongly to promote membrane fusion but in a Ca2+-independent manner , consistent with the fact that it does not contain a full set of the aspartate side chains that commonly form the Ca2+-binding sites of C2 domains ( Rizo and Sudhof , 1998; Brose et al . , 1995 ) .", "To investigate the mechanism underlying these findings , we first performed clustering experiments with T- and V-liposomes under the same conditions of Figures 5A , B with addition of only C1C2BMUN or C1C2BMUNC2C at different concentrations , and monitored the particle size after 3 min by DLS .", "Although these measurements reflect not only liposome clustering but also fusion , clustering should dominate the formation of large particles .", "These data indicated that C1C2BMUNC2C is somewhat more efficient in liposome clustering than C1C2BMUN , exhibiting substantial clustering activity at 50–100 nM concentration ( Figure 6—figure supplement 1 ) .", "Liposome co-floatation assays also suggested that the presence of the C2C domain might increase the affinity of C1C2BMUNC2C for liposomes , but it is not sufficient for stable liposome binding in the context of MUNC2C ( Figure 6—figure supplement 2 ) .", "However , it was unclear whether these effects of the C2C domain on clustering and liposome affinity are sufficient to explain those observed on membrane fusion .", "Interestingly , when we analyzed clustering of V- and T-liposomes separately , we found that C1C2BMUNC2C clustered V-liposomes only in the presence of Ca2+ whereas it clustered T-liposomes in the absence and presence of Ca2+ ( Figure 6—figure supplement 3 ) .", "These results contrast with those obtained with C1C2BMUN , which clusters V-liposomes even in the absence of Ca2+ ( Figure 2 ) , and suggest a delicate interplay between multiple lipid binding sites within these large protein fragments ( see discussion ) .", "Since C1C2BMUNC2C clustered mixtures of T- and V-liposomes more efficiently than C1C2BMUN ( Figure 6—figure supplement 1 ) , these data suggested that C1C2BMUNC2C may preferentially bridge V-liposomes to T-liposomes .", "To test this notion more rigorously , we analyzed liposome clustering after 3 min at 20°C to minimize contributions of liposome fusion to the DLS data .", "Lipid mixing assays confirmed that very little fusion occurs under these conditions ( Figure 6—figure supplement 4 ) .", "At 20°C , C1C2BMUNC2C again was able to cluster T-liposomes but not V-liposomes in the absence of Ca2+ , which required Ca2+ for clustering ( Figures 6A–D ) .", "To test whether populations of clustered and non-clustered liposomes can be distinguished by DLS , we used plain vesicles that did not contain PS or proteins and that , correspondingly , were not clustered by C1C2BMUNC2C ( Figure 6E ) .", "Indeed , a clearly bimodal distribution of clustered and non-clustered liposomes was observed by DLS when we added C1C2BMUNC2C to a 1:1 mixture of plain liposomes and T-liposomes ( Figure 6F ) .", "Importantly , only clustered vesicles were detectable by DLS analysis of a 1:1 mixture of V- and T-liposomes in the presence of C1C2BMUNC2C and the absence of Ca2+ ( Figure 6G , H ) .", "These data show that , whereas Ca2+-free C1C2BMUNC2C does not cluster V-liposomes ( Figure 6A , B ) , it can bridge V-liposomes to T-liposomes . 10 . 7554/eLife . 13696 . 016Figure 6 . The Munc13-1 C1C2BMUNC2C fragment bridges V-liposomes to T-liposomes .", "( A , C , E , G )", "Intensity autocorrelation curves measured by DLS after 3 min incubations at 20°C on samples containing: ( A ) V-vesicles alone or in the presence of C1C2BMUNC2C and 100 μM EGTA or 500 μM Ca2+; ( C ) T-vesicles alone or in the presence of C1C2BMUNC2C and 100 μM EGTA or 500 μM Ca2+; ( E ) plain vesicles containing no PS alone or in the presence of C1C2BMUNC2C and 100 μM EGTA or 500 μM Ca2+ , or a 1:1 mixture of plain vesicles and T-vesicles in the presence of C1C2BMUNC2C and 100 μM EGTA; ( G ) V-vesicles alone , T-vesicles alone , or 1:1 mixtures of V- and T-vesicles in the presence of C1C2BMUNC2C and 100 μM EGTA or 500 μM Ca2+ .", "( B , D , F , H )", "Bar diagrams showing the particle size distribution in samples containing: ( B ) V-vesicles in the presence of C1C2BMUNC2C and 100 μM EGTA; ( D ) T-vesicles alone or in the presence of C1C2BMUNC2C and 100 μM EGTA; ( F ) a 1:1 mixture of plain vesicles and T-vesicles in the presence of C1C2BMUNC2C and 100 μM EGTA; ( H ) a 1:1 mixture of V- and T-vesicles in the presence of C1C2BMUNC2C and 100 μM EGTA .", "These bar diagrams correspond to the autocorrelation curves of selected samples among those shown in ( A , C , E , G ) and are intended to illustrate that mixtures of clustered and non-clustered vesicles can be readily distinguished ( F ) , and that Ca2+-free C1C2BMUNC2C does not cluster isolated V-vesicles ( B ) but bridges V- to T-vesicles ( H ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01610 . 7554/eLife . 13696 . 017Figure 6—figure supplement 1 . Concentration dependence of the liposome clustering activity of Munc13-1 C1C2BMUN and C1C2BMUNC2C . The diagrams show intensity autocorrelation curves measured by DLS after 3 min incubations at 30°C for mixtures of V- and T-liposomes at the same concentrations used for lipid and content mixing assays ( 0 . 125 and 0 . 25 mM lipids , respectively ) in the presence of 0 . 1 mM EGTA and different concentrations of C1C2BMUN ( A ) or C1C2BMUNC2C ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01710 . 7554/eLife . 13696 . 018Figure 6—figure supplement 2 . Lipid binding to distinct Munc13-1 fragments monitored by liposome co-floatation assays .", "( A ) Analysis of Munc13-1 fragments that co-float with liposomes by SDS-PAGE and coomassie blue staining .", "The four lanes on the left correspond to the co-floatation assays .", "The four lanes on the right show loading controls ( 1 μg of protein ) for quantification .", "( B ) Relative binding of Munc13-1 C1C2BMUNC2C with respect to C1C2BMUN measured by co-floatation experiments .", "Band intensities were quantified with ImageJ and normalized with the corresponding control .", "The calculated values were further normalized with the average value obtained for C1C2BMUN .", "The diagram shows averages and standard deviations from triplicate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01810 . 7554/eLife . 13696 . 019Figure 6—figure supplement 3 . Ca2+-free C1C2BMUNC2C does not cluster V-liposomes .", "( A , B )", "Intensity autocorrelation curves measured by DLS after 3 min incubations at 30°C on samples containing: ( A ) V-vesicles alone or in the presence of C1C2BMUNC2C and 100 μM EGTA or 500 μM Ca2+; ( B ) T-vesicles alone or in the presence of C1C2BMUNC2C and 100 μM EGTA or 500 μM Ca2+ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 01910 . 7554/eLife . 13696 . 020Figure 6—figure supplement 4 . Minimal stimulation of lipid mixing between V- and T-liposomes in the presence of C1C2BMUNC2C at 20°C . Lipid mixing assays between V- and T-liposomes in the presence of C1C2BMUN and 100 μM EGTA were performed as in Figure 1 at 20 or 37°C without addition of Ca2+ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 020 Overall , this analysis suggests that the dramatic stimulation of membrane fusion caused by C1C2BMUNC2C compared to C1C2BMUN ( Figures 4 , 5 ) arises at least in part because of this preferential bridging of V- to T-liposomes by C1C2BMUNC2C , and perhaps because such bridging is more efficient ( Figure 6—figure supplement", "1 ) and/or longer lasting .", "Hence , these results further suggest that Munc13s play a role in bridging synaptic vesicles to the plasma membrane , which may contribute to its function in docking and facilitate the activity of the MUN in opening syntaxin-1 ( Figure 7 ) , and indicate that this bridging activity involves a synergy between the C1 , C2B and C2C domains .", "However , while the ability of Ca2+-free C1C2BMUNC2C to bridge V-liposomes to T-liposomes explains its stimulation of fusion between these liposomes in the absence of Ca2+ ( Figure 5 ) , it is unclear why the dramatic stimulation of fusion caused by C1C2BMUNC2C in the presence of Munc18-1 and NSF-αSNAP requires Ca2+ ( Figure 4B ) . 10 . 7554/eLife . 13696 . 021Figure 7 . Model of how bridging of synaptic vesicles to the plasma membrane by the highly conserved C-terminal region of Munc13s can create a cage-like environment and facilitate the activity of the MUN domain in promoting the transition from the syntaxin-1-Munc18-1 complex to the SNARE complex , thus favoring SNARE complex assembly . Syntaxin-1 ( Habc domain , orange; SNARE motif and N-terminus , yellow ) is shown in a closed conformation bound to Munc18-1 ( purple ) .", "Synaptobrevin is shown in red , SNAP-25 in green and the C-terminal region of Munc13-1 in brown .", "The model is inspired by the ability of C1C2BMUNC2C to bridge V- to T-liposomes ( Figure", "6 ) and assumes that the C1-C2B region binds to the plasma membrane while the C2C domain binds to the vesicle membrane .", "See text and the legend of Figure 7—figure supplement 1 for additional details . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 02110 . 7554/eLife . 13696 . 022Figure 7—figure supplement 1 . Speculative models of membrane bridging by C1C2BMUN and C1C2BMUNC2C . These models serve in part as a basis for the model proposed in Figure 7 and provide a rationalization for the liposomes clustering activities observed for C1C2BMUN and C1C2BMUNC2C .", "However , it is important to note that there are multiple potential explanations for these activities .", "The findings that PS is a major determinant of vesicle clustering by C1C2BMUN ( Figure", "2 ) without requiring Ca2+ ( Figure 2—figure supplement 1 ) , but C1C2BMUNC2C requires Ca2+ to cluster V-liposomes ( Figure 6A , B ) , suggest that there are multiple membrane binding sites in these large protein fragments that can cooperate in cis to interact with a single membrane or in trans to bind to two membranes .", "Indeed , the MUN , C1 and C2B domains contain several positive patches , the C1 domain binds to DAG , and the C2B domain binds to PIP2 weakly in the absence of Ca2+ and more strongly in the presence of Ca2+ ( Shen et al . , 2005; Shin et al . , 2010; Yang et al . , 2015 ) .", "The C2C domain is likely to have at least one lipid-binding site with moderate affinity that explains the stronger overall liposome clustering activity of C1C2BMUNC2C compared to C1C2BMUN ( Figure 6—figure supplement", "1 ) ( see discussion ) .", "Moreover , the sequence spanning residues 1517–1531 at the C-terminus of C1C2BMUN does not form part of the MUN domain structure ( Yang et al . 2015 ) and contains a highly hydrophobic sequence that could bind to membranes , but this sequence may become structured due to the presence of the C2C domain in C1C2BMUNC2C , which could render it unable to bind membranes .", "We speculate that this hydrophobic sequence together with positive patches in the C1-C2B region underlie the liposome clustering activity of Ca2+-free C1C2BMUN ( A ) , while in C1C2BMUNC2C the C2C domain provides a PS-binding site that cooperates with the C1-C2B region to favor binding in cis to the same membrane ( B ) .", "Ca2+ binding to the C2B domain may favor membrane binding of C1C2BMUNC2C in a different orientation that facilitates interaction of the C2C domain in trans with another membrane , which would explain why Ca2+-bound C1C2BMUNC2C can bridge V-liposomes; this orientation could also be favored by binding of the C2B domain to PIP2 and of the C1 domain to DAG in T-liposomes ( C ) , leading to the overall notion that the C1-C2B region binds to the plasma membrane and the C2C domain to synaptic vesicle membrane ( Figure 7 ) .", "Extensive studies will be required to test this and other plausible models compatible with the liposome clustering data . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 022 Attempts to test whether Ca2+ causes substantial additional clustering under these conditions were hindered by saturation of the DLS detector as the reaction progressed .", "We thus performed additional lipid and content mixing assays with all protein and liposome concentrations diluted 2-fold , 4-fold and 8-fold , and found that Ca2+ still stimulated fusion strongly even in the most diluted conditions , albeit at a somewhat slower rate ( Figure 4—figure supplement 4A , B ) .", "Analysis of the fusion reaction with the 8-fold dilution by DLS revealed that efficient clustering already occurred after 3 min in the absence of Ca2+ , and the particle size increased to a very little extent one minute after Ca2+ addition ( Figure 4E; Figure 4—figure supplement 4C–F ) , although the size did increase considerably as the reaction progressed to completion .", "These results suggest that the action of C1C2BMUNC2C in these experiments is not limited to bridging V- to T-liposomes but also involves activities downstream of such bridging .", "The use of the soluble Syt1 C2AB fragment in our assays allows analysis of the effects of including or omitting the Syt1 C2 domains on fusion , but in vivo Syt1 is anchored on synaptic vesicles .", "To study how membrane-anchoring of Syt1 affects fusion in our assays , we used liposomes containing synaptobrevin and a Syt1 fragment spanning its transmembrane ( TM ) and cytoplasmic regions ( VSyt1-liposomes ) .", "These liposomes contained a smaller percentage of PS ( 6 . 8% ) that resembles that of synaptic vesicles and prevents inhibition of fusion due to binding of Syt1 to the membrane where it is anchored ( Stein et al . , 2007 ) .", "The VSyt1-liposomes fused efficiently with T-liposomes in a Ca2+-independent manner , as described previously ( Stein et al . , 2007 ) , and the Munc13-1 C1C2BMUN fragment slightly increased the fusion efficiency , but Munc18-1 had no marked effect ( Figure 8A , B ) .", "However , as expected , fusion was abolished by NSF-αSNAP and , in their presence , fusion required Munc18-1 , C1C2BMUN and Ca2+ ( Figure 8C , D ) .", "These latter results are similar to those obtained in the experiments where the soluble Syt1 C2AB was added instead of incorporating Syt1 into the V-liposomes ( Figure 3C , D ) . 10 . 7554/eLife . 13696 . 023Figure 8 . Ca2+-independent membrane fusion between Syt1-containing V-liposomes and T-liposomes becomes Ca2+-dependent in the presence of Munc18-1 , Munc13-1 C1C2BMUN and NSF-αSNAP . Lipid mixing ( A , C ) between V-liposomes containing Syt1 ( VSyt1 ) and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B , D ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "The assays were performed in the presence of different combinations of Munc18-1 ( M18 ) , Munc13-1 C1C2BMUN and NSF-αSNAP as indicated .", "Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 023 Fusion assays between VSyt1- and T-liposomes in the presence of NSF-αSNAP , Munc18-1 and different Munc13-1 fragments ( Figure 9A , B and Figure 9—figure supplement", "1 ) also yielded similar results to those obtained with soluble Syt1 C2AB ( Figure 4C , D ) , as C1C2BMUNC2C was much more active than C1C2BMUN while MUN and MUNC2C remained inactive .", "Content mixing between VSyt1- and T-liposomes in the presence of NSF-αSNAP , Munc18-1 and C1C2BMUNC2C again required Ca2+ , even though there was lipid mixing before adding Ca2+ , and was very fast upon Ca2+ addition .", "Moreover , absence of either Munc18-1 or Munc13-1 C1C2BMUNC2C completely abolished fusion ( Figure 9C , D ) , in correlation with the absolute requirement of both proteins for neurotransmitter release in vivo . 10 . 7554/eLife . 13696 . 024Figure 9 . Fast , Ca2+-dependent membrane fusion between VSyt1- and T-liposomes in the presence of Munc18-1 , Munc13-1 C1C2BMUNC2C and NSF-αSNAP , which depends on Ca2+ binding to the Munc13-1 C2B domain . Lipid mixing ( A , C , E ) between V-liposomes containing Syt1 ( VSyt1 ) and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B , D , F ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes .", "In ( A , B ) , the assays were performed in the presence of Munc18-1 , NSF-αSNAP and distinct Munc13-1 fragments as indicated .", "In ( C , D ) , experiments were performed in the presence of NSF-αSNAP with or without addition of Munc18-1 and/or Munc13-1 C1C2BMUNC2C .", "In ( E , F ) , assays were performed in the presence of Munc18-1 , NSF-αSNAP and WT or D705N , D711N mutant Munc13-1 C1C2BMUNC2C .", "All experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 02410 . 7554/eLife . 13696 . 025Figure 9—figure supplement 1 . Quantification of lipid and content mixing experiments of Figure 9A , B . Panels ( A–B ) correspond to panels ( A–B ) of Figure 9 , respectively .", "Bars represent averages of the normalized fluorescence observed after 500 s ( 200 s after Ca2+ addition ) in experiments performed at least in triplicate .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 02510 . 7554/eLife . 13696 . 026Figure 9—figure supplement 2 . Quantification of lipid and content mixing experiments of Figure 9E , F . Panels ( A–B ) correspond to panels ( E–F ) of Figure 9 , respectively .", "Bars represent averages of the normalized fluorescence observed after 300 s ( before Ca2+ addition ) and 500 s ( i . e . 200 s after Ca2+ addition ) in experiments performed at least in triplicate .", "Error bars represent standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 02610 . 7554/eLife . 13696 . 027Figure 9—figure supplement 3 . Analysis of particle size during fusion assays between VSyt1- and T-liposomes in the presence of Munc18-1 , NSF-αSNAP and Munc13-1 C1C2BMUNC2C .", "( A–D )", "Lipid mixing ( A , C ) between V- and T-liposomes was measured from the fluorescence de-quenching of Marina Blue-labeled lipids and content mixing ( B , D ) was monitored from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the VSyt1-liposomes .", "The assays were performed in the presence of Munc18-1 , NSF-αSNAP and WT ( A , B ) or D705N , D711N mutant ( C , D ) C1C2BMUNC2C as in Figure 9E , F but with all protein and liposome concentrations divided by 2 , 4 or 8 ( C/2 , C/4 or C/8 , respectively ) .", "Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin , and Ca2+ ( 600 μM ) was added after 300 s .", "( E , F )", "Intensity autocorrelation curves measured by DLS for isolated VSyt1- or T-liposomes , or at different time points as indicated in fusion assays performed as in panels ( A–D ) with eight-fold dilution of all proteins and liposomes . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 027 The finding that the data obtained with NSF-αSNAP , Munc18-1 and Munc13-1 C1C2BMUNC2C do not depend strongly on Syt1 but exhibit a drastic Ca2+ dependence indicates that such Ca2+ dependence arises from the Munc13-1 C2B domain , which contains the only known Ca2+-binding sites in these proteins ( Shin et al . , 2010 ) .", "To test this idea , we analyzed fusion between VSyt1- and T-liposomes in the presence of NSF-αSNAP , Munc18-1 and a mutant Munc13-1 C1C2BMUNC2C fragment where two of the aspartate Ca2+ ligands were mutated to asparagine ( D705N , D711N ) to disrupt Ca2+ binding .", "We found that content mixing was strongly impaired by the D706N , D711N mutation while Ca2+-independent lipid mixing was not affected ( Figure 9E , F and Figure 9—figure supplement 2 ) , indicating that Ca2+ binding to the Munc13-1 C2B domain is critical for fusion under these conditions .", "To examine how these results are related to the membrane bridging activity of C1C2BMUNC2C , we analyzed the particle size in fusion reactions where all protein and liposome concentrations were diluted eight-fold , which still allows a strong Ca2+-induced stimulation of content mixing for WT C1C2BMUNC2C ( as observed in the experiments performed with the soluble Syt1 C2AB fragment; Figure 4—figure supplement 4A , B ) but not for the D706N , D711N mutant ( Figure 9—figure supplement 3A–D ) .", "DLS analysis of the 8-fold diluted fusion reactions including WT C1C2BMUNC2C showed that much of the liposome clustering had already occurred after 3 min in the absence of Ca2+ , while little changes were observed 1 min after adding Ca2+ and a moderate increase in particle size occurred as the reaction progressed to completion ( Figure 9—figure supplement 3E ) .", "In analogous reactions with the C1C2BMUNC2C D706N , D711N mutant , efficient clustering occurred after 3 min and did not increase further afterwards ( Figure 9—figure supplement 3F ) .", "These results suggest that , while Ca2+ binding to the C2B domain of WT C1C2BMUNC2C might contribute to more efficient bridging of V- to T-liposomes , it is unlikely that an effect on such bridging alone can explain the dramatic enhancement of content mixing induced by Ca2+ .", "Note also that the finding that the D706N , D711N mutation disrupts content mixing but not Ca2+-independent lipid mixing ( Figure 9E ) correlates with the observation that efficient content mixing in the presence of WT C1C2BMUNC2C requires Ca2+ , while there is substantial lipid mixing without Ca2+ ( Figures 9A , C , E ) .", "Indeed , quantification at 300 s , before Ca2+ addition , showed that the fluorescence increase reflecting lipid mixing was 28 . 9% of that observed 200 s after Ca2+ addition while content mixing was minimal at 300 s ( fluorescence 3 . 3% of that observed 200 s after adding Ca2+ ) ( Figure 9—figure supplement 2 ) .", "This difference is exacerbated by the fact that the maximal fluorescence associated with content mixing is expected to correspond to only one round of fusion , whereas additional rounds of fusion can contribute to the maximal fluorescence in the lipid mixing signal .", "Hence , these results suggest that C1C2BMUNC2C , NSF-αSNAP and Munc18-1 enable formation of a ‘primed state’ that is ready for membrane fusion and includes assembled trans-SNARE complexes , as lipid mixing can occur , but requires Ca2+ binding to the Munc13-1 C2B domain for fast full fusion that might be further accelerated by Ca2+ binding to Syt1 .", "Rescue experiments in which Munc13-1 fragments were overexpressed using the Semliki Forest virus in neurons from Munc13-1/2 double KO mice indicated that the MUN domain is sufficient to rescue neurotransmitter release ( Basu et al . , 2005 ) , but other functional studies in chromaffin cells and C . elegans indicated that the C2C domain is also critical to rescue Munc13 function ( Stevens et al . , 2005; Madison et al . , 2005 ) .", "To clarify the functional importance of different domains for Munc13-1 , we performed additional rescue experiments in autaptic neuronal cultures from Munc13-1/2 double KO mice ( Varoqueaux et al . , 2002 ) but expressing Munc13-1 fragments with a lentiviral expression vector , which does not overexpress proteins at such high levels as the Semliki Forest virus .", "Analysis of excitatory postsynaptic currents ( EPSCs ) revealed that a Munc13-1 fragment encompassing the C1C2BMUNC2C region rescued close to 50% of the EPSC amplitude compared to rescue with WT Munc13-1 , whereas Munc13-1 fragments spanning the MUN , MUNC2C or C1C2BMUN sequences led to only very small levels of rescue ( Figure 10A , B ) .", "Similar results were obtained when we analyzed the readily-releasable pool using hypertonic sucrose ( Figure 10C , D ) .", "These results need to be examined with caution because distinct expression levels were observed for the different fragments ( Figure 10—figure supplement 1 ) .", "However , all Munc13-1 fragments were expressed at higher levels than WT , and hence their levels should be sufficient to rescue release if the fragments are functional .", "Indeed , since there is no release in Munc13-1/2 double KO neurons ( Varoqueaux et al . , 2002 ) , the very small amounts of release observed for the rescues with MUN , MUNC2C and C1C2BMUN fragments imply that these fragments can perform Munc13-1 function to some degree , albeit with very low efficiency , and vast protein production may have compensated for functional deficiency in the previous rescues with Semliki Forest virus overexpression of the MUN domain ( Basu et al . , 2005 ) .", "Importantly , the robust rescue observed with the C1C2BMUNC2C fragment compared to C1C2BMUN ( Figure 10 ) shows that the Munc13-1 C2C domain indeed plays an important role in neurotransmitter release , in clear correlation with our reconstitution data .", "Moreover , the C1C2B region is also critical for Munc13-1 function , as the MUNC2C fragment is much less active than C1C2BMUNC2C in both the rescue experiments ( Figure 10 ) and in our fusion assays ( Figures 4 and 9 ) . 10 . 7554/eLife . 13696 . 028Figure 10 . The Munc13-1 C1 , C2B and C2C domains are critical for neurotransmitter release .", "( A ) Representatives traces of single AP-evoked EPSCs from Munc13-1/2 DKO hippocampal neurons rescued with Munc13-1 full length , or C-terminal Munc13-1 fragments , in response to 2 ms somatic depolarization .", "Depolarization artifacts and action potentials were blanked .", "( B ) Normalized summary plot of EPSC peak amplitudes from Munc13-1/2 DKO hippocampal neurons rescued with Munc13-1 full length or C-terminal fragments .", "Data were collected during 4 consecutive days of recording .", "Data were normalized to the mean value of the control group ( Munc13-1 full length ) .", "Error bars represent SEM .", "Normalized data were pooled from two independent cultures .", "Values that differ significantly from controls are indicated ( *p<0 . 05; ***p<0 . 001 ) by Non parametric Kruskal-Wallis test with a post hoc Dunn's Multiple comparison test .", "( C ) Representative traces of RRP sizes induced by 5 s hypertonic sucrose solution application , from Munc13-1/2 DKO hippocampal neurons rescued with Munc13-1 full length and C-terminal fragments .", "( D ) Normalized summary plot of RRP charge .", "For the rescue experiments , approximately equal numbers of green positive Munc13-1/2 DKO neuron rescues with Munc13-1 full length or C-terminal fragments were collected the same day .", "But due to the fact that neurons that lacked both Munc13-1 and Munc13-2 proteins show no evoked excitatory postsynaptic currents ( EPSCs ) , and no response with sucrose stimulation , EPSC and RRP that show no responses were not quantified in the plots .", "The following numbers of EPSC or RRP responses were observed out of the total green positive neurons for each condition: FL , 53/55; C1C2BMUNC2C , 45/54; C1C2BMUN , 6/50; MUN , 6/50; MUNC2C , 4/51 .", "EPSC means ± SEM ( nA ) excluding 0: FL , 1 . 963 ± 0 . 2511; C1C2BMUNC2C , 0 . 83120 ± 0 . 1427; C1C2BMUN , 0 . 03298 ± 0 . 008110; MUN , 0 . 05984 ± 0 . 0009355; MUNC2C , 0 . 05892 ± 0 . 0009125 .", "EPSC charge means ± SEM ( pC ) excluding 0: FL , 226 . 3 ± 42 . 04; C1C2BMUNC2C , 139 . 5 ± 23 . 90; C1C2BMUN , 5 . 090 ± 1 . 190; MUN , 1 . 847 ± 1 . 092; MUNC2C 8 . 259 ± 1 . 092 . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 02810 . 7554/eLife . 13696 . 029Figure 10—figure supplement 1 . Protein expression from Munc13-1/2 DKO hippocampal mass cultures infected with Munc13-1-Flag and C-terminal-Flag tagged fragments .", "( A ) Diagrams illustrating the pLenti/Syn/NLS-GFP/P2A/Munc13-1-Flag constructs and the Munc13-1 fragments used .", "( B ) Western blot .", "Lane 1 shows the expression of the two cleaved products expected from the NLS-GFP/P2A/Munc13-1-Flag after the 2A cleavage .", "A signal for anti-flag at 250kDa corresponds to the expected full length Munc13-1-flag , and the anti-GFP signal at around 30kDa indicates the second cleave product NLS-GFP .", "This indicates that the P2A fusion construct was cleaved successfully , producing the two translational products expected .", "Lane 2 shows the lack of expression of the protein Munc13-1-flag or NLS-GFP in untransfected Munc13-1/2 DKO neurons as a negative control .", "Lanes 3–6 show the protein expression of all Munc13-1 fragments used .", "All constructs tested presented bands for flag and GFP .", "In all lanes the band of molecular weights ( → ) 30 kDa corresponds to the translational products NLS-GFP protein after the 2A cleavage .", "Bands at 130 , 115 , 75 and 100 kDa corresponded to the cleavage products of C-terminal Flag tagged fragments ( * ) .", "Little amounts of uncleaved products ( → ) were also found .", "Note that the GFP signal increases with the shortness of the construct introduced while the Flag signal exhibits a different pattern that may arise from differences in protein instability . DOI: http://dx . doi . org/10 . 7554/eLife . 13696 . 029" ], [ "Great advances have been made to characterize the central components of the neurotransmitter release machinery , but fundamental questions remain about how these components work together to trigger Ca2+-dependent membrane fusion .", "Strong evidence indicates that Munc18-1 and Munc13s orchestrate SNARE complex formation in an NSF-SNAP-resistant manner ( Ma et al . , 2013 ) , and that the participation of Munc13s in SNARE complex formation underlies their key functions in vesicle docking and priming ( Weimer et al . , 2006; Hammarlund et al . , 2007; Imig et al . , 2014 ) .", "However , it was unclear whether Munc13s have additional roles upstream and/or downstream of SNARE complex assembly .", "Moreover , it was unknown how the functions of the different domains that form the highly conserved C-terminal region of Munc13s are integrated .", "Our results now show that both the C1C2B-region preceding the MUN domain and the C2C domain at the C-terminus are critical for neurotransmitter release , and suggest a strong functional synergy between these domains that arises because they help bridging the synaptic vesicle and plasma membranes , facilitating the activity of the MUN domain in mediating opening of syntaxin-1 ( Figure 7 ) .", "Our results also indicate that the neuronal SNAREs , Munc18-1 , Munc13 , NSF and α-SNAP are crucial to generate a ‘primed state’ that includes Munc13 as an integral component and is ready to release but needs Ca2+ to trigger fast membrane fusion .", "Our reconstitutions recapitulate many key features of synaptic vesicle fusion , providing an ideal system to investigate the mechanism of release .", "The total abrogation of neurotransmitter release observed in the absence of Munc18-1 and Munc13s ( Verhage et al . , 2000; Varoqueaux et al . , 2002 ) established that these two proteins are the most central factors of the membrane fusion apparatus together with the SNAREs; accordingly , membrane fusion depends strictly on Munc18-1 and Munc13-1 C1C2BMUNC2C in our reconstitutions , as shown in a compelling fashion by Figure 9D .", "Moreover , fusion exhibits a tight dependence on Ca2+ ( Figure 9D ) and removal of the C1-C2B region or the C2C domain of Munc13-1 markedly impairs fusion in our reconstitutions ( Figures 4B , D and 9B ) as well as neurotransmitter release in neurons ( Figure 10 ) .", "The dependence of fusion on DAG and PIP2 ( Figure 4—figure supplement", "2 ) correlates with the notion that these factors enhance neurotransmitter release in part via their respective interactions with the Munc13 C1 and C2B domains ( Rhee et al . , 2002; Shin et al . , 2010 ) .", "NSF and αSNAP are key for the strict requirement of Munc18-1 , Munc13-1 and Ca2+ for fusion ( Figures 4 , 5 ) , and for the dependence of lipid mixing on DAG and PIP2 ( Figure 1D , E ) , because they disassemble syntaxin-1-SNAP-25 heterodimers ( Weber et al . , 2000 ) , ensuring that vesicle docking , priming and fusion proceed through the Munc18-1-Munc13-dependening pathway ( Ma et al . , 2013 ) .", "Although our reconstitutions are incomplete ( see below ) , these multiple correlations with physiological data imply that the mechanisms of action of the proteins included are likely to be related at least in part to those operating in vivo .", "The finding that the Mun13-1 C1C2BMUNC2C fragment can bridge V-liposomes to T-liposomes ( Figure 6 ) is particularly revealing because it suggests a natural model for how the different domains that form the conserved C-terminal region of Munc13s cooperate to mediate synaptic vesicle docking and priming ( Figure 7 ) , providing an explanation for why this fragment rescues neurotransmitter release and stimulates liposome fusion much more efficiently than shorter fragments ( Figures 4 , 9 and 10 ) .", "In this model , we assume that NSF-αSNAP disassemble the syntaxin-1-SNAP-25 complex in the T-liposomes and Munc18-1 binds to the released syntaxin-1 folded into a closed conformation .", "Bridging of the two membranes through respective interactions with the C1-C2B region and the C2C domain at opposite ends of the highly elongated MUN domain creates a ‘cage-like’ environment to facilitate SNARE complex assembly , placing the MUN domain in an ideal position to exert its activity in accelerating the transition from the syntaxin-1-Munc18-1 complex to the SNARE complex ( Figure 7 ) .", "This model is consistent with studies that revealed an important function for Munc13s in docking using stringent vesicle-plasma membrane distance criteria [direct contact or <5 nm; ( Weimer et al . , 2006; Hammarlund et al . , 2007; Imig et al . , 2014 ) ] , and that suggested that docking is equivalent to priming , reflecting partial SNARE complex formation ( note that this notion may not be valid for more relaxed definitions of docking ) .", "Our model postulates that Munc13s function in docking and priming not only because they promote SNARE complex assembly but also because they participate in upstream interactions that provide a bridge between the two membranes .", "A function of Munc13s in docking in the traditional sense of bridging two membranes ( sometimes referred to as tethering when the intermembrane distances are longer ) seems natural given the architecture of their conserved C-terminal region , with an elongated MUN domain that is flanked by domains with demonstrated or putative membrane-binding properties and that is related to tethering factors involved in traffic at diverse compartments ( Li et al . , 2011 ) .", "Indeed , these tethering factors likely facilitate SNARE complex formation by similar mechanisms to that proposed here for Munc13-1 ( Yu and Hughson , 2010 ) .", "The presence of C1 domain and C2 domains adjacent to the MUN domain in Munc13s provides opportunities for modulation by factors that regulate release such as DAG , PIP2 and Ca2+ .", "In addition , Munc13-1 contains an N-terminal C2A domain that contributes to vesicle docking via interaction with αRIMs [ ( Dulubova et al . , 2005 ) ; M . Camacho and C . Rosenmund , unpublished results] , which underlies the finding that rescue of release by C1C2BMUNC2C is incomplete ( Figure 10 ) and further supports the notion of an overall role for Munc13s in docking .", "We also note that reconstitution studies had previously shown that Munc13-4 promotes docking of V- to T-membranes in a Ca2+-dependent manner ( Boswell et al . , 2012 ) , but it is unclear to what extent this activity is related to that describe here for Munc13-1 because the C-terminal C2 domain of Munc13-4 binds Ca2+ , whereas the Munc13-1 C1C2BMUNC2C is not predicted to bind Ca2+ ( Rizo and Sudhof , 1998 ) .", "Synaptic vesicle docking and priming occur before Ca2+ influx and hence the underlying interactions are not expected to require Ca2+ .", "Correspondingly , C1C2BMUNC2C can bridge V- to T-membranes in the absence of Ca2+ ( Figure 6 ) .", "The interactions that cause such bridging are still unclear and extensive studies will be required to characterize them because the distinct vesicle clustering properties of C1C2BMUN and C1C2BMUNC2C ( Figure 2 , Figure 2—figure supplement 1 , Figure 6 ) suggest that these large protein fragments contain multiple membrane binding sites that can cooperate in cis to interact with a single membrane or in trans to bind to two membranes ( Figure 7—figure supplement 1 ) .", "We have been unable to express the C2C domain alone and we have not detected specific interactions of this domain with the SNAREs in preliminary NMR experiments using the MUNC2C fragment .", "Although this fragment does not bind tightly to liposomes ( Figure 6—figure supplement 2 ) , it seems likely that the C2C domain contains a lipid-binding site ( s ) with moderate affinity , as this feature would explain the stronger overall liposome clustering activity of C1C2BMUNC2C compared to C1C2BMUN ( Figure 6—figure supplement", "1 ) and phospholipid binding is a characteristic property of C2 domains ( Rizo and Sudhof , 1998 ) .", "We speculate that Ca2+-free C1C2BMUNC2C may bridge T- and V-liposomes because the C1-C2B region binds to the DAG-PIP2-containing T-liposomes in a configuration that favors binding of the C2C domain in trans to another membrane , i . e . a V-liposome ( Figure 7; Figure 7—figure supplement 1C ) .", "Cooperation between multiple C2C domains could readily strengthen the V-liposome binding and hence the bridging activity .", "Ca2+ had generally small effects on vesicle clustering ( Figure 2—figure supplement 1 , Figure 6C , G , Figure 4E , Figure 9—figure supplement 3E ) except for a strong stimulation of V-liposome docking by C1C2BMUNC2C ( Figure 6A ) that is unlikely to have physiological relevance .", "Hence , an effect on docking cannot explain the dramatic effects of Ca2+ in the reconstitutions that include C1C2BMUNC2C , Munc18-1 and NSF-αSNAP ( Figures 4B , D , 9B , D ) .", "Note that the ability of C1C2BMUNC2C to stimulate both lipid and content mixing of V- and T-liposomes is largely independent of Ca2+ in the absence of Munc18-1 and NSF-αSNAP ( Figure 5A , B ) , as expected because Ca2+-free C1C2BMUNC2C clusters V- to T-liposomes ( Figure 6G , H ) .", "In the presence of C1C2BMUNC2C , Munc18-1 and NSF-αSNAP , there is substantial lipid mixing but practically no content mixing in the absence of Ca2+ , and both lipid and content mixing occur very fast upon Ca2+ addition ( Figures 4 and 9; Figure 9—figure supplement 2 ) .", "These observations indicate that partial SNARE complex formation already occurs in the absence of Ca2+ , resulting in the formation of a ‘primed state’ that is ready for fusion but only fuses ( and fast ) upon Ca2+ addition [note in this context that the lipid mixing observed before adding Ca2+ can occur through lipid transfer when the SNARE complex brings membranes transiently into proximity without the need for fusion or hemifusion ( Zick and Wickner , 2014 ) ] .", "Formation of this primed state requires Munc18-1 , Munc13-1 , NSF , αSNAP and the three SNAREs , but not Syt1 ( Figure 4A , B ) .", "Such requirements correlate with the findings that Munc18-1 and Munc13s are essential for vesicle priming ( Verhage et al . , 2000; Varoqueaux et al . , 2002 ) whereas Syt1 is not ( Geppert et al . , 1994; Bacaj et al . , 2015 ) , supporting the notion that the primed state formed in our reconstitutions resembles the primed state of synaptic vesicles .", "The nature of the primed protein complex underlying this state is unclear , but it is likely to include C1C2BMUNC2C and Munc18-1 since the MUN domain binds to membrane-anchored SNARE complexes ( Guan et al . , 2008 ) and Munc18-1 also binds to SNARE complexes ( Dulubova et al . , 2007; Shen et al . , 2007 ) .", "This proposal is consistent with data suggesting a role for Sec1p ( the yeast Munc18-1 ) after SNARE complex assembly ( Grote et al . , 2000 ) and with the observation that a constitutively open syntaxin-1 mutant fully rescues the docking defect observed in Unc13 nulls in C . elegans but rescues neurotransmitter release only partially , which also suggested a role for Unc13 downstream of SNARE complex formation ( Hammarlund et al . , 2007 ) .", "It is also possible that α-SNAP forms part of this primed complex ( Zick et al . , 2015 ) .", "Regardless of its composition , our data suggest that the primed state is metastable and requires Ca2+ binding to the Munc13-1 C2B domain for efficient content mixing ( Figure 9F ) either because the Ca2+ -bound Munc13-1 C2B domain contributes directly to facilitate membrane fusion or because Ca2+ binding releases an inhibitory interaction existing in the primed state .", "The finding that a mutation in a Ca2+ -binding loop of the Munc13-2 C2B domain increases release probability ( Shin et al . , 2010 ) is consistent with both possibilities and supports the notion that Munc13s form intrinsic part of the primed state of synaptic vesicles .", "Clearly , our reconstitutions raise many questions and are incomplete , as they do not incorporate other important proteins that control release such as CAPS , complexins , RIMs or Rab3s ( Rizo and Sudhof , 2012 ) .", "While Syt1 C2AB stimulates content mixing in reconstitutions with C1C2BMUN ( Figure 3 ) , the effects of Syt1 become masked with C1C2BMUNC2C ( Figure 4 ) , likely because this Munc13-1 fragment promotes fusion with high efficiency and no further acceleration can be observed in our experiments .", "Thus , effects of Syt1 may only be observable at faster time scales , as Syt1 is not essential for release but is key to trigger release with high speed upon Ca2+ influx ( Sudhof , 2013 ) .", "Approaches that allow faster measurements [e . g . single vesicle assays ( Lee et al . , 2010; Kyoung et al . , 2011 ) ; Diao et al . , 2012; Lai et al . , 2014] will be required to test this prediction .", "An additional advantage of these assays is that they allow distinction of the docking event from lipid and content mixing .", "Note also that the strong effect of the D705N , D711N mutation in the Munc13-1 C2B domain in our reconstitutions ( Figure 9E , F ) contrasts with the mild effects of an analogous mutation in the C2B domain of the related isoform Munc13-2 on evoked release ( Shin et al . , 2010 ) .", "However , it is plausible that these mild effects arise because this isoform has some functional differences with Munc13-1 ( Rosenmund et al . , 2002 ) or because there is some functional redundancy with another protein not included in our reconstitutions ( e . g . CAPS ) , and the increased release probability caused by the mutation in the Munc13-2 C2B domain Ca2+-binding loops ( Shin et al . , 2010 ) suggests a role as a Ca2+ sensor that might cooperate with Syt1 .", "Mutating the Munc13-2 Ca2+-binding sites did have marked effects on release during action-potential trains; hence , our reconstitution results , which were obtained in the presence of DAG and PIP2 , may be related to hyper-activated states present during these trains .", "Further research will be required to distinguish between these possibilities , but we would like to emphasize that our reconstitutions recapitulate many key features of neurotransmitter release .", "Hence , unexpected findings from our reconstitutions that appear to contradict physiological data may actually uncover novel mechanistic aspects of release that were not observed in physiological experiments because of functional redundancy or compensatory effects by factors not included in the reconstitutions ." ], [ "Expression and purification of full length rat syxtaxin-1A , full length human SNAP-25A ( with its four cysteines mutated to serines ) , full-length rat Synaptobrevin , full-length rat Munc18-1 , the rat synaptotagamin-1 C2AB fragment ( residues 131–421 ) , full length Cricetulus griseus NSF V155M mutant , full-length Bos taurus αSNAP , and rat Munc13-1 fragments spanning the MUN and MUNC2C regions ( residues 859–1531 and 859–1735 , respectively , both with residues 1408–1452 from a flexible loop replaced by the sequence EF ) , were described previously ( Ma et al . , 2011; 2013; Chen et al . , 2002; 2006; Dulubova et al . , 1999; Xu et al . , 2013 ) .", "A construct encoding TM and cytoplasmic regions of Syt1 ( residues 57–421 with the following cysteine mutations: C74S , C75A , C77S , C79I , C82L ) with a C-terminal His-tag within a Pet28a vector ( Mahal et al . , 2002 ) was a kind gift from Thomas Sollner .", "The protein was expressed in Escherichia coli BL21 ( DE3 ) cells in Terrific Broth media at 16°C for 18 hr with 0 . 4 mM isopropyl β-D-1-thiogalactopyranoside .", "Cells were re-suspended in a buffer containing 50 mM Hepes pH 7 . 4 and 600 mM KCl with a protease inhibitor mixture , and lysed using an Avestin EmulsiFlex-C5 homogenizer .", "The soluble fraction of the cell lysate was collected after centrifugation at 48 , 000 × g for 30 min; 1% β-OG was very slowly added to this soluble fraction and incubated on an orbital shaker for 4 hr at 4°C .", "This mixture was centrifuged at 48 , 000 × g for 30 min , and the soluble fraction was incubated with Ni-NTA resin ( Qiagen; Valencia , CA ) at 4°C for 2 hr .", "The resin was washed with a buffer containing 50 mM Hepes pH 7 . 4 , 600 mM KCl , 10 mM Imidazole and 1% β-OG .", "Nucleic acid contaminants were cleared with benzonase treatment of the resin using 40 units of benzonase per milliliter of solution .", "The Syt1 fragment was eluted using the washing buffer supplemented with 250 mM imidazole and further purified with size-exclusion chromatography on a Superdex 200 16/60 column using 20 mM Hepes pH 7 . 4 containing 600 mM KCl and 1% β-OG as the buffer .", "To express rat Munc13-1 fragments encoding the C1C2BMUN and C1C2BMUNC2C regions ( residues 529–1531 and 529–1735 , respectively , both with residues 1408–1452 from a flexible loop replaced by the sequence EF ) , the corresponding DNA sequences originating from full-length rat Munc13-1 ( Basu et al . , 2005 ) were cloned into the pFastBac vector ( the vector was modified by adding a GST tag and a TEV cleavage site in front of the EcoRI cloning site ) .", "The construct was used to generate a baculovirus using the Bac-to-Bac system ( Invitrogen ) .", "Insect cells ( sf9 ) were infected with the baculovirus , harvested about 72–96 hr post-infection , and re-suspended in lysis buffer ( 50 mM Tris pH8 . 0 , 250 mM NaCl , 1 mM TCEP ) .", "Cells were lysed and centrifuged at 18 , 000 rpm for 45 min , and the clear supernatant was incubated with GST agarose at room temperature for 2 hr .", "The beads were washed with:", "i ) lysis buffer;", "ii ) lysis buffer containing 1% TX-100;", "iii ) lysis buffer containing 1M NaCl; and", "iv ) lysis buffer .", "The protein was treated with TEV protease on the GST agarose at 22°C for 2 hr .", "The protein was further purified by ion exchange chromatography and gel filtration , and was concentrated to 1–4 mg/ml for storage in 10 mM Tris buffer ( pH 8 . 0 ) containing 10% glycerol , 5 mM TCEP and 250 mM NaCl .", "The C1C2BMUNC2C D705N , D711N mutant was generated by site-directed mutagenesis , and purified as the WT fragment .", "Lipids mixture containing 37 . 5% POPC , 18% POPE , 20% DOPS , 2% PIP2 , 2% DAG , 20% Cholesterol and 0 . 5% Rhodamine-PE were dried in glass tubes with nitrogen gas and kept under vacuum overnight .", "Lipid films were re-suspended in buffer ( 25 mM HEPES , pH 7 . 4 , 150 mM KCl , 10% glycerol ( v/v ) ) and vortexed for 5 min .", "The re-suspended lipid films were frozen and thawed for five times , then extruded through a 80 nm polycarbonate filter with an Avanti extruder for at least 19 times and the size of the liposomes was analyzed by DLS .", "Liposome solutions containing 2 mM lipids were incubated with 1 µM Munc13-1 fragments at room temperature for 1 hr .", "The liposomes and bound proteins were isolated by a co-floatation assay on a Histodenz density gradient ( 40%:35%:30% ) as described previously ( Guan et al . , 2008 ) .", "Samples from the top of the gradient were taken and analyzed by SDS-PAGE and Coomassie blue staining .", "These lipid mixing assays were performed as described in ( Ma et al . , 2013 ) with some modifications .", "Donor liposomes with synaptobrevin ( V ) contained 40% POPC , 20% DOPS , 17% POPE , 20% Cholesterol , 1 . 5% NBD PE , and 1 . 5% Liss Rhod PE .", "Donor liposomes with both synaptobrevin and Synaptotagmin-1 ( VSyt1 ) contained 40% POPC , 6 . 8% DOPS , 30 . 2% POPE , 20% Cholesterol , 1 . 5% NBD PE , and 1 . 5% Liss Rhod PE .", "Acceptor liposomes with syntaxin-1-SNAP-25 ( T ) contained 38% POPC , 18% DOPS , 20% POPE , 20% Cholesterol , 2% PIP2 and 2% DAG .", "Lipid mixtures were dried in glass tubes with nitrogen gas and kept under vacuum overnight .", "Lipid films were re-suspended and dissolved in buffer ( 25 mM HEPES , pH 7 . 4 , 150 mM KCl , 0 . 5 mM TCEP , 10% glycerol ( v/v ) ) with 1% β-OG .", "Purified SNARE proteins containing 1% β-OG were added to liposomes to make Syx:SNAP25:Lipid ratios = 1:5:800 for T-liposomes , Syb:Lipid = 1:500 for V-liposomes and Syt1:Syb:Lipid = 1:2:1000 for VSyt1 liposome .", "The mixtures were incubated at room temperature for 30 min and dialyzed against the reaction buffer ( 25 mM HEPES , pH 7 . 4 , 150 mM KCl , 0 . 5 mM TCEP , 10% glycerol ( v/v ) ) with 1 g/L Biobeads SM2 ( Bio-Rad; Hercules , CA ) 3 times .", "For lipid mixing assays , donor liposomes ( 0 . 125 mM lipids ) were mixed with acceptor liposomes ( 0 . 25 mM lipids ) with various additions of the other proteins in a total volume of 200 μl .", "For experiments with NSF-αSNAP , acceptor liposomes were first incubated with 0 . 8 μM NSF , 2 μM αSNAP , 2 . 5 mM MgCl2 , 2 mM ATP , 0 . 1 mM EGTA and 1 μM Munc18-1 at 37°C for 25 min , and then mixed with donor liposomes and 0 . 5 μM Munc-13 fragments , 1 μM C2AB fragment , 1 µM excess SNAP-25 , and 0 . 1 mM EGTA ( this amount of EGTA is critical to prevent effects from residual Ca2+ co-purified with the Munc13-1 fragments and is low enough to preserve the Zn2+-binding sites of the C1 domain ) .", "To measure the effects of Ca2+ , 0 . 6 mM Ca2+ was added after 300 s of the start of the reaction .", "The NBD-PE fluorescence probe was excited at 460 nm and the emission signal from NBD was monitored at 538 nm with a PTI Spectrofluorometer ( Edison , NJ ) .", "All experiments were performed at 37°C .", "At the end of each reaction , 1% w/v β-OG was added to solubilize the liposomes , and all the data were normalized to the maximum fluorescence signal achieved after addition of β-OG .", "All the experiments were repeated at least three times with a given preparation and the results were verified in multiple experiments performed with different preparations .", "For quantification , we calculated the average fluorescence at 500 s , expressed as percentage of the maximum fluorescence , and the corresponding standard deviation .", "Assays that simultaneously measured lipid mixing from de-quenching of the fluorescence of Marina Blue-labeled lipids and content mixing from the development of FRET between PhycoE-Biotin trapped in the T-liposomes and Cy5-Streptavidin trapped in the V-liposomes were performed as described ( Zucchi and Zick , 2011; Zick and Wickner , 2014 ) with some modifications .", "V-liposomes with synaptobrevin contained 39% POPC , 19% DOPS , 19% POPE , 20% Cholesterol , 1 . 5% NBD PE , and 1 . 5% Marine Blue PE .", "VSyt1-liposomes with both synaptobrevin and Synaptotagmin-1 contained 40% POPC , 6 . 8% DOPS , 30 . 2% POPE , 20% Cholesterol , 1 . 5% NBD PE , and 1 . 5% Marine Blue PE .", "T-liposomes with syntaxin-1-SNAP-25 contained 38% POPC , 18% DOPS , 20% POPE , 20% Cholesterol , 2% PIP2 and 2% DAG .", "Lipid mixtures were dried in glass tubes with nitrogen gas and under vacuum overnight .", "Lipid films were re-suspended and hydrated in buffer ( 25 mM HEPES , pH 7 . 4 , 150 mM KCl , 0 . 5 mM TCEP , 10% glycerol ( v/v ) ) with 1% β-OG by vortex and sonication .", "Purified SNARE proteins and fluorescence labeled proteins were added to lipid mixtures to make Syx:SNAP25:Lipid = 1:5:800 and PhycoE-Biotin 4 µM for T-liposomes; Syb:Lipids = 1:500 and Cy5-Streptavidin 8 µM for V-liposomea; and Syt1:Syb:Lipids = 1:2:1000 and Cy5-Streptavidin 8 µM for VSyt1 liposomes .", "The mixtures were incubated at room temperature for 30 min and dialyzed against the reaction buffer ( 25 mM HEPES , pH 7 . 4 , 150 mM KCl , 0 . 5 mM TCEP , 10% glycerol ( v/v ) ) with 1g/L Biobeads SM2 ( Bio-Rad ) 3 times at 4°C .", "The proteoliposomes were purified by floatation on a three-layer histodenz gradient ( 35% , 25% , and 0% ) and harvesting from the topmost interface .", "To simultaneously measure lipid mixing and content mixing , T-liposomes ( 0 . 25 mM lipids ) were mixed with V-liposomes ( 0 . 125 mM lipids ) with various additions in a total volume of 200 μl under analogous conditions as those described above for lipid mixing assays , including 100 μM EGTA .", "All experiments were performed at 30°C , and 0 . 6 mM Ca2+ was added at 300 s .", "The fluorescence signals from Marine Blue ( excitation at 370 nm , emission at 465 nm ) and Cy5 ( excitation at 565 nm , emission at 670 nm ) were recorded to monitor lipid and content mixing , respectively .", "At the end of each reaction , 1% w/v β-OG was added to solubilize the liposomes , and the lipid mixing data were normalized to the maximum fluorescence signal achieved after addition of β-OG .", "To measure content mixing without interference from vesicle leakiness , most experiments were performed in the presence of 5 μM streptavidin .", "Control experiments without streptavidin were performed to measure the maximum Cy5 fluorescence attainable upon detergent addition .", "However , there was a large variability in the maximum Cy5 fluorescence values observed , perhaps because of binding of the dye to the detergent .", "Since the average maximum values observed in detergent were similar to the maximum Cy5 fluorescence observed at the end of the most efficient fusion reactions ( those including the Munc13-1 C1C2BMUNC2C fragment , e . g . Figure 9B , blue trace ) and the latter was more reproducible , in practice we used averaged maximum values of these reactions for normalization .", "All the experiments were repeated at least three times with a given preparation and the results were verified in multiple experiments performed with different preparations .", "For quantification , we calculated the average fluorescence at 500 s , expressed as percentage of the maximum fluorescence , and the corresponding standard deviation .", "The clustering activity of Munc13-1 fragments was measured by DLS using a DynaPro instrument ( Wyatt Technology; Santa Barbara , CA ) basically as described ( Arac et al . , 2006 ) .", "For experiments with liposomes and Munc13-1 fragments , the conditions and liposome compositions were similar to those of the liposomes used for lipid mixing assays unless specifically indicated .", "Thus , 0 . 5 μM Munc13-1 fragments were mixed with T-liposomes ( 0 . 25 mM lipids ) and/or V-liposomes ( 0 . 125 mM lipids ) and incubated in a buffer containing 25 mM Hepes ( pH 7 . 4 ) , 150 mM KCl , 10% ( v/v ) glycerol , 0 . 5 mM TCEP , 2 . 5 mM MgCl2 , 0 . 1 mM EGTA at 25°C .", "For titrations with Munc13-1 fragments ( Figure 6—figure supplement 1 ) , different concentrations of the fragments were mixed with T-liposomes ( 0 . 25 mM lipids ) and V-liposomes ( 0 . 125 mM lipids ) and incubated in the same buffer at 30°C .", "For experiments to monitor particle size in parallel with lipid and content mixing , lipid and content mixing assays were performed at 30°C under the standard conditions described above but with all protein and liposome concentrations diluted 8-fold , and identical samples were analyzed by DLS as a function of time at 30°C .", "The cDNAs of Munc13-1 full length and C-terminal fragments ( C1C2BMUNC2C , aa529-1693; C1C2BMUN , aa529-1508; MUN domain , aa859-1508; and MUNC2C , aa859-1693 ) were generated from rat Munc13-1 ( Basu et al . , 2005 ) by PCR amplification .", "The reverse primer harbors a 3xFLAG sequence ( Sigma-Aldrich ) to allow expression analysis .", "The corresponding PCR products were fused to a P2A linker ( Kim et al . , 2011 ) after a nuclear localized GFP sequence into the lentiviral shuttle vector , which allows a bicistronic expression of NLS-GFP and the Munc13-1-Flag protein/fragment under the control of a human SYNAPSIN1 promoter .", "Concentrated lentiviral particles were prepared as described ( Lois et al . , 2002 ) .", "Animal welfare committees of Charité Medical University and the Berlin state government Agency for Health and Social Services approved all protocols for animal maintenance and experiments ( license no . T 0220/09 ) .", "Astrocytes were plated at a density of 5000 cells/cm2 onto microdots coated coverslips to allow them to grow onto the growth permissive substrate .", "Hippocampi were dissected from embryonic day 18 . 5 Munc13 1/2 DKO mouse and enzymatically treated with 25 units ml-1 of papain for 45 min at 37°C .", "After enzyme digestion , hippocampi were mechanically dissociated and the neuron suspension was plated onto the astrocytes microislands at a final density of 300 cells cm-2 .", "Neurons were incubated at 37°C and 5% CO2 for 13–16 days to mature before starting the experiments; 24 hr after plating , neurons were infected with lentiviral rescue constructs per 35 mm diameter well .", "Synaptic function was assayed by whole-cell voltage clamp .", "Synaptic currents were monitored using a Multiclamp 700B amplifier ( Axon instrument ) .", "The series resistance was compensated by 70% and only cells with series resistances <10 MΩ were analyzed .", "Data were acquired using Clampex 10 software ( Axon instrument ) at 10 kHz and filtered using a low-pass Bessel filter at 3 kHz .", "Recordings were done at room temperature in autaptic hippocampal Munc13- 1/2 DKO neurons at 13–16 days in vitro ( DIV ) .", "Borosilicate glass pipettes with a resistance between 2–3 . 5 MΩ were used .", "Internal pipette recording solution contained the following ( in mM ) : 136 KCl , 17 . 8 HEPES , 1 EGTA , 4 . 6 MgCl2 , 4 Na2ATP , 0 . 3 Na2GTP , 12 creatine phosphate , and 50 Uml-1 phosphocreatine kinase; 300 mOsm; pH 7 . 4 .", "Neurons were continuously perfused with standard extracellular solution including the following ( in mM ) : 140 NaCl , 2 . 4 KCl , 10 HEPES , 10 glucose , 2 CaCl2 , 4 MgCl2; 300 mOsm; pH 7 . 4 .", "Action potential-evoked EPSCs were triggered by 2 ms somatic depolarization from −70 to 0 mV .", "To determine the size of the readily-releasable pool ( RRP ) , hypertonic solution , 500 mM sucrose added to standard extracellular solution , was applied directly onto the isolated neuron for 5s using a fast application system .", "A transient inward current component that lasts for 2–3 s represents the RRP charge ( Rosenmund and Stevens , 1996 ) .", "Hippocampal neurons from E18 . 5 Munc13-1/2 DKO at a density of 10 . 000 / cm2 were plated into 6 well plates containing monolayer cultures of astrocytes .", "Neurons were lysed after 15 DIV at 4°C with 50 mM Tris·HCl , pH 7 . 9 , 150 mM NaCl , 5 mM EDTA , 1% Triton X-100 , 1% sodium deoxycholate , 250 μM phenylmethylsulfonyl fluoride , 1% Nonidet P-40 , and protease inhibitor cocktail-complete mini ( Roche Diagnostics , Berlin , Germany ) .", "Lysates were mixed with Laemmli Buffer containing 0 . 3 mM DTT , and boiled 10 min at 95°C .", "30 µg of protein lysates were used for the SDS-PAGE electrophoresis .", "After separation by SDS-PAGE proteins were transferred to a polyvinyl difluoride ( PVDF ) membrane .", "Membranes were blocked with 5% skim milk in TBST , and incubated at 4°C over night with primary antibodies: anti-Flag M2 ( F1804; Sigma-Aldrich ) , and anti-Living Colors GFP ( 632375; Clontech; Mountain View , CA ) .", "Secondary antibodies were horseradish peroxidase-conjugated ( Jackson ImmunoResearch; West Grove , PA ) .", "The immunoreactive proteins were detected by ECL Plus Western Blotting Detection Reagents ( GE Healthcare Biosciences; Pittsburgh , PA ) in a Fusion FX7 detection system ( Vilber Lourmat , Eberhardzell , Germany ) ." ] ]
[ "Neurotransmitter release requires SNARE complexes to bring membranes together , NSF-SNAPs to recycle the SNAREs , Munc18-1 and Munc13s to orchestrate SNARE complex assembly , and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion .", "However , it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly , and how the actions of their multiple domains are integrated .", "Reconstitution , liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1 , C2B and C2C domains of Munc13-1 , indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain .", "Our reconstitution data also suggest that Munc18-1 , Munc13-1 , NSF , αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx .", "Overall , our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking , priming and fusion ." ]
[ "In the brain , neurons communicate with each other using small molecules called neurotransmitters .", "Electrical signals in one neuron trigger the release of the neurotransmitters , which then bind to receptor proteins on another neuron nearby .", "Neurotransmitters are packaged into small compartments called synaptic vesicles and are released from the neuron when these vesicles fuse with the membrane that surrounds the cell .", "Many proteins are involved in regulating this process to ensure that neurotransmitters are released at the right place and time .", "A large protein called Munc13 plays an important role in the release of neurotransmitters .", "It contains many different regions , including a long domain called MUN and three additional domains called C1 , C2B and C2C among others .", "However , it is not clear how all these domains work together to control neurotransmitter release .", "Here Liu , Seven et al . address this question using purified proteins inserted into membranes as well as experiments in neurons from mice .", "The experiments show that the C1 , C2B and C2C domains all play key roles in neurotransmitter release .", "Together with the MUN domain , these three domains help to form bridges between synaptic vesicles and the membrane surrounding the neuron .", "These bridges could help other proteins involved in neurotransmitter release to form a group that induces vesicle fusion .", "Liu , Seven et al . ’s findings also suggest that Munc13 proteins cooperate with other proteins to form a 'primed' state in which a synaptic vesicle is ready to rapidly fuse with a neuron’s membrane when triggered to do so by an electrical signal .", "A future challenge is to find out how the proteins that form this primed state promote vesicle fusion ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "medicine", "cancer biology" ]
A nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis
elife-15691-v3
[ [ "We recently found that Lactic acidosis could effectively protect cancer cells against glucose starvation or deprivaiton ( Wu et al . , 2012; Xie et al . , 2014 ) .", "First , lactic acidosis dramatically reduces glycolysis rate with little wasting glucose to lactate , as such , a limited amount of glucose could support cancer cells for a relatively long time otherwise would be exhausted quickly .", "Second , when glucose was deprived , lactic acidosis transformed cancer cells to a ‘dormant’ state , via arresting cells at G0/G1 phase , initiating autophagy , inhibiting apoptosis , etc .", "The protective function relies on co-presence of lactate and proton , depriving either of which would abolish the function ( Wu et al . , 2012; Xie et al . , 2014 ) .", "When converting lactic acidosis to lactosis by a base , the protective function is gone; similarly , removing lactate , acidosis conferred cancer cells with little resistance to glucose deprivation .", "The significance of intratumoral lactic acidosis in tumor biology has been extensively revealed by many other investigators .", "Clinical studies showed that high level of lactate was a strong prognostic indicator of increased metastasis and poor overall survival ( Gatenby and Gillies , 2004; Brizel et al . , 2001; Walenta et al . , 2000; Schwickert et al . , 1995; Walenta et al . , 1997; Yokota et al . , 2007; Paschen et al . , 1987 ) .", "The work of Gillies and Gatenby group demonstrated that systematic and tumor pHe alkalization could inhibit carcinogenesis , tumor invasion and metastasis , and they also provided integrated models that can predict the safety and efficacy of buffer therapy to raise tumour pHe ( Silva et al . , 2009; Robey et al . , 2009; Ibrahim-Hashim et al . , 2012 ) and related theoretical work ( Martin et al . , 2012 , Martin et al . , 2011 ) .", "Furthermore , many studies reported that lactic acidosis played multifaceted roles in skewing macrophages ( Colegio et al . , 2014 ) and inhibiting the function of cytotoxic T cells ( Haas et al . , 2015 ) , altering cancer cell metabolism ( Chen et al . , 2008; Sonveaux et al . , 2008 ) , inducing chromosomal instability ( Dai et al . , 2013 ) , and promoting tumor angiogenesis ( Gatenby and Gillies , 2004; Végran et al . , 2011 ) .", "According to the guideline of Barcelona Clinic Liver Cancer ( BCLC ) staging and treatment strategy , HCC larger than 3 cm in diameter is not suitable for curative therapy ( surgical resection , liver transplantation , and ablation ) and the recommended treatment is TACE ( Forner et al . , 2012; El-Serag , 2011; Knox et al . , 2015 ) .", "But sadly , it is recognized that TACE is not effective to treat large tumors ( Sieghart et al . , 2015 ) .", "This leaves the patients with large HCC without choice of effective therapy , as also pointed out by Sieghart et al , “maximal restriction of patients selection for TACE would otherwise only improve the results of the treatment modality per se but again would leave those more advanced patients within the intermediate stage without treatment options . ” ( Sieghart et al . , 2015 ) TACE is for local control of the targeted tumor .", "TEX equation checkTACE kills HCC via 2 mechanisms , delivering concentrated anticancer drugs locally into tumor and occluding tumor feeding arteries to deprive nutrients to starve cancer cells .", "We would focus on the second mechanism .", "Occluding tumor feeding arteries effectively deprive nutrients including glucose .", "The problem is that embolization-created hypoxia condition would stimulate cancer cells to emit strong signals to initiate angiogenesis ( Knox et al . , 2015 ) to reestablish tumor vasculature to bypass the occluded tumor feeding arteries .", "If the tumor cells cannot be rapidly eliminated , tumor vasculature would be re-established , and a certain amount of tumor ( ranging from a few percent of the original tumor to even a larger tumor known as progressive disease ) would survive and thrive .", "The lactate concentrations in HCC biopsies were around 20 mM ( unpublished data ) , suggesting a lactic acidosis condition .", "After TACE , lactic acidosis would be trapped in embolized tumor and it would potentially attenuate the therapeutic efficacy of TACE .", "If it were true , locally infusion of bicarbonate to neutralize it would result in a severer necrosis in the embolized area ." ], [ "TACE is to control the targeted tumors rather than to systematically control the disease .", "As such , to measure the necrosis of the targeted tumors can quantitatively reflect the therapeutic efficacy of TACE .", "It is known that large size of HCC is a major obstacle to limit the therapeutic efficacy of TACE ( Sieghart et al . , 2015 ) , the larger the tumor size , the poorer the efficacy of TACE .", "Thus , the large HCC is a perfect tumor model to test our hypothesis .", "If intratumoral lactic acidosis is responsible for the therapeutic limitation , locally neutralizing it should significantly improve the anticancer activity , which can be quantitatively measured by the viable tumor residues .", "To the best of our knowledge , assessment of the necrosis of tumor after the first treatment is probably most objective with least interferences of known and unknown factors .", "Unless otherwise indicated , the data presented below are the response to the first TACE treatment .", "We retrieved 27 patients with large HCC ( ranging 5 . 0–14 . 6 cm ) from the pool of patients treated with cTACE in the hospital between 2010 and 2012 according to the inclusion and exclusion criteria as specified in the Materials and methods section .", "The amount of viable tumor residues after cTACE treatment was calculated .", "We then recruited 30 patients for the TILA-TACE group using the same inclusion and exclusion criteria between 2012 and 2013 .", "Most of patients’ demographic and clinicopathologic characteristics between the two treatment groups cTACE and TILA-TACE were generally comparable ( Table 1 ) .", "However , patients in the cTACE group were more likely to have multifocal tumors .", "The geometric mean of VTR after the first treatment was 45 . 1% ( 95% CI: 30 . 3%–67 . 0% ) in the cTACE group , significantly greater than that in the TILA-TACE ( 7 . 1% [95% CI: 4 . 4%–11 . 5%]; p<0 . 0001 ) ( Table 2 and Figure 1 ) .", "We further evaluated whether treatment effects ( VTR ) were confounded by some covariates such as age , BCLC tumor stage , extra-hepatic metastasis , HBV DNA copy numbers , viable tumor volume before treatment , macrovascular invasion , and tumor multifocality using general linear models .", "In this study , none of these clinical covariates were significantly associated with VTR ( data not shown ) .", "Adjustment of these variables did not appreciably alter the results ( Table 2 ) .", "We then calculated the relative therapeutic improvement by TILA-TACE as described in Materials and methods .", "TILA-TACE achieved a 81 . 1% therapeutic improvement relative to cTACE .", "We also categorized tumor responses to treatment into four categories from complete response ( CR ) to progressive disease ( PD ) according to EASL criteria .", "Complete or partial responses to treatment in the TILA-TACE group were significantly higher than that in the cTACE group .", "The percentage of CR , PR , SD , and PD in the cTACE group was 0% , 44 . 4% , 33 . 3% , and 22 . 2% ( Figure 2A ) , respectively , compared with 23% , 77% , 0% , and 0% , respectively , in the TILA-TACE group ( Figure 2B ) .", "The total objective response rate ( complete or partial responses ) in the cTACE group was 44 . 4% , whereas it was 100% in the TILA-TACE group ( Figure 2C ) .", "The observed greater responses to treatment in the TILA-TACE than in the cTACE persisted after accounting for tumor volume prior to treatment using the proportional odds model ( p<0 . 0001 ) .", "Because of the marked therapeutic improvement by TILA-TACE , the sample size required for the subsequent RCT to evaluate tumor responses to treatment was rather small .", "When patient number was 10 for each group , the estimated power value reached 0 . 826 .", "In 2014 , we recruited and randomly assigned twenty patients with large HCC ( 5 . 0 – 13 . 5 cm ) into cTACE ( n=10 ) or TILA-TACE ( n=10 ) .", "Again , most of the selected clinicopathologic characteristics were well matched between two treatment groups ( Table 3 ) .", "After completion of the first treatment , the VTR in the TILA-TACE group was 5 . 5-fold lower than that in the cTACE group ( 4 . 6% [95% CI: 1 . 8%–11 . 4%] vs . 25 . 4% [95% CI: 10 . 1%–64 . 0%]; p=0 . 008 ) ( Figure 3 and Table 4 ) .", "We also evaluated whether the treatment effect was confounded by aforementioned clinical parameters in the RCT .", "Among them , only viable tumor volume before treatment was significantly associated with VTR .", "Adjustment for tumor volume before treatment slightly changed the results , with the VTR in the TILA-TACE of 4 . 1% ( 2 . 0%–8 . 4% ) vs . 28 . 1% ( 13 . 9%–56 . 8% ) in the cTACE ( p=0 . 0009 ) .", "The relative therapeutic improvement by bicarbonate in the RCT was 80 . 1% , similar to that observed in the non-randomized cohort , 81 . 1% .", "If assessed by tumor objective response rate according to the EASL criteria , in TILA-TACE group , out of 12 targeted tumors , 4 achieved CR and 8 PR ( Figure 4 ) , whereas in cTACE group , out of 11 targeted tumors , 1 achieved CR , 6 PR , 2 SD , and 2 PD ( Figure 4 ) , with P value of 0 . 003 after adjustment for tumor volume prior to treatment .", "A similar result was found when assessing treatment effects for the largest tumor ( p=0 . 003 ) .", "In this RCT , the ORR in TILA-TACE group was 100% and the ORR in cTACE group was 63 . 6% .", "In the nonrandomized cohort of study , the 1- , 2- , 3-year survival of 27 patients treated with cTACE retrieved from our database were 66 . 7% ( 95% CI 45 . 7%–81 . 1% ) , 40 . 7% ( 95% CI 22 . 5%–58 . 2% ) , and 25 . 9% ( 95% CI 11 . 5%–43 . 1% ) , respectively , with a median survival of 14 months ( Figure 5A ) , and the 1- , 2- , 3-year survival of 30 patients treated with TILA-TACE were 82 . 8% ( 95% CI 63 . 4%–92 . 8% ) , 67 . 7% ( 95% CI 47 . 0%–81 . 8% ) , and 61 . 8% ( 95% CI 39 . 7%–77 . 8% ) , respectively , with a median survival beyond 41 months ( Figure 5A ) .", "The survival difference between TILA-TACE and cTACE was statistically significant ( p=0 . 0052 ) .", "In the randomized study , 4 patients initially assigned in and treated with cTACE group subsequently requested to cross over to TILA-TACE treatment .", "Although the cross-over was ethically warranted , this somehow blurred the overall survival difference between cTACE and TILA-TACE ( Figure 5B ) .", "In cTACE group , 3 deaths occurred in 6 patients who solely received cTACE treatment , and 4 patients who initially received cTACE treatment and subsequently crossed over to TILA-TACE treatment were alive .", "In TILA-TACE group of 10 patients , 3 deaths occurred and 7 patients live .", "There was no apparent difference in overall survival between two treatment groups in the intent-to-treat ( ITT ) analysis ( Figure 5B , left panel ) .", "However , a survival advantage appears in TILA-TACE treatment over cTACE treatment in the per-protocol ( PP ) analysis ( Figure 5B , right panel ) , but statistically not significant .", "Overall , the RCT was limited by the small sample size and the result of the PP analysis was potentially confounded by the crossover of patients from cTACE group to TILA-TACE .", "After pooling all patients together , there was a significant difference of survival between cTACE and TILA-TACE group ( Figure 5C ) .", "The adverse effect between cTACE and TILA-TACE group were comparable ( Tables 5 and 6 ) ." ], [ "In this clinical investigation , we carried out 2 studies sequentially , the first one is a nonrandomized controlled study , which demonstrated a remarkable therapeutic improvement of TILA-TACE , based on which , a randomized controlled study was designed and carried out , and again it demonstrated a superior anticancer activity of TILA-TACE .", "The most striking point is that the numbers reflecting the therapeutic improvements by TILA-TACE from the 2 studies were nearly identical ( 81 . 1% and 80 . 1% ) .", "This confirms the consistency of anticancer activities of cTACE as well as TILA-TACE with respect to local control of large HCC .", "We compared the ORR in our cTACE practice with those reported globally .", "The average objective tumor response to TACE is 35% ( range , 16%–61% ) , as systematically reviewed by LIovet and Bruix for the Barcelona-Clinic Liver Cancer Group in 2002 ( Llovet and Bruix , 2003 ) .", "In 2012 , Forner , LIovet , and Bruix summarized that more than 50% of patients had an objective response to TACE ( Forner et al . , 2012 ) , suggesting that the objective tumor response to TACE in the 10-year period ( 2002–2012 ) had been increased for about 15% .", "The complete tumor response to TACE is rare ( 0-4 . 8% ) ( Jansen et al . , 2005 ) .", "Obviously , the results obtained from our cTACE practice were similar to those reported globally .", "TACE is for local control of the targeted tumor .", "Many previous studies have confirmed that better local control was an independent prognostic indicator for patient survival ( Kim et al . , 2015; Jung et al . , 2013; Kim et al . , 2013; Shim et al . , 2012; Riaz et al . , 2011; Gillmore et al . , 2011; Riaz et al . , 2010 ) .", "Kim et al and Shim et al ( Kim et al . , 2015; Shim et al . , 2012 ) further demonstrated a clear prognostic difference between CR , PR , stable disease and progressive disease .", "The current study demonstrated that TILA-TACE achieved a remarkable improvement of local tumor control and suggested an early sign of improved survival for patients with large HCCs ( Figure 5A ) .", "It is noted that , during follow up , 16 patients in the TILA-TACE arm ( Figure 5A ) , eventually exhibited progressive disease , including 11 patients with new foci in the liver , 1 with new liver foci and lung metastasis , and 3 with lung metastasis , and 1 with bone metastasis , all of which may account for the death ( Supplementary file 2 ) .", "These observations suggest that fast CR and timely control of recurrent tumors in the liver would likely improve the survival of patients , especially those with large tumor burden and low liver reserve .", "Nevertheless , the work is limited by the study design .", "The randomized controlled study designed in this investigation was for local tumor control , not for survival .", "Although the small sample size allowed us to evaluate the local tumor control , we did not expect that such small sample size would yield statistically significant data for cumulative survival .", "Evaluation of survival is a matter much more complicated than evaluation of local control .", "There are many more factors affecting the survival of patients than those affecting the local tumor control , e . g . , tumor characteristics , liver function reserve , tumor staging , disease complications , vascular invasion , metastasis , etc . , all of which must be well controlled .", "While we acknowledged these limitations of the present small RCT , the preliminary survival data allowed us to rationally calculate the sample size for a subsequent large RCT for evaluating the survival difference between cTACE and TILA-TACE .", "We are planning a large-scale RCT to further confirm the therapeutic advantage of TILA-TACE with respect to overall and progression-free survival of patients with large HCCs .", "There was no significant difference of adverse effect between TILA-TACE and cTACE ( Tables 3 and 4 ) , i . e . , TILA-TACE was as tolerable as cATCE .", "It was within our expectation , as locally administration of bicarbonate into tumor is safe .", "Taken together , this pilot study demonstrated that bicarbonate infusion locally into HCC can markedly enhance anticancer activity of TACE , supporting the notion that neutralizing intratumoral lactic acidosis combined with glucose deprivation may deliver an effective approach to control tumor ." ], [ "The study was performed with patients’ written informed consent and with the approval of hospital’s Institutional Review Board .", "Patients’ consent to publish is obtained .", "The study is composed of 2 parts ( Reporting standard 1 ) .", "In the first part , twenty seven patients treated with cTACE were retrospectively retrieved ( February 2010 – March 2012 ) ( Supplementary file 1 ) and the viable tumor residues were calculated , and 30 patients ( January 2012 – September 2013 ) ( Supplementary file 2 ) with large HCC were recruited and treated with TILA-TACE ( targeting-intratumoral-lactic-acidosis TACE , in this modality , bicarbonate was infused into tumor to neutralizing intratumoral lactic acidosis ) .", "In the second part , the data generated from part 1 were used to estimate the sample size for a subsequent randomized controlled study .", "Twenty patients ( March 2014 – August 2014 ) ( Supplementary file 3 ) were randomly assigned to either TILA-TACE or cTACE treatment with ratio 1:1 ( registration number: ChiCTR-IOR-14005319 in Chinense Clinical Trial Registry; protocol [Supplementary file 5] can be accessed at http://www . medresman . org/ ) , according to the method of random number table .", "Patient inclusion criteria are: a diagnosis of HCC based on EASL or histological evidence , Barcelona Clinic Liver Cancer ( BCLC ) stage B or C , Child-pugh A or B , adult patients of age ≥ 20 , hypervascular lesion as evaluated by triphasic MRI and digital subtraction angiography ( DSA ) .", "Patient exclusion criteria are: BCLC stage 0 , A or D , Child pugh C , evidence of combined A-V shunt .", "Sample size calculation was determined by the values of power and alpha: power ( 0 . 8 ) and alpha ( 0 . 05 ) can properly tell the statistical significance and minimize the number of patients to receive false choice of therapy .", "In order to calculate sample size for RCT , we calculate the viable tumor residues of 30 patients treated with TILA-TACE and 27 patients treated with cTACE as described below ( Evaluation of tumor response , Calculation of total tumor volume , viable tumor volume , and necrotic tumor volume ) .", "Based on these data , we assessed the minimal number of patients required in a subsequent RCT .", "We assume the probability of making a Type I error to be 0 . 05 ( α , two-sided ) , and the probability of making a Type II error to be 0 . 20 ( β level ) , so the power of this RCT is 0 . 8 ( 1-β ) .", "The sample size was calculated according to Schouten’s general formula:n1 ≥ ( z1−α2+zβ ) 2 ( τ+γ ) σ12γ ( μ1−μ2 ) 2+ ( τ2+γ3 ) z1−α/222γ ( τ+γ ) 2 Where n1 and n2 represent the number of patients assigned to cTACE and TILA-TACE group , γ=n1/n2 , μ1 ( cTACE ) and μ2 ( TILA-TACE ) is the mean viable tumor residues , and σ1 and σ2 are the corresponding standard deviation , τ=σ22/σ12 , and Z is the normal deviate for alternative hypothesis at a level of significance .", "In practice , we used PASS software ( version 11 ) to calculate the power and sample size , using the parameters mentioned above .", "As can been seen in Supplementary file 4 , 9 patients in each group already satisfy the power and alpha value .", "For a pilot study , the sample size of 10 patients for each group was appropriate , as the estimated power value reached 0 . 826 .", "Twenty patients were randomly allocated in a ratio of 1:1 to each group for the RCT .", "As the study was designed for observing the local tumor control , the primary endpoint was tumor objective response rate to the first treatment , measured by viable tumor residue ( VTR ) , and the secondary endpoint was the overall survival .", "Relative therapeutic improvement by bicarbonate is defined as [ ( μ1-μ2 ) /μ1] × ( 100% ) , where μ1 is the mean percentage of viable tumor residues after treatment ( cTACE ) and μ2 the mean percentage of viable tumor residues after treatment ( TILA-TACE ) .", "The maximum therapeutic improvement is 100% .", "The enhanced area and nonenhanced area in every slice of a tumor ( 2 mm thichness of each image if scanned by 3 . 0T MRI Discovery 750 , GE Medical Systems or 3 . 5 mm thichness of each image if scanned by 3 . 0T MRI Signa Excite HD , GE Medical Systems ) , which was confirmed by 2 radiologists , was integrated according to MIPAV software ( Partecke et al . , 2011 ) ( http://mipav . cit . nih . gov/ ) .", "Summation of integrated enhanced and nonenhanced area of all slices of a tumor gave the viable and necrotic volumes of a tumor .", "Total tumor volume was the sum of viable and necrotic volume .", "Viable tumors were assessed by MRI according to EASL criteria .", "The enhanced and non-enhanced areas represent viable and necrotic tumors .", "MRI examinations were performed on a 3 . 0T MRI ( Signa Excite HD , GE Medical Systems , USA ) or 3 . 0T MRI ( Discovery 750 , GE Medical Systems , USA ) .", "We assessed the necrotic and non-necrotic volume using T1 post gadolinium .", "In some cases , when the T1 post gadolinium image of the lesions did not show typical enhancement , we confirmed these lesions using imaging sequence of DWI and T2W .", "The parameters of 3 . 0T MRI ( Signa Excite HD , GE Medical Systems，USA ) were as follows: T1WI fast gradient echo sequence TR/TE 180/2 . 4 , FOV 40 × 36 cm , slice thickness 7 mm; T2WI fast spin echo sequence , TR/TE 6000/104 . 2 , FOV 40 × 28 cm , slice thickness 7 mm; DWI single-shot spin-echo echo-planar imaging , SS-SE-EP , b value 600 sec/mm2 , TR/TE 1300/52 . 3 , FOV 40 × 40 cm , slice thickness 7 mm; Dynamic contrast-enhanced LAVA sequence , inversion time 5 . 0 s , TR/TE 2 . 7/1 . 3 , FOV 40 × 40 cm; slice thickness 4 mm .", "The parameters of 3 . 0T MRI ( Discovery 750 , GE Medical Systems , USA ) were: T1WI fast gradient echo sequence , TR/TE 4 . 2/1 . 9 , FOV 36 36 cm , slice thickness 4 mm; T2WI fast spin echo sequence , TR/TE 6666/65 . 3 , FOV 36 × 36 cm , slice thickness 5 mm; DWI single-shot spin-echo echo-planar imaging , SS-SE-EP , b value 800 sec/mm2 , TR/TE 6000/53 . 1; FOV 36 × 36 cm , slice thickness 5 mm; Dynamic contrast-enhanced LAVA sequence , inversion time 5 . 0 s , TR/TE 3 . 8/1 . 6 , FOV 36 × 36 cm , slice thickness 4 mm .", "The targeted-tumor response to treatment was assessed 30 days after the first treatment according to EASL criteria ( Bruix et al . , 2001 ) and defined as below: complete response ( CR ) , no obvious viable residues; partial response ( PR ) , viable residues <50%; stable disease ( SD ) , viable tumor residues between >50% but ≤100%; and progressive disease ( PD ) , viable tumors >100% .", "Overall survival was measured from the date of the first treatment until the date of death or the final follow up visit .", "TILA-TACE was performed through the transfemoral route using a 5-Fr catheter ( Shepherd-hook modified Angiographic Catheter , HANACO Medical , Tian Jin , China ) that was advanced to celiac artery .", "The tumor feeding arteries were defined by digital subtraction angiography .", "Then , a coaxial microcatheter ( 2 . 8 Fr Marguerite II , ASAHI INTECC GMA CO . , LTD , Nagoya , Japan ) was selectively inserted through a 5-Fr catheter into the tumor feeding artery , into which , 5% sodium bicarbonate ( 5% Sodium Bicarbonate Injection , Hunan Kelun Pharmaceutical , Ltd . , Hunan , China ) was infused alternatively with doxorubicin-lipiodol emulsion and oxaliplatin/homocamptothecin with the dose adjusted to tumor size .", "The dose of bicarbonate ranged between 50 and 250 ml corresponding to tumor sizes between 5 and 14 cm .", "Finally , the artery was permanently embolized with PVA of proper sizes ( Embosphere , BioSphere Medical , Paris , France ) and microcoil ( Tornado , COOK Medical , USA ) .", "The following example may give a clearer description of the procedure: If a tumor ( 10 cm ) is to be treated , according to our experience , we would prepare 150 ml 5% bicarbonate , 60 ml lipiodol doxorubicin emulsion ( 40 mg doxorubicin dissolved in 30 ml contrast medium and mixed with 30 ml lipiodol ) , oxaliplatin 150 mg in 20 ml 5% glucose , 40 mg homocamptothecin in 20 ml saline , PVA particles ( 100–300 , 300–500 , 500–700 , or 700–900 μm ) , and microcoil .", "The TILA-TACE procedure would be as follows cTACE was performed the same as above , except no bicarbonate .", "Retreatment was based on the evidence of viable tumor residues .", "The average sessions of treatment were 4 ( range 1–13 ) .", "The following adverse events which might occur during and after TACE procedure were monitored: blood pressure and oxygen saturation during TACE , pain , fever , and signs of liver decompensation after treatment , and biliary system .", "Differences in viable tumor residues after the first treatment ( the primary endpoint of the study ) between the two treatment groups cTACE and TILA-TACE were examined using unpaired t-tests and were further adjusted for viable tumor volume before treatment and other potential confounding factors using the general linear model .", "Log-transformation was conducted to normalize the distribution of viable tumor residues and viable tumor volume before treatment in parametric analyses , with assigning 1 to viable tumor residues if the measured value ( ranging 0–216 . 5% ) was 0 .", "We also categorized tumor responses to treatment into four categories according to EASL criteria .", "Differences in the distribution of categories of tumor responses to treatment between two treatment groups were examined using the proportional odds model after adjustment for viable tumor volume before treatment .", "Overall survival time was calculated from the date of the first treatment to the date of death from any cause or the last follow-up visit ( October 31 , 2015 ) .", "Distributions of overall survival were charted by Kaplan-Meier method and compared with the log-rank test .", "The overall survival in the RCT was assessed using both the intent-to-treat and per-protocol methods .", "A two-sided alpha of 0 . 05 was used for all tests ." ] ]
[ "Previous works suggested that neutralizing intratumoral lactic acidosis combined with glucose deprivation may deliver an effective approach to control tumor .", "We did a pilot clinical investigation , including a nonrandomized ( 57 patients with large HCC ) and a randomized controlled ( 20 patients with large HCC ) study .", "The patients were treated with transarterial chemoembolization ( TACE ) with or without bicarbonate local infusion into tumor .", "In the nonrandomized controlled study , geometric mean of viable tumor residues ( VTR ) in TACE with bicarbonate was 6 . 4-fold lower than that in TACE without bicarbonate ( 7 . 1% [95% CI: 4 . 6%–10 . 9%] vs 45 . 6% [28 . 9%–72 . 0%]; p<0 . 0001 ) .", "This difference was recapitulated by a subsequent randomized controlled study .", "TACE combined with bicarbonate yielded a 100% objective response rate ( ORR ) , whereas the ORR treated with TACE alone was 44 . 4% ( nonrandomized ) and 63 . 6% ( randomized ) .", "The survival data suggested that bicarbonate may bring survival benefit .", "Bicarbonate markedly enhances the anticancer activity of TACE .", "Funded by National Natural Science Foundation of China .", "ChiCTR-IOR-14005319 ." ]
[ "Surgery is the main treatment for liver cancer , but the most common liver cancer – called hepatocellular carcinoma – can sometimes become too large to remove safely .", "An alternative option to kill the tumor is to block its blood supply via a process called embolization .", "This procedure deprives the tumor cells of oxygen and nutrients such as glucose .", "However , embolization also prevents a chemical called lactic acid – which is commonly found around tumors – from being removed .", "Lactic acid actually helps to protect cancer cells and also aids the growth of new blood vessels , and so the “trapped” lactic acid may reduce the anticancer activity of embolization .", "Previous works suggested that neutralizing the acidic environment in a tumor while depriving it of glucose via embolization could become a new treatment option for cancer patients .", "Chao et al . now report a small clinical trial that tested this idea and involved patients with large hepatocellular carcinomas .", "First , a group of thirty patients received the embolization treatment together with an injection of bicarbonate – a basic compound used to neutralize the lactic acid – that was delivered directly to the tumor .", "The neutralization killed these large tumors more effectively than what is typically seen in patients who just undergo embolization Chao et al . then recruited another twenty patients and randomly assigned them to receive either just the embolization or the embolization with bicarbonate treatment .", "This randomized trial showed that the tumors died more and patients survived for longer if they received the bicarbonate together with the embolization treatment compared to those patients that were only embolized .", "In fact , four patients initially assigned to , and treated in , the embolization-only group subsequently asked to cross over to , and indeed received , the bicarbonate treatment as well .", "These data indicate that this bicarbonate therapy may indeed be effective for patients with large tumors that are not amenable to surgery .", "In future , larger clinical trials will need to be carried out to verify these initial findings ." ]
2016
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "chromosomes and gene expression", "genetics and genomics" ]
A spontaneous genetically induced epiallele at a retrotransposon shapes host genome function
elife-65233-v2
[ [ "More than 10% of the mouse genome is made up of endogenous retroviruses ( ERVs ) ( Smit et al . , 2015 ) .", "ERVs are transposable elements ( TEs ) containing retrotransposition-enabling genes flanked by non-coding identical 5′ and 3′ long terminal repeats ( LTRs ) .", "Although most ERVs have lost their mobilisation potential due to mutational decay , they retain the ability to modulate host genome function through the use of transcriptional regulation motifs contained in their sequences .", "These include transcription factor binding sites , polyadenylation signals , and splice acceptor sites ( Maksakova et al . , 2006 ) .", "For instance , solo LTRs produced via inter-LTR homologous recombination make up a large fraction of mammalian ERV sequences and can influence host gene expression through their promoter activity ( Nellåker et al . , 2012; Ruda et al . , 2004; Subramanian et al . , 2011 ) .", "Mammals have evolved a range of mechanisms to mitigate the deleterious effects of ERV retrotransposition and transcriptional disruption .", "The vast majority of mouse ERVs exhibit high levels of DNA methylation , and loss of DNA methyltransferase activity causes an increase in ERV transcription in embryos and in the germline ( Barau et al . , 2016; Bourc'his and Bestor , 2004; Jain et al . , 2017; Walsh et al . , 1998 ) .", "In addition , the deposition of histone H3 lysine nine trimethylation ( H3K9me3 ) by the methyltransferase SETDB1 ( or ESET ) in early development is crucial for ERV repression ( Matsui et al . , 2010 ) .", "SETDB1 is recruited to ERVs following the binding of KAP1 ( or TRIM28 ) to KRAB zinc finger proteins ( KZFPs ) that recognise specific sequence motifs within ERV elements ( Ecco et al . , 2017 ) .", "RNA-mediated mechanisms such as the piRNA pathway also play key roles in silencing mammalian ERVs , especially in the male germline ( Aravin et al . , 2007; Carmell et al . , 2007 ) .", "Intracisternal A-particles ( IAPs ) are murine-specific ERVs .", "They are young and highly active elements , exhibiting extensive insertional polymorphism across inbred mouse strains and accounting for more than 10 , 000 ERVs in the C57BL/6J ( B6 ) inbred strain ( Nellåker et al . , 2012; Smit et al . , 2015 ) .", "IAP insertions are responsible for the majority of documented insertional mutations in the mouse , most of which disrupt host gene function by generating aberrant or fusion gene transcripts ( Gagnier et al . , 2019 ) .", "In some cases , the phenotypic severity of the mutation is associated with the methylation status of the IAP .", "For example , the IAP LTR promoter at the Agouti viable yellow ( Avy ) allele drives ectopic expression of the downstream coat colour gene Agouti when unmethylated .", "By mechanisms not yet fully understood , the Avy IAP exhibits variable DNA methylation levels across genetically identical individuals , which results in inbred mice displaying a range of coat colours ( Duhl et al . , 1994; Morgan et al . , 1999 ) .", "We previously carried out a genome-wide screen to probe the generalisability of inter-individual methylation variability at IAPs ( Elmer et al . , 2021; Kazachenka et al . , 2018 ) .", "We identified dozens of novel variably methylated IAPs ( VM-IAPs ) in the B6 genome and showed that their characteristic methylation variability is recapitulated from generation to generation irrespective of parental methylation level .", "Our screen for VM-IAPs identified the IAP-Pgm2 element , named after its closest annotated coding gene Phosphoglucomutase-2 ( Pgm2 ) .", "It is a fully structured 7 . 5 kb IAP located on Chromosome 5 containing identical 5′ and 3′ LTRs of the IAPLTR2_Mm subclass ( mm10 coordinates: chr5:64 , 030 , 834–64 , 038 , 297; Figure 1—figure supplement 1 ) .", "Here we show that IAP-Pgm2 , in sharp contrast to VM-IAPs , exhibits two distinct DNA methylation states which are stably inherited from parent to offspring within the B6 strain .", "Sequencing of the locus reveals that the epiallele is genetically conferred , where the methylated variant of IAP-Pgm2 is a full-length IAP matching the B6 reference genome ( renamed Iap5-1full ) and the unmethylated variant is a solo LTR produced from an inter-LTR recombination event ( renamed Iap5-1solo ) .", "The absence of DNA methylation at Iap5-1solo is accompanied by a loss of H3K9me3 marks .", "We find that differential modification of the two variants is established during early preimplantation development and identify candidate KZFPs responsible for the acquisition of contrasting epigenetic states .", "We report that Iap5-1solo is unresponsive to dietary methyl supplementation but highly susceptible to genetic background , becoming a bona fide VM-IAP in an F1 hybrid context .", "In addition , we demonstrate that formation of the Iap5-1solo allele is associated with tissue-specific changes in neighbouring gene expression and a decrease in fasting plasma glucose and triglyceride concentrations .", "Our study establishes the Iap5-1 locus as a naturally occurring and biologically relevant model in the widely studied reference mouse strain to investigate the mechanisms underlying TE repression , inter-individual methylation variability , and TE-induced disruptions to host genome function ." ], [ "Following our genome-wide screen for VM-IAPs in the B6 genome , DNA methylation at the distal CpGs of the 5′ LTR of each candidate was validated using genomic DNA ( gDNA ) extracted from adult inbred B6 mice ( Elmer et al . , 2021; Kazachenka et al . , 2018 ) .", "While VM-IAP DNA methylation levels display continuous probability distributions in the B6 population , the IAP-Pgm2 5′ LTR exhibited three distinct states: high ( >85% ) , low ( <20% ) , and intermediate ( 60–70% ) methylation ( Figure 1A and B ) .", "This pattern was observed in both sexes ( Figure 1—figure supplement 2 ) .", "In addition , 5′ and 3′ LTR methylation levels were consistent with one another within an individual ( Figure 1A and B ) .", "Methylation quantification of unique non-repetitive DNA immediately up- and downstream of IAP-Pgm2 showed that the three distinct methylation states become less defined as the distance from the LTR borders increases , ultimately collapsing approximately 500 bp from either side of the IAP ( Figure 1C ) .", "This provides evidence for short-distance spreading of DNA methylation levels from IAP-Pgm2 into bordering DNA and suggests that the methylation differences observed between individuals are intrinsic to the IAP-Pgm2 element rather than a reflection of differential methylation of the insertion site prior to integration .", "One of the characteristic properties of VM-IAPs is the reconstruction of inter-individual methylation variability from one generation to another regardless of parental methylation level ( Kazachenka et al . , 2018 ) .", "To test whether this phenomenon occurs at IAP-Pgm2 , specific parental combinations were set up for breeding and IAP-Pgm2 5′ LTR methylation levels were quantified in the offspring .", "In stark contrast to VM-IAPs , IAP-Pgm2 exhibited stable inheritance of methylation levels .", "Offspring born to highly methylated parents were all highly methylated and offspring born to lowly methylated parents were all lowly methylated ( Figure 1D ) .", "When one parent was highly methylated and the other lowly methylated , all offspring were intermediately methylated ( Figure 1D ) .", "This indicates that high and low methylation states are allelic variants of IAP-Pgm2 ( designated IAP-Pgm2HH and IAP-Pgm2LL ) , with intermediate methylation representing co-dominant epigenetic heterozygosity ( IAP-Pgm2HL ) .", "Additional crosses confirmed this inheritance pattern: an IAP-Pgm2HL intercross produced IAP-Pgm2HH , IAP-Pgm2LL , and IAP-Pgm2HL offspring; an IAP-Pgm2HH x IAP-Pgm2HL cross produced IAP-Pgm2HH and IAP-Pgm2HL offspring; and an IAP-Pgm2LL x IAP-Pgm2HL cross produced IAP-Pgm2LL and IAP-Pgm2HL offspring ( Figure 1D ) .", "These results additionally demonstrate that the methylation state of one IAP-Pgm2 variant does not influence the methylation state of the other in a heterozygous context .", "The stable inheritance of IAP-Pgm2 methylation is indicative of a spontaneous genetic mutation in the B6 population , either in the IAP element itself or in a gene involved in its epigenetic regulation .", "To investigate the former possibility , we designed PCR primers amplifying the entirety of IAP-Pgm2 from IAP-Pgm2HH and IAP-Pgm2LL gDNA ( Figure 2A ) .", "Nested primer pairs N1 and N2 amplified two overlapping fragments , each containing half of the IAP-Pgm2 element ( Figure 2A ) .", "Agarose gel electrophoresis of the PCR products revealed amplification of both fragments from IAP-Pgm2HH gDNA but no amplification of either fragment from IAP-Pgm2LL gDNA ( Figure 2B and C ) , pointing to a substantial genetic difference between IAP-Pgm2HH and IAP-Pgm2LL individuals .", "The nested primer pair N3 was designed to target the unique bordering regions on either side of the IAP element , amplifying an 8 . 5 kb fragment based on the GRC38/mm10 ( mm10 ) genome assembly ( Figure 2A ) .", "While no DNA bands were observed following the use of this primer pair on IAP-Pgm2HH gDNA , a 1 . 5 kb band was amplified from both IAP-Pgm2LL and IAP-Pgm2HL gDNA ( Figure 2D ) .", "The smaller amplicon is indicative of a 7 kb deletion on the IAP-Pgm2L allele between the two primer annealing sites , while the lack of amplification from the IAP-Pgm2H allele is likely due to the technical challenges associated with amplifying a large repetitive DNA fragment .", "To determine the location of the 7 kb deletion , we purified and sequenced the PCR products and aligned the assembled IAP-Pgm2H and IAP-Pgm2L sequences .", "The alignment revealed that the IAP-Pgm2H allele is an identical match to the full-length IAP-Pgm2 sequence from the mm10 reference , while the IAP-Pgm2L allele is a solo LTR ( Figure 2E ) .", "The IAP-Pgm2L solo LTR and the IAP-Pgm2H full-length IAP are both flanked by the same target site duplications ( TSDs ) and the IAP-Pgm2L solo LTR exhibits 100% sequence identity to both the 5′ and 3′ LTRs of the full-length IAP-Pgm2H ( Figure 2E ) .", "Therefore , a recent inter-LTR homologous recombination event in the inbred B6 mouse strain gave rise to the solo LTR variant .", "In accordance with nomenclature guidelines from the International Committee on Standardized Genetic Nomenclature for Mice ( ICSGNM ) , IAP-Pgm2H and IAP-Pgm2L are hereafter referred to as Iap5-1full and Iap5-1solo , respectively .", "Inter-LTR recombination events occur when identical or near-identical LTR sequences engage in ectopic homologous recombination , resulting in the formation of solo LTRs ( Jern and Coffin , 2008 ) .", "These can occur both intra- and inter-chromosomally ( Figure 2—figure supplement 1 ) .", "Although we identified both Iap5-1 allelic variants in our own B6 colony , we also detected them in B6 samples sent to us from mainland Europe and North America ( data not shown ) , so the recombination event most likely occurred at a B6-distributing facility .", "This type of proviral excision event is common: approximately half of the IAPs in the mouse genome are solo LTRs ( Nellåker et al . , 2012 ) .", "However , the vast majority of the ~5 , 000 solo LTRs in the B6 genome are highly methylated ( Shimosuga et al . , 2017 ) , making Iap5-1solo unique from a regulatory perspective and raising questions regarding the functional consequences of a solo LTR left unmodified .", "Using clonal bisulphite sequencing , we confirmed that the entirety of the Iap5-1solo solo LTR is unmethylated ( bar the occasional methylated CpGs at the 5′ end , consistent with the pyrosequencing data ) while all CpGs in both the 5′ and 3′ LTRs of Iap5-1full are highly methylated ( Figure 2—figure supplement 2 ) .", "The experiments described thus far have focused on adult somatic Iap5-1 DNA methylation within and across generations .", "Given the dynamic nature of DNA methylation during mammalian development and considering previous reports on the resistance of IAPs to genome-wide methylation erasure ( Lane et al . , 2003; Seisenberger et al . , 2012 ) , we sought to examine and compare DNA methylation levels of the Iap5-1full and Iap5-1solo variants in the germline and during early embryonic development .", "Germinal vesicle ( GV ) oocytes and mature sperm were collected from Iap5-1full and Iap5-1solo adult B6 females and males .", "Oocytes collected from Iap5-1full and Iap5-1solo females were highly and lowly methylated at the Iap5-1 locus , respectively ( Figure 2F ) .", "Thus , oocyte Iap5-1 methylation levels are reflective of somatic Iap5-1 methylation levels .", "In contrast , both variants were hypermethylated in sperm ( Figure 2F ) , indicating that both alleles are targeted for repression during spermatogenesis by mechanism ( s ) that are distinct from those operating on Iap5-1 in the soma .", "Together , these experiments indicate that the maternal and paternal Iap5-1solo alleles are differentially methylated upon fertilisation in the early zygote .", "We examined the behaviour of Iap5-1 methylation levels during early development in blastocysts ( embryonic day 3 . 5 , E3 . 5 ) collected from the uteri of Iap5-1full and Iap5-1solo B6 females bred to Iap5-1full and Iap5-1solo B6 males , respectively .", "Blastocysts generated from Iap5-1solo parents exhibited low methylation levels at Iap5-1 , suggesting that the paternally inherited hypermethylated Iap5-1solo allele becomes demethylated shortly after fertilisation ( Figure 2F ) .", "By comparison , blastocysts generated from Iap5-1full parents exhibited methylation levels around 70% ( Figure 2F ) .", "It is unclear whether Iap5-1full is demethylated after fertilisation and rapidly methylated again by the blastocyst stage , or whether Iap5-1full is generally resistant to epigenetic reprogramming during preimplantation development .", "Nonetheless , these results demonstrate that methylation patterns at Iap5-1full and Iap5-1solo are specified prior to implantation .", "Despite the marked distinction in methylation states between Iap5-1full and Iap5-1solo blastocysts , Iap5-1full methylation levels are lower at the blastocyst stage than in adult somatic tissue .", "It is possible that this incomplete methylation is symptomatic of lower methylation levels in the developing trophectoderm which counteract the higher methylation levels in the inner cell mass ( ICM ) .", "In support of this , DNA methylation levels in embryonic stem ( ES ) cell lines derived from the ICM of Iap5-1full and Iap5-1solo blastocysts closely matched those observed in adult somatic tissues , with ES cells derived from Iap5-1full blastocysts nearing 100% methylation ( Figure 2F ) .", "In addition , we found that Iap5-1full methylation levels in E16 . 5 placentas were lower than those in E16 . 5 embryonic tissue ( Figure 2F ) , consistent with reduced global DNA methylation in the placenta ( Ehrlich et al . , 1982; Schroeder et al . , 2015 ) .", "These results provide evidence for differential methylation between ICM- and trophectoderm-derived lineages at the Iap5-1 locus .", "Of note , even though placental Iap5-1 methylation levels are less methylated compared to their embryonic counterparts , the two variants retain a pronounced difference in DNA methylation levels in this tissue .", "To determine whether loss of DNA methylation at Iap5-1solo is associated with changes in histone modifications , we quantified H3K9me3 enrichment in Iap5-1full and Iap5-1solo adult liver samples via ChIP-qPCR .", "The retrotransposons IAP-Asxl3 and SINE-Rbak , used as positive controls , exhibited equivalent H3K9me3 enrichment between Iap5-1full and Iap5-1solo individuals ( Figure 3A ) .", "The Gapdh promoter was used as a negative control .", "Because the sequence of Iap5-1solo is identical to that of the 5′ and 3′ LTRs of Iap5-1full , the same primers were used to probe H3K9me3 enrichment at the borders of both variants .", "H3K9me3 enrichment at the 5′ and the 3′ borders was significantly decreased in Iap5-1solo samples compared to Iap5-1full samples ( Figure 3A ) .", "Iap5-1full showed comparable H3K9me3 levels to those observed at the positive controls ( Figure 3A ) .", "We suggest that the slightly higher H3K9me3 enrichment at the 3′ end compared to the 5′ end in Iap5-1solo samples is due to the presence of an ERV element immediately downstream of Iap5-1 .", "Heterochromatin formation at mammalian TEs occurs in early development following their sequence-specific recognition by KZFPs .", "KZFPs recruit the scaffold protein KAP1 , which in turn recruits the H3K9 methyltransferase SETDB1 as well as de novo DNA methyltransferases ( Ecco et al . , 2017 ) .", "We reasoned that Iap5-1full may be targeted for repression in the early embryo by KZFP ( s ) whose binding sites are located in the internal proviral portion of the IAP which is no longer present in the Iap5-1solo variant .", "To explore this hypothesis , we analysed previously published ChIP-seq ( chromatin immunoprecipitation followed by high-throughput sequencing ) binding profiles of more than 60 murine KZFPs ( Wolf et al . , 2020 ) and identified two Iap5-1full-binding candidates , Gm14419 and Gm8898 ( Figure 3B ) .", "The ChIP-seq profiles for Gm14419 and Gm8898 in B6 ES cells displayed peaks immediately downstream and 1 . 5 kb downstream of the 5′ LTR , respectively ( Figure 3B ) .", "We observed KAP1 occupancy at the Gm14419 binding site , rendering Gm14419 a particularly promising candidate for future mechanistic research ( Figure 3B ) .", "The Gm14419 peak overlaps with the primer binding site ( PBS ) just downstream of the 5′ LTR ( Figure 3B ) , a retroviral sequence often bound by KZFPs to effectuate repression ( Wolf and Goff , 2007 ) .", "Furthermore , it is likely that additional as yet unidentified KZFP ( s ) bind to Iap5-1full , as evidenced by a second KAP1 peak in the Iap5-1full internal region which does not overlap with the binding site of any of the KZFPs identified in our analysis ( Figure 3B ) .", "This is consistent with the redundant nature of KZFP-mediated ERV repression ( Imbeault et al . , 2017; Wolf et al . , 2020 ) .", "In addition , we detected ZFP429 binding peaks at the 5′ and 3′ LTRs of Iap5-1full ( Figure 3B ) .", "Given the shared sequence between the Iap5-1full LTRs and Iap5-1solo , this observation suggests that ZFP429 does not recruit heterochromatin factors as effectively as other KZFPs .", "This is in line with our previous finding that ZFP429 is enriched at VM-IAPs compared to fully methylated IAPs of the same subclass ( Bertozzi et al . , 2020 ) .", "Previous studies have shown that in some cases IAP methylation is modulated by genetic background ( Bertozzi et al . , 2020; Elmer and Ferguson-Smith , 2020; Rakyan et al . , 2003; Wolff , 1971 ) .", "To assess whether this is the case for Iap5-1 methylation , we carried out reciprocal crosses between B6 and wild-derived CAST/EiJ ( CAST ) mice ( BC , B6 female × CAST male; CB , CAST female × B6 male ) .", "F1 hybrid offspring carry a single copy of Iap5-1 inherited from their B6 parent because the Iap5-1 insertion is absent from the CAST genome ( Figure 4A ) .", "In line with our breeding experiments in pure B6 mice , hemizygous offspring born to a Iap5-1full B6 parent were highly methylated and those born to a Iap5-1solo B6 parent were lowly methylated ( Figure 4B ) .", "However , although Iap5-1solo methylation levels remained low in BC and CB F1 hybrids , they were significantly higher compared to those in pure B6 individuals ( Figure 4C ) , suggesting that CAST-derived modifier ( s ) act on Iap5-1solo in trans .", "In addition , CB offspring displayed higher and more variable Iap5-1solo methylation levels compared to BC offspring , indicative of a genetic background-specific maternal effect similar to those recently reported at VM-IAPs ( Bertozzi et al . , 2020 ) .", "We further interrogated the strain-specific maternal effect by backcrossing Iap5-1solo CB hybrid males to pure CAST females .", "This resulted in a cumulative effect , whereby N1 Iap5-1solo mice showed even higher and more variable methylation levels compared to F1 Iap5-1solo mice ( Figure 4C ) .", "Therefore , the CAST content of the inherited paternal genome and passage through a CAST egg may compound each other .", "The subsequent N2 backcrossed generation did not cause a further increase in methylation , and backcrossing N5 males to B6 females resulted in a reversion to the original B6 methylation state ( Figure 4C ) .", "These results are reminiscent of strain-specific behaviours of transgene methylation reported decades ago and support a role for strain-specific oocyte factors ( possibly polymorphic KZFPs ) in driving these genetic–epigenetic interactions ( Allen et al . , 1990; Bertozzi et al . , 2020; Kearns et al . , 2000 ) .", "The IAPs located at the Avy and AxinFu VM-IAPs exhibit increased DNA methylation following gestational methyl supplementation , resulting in shifts in the associated coat colour and tail morphology phenotypes , respectively ( Waterland et al . , 2006; Waterland and Jirtle , 2003 ) .", "However , it remains unclear whether susceptibility to this environmental exposure is conferred by variable or by incomplete DNA methylation at these epialleles ( or neither ) .", "The unmethylated Iap5-1solo variant provides an opportunity to test the latter .", "To examine whether Iap5-1solo is responsive to methyl supplementation , Iap5-1solo females were put on a methyl-supplemented diet 2 weeks prior to mating and were kept on the same diet throughout pregnancy and lactation .", "Control females were fed standard chow throughout and all pups in the experiment were weaned onto standard chow .", "DNA methylation levels at the Iap5-1solo LTR were quantified in 8-week-old offspring liver samples .", "Unlike Avy and AxinFu loci , Iap5-1solo remained unmethylated in the methyl-supplemented offspring ( Figure 4—figure supplement 1 ) , indicating that complete lack of methylation at IAPs does not go hand-in-hand with susceptibility to dietary methyl supplementation .", "IAP insertions can influence neighbouring gene expression .", "Intergenic IAPs can induce the formation of chimeric transcripts initiated at the promoter in the IAP LTR and may also act as enhancers ( Gagnier et al . , 2019 ) .", "These effects are sometimes dependent on the epigenetic properties of the IAP , as illustrated by the Avy and AxinFu IAPs which modulate Agouti or Axin expression in a methylation-dependent manner .", "We asked whether allelic variation at the Iap5-1 locus influences the expression of neighbouring genes by quantifying expression of the four closest genes ( Figure 5A ) in liver , cortex , thymus , and placental tissues collected from Iap5-1full and Iap5-1solo individuals .", "Protein-coding genes Pgm2 , TBC1 domain family , member 1 ( Tbc1d1 ) , and RELT-like protein 1 ( Rell1 ) were expressed in all tissues examined; long non-coding RNA ( lncRNA ) 5830416l19Rik transcripts were only detected in the thymus .", "Pgm2 and Rell1 expression levels in the thymus and placenta were significantly higher in Iap5-1solo than in Iap5-1full individuals , indicating that in these tissues the unmethylated solo LTR is associated with increased expression ( Figure 5B ) .", "Tbc1d1 , the furthest in distance from Iap5-1 , did not display significant differences in expression in any of the tested tissues , suggesting that proximity to the Iap5-1 locus is predictive of its transcriptional effect ( Figure 5B ) .", "5830416l19Rik expression in the thymus was barely detected in Iap5-1full samples , showing a highly significant increase in Iap5-1solo samples ( Figure 5B ) .", "Rell1 was the only gene to show a significant difference in expression in liver tissue and the directionality of the effect was inversed , with lower expression levels observed in Iap5-1solo individuals ( Figure 5B ) .", "No significant differences in expression were observed for any of the genes in cortex samples ( Figure 5B ) .", "In thymus and cortex , however , we detected transcription of the unique non-coding regions bordering Iap5-1solo , which was not observed for the equivalent regions bordering Iap5-1full ( Figure 5—figure supplement 1 ) .", "Together , these data show that the Iap5-1 polymorphism is associated with tissue-specific altered adjacent gene expression .", "Little is known about the biological functions of Rell1 and 5830416l19Rik , but Pgm2 is better characterised .", "Pgm2 is the closest coding gene to Iap5-1 , lying 55 kb downstream of the 3′ LTR .", "PGM2 catalyses the interconversion between glucose 1-phosphate and glucose 6-phosphate and has a secondary role to that of the predominant PGM isozyme , PGM1 ( Geer et al . , 2010 ) .", "PGM1 deficiency in humans is associated with glycogen storage disease and a congenital disorder of glycosylation ( Beamer , 2015 ) .", "PGM2 has been associated with metabolic disease in GWAS studies ( Timmons et al . , 2018 ) .", "Considering the established role of Pgm2 in metabolic pathways , we investigated potential metabolic effects resulting from the formation of the Iap5-1solo allele .", "We screened plasma samples collected from Iap5-1full and Iap5-1solo adult males for a range of metabolic biomarkers .", "While most assays revealed no difference between the two variants ( Figure 5—figure supplement 2A ) , we found that fasting plasma glucose and triglyceride concentrations were significantly lower in Iap5-1solo mice ( Figure 5C ) .", "Glucose tolerance tests did not show additional metabolic disparities between the two variants ( Figure 5—figure supplement 2B ) .", "These findings suggest that the emergence of a derepressed solo LTR near a gene involved in glucose metabolism has phenotypic implications , providing a basis for further functional characterisation of this model ." ], [ "Historically , inter-LTR recombination events have been identified due to phenotypic reversions of ERV-induced mutations , whereby the phenotypic effect of the insertion is reversed following proviral excision and solo LTR formation ( Bultman et al . , 1994; Seperack et al . , 1988; Stoye et al . , 1988 ) .", "In this study , we identified one such chromosomal event based on its epigenetic rather than phenotypic outcome , revealing Iap5-1 as a genetically induced epiallele in the ostensibly isogenic B6 mouse strain .", "We demonstrated that the Iap5-1full and Iap5-1solo variants display distinct DNA and H3K9 methylation profiles which are associated with differential adjacent gene expression and altered metabolism , establishing the Iap5-1 locus as a valuable endogenous system to study the mechanisms , evolution , and functional implications of TE repression in both the germline and soma .", "The unmethylated status of Iap5-1solo sets it apart from other solo LTRs in the B6 genome , the vast majority of which ( >93% ) are highly methylated ( Shimosuga et al . , 2017 ) .", "Shimosuga and colleagues quantified DNA methylation at more than 8000 B6 IAP LTRs and only 14 exhibited less than 20% methylation in tail samples .", "Ten of these were solo LTRs , mostly of the same subtype as the Iap5-1 LTR ( IAPLTR2_mm ) .", "While the authors classified the Iap5-1 LTR as a hypomethylated 3′ LTR of a full-length IAP , in all likelihood they unknowingly detected the hypomethylated Iap5-1solo allele in their particular samples .", "It is conceivable that some of the other documented hypomethylated LTRs of full-length IAPs are in fact solo LTRs formed from recent recombination events .", "In comparing tail and sperm DNA methylation , the same study showed that individual hypomethylated IAPs in either tail or sperm were non-overlapping .", "This is consistent with the hypermethylation of Iap5-1solo that we observed in sperm and highlights the divergence of TE silencing mechanisms between the soma and the germline .", "Ancestral variants of spontaneous mutations in inbred mouse strains are easily lost from the population .", "The fortuitous timing of our discovery and the resultant availability of both Iap5-1full and Iap5-1solo mice allowed us to quantitatively explore the functional repercussions of a derepressed solo LTR .", "We showed that the Iap5-1solo variant is associated with increased expression of adjacent genes in thymic and placental tissue .", "This may reflect the use of newly accessible regulatory sequences in the solo LTR or , alternatively , a secondary effect stemming from heterochromatin loss at a neighbouring region .", "The transcriptional consequences were tissue-specific: Rell1 expression was lower in Iap5-1solo compared to Iap5-1full livers , and no gene expression differences were observed in cortex samples .", "The lncRNA 5830416l19Rik , identified from the sequencing of adult male thymus and aorta cDNA libraries ( Kawai et al . , 2001 ) , was only expressed in thymus samples collected from Iap5-1solo individuals; it was nearly undetectable in Iap5-1full thymic samples and not expressed at all in any of the other tested tissues .", "We speculate that the formation of Iap5-1solo gave rise to 5830416l19Rik as a novel tissue-specific ( perhaps immune-specific ) lncRNA .", "Indeed , IAP transcripts are rarely detected in somatic tissues outside of tumours and early embryogenesis , but thymic and activated splenic cells are notable exceptions ( Kuff and Lueders , 1988 ) .", "In addition , young ERVs have recently been implicated in shaping the evolution of transcriptional networks underlying innate immunity ( Chuong et al . , 2016; Tie et al . , 2018; Ye et al . , 2020 ) , and our group recently identified individual IAP elements that exhibit inter-individual methylation variability exclusively in B cells ( Elmer et al . , 2021 ) .", "Along with the transcriptional differences observed at the metabolic gene Pgm2 , we found that Iap5-1solo individuals exhibit lower plasma glucose and triglyceride concentrations compared to Iap5-1full individuals .", "While the effect sizes were small , we note that these measurements were taken in an unaltered environment .", "Administration of a long-term metabolic challenge such as a high-fat diet and subsequent metabolic phenotyping will aid in further elucidating the biological significance of these results .", "Nevertheless , our work adds to the body of work suggesting that TE insertions and their epigenetic modification can have quantifiable consequences on host metabolic pathways ( Du et al . , 2016; Kuehnen et al . , 2012; Scherneck et al . , 2009; Yen et al . , 1994 ) .", "We have shown that Iap5-1solo is susceptible to genetic background effects , whereby passage through a CAST egg promotes methylation of the B6-derived solo LTR .", "In a recent study we reported the same effect at a number of VM-IAPs and showed that KZFP diversification is associated with strain-specific IAP methylation ( Bertozzi et al . , 2020 ) .", "This combined with the identification of candidate KZFPs capable of binding the Iap5-1 variants suggests that the differential recruitment of heterochromatin factors by KZFPs is involved in both driving the epigenetic differences between Iap5-1full and Iap5-1solo , and for the strain-specific methylation of Iap5-1solo .", "In fact , by definition , the wide range of methylation levels observed at the Iap5-1solo allele across genetically identical CB individuals renders this locus a bona fide VM-IAP ( or metastable epiallele ) in this hybrid context ( Bertozzi and Ferguson-Smith , 2020; Rakyan et al . , 2002 ) .", "Therefore , we expect the new Iap5-1solo variant to be a useful tool for the study of epigenetic stochasticity in the absence of genetic variation , a phenomenon for which the underlying mechanisms remain poorly understood .", "The identification of Iap5-1solo demonstrates that cryptic genetic diversity in an inbred mouse population can have functional repercussions with important implications for experimental outcomes .", "It serves as a cautionary tale for researchers working with inbred mouse colonies , particularly in the field of epigenetics where ruling out genetic effects is often paramount .", "We are reminded that the current mouse reference genome harbours large gaps and inaccuracies , largely due to mapping difficulties associated with the repeat genome .", "Inter-LTR recombination events are surely an underappreciated source of genetic variation considering that a lack of uniquely mapped reads in internal portions of TEs is more likely to be attributed to technical rather than biological limitations .", "A recent study in humans developed a computational pipeline to capture dimorphic human ERVs ( HERVs ) resulting from inter-LTR recombination events and detected dozens of previously unidentified candidates ( Thomas et al . , 2018 ) .", "The advent of such analytical tools as well as routine long-read sequencing of whole genomes will be highly beneficial in addressing these issues .", "In summary , we have identified and characterised a recent spontaneous inter-LTR recombination event at the Iap5-1 locus , introducing a genetic variant in the commonly investigated and supposedly inbred B6 mouse strain .", "The ancestral variant , Iap5-1full , is a full-length IAP repressed by DNA methylation and H3K9 trimethylation , as is typical for this class of evolutionary young ERV .", "The recently formed solo LTR variant , Iap5-1solo , lacks these silencing marks and is associated with metabolic changes and differential neighbouring gene expression .", "Our study lays the foundation for further comparative studies between the Iap5-1full and Iap5-1solo variants aimed at better understanding the epigenetic and functional consequences of TE structural variation and evolution ." ], [ "Mouse work was carried out in accordance with the Animals ( Scientific Procedures ) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body ( Home Office project license # PC213320E ) .", "C57BL/6J ( RRID:IMSR_JAX:000664 ) and CAST/EiJ ( RRID:IMSR_JAX:000928 ) mice were obtained from Charles River and the MRC Harwell Institute , respectively , and maintained under a 12 hr light–dark cycle in temperature- and humidity-controlled conditions .", "Mice were fed a standard chow diet ( RM3 ( E ) ; Special Diet Services ) ad libitum unless otherwise noted .", "For pure and hybrid breeding experiments , mice were mated at 8–12 weeks of age and 10 day old pups were ear notched for DNA methylation quantification .", "Male and female offspring from multiple litters per breeding pair were included in the analyses .", "Adult Iap5-1solo females were randomly placed on control diet ( RM3 ( E ) ; Special Diet Services ) or methyl supplemented diet ( RM3 ( E ) supplemented with 15 g Choline , 15 g Betaine , 15 mg Folic acid , 1 . 5 mg Vitamin B12 , 7 . 5 g L-methionine , 150 mg Zinc; Special Diet Services ) 2 weeks prior to mating with Iap5-1solo males and kept on their respective diets throughout pregnancy and lactation .", "Eight dams were used for each dietary group .", "The methyl supplemented diet recipe matches the 3SZM diet in Wolff et al . , 1998 .", "All offspring were weaned onto the control diet and culled at 8 weeks of age .", "DNA methylation was quantified at the distal CpGs of the 5′ end of Iap5-1solo in all offspring livers ( control diet: 38 born from eight litters; methyl supplemented diet: 41 born from eight litters ) .", "Statistics were carried out on litter averages because the dietary intervention was done on the dams , not the offspring .", "Blood was sampled from 4-month-old Iap5-1full and Iap5-1solo males via cardiac puncture following isoflurane-induced deep terminal anaesthesia .", "Blood samples were placed in EDTA-coated tubes and centrifuged at 1500 × g for 15 min at 4°C .", "Plasma was collected from the separated upper phase and stored at −80°C before sending to the Core biochemical assay laboratory ( CBAL ) at the Cambridge University Hospitals for analysis .", "Assays performed on the plasma samples included adiponectin ( ng/ml ) , albumin ( g/l ) , ALT ( U/l ) , cholesterol ( mmol/l ) , corticosterone ( ng/ml ) , glucagon ( pg/ml ) , glucose ( mmol/l ) , HDL ( mmol/l ) , insulin ( µg/l ) , LDL ( mmol/l ) , leptin ( pg/ml ) , NEFA ( µmol/l ) , and triglycerides ( mmol/l ) .", "Glucose tolerance tests were conducted on Iap5-1full and Iap5-1solo 16-week-old males ( n = 12 per genotype ) .", "Following an overnight fast of 15 hr , mice were weighed and baseline blood glucose levels ( time 0 ) were measured using the Glucomen Areo glucometer .", "A glucose dosage of 2 g/kg was calculated for each mouse based on weight and administered via intraperitoneal injection .", "Tail blood glucose levels were measured 15 , 30 , 45 , 60 , 90 , and 120 min post injection .", "Blood glucose levels were compared between Iap5-1full and Iap5-1solo mice at each time point using multiple unpaired t-tests corrected for multiple comparisons using the Holm-Šidák method .", "For somatic tissues and ES cells , samples were treated with RNase A at 37°C for 60 min and digested with Proteinase K at 55°C overnight in lysis buffer ( 10 mM EDTA , 150 mM NaCl , 10 mM Tris-HCl pH 8 , 0 . 1% SDS ) .", "gDNA was isolated the next day using a standard phenol–chloroform extraction and ethanol precipitation protocol .", "The same protocol was followed for sperm gDNA extraction except that the Proteinase K digestion was carried out in equal volumes of Solution A ( 75 mM NaCl pH 8; 25 mM EDTA ) and Solution B ( 10 mM Tris-HCl pH 8; 10 mM EDTA; 1% SDS; 80 mM DTT ) following centrifugation and removal of PBS from thawed sperm .", "To extract oocyte and blastocyst gDNA , pooled GV oocytes or blastocysts were incubated for 1 hr at 37°C in 14 µl of ddH2O , 1 µl of 1 mg/ml Carrier RNA ( QIAGEN ) , 1 µl of 10% SDS , and 1 µl of 10 mg/ml Proteinase K . After a 15 min incubation at 98°C , samples was directly bisulphite-converted as described below .", "ESC lines were generated as previously described ( Nichols and Jones , 2017 ) with the following modifications .", "The concentration of MEK inhibitor PDO325901 used in the KSOM+2i and N2B27+2i+LIF media was reduced to 0 . 2 µM for the initial stages of the protocol and increased to 1 µM following disaggregation of the ICM .", "The zona pellucide was removed from unhatched embryos using a 10 min pronase digestion at 37°C rather than using acidic Tyrode’s solution , and rat serum was used as a source of complement instead of guinea pig serum .", "Laminin-coated wells were used to ensure proper cell attachment and Accutase solution was used to detach cells prior to passaging .", "Only male embryos were kept for the generation of ESC lines .", "Established lines were screened for mycoplasma contamination using the PCR Mycoplasma Test Kit I/C ( PromoCell ) .", "Ear notches were used for Iap5-1 allelic variant genotyping .", "Ear notch gDNA was extracted using the PCRBIO Rapid Extract lysis kit ( PCR Biosystems ) and 1 μl of 1:10 diluted DNA was used as a template for PCR using the REDTaq ReadyMix PCR Reaction Mix ( Sigma-Aldrich ) .", "The PCR conditions were as follows: ( 1 ) 95°C for 4 min 30 s; ( 2 ) 94°C for 30 s , optimised T°C for 30 s , 72°C for 30 s , 40 cycles; ( 3 ) 72°C for 5 min .", "For trophectoderm sex-genotyping , trophectoderm lysates were placed in PCR buffer with Proteinase K ( 50 mM KCl , 10 mM Tris-HCl pH 8 . 3 , 2 . 5 mM MgCl2 , 0 . 1 mg/ml gelatin , 0 . 45% NP40 , 0 . 45% Tween 20 , 200 µg/ml Pro K ) and incubated at 55°C for 1 hr followed by 95°C for 10 min .", "Trophectoderm gDNA samples were sex-genotyped by PCR using HotStarTaq DNA Polymerase ( QIAGEN ) and in the following conditions: ( 1 ) 95°C for 3 min; ( 2 ) 94°C for 30 s , 56°C for 30 s , 72°C for 55 s , 40 cycles; ( 3 ) 72°C for 5 min .", "Amplified DNA was evaluated by agarose gel electrophoresis .", "Genotyping primers for Iap5-1 allelic variants and the Y-linked Sry gene are listed in Supplementary file 1 .", "The PCR primer pairs P1 , P2 , and P3 were designed to amplify the 5′ half , 3′ half , and the entirety of IAP-Pgm2 prior to Sanger sequencing , respectively ( Figure 2A , Supplementary file 1 ) .", "PCR amplification was carried out using the Expand Long Template PCR System ( Roche ) with the following thermocycler conditions: ( 1 ) 94°C for 2 min; ( 2 ) 94°C for 10 s , optimised T°C for 30 s , 68°C for 6 min , 10 cycles; ( 3 ) 94°C for 15 s , optimised T°C for 30 s , 68°C for 6 min + 20 s each successive cycle , 20 cycles; ( 4 ) 68°C for 7 min .", "Nested PCRs with primer pairs N1 , N2 , and N3 were performed using HotStarTaq DNA Polymerase ( QIAGEN ) and a 1:20 dilution of the first PCR products as templates ( Figure 2A , Supplementary file 1 ) .", "Conditions for the nested PCRs were as follows: ( 1 ) 95°C for 3 min; ( 2 ) 94°C for 30 s , optimised T°C for 30 s , 72°C for 55 s , 40 cycles; ( 3 ) 72°C for 5 min .", "Nested PCR products were purified by gel extraction using the QIAquick Gel Extraction Kit ( QIAGEN ) according to the manufacturer’s instructions and DNA was eluted in ddH2O .", "Sanger sequencing was carried out by Source BioScience .", "Sequencing primers were interspersed regularly across the IAP element ( Supplementary file 1 ) .", "Sequence traces were visually examined , and reliable sequences were merged using the EMBOSS merger tool .", "The resulting sequences for the two Iap5-1 alleles were aligned to the GRCm38/mm10 reference sequence using CLC Sequence Viewer 6 .", "The base-resolution sequence for the Iap5-1solo allele has been uploaded to GenBank ( accession number: MW308129 ) .", "Bisulphite conversions were carried out using the two-step modification procedure of the Imprint DNA Modification Kit ( Sigma-Aldrich ) according to the manufacturer’s instructions .", "1 µg gDNA was used per conversion with the exception of the oocyte and blastocyst experiments .", "PyroMark Assay Design SW 2 . 0 software ( QIAGEN ) was used to design the pyrosequencing assays ( primers listed in Supplementary file 1 ) .", "Target regions were PCR-amplified in technical triplicates from bilsulphite-converted DNA using a biotinylated forward or reverse primer and HotStarTaq DNA Polymerase ( QIAGEN ) .", "PCR conditions were as follows: ( 1 ) 95°C for 3 min; ( 2 ) 94°C for 30 s , optimised T°C for 30 s , 72°C for 55 s , 40 cycles; ( 3 ) 72°C for 5 min .", "For low-input pyrosequencing of oocyte and blastocyst DNA , two rounds of PCRs were performed: the first PCR used non-biotinylated primers and 20 amplification cycles ( other conditions remained the same ) ; the second PCR used 1 µl of the product from the first PCR as template as well as a biotinylated forward or reverse primer .", "Following PCR , Streptavidin Sepharose High Performance beads ( GE healthcare ) were bound to the product in binding buffer ( 10 mM Tris-HCl pH 7 . 6 , 2 M NaCl , 1 mM EDTA , 0 . 1% Tween-20 ) at 1400 rpm for 5 min .", "The bead-bound biotinylated strands were washed consecutively in 70% ethanol , denaturation solution ( 0 . 2 M NaOH ) , and wash buffer ( 10 mM Tris-acetate , pH 7 . 6 ) using the PyroMark Q96 Vacuum Workstation ( QIAGEN ) .", "Purified DNA resuspended in annealing buffer ( 20 mM Tris-acetate pH 7 . 6 , 2 mM magnesium acetate ) was incubated with the sequencing primer at 85°C for 4 min .", "Pyrosequencing was performed on the PyroMark Q96 MD pyrosequencer ( QIAGEN ) with PyroMark Gold Q96 Reagents and HS Capillary Tips ( QIAGEN ) according to the manufacturer’s instructions .", "Per cent CpG methylation was calculated by Pyro Q-CpG 1 . 0 . 9 software ( Biotage ) using the ratio of C-to-T at each site .", "Technical triplicates were averaged , and samples were kept for subsequent analysis if the standard deviation of technical triplicates did not exceed 5% .", "Where indicated , methylation levels were averaged across CpGs for each individual at each locus .", "Bisulphite-converted liver DNA was amplified by PCR using the primers listed in Supplementary file 1 and HotStarTaq DNA Polymerase ( QIAGEN ) .", "PCR conditions were as follows: ( 1 ) 95°C for 10 min; ( 2 ) 94°C for 30 s , 60°C for 30 s , 72°C for 30 s , 40 cycles; ( 3 ) 72°C for 5 min .", "PCR products were purified by gel extraction using the QIAquick Gel Extraction Kit ( QIAGEN ) and cloned into the pGEM-T Easy vector ( Promega ) using Stellar Competent Cells ( Takara Bio ) .", "Sanger sequencing of individual clones was carried out by Source BioScience .", "QUMA software ( Kumaki et al . , 2008 ) was used to analyse the sequencing data and generate figures .", "ChIP was carried out as previously described with modifications for use on frozen tissue ( Imbeault et al . , 2017 ) .", "100 mg of manually powdered frozen liver tissue dissected from adult Iap5-1full and Iap5-1solo males was cross-linked in 1% formaldehyde for 10 min at room temperature ( RT ) and quenched in 250 mM Tris-HCl pH 8 for 10 min at RT on a rotating wheel .", "Quenched samples were washed twice in 1× PBS supplemented with EDTA-free protease inhibitor cocktail cOmplete ( Sigma Aldrich ) , flash frozen in liquid nitrogen , and stored at −80°C .", "Fixed liver cells were thawed on ice and sequentially lysed in the following buffers for 10 min at 4°C: LB1 buffer ( 50 mM HEPES-KOH pH 7 . 4 , 140 mM NaCl , 1 mM EDTA , 0 . 5 mM EGTA , 10% glycerol , 0 . 5% NP-40 , 0 . 25% Triton-X-100 , 1× EDTA-free cOmpleteTM; one wash ) , LB2 buffer ( 10 mM Tris-HCl pH 8 . 0 , 200 mM NaCl , 1 mM EDTA , 0 . 5 mM EGTA , 1× EDTA-free cOmpleteTM; one wash ) , and SDS shearing buffer ( 10 mM Tris-HCl pH 8 , 1 mM EDTA , 0 . 15% SDS , 1× EDTA-free cOmpleteTM; three washes ) .", "Samples were centrifuged at 1700 × g for 5 min at 4°C after every wash .", "The resulting chromatin was sonicated for eight cycles ( one cycle: 30 s on and 30 s off ) at 4°C using a Bioruptor .", "The sonicated lysate was cleared by centrifugation at maximum speed for 10 min at 4°C and 10% of the input for each ChIP was stored at −20°C .", "50 µl magnetic beads ( Protein G Dynabeads , Invitrogen ) were pre-blocked in fresh blocking buffer ( 0 . 5% BSA in PBS ) and incubated with 2 . 5 µl of polyclonal H3K9me3 antibody ( RRID:AB_2532132 , Active Motif ) or Rabbit IgG ( negative control ) for 4 hr at 4°C .", "Antibody-bound beads were washed twice in blocking buffer using a magnetic stand at 4°C .", "The cleared lysate was topped up to 1 ml SDS shearing buffer + 150 mM NaCl and 1% Triton-X-100 and incubated with the antibody-bound beads on a rotating wheel overnight at 4°C .", "Non-specifically bound proteins were removed with the following sequential washes at 4°C: low salt buffer ( 10 mM Tris-HCl pH 8 . 0 , 150 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 15% SDS , 1 mM PMSF; two washes ) , high salt buffer ( 10 mM Tris-HCl pH 8 . 0 , 500 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 15% SDS , 1 mM PMSF; one wash ) , LiCl buffer ( 10 mM Tris-HCl pH 8 . 0 , 1 mM EDTA , 0 . 5 mM EGTA , 250 mM LiCl , 1% NP40 , 1% Na- deoxycholate , 1 mM PMSF; one wash ) , and 10 mM Tris pH 8 . 0 ( one wash ) .", "ChIP samples were resuspended in elution buffer ( 10 mM Tris pH 8 . 0 , 1 mM EDTA , 1% SDS , 150 mM NaCl ) , treated with RNaseA at 37°C for 1 hr at 1100 rpm , and reverse cross-linked overnight at 65°C at 1100 rpm .", "The eluted samples were treated with Proteinase K and DNA was purified using the Monarch PCR and DNA Cleanup Kit ( NEB ) .", "20–30 mg of thymus , liver , cortex , or placenta tissues was homogenised using the MagNA Lyser ( Roche ) at 6000 × g for 40 s .", "Thymus and placenta tissues were selected due to the specific expression of 5830416l19Rik in the thymus and the high expression of Rell1 and Pgm2 in the placenta , as reported by NCBI ( Geer et al . , 2010 ) .", "Liver and cortex were randomly selected to increase the number of tested tissue types .", "Total RNA was extracted using the AllPrep DNA/RNA Mini Kit ( QIAGEN ) .", "Tissue DNA was digested on the RNeasy spin column membrane using the RNase-Free DNase Set ( QIAGEN ) and RNA integrity was confirmed by agarose gel electrophoresis .", "cDNA was synthesised from 5 µg RNA using random hexamer primers and the RevertAid H Minus First Strand cDNA Synthesis kit ( Thermo Scientific ) .", "Quantitative PCR ( qPCR ) primers were designed using Primer3 software ( Supplementary file 1 ) .", "Each reaction was carried out in technical triplicates with Brilliant III Ultra-Fast SYBR Green QPCR Master Mix ( Agilent ) on the LightCycler 480 Instrument ( Roche ) under the following conditions: ( 1 ) 95°C for 5 min; ( 2 ) 95°C for 10 s , 60°C for 10 s , 72°C for 10 s , 45 cycles; ( 3 ) melting curve analysis of 65–95°C .", "For RT-qPCR , minus RT and no template controls were run for each sample and each primer pair , respectively .", "Relative expression was normalised to Hprt1 and β-actin expression and calculated using the ΔCt method .", "For ChIP-qPCR , no template controls and 10% ChIP input were run alongside the H3K9me3 and Rabbit IgG ChIP samples for each primer pair .", "Enrichment was calculated as per cent input .", "ChIP-seq data sets were downloaded in Bigwig format from the GEO database and visualised in the Integrative Genomics Viewer using the NCBI37/mm9 mouse reference genome ( Thorvaldsdóttir et al . , 2013 ) .", "The three KAP1 ChIP-seq biological replicate tracks were summed in IGV before importing into Adobe Illustrator CC 2020 v24 . 0 for figure design .", "GEO accession numbers are listed in Supplementary file 1 .", "B6 and CAST DNA sequences at the Iap5-1 insertion site were extracted from the GRCm38/mm10 and CAST_EiJ_v1 assemblies , respectively , accessed through the UCSC genome browser .", "The sequence alignment was generated using CLUSTAL OMEGA .", "All statistical tests in this study were carried out using GraphPad Prism 8 software as indicated in the figure legends ." ] ]
[ "Intracisternal A-particles ( IAPs ) are endogenous retroviruses ( ERVs ) responsible for most insertional mutations in the mouse .", "Full-length IAPs harbour genes flanked by long terminal repeats ( LTRs ) .", "Here , we identify a solo LTR IAP variant ( Iap5-1solo ) recently formed in the inbred C57BL/6J mouse strain .", "In contrast to the C57BL/6J full-length IAP at this locus ( Iap5-1full ) , Iap5-1solo lacks DNA methylation and H3K9 trimethylation .", "The distinct DNA methylation levels between the two alleles are established during preimplantation development , likely due to loss of KRAB zinc finger protein binding at the Iap5-1solo variant .", "Iap5-1solo methylation increases and becomes more variable in a hybrid genetic background yet is unresponsive to maternal dietary methyl supplementation .", "Differential epigenetic modification of the two variants is associated with metabolic differences and tissue-specific changes in adjacent gene expression .", "Our characterisation of Iap5-1 as a genetically induced epiallele with functional consequences establishes a new model to study transposable element repression and host-element co-evolution ." ]
[ "Our genome provides a complete set of genetic instructions for life .", "It begins by directing the growth and development of the embryo , and subsequently supports all the cells of the adult body in their daily routines .", "Yet approximately 10% of the DNA in mammalian genomes is made up of sequences originating from past retroviral infections , leaving a calling card in our genetic code .", "While these segments of retroviral DNA can no longer produce new infectious viruses , some of them retain the ability to copy themselves and jump into new parts of the genome .", "This can be problematic if they jump into and disrupt an important piece of genetic code .", "To protect against this , our bodies have evolved the ability to chemically strap down retroviral sequences by adding methyl groups to them and by modifying the proteins they are wrapped around .", "However , some of these endogenous retroviruses can dodge such so-called epigenetic modifications and disrupt genome function as a result .", "Studying a population of widely used inbred laboratory mice , Bertozzi et al . have identified a retroviral element that evades these epigenetic restraints .", "They discovered that some mice carry a full-length retroviral sequence while others have a shortened version of the same element .", "The shorter sequence lacked the repressive epigenetic marks found on the longer version , and this affected the expression of nearby genes .", "Moreover , the repressive marks could be partially restored by breeding the short-version mice with a distantly related mouse strain .", "Bertozzi et al . highlight an important issue for research using mouse models .", "Inbred laboratory mouse strains are assumed to have a fixed genetic code which allows scientists to conclude that any observed differences in their experiments are not a product of background genetic variation .", "However , this study emphasizes that this assumption is not guaranteed , and that hidden genetic diversity may be present in ostensibly genetically identical mice , with important implications for experimental outcomes .", "In addition , Bertozzi et al . provide a new mouse model for researchers to study the evolution and regulation of retroviral sequences and the impact of these processes on cell function ." ]
2021
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "developmental biology", "neuroscience" ]
Slow oscillation-spindle coupling predicts enhanced memory formation from childhood to adolescence
elife-53730-v1
[ [ "Active system memory consolidation theory proposes that sleep-dependent memory consolidation is orchestrated by three cardinal sleep oscillations ( Diekelmann and Born , 2010; Helfrich et al . , 2019; Klinzing et al . , 2019; Mölle et al . , 2011; Piantoni et al . , 2013; Rasch and Born , 2013; Staresina et al . , 2015 ) : ( 1 ) Hippocampal sharp-wave ripples represent the neuronal substrate of memory reactivation ( Vaz et al . , 2019; Wilson and McNaughton , 1994; Zhang et al . , 2018 ) , ( 2 ) thalamo-cortical sleep spindles are thought to promote long-term potentiation ( Antony et al . , 2018; De Gennaro and Ferrara , 2003; Rosanova and Ulrich , 2005; Schönauer , 2018; Schönauer and Pöhlchen , 2018 ) , while ( 3 ) neocortical SOs provide temporal reference frames where memory can be replayed , potentiated and eventually transferred from the short-term storage in the hippocampus to the long-term storage in the neocortex , rendering memories increasingly more stable ( Chauvette et al . , 2012; Diekelmann and Born , 2010; Frankland and Bontempi , 2005; Rasch and Born , 2013 ) .", "Importantly , these three oscillations form a temporal hierarchy , where ripples and spindles are nested in SO peaks , with ripples also being locked to spindle troughs .", "This hierarchy likely constitutes an endogenous timing mechanism to ensure that the neocortical system is in an optimal state to consolidate new hippocampus-dependent memories ( Chauvette et al . , 2012; Clemens et al . , 2011; Helfrich et al . , 2019; Klinzing et al . , 2016; Klinzing et al . , 2019; Latchoumane et al . , 2017; Niethard et al . , 2018; Piantoni et al . , 2013; Staresina et al . , 2015 ) .", "Recent findings indicate that the precise temporal coordination of SO-spindle coupling is deteriorating over the lifespan , which contributes to age-related memory decline ( Helfrich et al . , 2018b; Muehlroth et al . , 2019; Winer et al . , 2019 ) .", "It is currently unclear if similar principles apply to brain maturation and how the dynamic interplay of SOs and spindles is initiated .", "Critically , the transition from childhood to adolescence is marked by considerable changes in sleep architecture and cognitive abilities similar to the transition from young adulthood to old age ( Carskadon et al . , 2004; Huber and Born , 2014; Iglowstein et al . , 2003; Ohayon et al . , 2004; Shaw , 2007; Shaw et al . , 2006 ) .", "Previous research mainly focused on the individual development of SOs and sleep spindles across brain maturation , showing that these cardinal sleep oscillations undergo a substantial evolution in their defining features such as amplitude , frequency , distribution and occurrence ( Campbell and Feinberg , 2009; Campbell and Feinberg , 2016; Goldstone et al . , 2019; Hahn et al . , 2019; Kurth et al . , 2010; Nicolas et al . , 2001; Purcell et al . , 2017; Shinomiya et al . , 1999; Tarokh and Carskadon , 2010 ) .", "Currently , two major obstacles hamper our understanding of how the precise temporal interplay between SOs and spindles predicts brain development and memory formation .", "First , pronounced changes in sleep oscillatory activity pose major methodological challenges for assessing and comparing SOs and sleep spindles across the age spectrum ( Muehlroth and Werkle-Bergner , 2020 ) .", "Second , memory performance was rarely tested in developmental sleep studies , thus , impeding our understanding of the functional significance of temporal SO-spindle coupling for memory formation .", "Here , we leverage a unique longitudinal study design from childhood to adolescence to investigate how SO-spindle coupling emerges during development and infer its functional significance for developing memory networks .", "To account for the substantial morphological alterations of SO and spindle morphology across brain maturation , we developed a principled methodological approach to assess SO-spindle coupling .", "We utilized individualized cross-frequency coupling analyses , which enable a clear demonstration of SO-sleep spindles coupling during both developmental stages .", "Critically , over the course of brain maturation from childhood to adolescence , more spindles are tightly coupled to SOs , which directly predicts improved memory formation ." ], [ "To investigate whether SO-spindle coupling accounts for enhanced memory formation from childhood to adolescence , we first assessed the oscillatory signatures of NREM ( 2 and 3 ) sleep .", "We compared spectral estimates during childhood and adolescence using cluster-based permutation tests ( Maris and Oostenveld , 2007 ) across frequencies from 0 . 1 to 20 Hz ( Figure 2A; at electrode Cz ) .", "We found that EEG power significantly decreased from childhood to adolescence between 0 . 1 to 13 . 6 Hz ( cluster test: p<0 . 001 , d = −2 . 74 ) and 14 . 6 to 20 Hz ( cluster test: p<0 . 001 , d = −1 . 60; Figure 2A ) .", "However , inspection of the underlying spectra revealed that this effect was driven by ( I ) an overall offset of the 1/f component of the power spectrum on the y-axis and ( II ) by a shift of the peak frequency in the spindle band .", "In order to mitigate the prominent power difference , we first z-normalized the signal in the time domain , which alleviated the differences above ~15 Hz ( Figure 2B ) .", "This analysis showed increased spectral power during childhood from 0 . 3 to 8 . 4 Hz ( cluster test: p=0 . 002 , d = −084 ) , which was broadband and not oscillatory in nature .", "In addition power differences between 10 . 6 to 12 . 8 Hz ( cluster test: p=0 . 040 , d = −1 . 07 ) and 13 . 4 and 14 . 8 Hz ( cluster test: p=0 . 046 , d = 1 . 12 ) directly reflected the spindle peak frequency shift from childhood to adolescence .", "To account for the differences in broadband 1/f and oscillatory components , we disentangled the 1/f fractal component ( Figure 2C ) from the oscillatory residual ( Figure 2D ) by means of irregular-resampling auto-spectral analysis ( IRASA Helfrich et al . , 2018b; Wen and Liu , 2016 ) .", "We found that a significant decrease in the fractal component between 0 . 3 and 10 . 8 Hz from childhood to adolescence ( Figure 2C; cluster test: p<0 . 013 , d = −0 . 90 ) , accounted for the prominent broadband power differences as observed in Figure 2A .", "To assess true oscillatory brain activity , we subtracted the fractal component ( Figure 2C ) from the normalized power spectrum ( Figure 2B ) , to isolate SO and spindle oscillations in the frequency domain ( Figure 2D ) .", "Based on the oscillatory residuals , we then extracted the individual peak frequency and the corresponding amplitude in the SO and sleep spindle range for each electrode in every participant during childhood and adolescence .", "After discounting 1/f effects , we found that spindle amplitude ( Figure 2E ) increased in a centro-parietal cluster ( cluster test: p=0 . 005 , d = 0 . 63 ) , whereas spindle peak frequency ( Figure 2F ) accelerated at all channels from childhood to adolescence ( cluster test: p<0 . 001 , d = 1 . 57 ) .", "SO amplitude and frequency decreased from childhood to adolescence ( Figure 2—figure supplement 1A , B ) .", "Both , SO and spindle features have been previously related to memory formation ( Gais et al . , 2002; Huber et al . , 2004; Lustenberger et al . , 2017; Schabus et al . , 2004; Schabus et al . , 2006 ) .", "However , neither spindle nor SO amplitude or peak frequency changes explained the behavioral differences ( Figure 2—figure supplement 1C , D ) .", "Note , we also observed a peak in the theta band , which was unrelated to behavior ( for theta peak frequency and amplitude correlations with behavior see Figure 2—figure supplement 1E ) .", "After having established the cardinal features of SO and spindle oscillations during childhood and adolescence , we then individually adjusted previously used SO and spindle detection algorithms ( Helfrich et al . , 2018b; Mölle et al . , 2011; Staresina et al . , 2015 ) according to the individual peak frequencies ( Bódizs et al . , 2009; Ujma et al . , 2015 ) .", "We considered the possibility that two distinct spindle frequency peaks exist ( Anderer et al . , 2001; Werth et al . , 1997 ) , but inspecting the oscillatory residuals did not indicate two clearly discernable peaks in individual electrodes of the majority of participants ( for exemplary oscillatory residuals see Figure 2—figure supplement 2A ) .", "Because we observed the typical antero-posterior spindle frequency gradient ( Cox et al . , 2017; De Gennaro and Ferrara , 2003; Zeitlhofer et al . , 1997 ) with slower frontal and faster posterior spindles ( Figure 2—figure supplement 2B ) , we used the highest peak in the spindle range at every electrode as the most representative oscillatory event for the detection algorithm .", "Importantly , individualized SO and spindle event detections closely followed spectral sleep patterns during childhood and adolescence ( Figure 3A , B; event detections are superimposed in white ) .", "Next , we quantified how many separate SO and spindle event detections co-occurred within a 2 . 5 s time window ( reflecting ±2 SO cycles around the spindle peak; Helfrich et al . , 2019 ) .", "Note that the co-occurrence rate does not actually indicate coupled SO-spindle events but directly reflects the percentage of detected spindle events that are concomitant with detected SO events .", "Co-occurrence rate was higher in NREM3 than NREM2 sleep during childhood and adolescence ( Figure 3C; F1 , 32 = 2334 . 19 , p<0 . 001 , η2 = 0 . 99 ) .", "Subsequently we restricted our analyses to NREM3 sleep to avoid spurious cross-frequency coupling estimates caused by the lack of simultaneous detections during NREM2 sleep ( Aru et al . , 2015; for circular plots including NREM2 sleep see Figure 4—figure supplement 1D ) .", "To ensure reliable coupling estimates , we further Z-normalized individual spindle-locked data epochs in the time domain ( Figure 3D ) for all subsequent analyses to avoid possible confounding amplitude differences ( Aru et al . , 2015; Cole and Voytek , 2017; Helfrich et al . , 2018b ) .", "Differences in the grand average spindle time-lock directly reflect the enhanced SO-spindle coupling , which becomes visible in the time domain when more events are precisely coupled to the SO ‘up-state’ ( positive SO-peak ) .", "This effect can also be appreciated in single subject spindle-locked data ( Figure 3E , F , left ) .", "Note that we found no differences in the underlying SO-component around the spindle peak ( 0 s , time point of phase readout ) , thus , confirming that the Z-normalization alleviated possible amplitude differences ( Figure 3D , inset; for non-normalized spindle-locked data see Figure 3—figure supplement 1 ) .", "To further elucidate the interaction between SO phase and spindle activity , we also assessed this effect in the time-frequency domain by calculating SO-trough-locked time-frequency representations ( Figure 3G , H , left ) .", "The alternating pattern ( i . e . spindle power decreases during the ‘down-state’ and increases during the ‘up-state’ ) within the spindle frequency range during childhood and adolescence indicated an influence of SO phase on spindle activity .", "To quantify the interplay of SO and spindle oscillations , we employed event-locked cross-frequency analyses ( Dvorak and Fenton , 2014; Helfrich et al . , 2018b; Staresina et al . , 2015 ) .", "While this method is mainly equivalent to other frequently used methods to assess cross-frequency coupling ( Helfrich et al . , 2018a ) , it can be similarly impacted by their pitfalls ( Aru et al . , 2015 ) .", "Therefore , we adopted a conservative approach by first alleviating power differences ( Figure 2B and Figure 3D ) and establishing the presence of oscillations in the signal ( Figure 2D and Figure 3C ) .", "Next , we extracted the instantaneous SO phase during every spindle peak at every electrode and for every subject .", "Then we calculated the preferred phase ( circular mean direction ) and coupling strength ( phase-locking value , plv ) separately during childhood and adolescence for all events at a given electrode ( see Figure 3E , F right for exemplary phase histograms ) .", "We confirmed that both markers of SO-spindle coupling were not confounded by differences in the total number of detected events using a bootstrapping procedure ( Figure 4—figure supplement 1C ) .", "First , we assessed in which phase of the SO spindles preferably occur and how maturation affects the preferred coupling direction .", "We found that coupling direction did not change at frontal electrodes ( Figure 3G , H , right ) , where spindles were locked to the SO peak during childhood ( 33 . 5°±44 . 8°; circular mean ± SD ) and adolescence ( 34 . 2°±35 . 8°; circular mean ± SD ) .", "However , we detected differences in centro-posterior clusters ( central: p<0 . 001 , d = −0 . 84 , parieto-occipital: p<0 . 001 , d = 0 . 74; Figure 4—figure supplement 1A ) , which did not correlate with behavior ( Figure 4—figure supplement 1B ) .", "After showing that sleep spindles are preferably locked to the SO peaks , we subsequently quantified how precisely spindles are embedded in the preferred SO phase by assessing the respective coupling strength ( 1 – circular variance ) .", "Coupling strength increased from childhood to adolescence across all electrodes except P4 ( cluster test: p<0 . 001 , d = 0 . 74; Figure 4A ) , indicating that frontal sleep spindles become more tightly locked to their respective preferred SO phase as the phase on frontal sensors remained stable across maturation .", "To further illustrate the impact of the coupling strength increase on spindle-SO-events , we calculated the percentage of spindle events that peaked at the preferred phase ( ±22 . 5° ) as compared to all detected events ( Figure 4B , left ) .", "The resulting metric is directly related to the coupling strength and was solely computed to further illustrate and highlight the observed effects .", "Concurrent with our coupling strength analyses , percentage of spindles within the preferred phase bin increased in a fronto-parietal cluster ( p<0 . 001 d=0 . 74; Figure 4B , right ) however , decreased in an occipital cluster from childhood to adolescence ( p=0 . 007 , d = 0 . 78 ) .", "These results reveal an overall increase in SO-spindle coupling precision from childhood to adolescence .", "After having established SO-spindle coupling properties and demonstrating their qualitative enhancement from childhood to adolescence , we next tested the hypothesis that these changes also predict maturational differences in recall performance and sleep-dependent memory consolidation .", "We utilized cluster-based correlation analyses to relate differences in coupling strength to differences in recall performance ( delayed recalladolescence – delayed recallchildhood ) .", "We observed a significant frontal cluster ( p=0 . 0050 , mean rho = 0 . 48; Figure 4C; left ) showing that a stronger increase in SO-spindle coupling strength from childhood to adolescence related to improved recall performance from childhood to adolescence .", "This relationship was most pronounced at electrode F3 ( rho = 0 . 57 , p<0 . 001; Figure 4C; right ) .", "Notably using a non-individualized approach with a fixed frequency band ( 11–15 Hz ) attenuated the test statistic ( rho = 0 . 50 , p=0 . 035 , cluster-corrected; Figure 4—figure supplement 1E ) .", "Next , we only considered spindle events that co-occur with detected SO-events to ( 1 ) ensure more precise coupling metrics by guaranteeing the presence of oscillations and ( 2 ) to follow the notion that the temporal proximity of these events is crucial for sleep-dependent memory consolidation ( Diekelmann and Born , 2010; Helfrich et al . , 2018b; Muehlroth et al . , 2019; Rasch and Born , 2013 ) .", "Participants with a stronger coupling strength increase also showed enhanced sleep-dependent memory consolidation ( delayed recall - immediate recall ) from childhood to adolescence ( rho = 0 . 54 , p=0 . 0011; Figure 4D , electrode F3 ) .", "To validate whether the SO-spindle coupling strength predicts sleep specific memory formation , we correlated coupling strength during adolescence with the difference in memory consolidation between the sleep and wake condition ( [ ( delayed recallsleep –immediate recallsleep ) - ( delayed recallwake –immediate recallwake ) ] ) .", "However , we could not find evidence for this relationship ( Figure 4—figure supplement 1F ) .", "Previously , we and others reported that spindle density correlates with memory formation ( Gais et al . , 2002; Hahn et al . , 2019 ) .", "In order to test whether spindle densities potentially confound metrics of cross-frequency coupling , we first correlated the two but found no significant correlation ( rhochildhood = 0 . 08 , p=0 . 664; rhoadolescence = −0 . 115 , p=0 . 400; rhoadolescence-childhood = −0 . 02 , p=0 . 894 ) .", "Next , we partialed out the influence of spindle density on the original coupling strength-correlations , which left the significant correlation unchanged ( Recall Performance: p . rho = 0 . 57 , p<0 . 001; Sleep-dependent memory consolidation: p . rho = 0 . 54 , p=0 . 0013 ) .", "Finally , we directly compared the strengths of the dependent correlations ( coupling to memory vs . spindle density to memory formation ) using a percentile bootstrap approach ( Wilcox , 2016 ) and found that behavioral memory metrics were significantly better predicted by coupling strength than spindle density correlations ( Recall performance: Z = 2 . 20 , p=0 . 028 , CI95 = [0 . 048 0 . 99]; Sleep-dependent memory consolidation: Z = 2 . 17 , p=0 . 030; CI95 = [0 . 040 1 . 018] ) .", "Furthermore , all reported correlations were not driven by demographic parameters or differences in immediate recall scores as shown by partial correlations ( correlation of coupling strength and recall performance with the following confounding variables: p . rhoage difference = 0 . 63 , p<0 . 001; p . rhopubertal stage = 0 . 58 , p<0 . 001 , p . rhoIQ = 0 . 56 , p<0 . 001; p . rhoimmediate recall = 0 . 46 , p=0 . 009; correlation of coupling strength and sleep-dependent memory consolidation: p . rhoage difference = 0 . 54 , p=0 . 001 , p . rhopubertal stage = 0 . 54 , p=0 . 001 , p . rhoIQ = 0 . 55 , p=0 . 001 , p . rhoimmediate recall = 0 . 53 , p=0 . 002 ) .", "We also confirmed that non-individualized preferred phase and coupling strength values did not predict sleep-dependent memory consolidation enhancements ( Figure 4—figure supplement 1D , E; for how analytical choices impacted the correlations see Figure 4—figure supplement 1G ) ." ], [ "In a longitudinal sleep and memory study , we show that SO and spindles become more precisely coupled during brain maturation from childhood to adolescence .", "Crucially , this increase indicated improved recall performance and sleep-dependent consolidation in a declarative memory task .", "Collectively , our findings suggest that the emergence of precise temporal SO-spindle coordination might index the development of hippocampal-neocortical memory systems .", "Here , we employed an individualized cross-frequency coupling approach , since profound changes in the network organization during development were apparent , which potentially confound cross-frequency coupling estimates ( Aru et al . , 2015; Helfrich et al . , 2018b ) : In particular , we observed ( 1 ) changes in non-oscillatory 1/f background activity ( Figure 2C ) and ( 2 ) low frequency power ( Figure 2D and Figure 2—figure supplement 1A , B ) as well as ( 3 ) a peak frequency shift in the spindle-band ( Figure 2E , F ) .", "While some SO and spindle activity markers , such as amplitude and density have been previously associated with memory consolidation ( Gais et al . , 2002; Huber et al . , 2004; Schabus et al . , 2004; Schabus et al . , 2006 ) , our findings now could provide a mechanistic explanation how improved network coordination subserves memory formation .", "Furthermore , our results highlight the critical need to account for individual oscillatory features and to discount non-oscillatory broadband effects , which are known to confound cross-frequency coupling analyses ( Figure 4—figure supplement 1D , E , G; Aru et al . , 2015; Bódizs et al . , 2009; Cole and Voytek , 2017; Ujma et al . , 2015; Voytek et al . , 2015 ) .", "While our findings replicate the pattern that parietal spindles are faster than frontal spindles , we did not find reliable evidence for the presence of two distinct spindle peaks in individual electrodes ( Figure 2—figure supplement 2 ) .", "Importantly , SO-spindle coupling is one of the main building blocks of the active system memory consolidation theory , which posits that sleep-dependent memory consolidation is coordinated by a temporal hierarchy of SO , sleep spindles and hippocampal ripples ( Clemens et al . , 2011; Diekelmann and Born , 2010; Helfrich et al . , 2019; Helfrich et al . , 2018b; Klinzing et al . , 2016; Klinzing et al . , 2019; Mölle et al . , 2002; Mölle et al . , 2006; Muehlroth et al . , 2019; Niethard et al . , 2018; Rasch and Born , 2013; Staresina et al . , 2015 ) .", "Recently , it has been shown , that only SO coupled spindles , not isolated spindles trigger hippocampal-neocortical information transfer , suggesting that SO-spindle coupling might be a proxy of this subcortical-cortical network communication that is measurable on the scalp level ( Helfrich et al . , 2019 ) .", "While we did not measure hippocampal activity in the current study , our results still provide direct evidence for the behavioral relevance of the temporal hierarchy of SO and sleep spindles .", "We demonstrate that improved temporal coordination between prefrontal SOs and sleep spindles indexes memory network maturation .", "Notably , we observed that the precise coupling phase over frontal EEG sensors was already determined during childhood , but over time even more spindles became precisely locked to the preferred SO phase ( ~0°; SO ‘up-state’: Figure 3G , H & Figure 4—figure supplement 1A ) .", "This might indicate that SO-spindle coupling is an inherent feature of the human memory system .", "Previous research has emphasized the importance of a preferred phase closer to the SO up-state ( Helfrich et al . , 2018b; Muehlroth et al . , 2019; Winer et al . , 2019 ) .", "However , those studies focused on the detrimental impact of aging on memory consolidation .", "Now our findings reveal that mechanisms of maturation and aging are different .", "While maturation reduces the circular variance by increasing the coupling strength , ( Figure 4A , B ) , aging leads to a temporal dispersion of the coupling away from the SO ‘up-state’ with almost constant circular variance ( Helfrich et al . , 2018b; Muehlroth et al . , 2019 ) .", "In both instances , only changes in frontal cross-frequency coupling were predictive of behavior , which is in line with the hypothesized origin of SOs in the medial prefrontal cortex ( Massimini et al . , 2004; Murphy et al . , 2009 ) .", "Critically , precise prefrontal SO-spindle coupling governs over hippocampal-neocortical network communication and actively initiates the information transfer within the network ( Helfrich et al . , 2019 ) .", "Therefore , our results indicate that brain maturation leads to more fluent communication within memory networks .", "A multitude of studies suggested a general positive effect of sleep on memory consolidation ( Backhaus et al . , 2008; Diekelmann and Born , 2010; Huber et al . , 2004; Klinzing et al . , 2019 ) .", "Accordingly , our data provide additional support for this consideration by showing that consolidation is superior after sleep retention than after a wake period during adolescence ( Figure 1—figure supplement 1C ) .", "In the present study , SO-spindle coupling strength did not predict sleep specific memory improvements ( Figure 4—figure supplement 1F ) .", "Two likely explanations possibly contributed to this observation: ( 1 ) Performance in the word pair task was close to ceiling level in the adolescent group .", "Considering , that most evidence for a relationship between SO-spindle coupling and memory formation stems from studies comparing across relatively wide age ranges ( Helfrich et al . , 2018b; Muehlroth et al . , 2019 ) , an overall high performance level could lead to less variance that might mitigate detecting a relationship between coupling strength and sleep specific memory benefits .", "( 2 ) Apart from memory consolidation , the coupling strength increase is also related to maturation of general cognitive abilities ( Figure 1C and Figure 4C; also see Hahn et al . , 2019 ) , which further complicates disentangling sleep-specific memory benefits since both processes exhibit a substantial overlap ( Hoedlmoser et al . , 2014; Schabus et al . , 2004; Schabus et al . , 2006 ) .", "Nonetheless , several recent findings demonstrated a causal relationship between SO-spindle coupling and memory formation .", "For example , ripple-triggered electrical stimulation ( Maingret et al . , 2016 ) or optogenetic manipulation boosted SO-spindle coupling and memory performance the next day in rodents ( Latchoumane et al . , 2017 ) .", "In humans , it has been shown that electrical stimulation at ~0 . 75 Hz induces both SO and spindle power as well as improved memory performance ( Marshall et al . , 2006 ) .", "However , recent replication attempts provided contradictory findings ( Bueno-Lopez et al . , 2019; Lafon et al . , 2017; Sahlem et al . , 2015 ) .", "Apart from electrical stimulation , slow rocking motions ( Perrault et al . , 2019 ) and auditory stimulation ( Ngo et al . , 2013 ) might be effective in entraining SOs and concomitant spindles to improve memory performance .", "Furthermore , targeted memory reactivation triggers SO-spindle coupling , which enabled successful decoding of mnemonic information ( Antony et al . , 2018; Antony et al . , 2019; Cairney et al . , 2018; Schönauer , 2018; Schönauer et al . , 2017 ) .", "Taken together , several lines of research converge on the notion that precise SO-spindle coupling constitutes to a key mechanism for memory formation .", "This is of immediate relevance for future studies , since it has been shown that for example tau pathology in the medial temporal lobe , a precursor of Alzheimer’s disease , also impairs prefrontal SO-spindle coupling ( Winer et al . , 2019 ) .", "Thus , cross-frequency coupling might potentially constitute a novel pathway to understand age- or disease-related cognitive decline , amendable to intervention .", "In future , it might be possible to entrain SO-spindle synchrony through electrical ( Lustenberger et al . , 2016 ) or auditory stimulation ( Ngo et al . , 2013 ) to alleviate memory deficits ( for a recent review see Hanslmayr et al . , 2019 ) ." ], [ "Initially , 63 subjects ( mean ± SD age , 9 . 56 ± 0 . 76 years; 28 female , 35 male ) were recruited during childhood from public elementary schools ( Hoedlmoser et al . , 2014 ) .", "Seven years later , 36 healthy subjects agreed to participate again in the current follow-up study .", "Two participants were excluded because of technical issues during polysomnography ( PSG ) .", "One participant was excluded because of insufficient amount of NREM3 sleep ( 2 . 63% ) .", "All analyses are based on 33 healthy participants ( 23 female ) during childhood ( mean ± SD age , 9 . 5 ± 0 . 8 years ) and during adolescence ( mean ± SD age , 16 ± 0 . 9 years ) .", "Participants and their legal custodian provided written informed consent before entering the study .", "The study protocol was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the University of Salzburg ( EK-GZ:16/2014 ) .", "All participants were screened for possible sleep disorders using established questionnaires at both time points ( Children’s Sleep Habits Questionnaire [Owens et al . , 2000]; Pittsburgh Sleep Quality Index [Buysse et al . , 1989] ) .", "To determine the pubertal stage of the participants we used the Pubertal Development Scale ( Carskadon and Acebo , 1993 ) .", "Cognitive abilities were assessed by the Wechsler intelligence Scale for Children ( Petermann and Petermann , 2007 ) and the Wechsler Adult Intelligence Scale ( Wechsler , 1997 ) .", "Participants maintained a regular sleep rhythm during the study as verified by wrist actigraphy ( Cambridge Neurotechnology Actiwatch , Cambridge , UK ) and a sleep log ( Saletu et al . , 1987 ) .", "To guarantee a habitual sleep environment , full-night ambulatory polysomnography was recorded at the participants’ homes .", "Sleep was recorded during two nights during both childhood and adolescence .", "The first night was used for adaptation purposes .", "The experimental night served as retention interval for the word pair task .", "To satisfy the differences in sleep need during maturation , participants had a scheduled time in bed of 10 hr during childhood ( 8 . 30 pm – 6 . 30 am ) and 8 hr during adolescence ( 11 . 00 pm – 7 . 00 am ) .", "The word pair task at the experimental night consisted of word pair encoding followed by an immediate recall after a short delay ( 10 min ) in the evening and a delayed recall after a sleep retention interval in the morning ( Figure 1A ) .", "To confirm that sleep has a beneficial effect on memory , 31 participants performed the word pair task in a counterbalanced wake condition during adolescence .", "In the wake condition , participants encoded new word pairs at 8 . 00 am in the morning and recalled them after 10 hr of wakefulness ( Figure 1—figure supplement 1C ) .", "Participants performed a previously established declarative memory task ( Figure 1B ) , where they encoded and recalled non-associated word pairs ( Gais et al . , 2002; Hoedlmoser et al . , 2014; Schabus et al . , 2004; Schabus et al . , 2008 ) .", "To alleviate the impact of enhanced cognitive abilities across maturation on task performance , we adjusted word pair count and timing parameters in order to increase the difficulty during adolescence .", "Participants had to encode 50 pairs during childhood and 80 word pairs during adolescence .", "The word pair task was performed using Presentation software ( Version 18 . 2 , Neurobehavioral Systems , Inc , Berkeley , CA , www . neurobs . com ) .", "During childhood , each pair was visible for 5 s followed by a fixation cross for 3 s .", "During adolescence , each pair was presented for 6 . 5 s followed by a fixation cross for 3 . 5 s .", "Participants were advised to imagine a visual connection between the two words in order to control for different mnemonic strategies .", "All word pairs were presented twice in randomized order .", "During recall , only the first word of the word pair was presented .", "Participants had 10 s to recall the corresponding missing word during childhood and 6 . 5 s during adolescence .", "If the participants recalled the corresponding missing word , they had to press the mouse button and name the word .", "A button press or running out of recall time was followed by a fixation cross for 1 . 5 s during childhood and 3 . 5 s during adolescence as a reference interval .", "It was not allowed to name already disappeared word pairs .", "Participants received no feedback about their performance .", "Words were presented in randomized order in the immediate and delayed recall block .", "Ambulatory PSG was recorded with an Alphatrace , Becker Meditec ( Karlsruhe , Germany ) portable amplifier system using gold-plated electrodes ( Grass Technologies , AstroMed GmbH , Germany ) at a sampling rate of 512 Hz .", "Eleven EEG-electrodes were placed on the scalp according to standard 10–20 system .", "Two electromyogram electrodes were placed at left and right musculus mentalis .", "Two horizontal electrooculogram electrodes were placed above the right outer canthus and below the left outer canthus , with two additional vertical electrooculogram electrodes above and below the right eye as well as two electrodes placed on bilateral mastoids .", "The EEG signal was referenced online against Cz and re-referenced offline to a common average reference .", "For sleep staging , electrodes were re-referenced to contra lateral mastoids .", "Sleep stages were automatically scored in 30 s bins ( Somnolyzer 24 × 7 , Koninklijke Philips N . V . ; Eindhoven , The Netherlands ) and visually controlled by an expert scorer according to standard sleep staging criteria ( Iber et al . , 2007 ) .", "Recall performance ( Figure 2C ) was calculated as percentage by dividing the number of correctly recalled word pairs and semantically correct word pairs by the total count of word pairs .", "Semantically correct word pairs were weighted by 0 . 5 .", "A word pair was rated as semantically correct whenever the answer was unambiguously related to the correct answer ( e . g . ‘boot’ instead of ‘shoe’ ) .", "Recall performance development was subsequently calculated by subtracting delayed recall performance during childhood from the performance during adolescence .", "Sleep-dependent memory consolidation was calculated by subtracting immediate recall scores from delayed recall scores .", "The developmental change of sleep-dependent memory consolidation was calculated by subtracting values during childhood from values during adolescence .", "EEG data were visually inspected using BrainVision Analyzer 2 ( Brain Products GmbH , Germany ) .", "Artefactual activity was marked for every 5 s bin of continuous data for further processing in FieldTrip ( Oostenveld et al . , 2011 ) and EEGlab ( Delorme and Makeig , 2004 ) .", "Segments containing artifacts were rejected for all following analyses .", "Average power spectra from 0 . 1 to 30 Hz were calculated by means of a Fast Fourier Transform ( FFT ) after applying a Hanning window on continuous 15 s NREM sleep data ( i . e . NREM2 and NREM3; Figure 2A , B ) in 1 s sliding steps .", "All power values are log transformed .", "To mitigate power differences between childhood and adolescence ( Figure 2A ) we z-normalized the continuous signal on every channel in the time domain ( Figure 2B ) .", "To disentangle the 1/f fractal component from the true oscillatory components we applied irregular auto-spectral analysis ( IRASA , Wen and Liu , 2016 ) on the normalized data from 0 . 1 to 30 Hz in a sliding window of 15 s in 1 s steps .", "In brief , the EEG signal in each window is stretched by a non-integer resampling factor ( rf; e . g . 1 . 1 ) and subsequently compressed by a corresponding rf* ( e . g . 0 . 9 ) .", "Resampling was repeated with factors from 1 . 1 to 1 . 9 in 0 . 05 steps whereby the corresponding rf* is calculated by 2-rf .", "This resampling causes peak shifts of the oscillatory components in the frequency domain .", "The 1/f component of the signal however remains unchanged .", "Because resampling is done in a pair-wise fashion , median averaging across resampled FFT segments extracts the fractal 1/f component power spectrum ( Figure 2C ) by averaging out oscillatory components .", "Finally , to obtain the true oscillatory power spectrum ( Figure 2D ) we subtracted the extracted fractal component power spectrum from the mixed ( i . e . containing fractal and oscillatory parts ) power spectrum in the semi-log space ( Figure 2B ) .", "Based on the oscillatory power spectrum we detected slow oscillation ( <2 Hz ) and sleep spindle ( 10–18 Hz ) frequency peaks ( Figure 2E , F ) with their corresponding amplitude .", "Note however , that the extracted amplitude was 1/f corrected .", "We repeated this step for every subject during both time points in both nights on every channel in order to obtain individual SO and sleep spindle frequency peaks .", "These values were used for individualized event detections of SOs and sleep spindles in the subject-time-night-channel domain .", "We also considered the possibility of two distinct spindle frequency peaks .", "However , most of the participants only expressed a single frequency peak in the spindle range ( Figure 2—figure supplement 2A ) even though we also observed the expected fronto-posterior frequency gradient ( Figure 2—figure supplement 2B ) .", "Therefore , we only considered the highest peak in the power spectrum after discounting the fractal component as the most representative spindle frequency at the corresponding electrode .", "We devised individualized event detection on every channel separately by adjusting previously established algorithms ( Helfrich et al . , 2018b; Mölle et al . , 2011; Staresina et al . , 2015 ) based on the obtained individual SO and spindle features described above .", "For SO detection , the continuous EEG signal was high-pass filtered at 0 . 16 Hz and subsequently low-pass filtered at 2 Hz .", "Next , we detected all zero-crossings of the filtered signal .", "A zero-crossing was considered a slow oscillation if it fulfilled the time criterion ( length 0 . 8–2 s ) and exceeded the 75% percentile threshold of the amplitude .", "Valid slow oscillation epochs were extracted ±2 . 5 s around the trough of artifact free data .", "For sleep spindle detection , we bandpass filtered the continuous signal ±2 Hz around the individual spindle peak frequency .", "Next , we extracted the instantaneous amplitude by a Hilbert transform and subsequently smoothed the signal with a 200 ms moving average .", "We considered a valid spindle event if the signal exceeded the 75% percentile threshold of the amplitude for 0 . 5 to 3 s .", "Sleep spindle epochs were extracted ±2 . 5 s around the peak of artifact free data .", "To alleviate the prominent power differences ( Figure 2A ) we further z-normalized all detected SO and spindle events within each subject for subsequent analyses ( Figure 3D and Figure 3—figure supplement 1 ) .", "Spindle density was computed as the mean spindle number per 30 s NREM3 epoch .", "For the full-night time-frequency representations ( Figure 3A , B ) , we first segmented the data in 30 s epochs with an 85% overlap .", "Then we applied a multi-taper spectral analysis using 29 discrete prolate slepian sequences ( dpss ) tapers with a frequency smoothing of ±0 . 5 Hz from 0 . 5 to 30 Hz in 0 . 5 Hz steps ( Mitra and Pesaran , 1999; Prerau et al . , 2017 ) .", "To obtain the co-occurrence rate of detected sleep spindles and slow oscillations ( Figure 3C ) , we quantified how many SO events coincided with a sleep spindle peak within a 2 . 5 s time interval and subsequently divided the measure by overall detected spindle events .", "We chose the 2 . 5 s time window to capture ±2 SO cycles around the spindle peak ( Helfrich et al . , 2019 ) .", "Because of the low co-occurrence rate during NREM2 we conducted all following analyses on NREM3 event detections only .", "For event-locked time-frequency representations ( Figure 3G , H , left ) , we first applied a 500 ms Hanning window on normalized SO-trough locked segments ( −2 to 2 s , in 50 ms steps ) .", "Frequency power was analyzed from 5 to 30 Hz in 0 . 5 Hz steps .", "Next , we baseline corrected the spectral estimates by constructing a bootstrapped baseline distribution based on the −2 to −1 . 5 epoch of all trials ( 10000 iterations ) .", "All values were subsequently z-transformed relative to the obtained means and standard deviations of the bootstrapped distribution .", "To assess event-locked cross-frequency coupling ( Dvorak and Fenton , 2014; Helfrich et al . , 2018b; Staresina et al . , 2015 ) we focused our analyses on NREM3 sleep given that we observed a higher co-occurrence of sleep spindles and slow oscillations during this sleep stage ( Figure 3C ) .", "Based on the individualized and normalized spindle peak-locked epochs , we first low-pass filtered the time-locked data by 2 Hz and subsequently extracted the phase angle of the SO-component corresponding to the spindle amplitude peak using a Hilbert transform .", "To obtain the mean direction of the phase angles ( preferred phase , Figure 3E–H ) and coupling strength ( i . e . phase-locking value , Figure 4A ) of all NREM3 events , we used the CircStat Toolbox functions circ_mean and circ_r .", "However , there are no repeated measures statistical tests for circular data .", "Therefore , to allow for repeated measure testing , we calculated the absolute circular distance ( circ_dist ) of the individual preferred phase to the SO peak at 0° ( ‘up-state’ ) , where spindles are preferentially locked to Mölle et al . , 2011; Mölle et al . , 2002; Staresina et al . , 2015 .", "This transformation rendered our circular variable linear .", "The spindles within preferred phase metric ( Figure 4B ) was calculated by dividing the number of spindles within ±22 . 5° of the preferred phase ( i . e . 45° radius ) by the total number of spindle events .", "To control for differences in event number , we used bootstrapping by randomly drawing 500 spindle events 100 times and recalculating the mean direction and coupling strength ( Figure 4—figure supplement 1C ) .", "We calculated two-factorial repeated measure ANOVAs to assess the differences in recall performance between childhood and adolescence ( Figure 1C ) and to investigate the co-occurrence of sleep spindle- and SO-events during NREM2 and NREM3 sleep ( Figure 3C ) .", "We used cluster-based random permutation testing ( Maris and Oostenveld , 2007 ) to correct for multiple comparison ( Monte-Carlo method , cluster alpha 0 . 05 , max size criterion , 1000 iterations , critical alpha level 0 . 05 two-sided ) .", "We clustered the data in the frequency ( Figure 2A–C ) , space ( Figure 2E , F Figure 4A–C , Figure 2—figure supplement 1 , Figure 4—figure supplement 1A , B , E ) and time domain ( Figure 3D ) .", "For correlational analyses we calculated spearman rank correlations ( Figure 4C , D , Figure 4—figure supplement 1B , E , F ) , circular-linear correlations ( Figure 4—figure supplement 1B , E middle column ) and circular-circular correlations ( as implemented in CircStat toolbox ( Berens , 2009; Figure 4—figure supplement 1C ) .", "For cluster-corrected correlations we transformed the correlation coefficients ( rho ) to t-values ( p<0 . 05 threshold ) .", "Watson-Williams-Test ( circular ANOVA ) was not appropriate for repeated measure designs , therefore we transformed circular measures ( phase ) to parametric values by calculating the absolute distance to 0° ( i . e . SO ‘up-state’ ) to allow for repeated measure testing ( Figure 4—figure supplement 1A , topographical plot ) .", "Partial eta squared ( η2 ) , Cohen’s d ( d ) and spearman correlation coefficients ( rho ) are reported for effect sizes .", "We estimated cluster effect size by calculating the effect size for every data point within the significant cluster ( in frequency , space and time ) separately , followed by averaging across all obtained effect sizes .", "To compare correlations , we used a percentile bootstrap method for overlapping correlations ( Wilcox , 2016 ) .", "In brief we randomly drew participants with replacement , while keeping the dependency between the observation pairs of the correlations .", "Next we calculated two spearman correlation coefficients and their difference .", "This step was repeated 1000 times .", "Based on the resulting distribution we computed the 95% confidence interval ( CI95 ) of the difference and p-value .", "Data were analyzed with MatLab 2015a ( Mathworks Inc ) , utilizing functions from the Fieldtrip toolbox ( Oostenveld et al . , 2011 ) , EEGlab toolbox ( Delorme and Makeig , 2004 ) and CircStat toolbox ( Berens , 2009 ) as well as custom written code .", "For filtering we used the EEGlab function eegfilt . m and the FieldTrip function ft_preprocessing . m .", "For time domain analyses we used ft_timelockanalysis . m .", "Frequency and time-frequency domain analyses were conducted with the ft_freqanalysis . m function .", "For irregular auto-spectral analysis ( IRASA Wen and Liu , 2016 ) we used code published in the original research paper .", "Cluster-based permutation tests were carried out with the ft_freqstatistics . m function .", "Circular statistics were computed with the CircStat functions circ_mean . m ( preferred phase ) , circ_sd . m ( circular standard deviation of the preferred phase ) , circ_r . m ( plv , coupling strength ) , circ_dist . m ( circular distance ) , circ_corrcc . m ( circular-circular correlation ) and circ_corrcl . m ( circular-linear correlation ) .", "Results were plotted using circ_plot . m ( circular plots , CircStat ) , topoplot . m ( topographical plots; EEGlab ) and MatLab functions ( plot . m , imagesc . m ) ." ] ]
[ "Precise temporal coordination of slow oscillations ( SO ) and sleep spindles is a fundamental mechanism of sleep-dependent memory consolidation .", "SO and spindle morphology changes considerably throughout development .", "Critically , it remains unknown how the precise temporal coordination of these two sleep oscillations develops during brain maturation and whether their synchronization indexes the development of memory networks .", "Here , we use a longitudinal study design spanning from childhood to adolescence , where participants underwent polysomnography and performed a declarative word-pair learning task .", "Performance on the memory task was better during adolescence .", "After disentangling oscillatory components from 1/f activity , we found frequency shifts within SO and spindle frequency bands .", "Consequently , we devised an individualized cross-frequency coupling approach , which demonstrates that SO-spindle coupling strength increases during maturation .", "Critically , this increase indicated enhanced memory formation from childhood to adolescence .", "Our results provide evidence that improved coordination between SOs and spindles indexes the development of sleep-dependent memory networks ." ]
[ "Sleep is essential for consolidating the memories that we made during the day .", "As we lie asleep , unconscious , our brain is busy processing the day’s memories , which travel through three parts of the brain before they are filed away .", "First , the hippocampus , the part of the brain that stores memories temporarily , replays the memories of the day .", "Then the reactivated memories pass through the thalamus , a central crossroads in the brain , so they can be embedded in the neocortex for long-term storage .", "Neuroscientists can eavesdrop on the brain at work , day or night , using a technique called EEG .", "Short for electroencephalogram , an EEG detects brain waves like the bursts of electrical activity known as sleep spindles and slower sleep waves called slow oscillations .", "These two brain wave patterns represent how the brain processes memories as people sleep – and it is all about timing .", "If the two patterns are running in sync , then the brain’s memory systems are thought to be communicating well and memories are more likely to be stored .", "But patterns of sleep spindles and slow oscillations change dramatically between childhood and adolescence .", "Memory consolidation also improves in those formative years .", "Still , it is not yet known if better synchronization between sleep spindles and slow oscillations explains how memory formation improves during this period; that is the going theory .", "To test it out , Hahn et al . completed a unique study examining how well a group of 33 children could store memories , and then again when the same group were teenagers .", "Both times , the group was asked to memorise and then recall a set of words before and after a full night’s sleep .", "Hahn et al . measured how much their memory recall improved and whether their brain wave patterns were in sync , looking for any changes between childhood and adolescence .", "This showed that children whose sleep spindles stacked better with their slow oscillations had improved memory formation once they became teenagers .", "This work highlights how communication between memory systems in the brain improves as children age , and so does memory .", "Moreover , it suggests that if disturbances were to be detected in patterns of sleep spindles and slow oscillations , there might be some problem with memory storage .", "It also points to brain stimulation as a possible treatment option for such problems in the future ." ]
2020
[ "Introduction", "Results", "Discussion", "Materials and methods" ]
[ "neuroscience" ]
Female mice ultrasonically interact with males during courtship displays
elife-06203-v1
[ [ "Vocalizations are a crucial part of courtship and mating in a wide variety of species ( Bradbury and Vehrencamp , 1998 ) .", "Gazelles roar during mating displays ( Blank et al . , 2014 ) , songbirds stimulate reproductive activity in females by singing ( Bentley et al . , 2000 ) , sac-winged bats produce complex serenades during sexual displays ( Behr and von Helversen , 2004 ) , and anurans phonotaxis towards ultrasonic courtship vocalizations ( Shen et al . , 2008 ) .", "In mice ( Mus musculus ) , several indirect observations suggest that ultrasonic vocalizations play a role in courtship .", "For example , USVs occur during mating ( Sewell , 1972; Hanson and Hurley , 2012 ) , urine from females but not males stimulates vocal production in males ( Nyby et al . , 1979; Musolf et al . , 2010 ) , female mice spend more time near vocalizing males than mute males ( Pomerantz et al . , 1983 ) , and female mice preferentially approach a speaker playing male vocalizations rather than a speaker playing background noise ( Musolf et al . , 2010 ) or whistle-like control sounds ( Hammerschmidt et al . , 2009 ) .", "When examining the ultrasonic vocal behavior of adult mice , most experimental paradigms use pairs of mice ( Bean et al . , 1981; Choi et al . , 2011; D'Amato and Moles , 2001; Hammerschmidt et al . , 2012b; Nunez et al . , 1978; Nyby et al . , 1976; Ogawa et al . , 2000; Pomerantz and Clemens , 1981; Rotschafer et al . , 2012; Scattoni et al . , 2008; Warburton et al . , 1989; White et al . , 1998 ) .", "However , mice are adaptable and opportunistic animals that thrive in diverse conditions .", "Mice can occupy large and sparsely populated territories , where dyadic interactions would be most common , or small , densely inhabited regions where multiple individuals would frequently interact ( Anderson , 1961; Berry and Bronson , 1992 ) .", "Portfors ( 2007 ) reported that USV rate and complexity increase when mice are housed in large , mixed sex groups .", "Moreover , mice living in large populations for extended periods exhibit social interactions that are affected by the dynamics of multiple group members ( Shemesh et al . , 2013 ) and complex dominance hierarchies ( Weissbrod et al . , 2013 ) .", "These interactions may have reproductive consequences that impact courtship by forcing females to choose between males ( Silk , 2007 ) .", "Therefore , examining the vocal repertoire of groups of mice housed together is a unique , untapped opportunity to examine USV production during complex social contexts .", "These conditions may provide insight into the role vocalizations play in mouse social behavior .", "Identifying the source of a mouse ultrasonic vocalization during social interactions between multiple potential vocalizers is challenging .", "When mice produce USVs , there are no visible movements associated with these signals ( Chabout et al . , 2012 ) and the vocalizations are inaudible to humans .", "Males are believed to produce the majority of vocalizations during male-female encounters .", "Previous reports have shown that female mice rarely vocalize in the presence of an anesthetized or devocalized male , and more often than not are completely silent ( Whitney et al . , 1973; Warburton et al . , 1989 ) .", "However , females have been reported to vocalize at high rates during same sex interactions ( Maggio and Whitney , 1985; Moles et al . , 2007 ) .", "Therefore , females may be vocalizing during encounters with males when animals are intact and freely behaving .", "To further complicate our ability to identify the source of a vocalization , these signals are similar across individuals , both within and between sexes ( Hammerschmidt et al . , 2012a ) .", "These complications require a technological advance to unmask the vocal contribution of each individual mouse .", "To investigate the role of ultrasonic vocalizations during social behavior , we developed a microphone-array-based system for localizing the source of USVs in socially interacting groups of mice .", "The system has three components: ( 1 ) estimating the likelihood that the vocalization was produced at any position in the cage , ( 2 ) automatically determining the spatial position of every mouse in the cage at the time of the vocalization , and ( 3 ) combining the positional information of the mice and sound source estimate to assign a vocalization to a specific mouse .", "First , we show that the system precisely and accurately assigns vocalization to mice .", "After determining the resolution of the system , we investigated the vocal behavior of groups of male and female mice during courtship .", "Using the array , we revealed that both sexes vocalize , contrary to the belief that courtship vocalizations are male-specific .", "Furthermore , we discovered a temporal correlation between male and female vocalizations .", "Vocal interactions were observed between all possible male-female pairs in the colony .", "Moreover , these vocal exchanges were predominantly detected during chases .", "Finally , females that vocally interacted with pursuing males had lower chase speeds than silent females , suggesting a role for these exchanges in communicating female receptivity ." ], [ "A four-channel ultrasonic microphone array based system was developed to identify vocalizing mice using a procedure modified from Zhang et al . ( 2008 ) .", "Video and audio data were synchronously recorded during experiments ( Figure 1A–B ) .", "Following the experiment , the video-based movement trajectory was determined for each subject ( Ohayon et al . , 2013 ) .", "Vocal signals were automatically extracted ( see ‘Materials and methods’; Figure 1B–C ) .", "To estimate the location of a vocal signal , each signal was partitioned into time-frequency snippets—filtered pieces of the signal 5 ms long and 2 kHz wide ( Figure 1D ) .", "For each snippet , a single location was found that best explained the different time delays observed between all possible microphone pairs ( Figure 1E–F ) .", "Multiple estimates from the same vocal signal were then averaged to estimate the location of the sound source ( Figure 1G ) . 10 . 7554/eLife . 06203 . 003Figure 1 . Illustration of sound-source localization procedure .", "( A ) Image shows the location of a mouse in the behavioral arena during one video frame .", "Microphone locations are indicated by numbered microphone symbols ( a circle with a tangent line segment ) .", "Yellow quadrilateral indicates the floor boundaries .", "( B ) Vocal signal recorded at the same time as the frame in panel A . The number of each signal corresponds to the microphone in A . Vertical lines indicate the start and end of the signal extracted by the audio segmentation software .", "( C ) Spectrograms of the signals in B . Numbers in upper-right corner indicate the corresponding microphone .", "In the fourth microphone spectrogram , the large green rectangle indicates the time- and frequency-bounding box determined by the audio segmentation software .", "( D ) Smaller rectangles indicate the ‘snippets’ calculated from the segment and the associated frequency contour .", "Small red rectangles indicate snippets that were eventually discarded ( see below ) .", "Small magenta rectangle is the snippet highlighted in panel E . ( E ) Correlation coefficient maps determined from each microphone pair .", "In each map , the color represents the correlation coefficient between the two microphone signals once each is time-shifted appropriately for that position .", "Thus , deep blue/red points represent likely/unlikely source locations , given the information just in this snippet , for just this microphone pair .", "Plus symbol ( + ) represents the source location eventually estimated from this individual snippet ( see below ) .", "Mouse icon represents mouse location .", "Inset is an enlargement of the area indicated in the upper left map , to show closely intercalated red and blue bands .", "Map boundaries correspond to the floor outline indicated in A . ( F ) Reduced steered response power ( RSRP ) map for the example snippet .", "Plus symbol ( + ) represents the location estimate for this snippet , and corresponds to the highest ( positive ) value in the map .", "Black boundary corresponds to the floor outline in A , and microphone locations are indicated by numbered microphone symbols .", "( G ) Consensus estimate from all snippets .", "Plus symbols ( + ) and open circles represent single-snippet estimates from all snippets for this segment .", "Open circles are those snippets determined to be outliers , and non-outlier snippets are pluses .", "Closed circle indicates the mean of the non-outlier estimates .", "Gray shading is the probability density of a Gaussian distribution with the mean and covariance matrix of the non-outlier estimates .", "Mouse probability index value that the vocalization came from the actual mouse was determined to be approximately 1 , and from three randomly located virtual mice ( gray mouse icons ) were 10−11 , 10−56 , and 10−75 .", "To generate three virtual mouse positions , we picked three random points within the floor of the cage .", "Black boundary corresponds to the floor outline in A , and microphone locations are indicated by numbered microphone symbols . DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 003 Determining the vocal contribution of a specific mouse when recording from groups of animals requires a systematic way to assign the vocal signal to an individual .", "To achieve this , we developed a method that combined the positions of the mice at the time of the signal with the likelihood that the signal was emitted at any given position in the cage .", "Since every vocal signal used multiple estimates to determine the location of the sound source , we were able to calculate the probability density across the cage .", "Each mouse was assigned a density from the probability density function depending on the position of the mouse at the time of the vocal signal .", "Based on the assumption that a vocal signal was emitted from one of the recorded mice , a mouse probability index ( MPI ) was calculated for each mouse using the following formula:MPIn=Dn∑i=1MDi , where n = mouse index and M = total number of mice .", "Consequently , we were able to calculate the likelihood that any animal in the arena emitted the vocal signal by combining information from the microphone array and the mouse position trajectories .", "The accuracy and resolution of the system was evaluated by recording female-urine-elicited USVs from individual male mice .", "This approach ensured that all vocal signals were from an identified source and that the position of the source was known at the time of every vocal signal .", "In six recording sessions , we detected a total of 3724 vocal signals , of which 2590 were localizable .", "Localization was restricted to signals that contained at least three snippets , which provided the minimum amount of data to calculate the probability density function across the cage .", "We then created a simulated social environment by randomly generating the positions of three virtual mice at the time of each vocal signal .", "The locations of the virtual mice were confined to an area within the boundaries of the cage .", "Using this technique directly allowed us to quantify the accuracy and precision of the system under tightly controlled conditions .", "The level of certainty associated with assigning a vocal signal to a mouse is related to the positions of the mice and the location of estimated sound source .", "Since the MPI describes the relative likelihood that a mouse emitted the vocal signal , the animal closest to the estimated sound source should in theory have the largest MPI .", "To examine this relationship , we compared each real and virtual animal’s MPI with the error between the estimated sound source location and the animal .", "Error was defined as the distance between the estimated sound source location and the true source position .", "Figure 2A shows a negative correlation between MPI and error ( r = −0 . 66; p < 10−5 ) , indicating that mice closer to the estimated sound source location have higher MPI values . 10 . 7554/eLife . 06203 . 004Figure 2 . Accuracy and precision of sound-source localization system .", "( A ) Heat map shows inverse relationship between MPI and the distance between the mouse and estimated sound source location ( r = -0 . 66; p < 10-5 ) .", "The heat map is plotted in log counts with red indicating the maximum .", "Data for real and virtual mice are included in the plot .", "( B ) Left panel shows the MPI threshold plotted relative to the percent of localized signals that were assigned .", "Right panel indicates the percentage of accurately assigned vocal signals plotted as a function of MPI threshold .", "( C ) Distribution of the errors between the location of the estimated sound source and real source for assigned vocalizations .", "Median error is plotted in red .", "Inset shows the cumulative probability histogram of the errors .", "( D ) Distribution of distances between the mouse assigned the vocalization and the animal closest to the assigned mouse .", "( E ) Heat map showing sound source estimates ( n = 2590 ) relative to mouse position ( nose shifted to origin and rotated upwards ) .", "Gray sector shows mouse body .", "The large gray square , which is divided into smaller squares , indicates the regions of interest used to determine the precision of sound source localization system .", "Yellow key located at the bottom left indicates numbering scheme for smaller regions of interest .", "( F ) Bar plot showing the number of sound source localization estimates in each of the smaller regions of interest .", "Region 5 includes the head of the mouse .", "Red line shows the expected counts based on a uniform distribution .", "The distribution of predicted sound source locations was significantly different between regions ( χ0 . 05 , 82 = 2305 . 7 , p < 10−5 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 004 The accuracy of the system is directly related to the level of certainty associated with assigning a vocal signal to a mouse .", "To determine the effect of MPI magnitude on the proportion of localizable signals that were correctly assigned , we incorporated a threshold prior to assigning the vocal signal to either a real or virtual mouse .", "Figure 2B shows that increasing the MPI threshold increased the accuracy of the system , with a threshold of 0 . 95 causing the vocal signal to correctly be assigned to the real mouse 97 . 0% of the time .", "However , the MPI threshold was inversely related to the number of assigned vocalizations , indicating that the increase in accuracy came at the cost of a decrease in the number of assigned vocalizations ( Figure 2B; see ‘Materials and methods’ for additional details ) .", "The resolution of the system was evaluated on vocal signals that were assigned to individual mice after meeting our inclusion criteria .", "When determining the system's resolution , the median error between the location of the actual mouse and estimated sound source was 3 . 87 cm ( Figure 2C ) .", "34 . 8% of the vocal signals were localized within 3 cm of the mouse and 63 . 0% were localized within 5 cm ( Figure 2C; inset ) .", "When the heads of two mice are in close proximity , one would expect an increase in type-2 errors ( i . e . , assigning the vocalization to the incorrect or non-vocalizing mouse ) .", "We therefore examined the distance between the mouse assigned the vocalization ( mouse", "1 ) and the animal closest to the assigned mouse ( mouse 2 ) .", "Figure 2D shows that mouse 1 and mouse 2 were rarely close together ( <3 cm ) when assigning a vocalization .", "These results indicate that our system is precise and , of utmost importance , accurate when a vocalization is assigned to a mouse .", "Next , we examined the relative position of the estimated sound source locations and the actual mouse position .", "This was achieved by translating the coordinates of each sound source estimate and the position of the mouse into a mouse-centered reference frame .", "The results revealed that the estimated sound source locations were clustered around the head of the mouse ( Figure 2E–F ) .", "To quantify these results , we examined an area surrounding the real mouse that was 225 cm2 and centered on the mouse's nose .", "The large region of interest was then divided into 9 equivalently sized smaller regions ( 5 cm × 5 cm squares ) .", "Region 5 was centered on the mouse's nose and the other 8 regions were located along the periphery of this square ( Figure 2E ) .", "The distribution of predicted sound source locations was significantly different between regions ( Figure 2F; χ0 . 05 , 82 = 2305 . 7 , p < 10−5 ) .", "Based on a uniform distribution , there were more estimated sound source locations in region 5 than expected , whereas the peripheral regions had fewer .", "These results indicate that the system precisely and accurately estimates the location of the sound source .", "Ultrasonic vocalizations are emitted when two mice approach each other; however , the majority of vocalizations are produced when the animals are in close proximity ( Sewell , 1972; Portfors and Perkel , 2014 ) .", "To control for this behavioral phenomenon , we ran a second more restrictive simulation .", "Instead of using 3 virtual mice at random locations within the cage , the simulation randomly generated the position of a single virtual mouse that was located within a 10 cm radius of the true source .", "Since vocalizations occur across a range of relative distances between two mice , this control analysis will overestimate the error rate and provide a lower bound for the accuracy .", "When applying our inclusion criteria , the vocal signal was assigned to the real mouse 89 . 5% of the time and 40 . 4% of the localized signals were assigned .", "The decreased assignment rate indicates the conservative approach we used when assigning the signals to mice .", "Consequently , the system allows us to precisely distinguish the sex-specific vocal and physical contributions of each individual mouse during complex social interactions .", "The vocal contributions of male and female mice were examined in conditions that increase the complexity of vocalizations and social interactions .", "Groups of mice ( 7 groups each consisting of two males and two females ) were recorded for five continuous hours .", "Because group housing affects social behavior in males ( Jones and Nowell , 1989 ) and females ( König , 1994 ) , all mice were singly housed for at least 14 days before the experiment in an attempt to maximize the amount of social information exchanged after the animals were introduced to each other .", "Our system detected a total of 255 , 396 vocal signals , of which 199 , 288 were localized to a distinct position in the cage .", "The localized signals were further refined to 37 , 371 signals that could be unambiguously assigned to individual mice ( Figure 3 ) .", "Figure 3A shows an example of a signal assigned to one of the male mice that was spatially isolated from the other mice in the cage .", "The 47 . 1 ms signal was partitioned into 25 snippets and the estimated position of each snippet was located near the tip of the nose of the solitary animal , suggesting that the signal was emitted from that male mouse .", "Figure 3B–C shows additional examples of vocal signals that were assigned to a male mouse .", "Note that the nose of the vocalizing male in Figure 3C is in close proximity to the anogenital region of a female .", "Supporting previous work ( Sewell , 1972; Nyby , 1983; Chabout et al . , 2012 ) , we directly showed that male mice vocalize in the presence of freely moving female mice . 10 . 7554/eLife . 06203 . 005Figure 3 . Microphone array reveals both male and female mice vocalize during social interactions .", "( A–C )", "Examples of sound source assigned to a male mice in the presence of multiple mice .", "( D–F )", "Examples of sound source assigned to female mice when groups of mice are present .", "( G ) Male ( blue ) and female ( orange ) vocal signal counts during each recording session ( groups displayed in ascending order of total call number ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 005 Males were not the only sex emitting vocalizations .", "Female mice also vocalized when freely moving male mice were present ( Figure 3D–F ) .", "Figure 3D depicts a sound source localization example for a 17 . 6 ms vocal signal .", "The signal was partitioned into 8 snippets that were confined to the nose of a female mouse while the other three mice were positioned in remote locations in the cage .", "As shown in Figure 3F , vocal signals can be assigned to a female mouse when a male is positioned behind the female .", "When combining the data from all the recording sessions , we observed that females produced 18% of the assigned vocalizations ( Figure 3G ) , indicating that both males and females vocally contribute while in the presence of the opposite sex .", "A possibility exists that the sound source localization system introduces a selection bias because the majority of vocalizations are unassigned .", "To address this possibility , we examined whether any consistent differences were detected between assigned and unassigned vocalizations as a function of time or space ( Figure 4 ) .", "In all groups , both assigned and unassigned vocalizations occurred throughout the experiment ( Figure 4A ) .", "Moreover , the spatial distribution was similar for assigned and unassigned vocalizations ( Figure 4B ) .", "The evidence from these analyses demonstrates that there is no temporal or spatial bias in vocalization assignment .", "We next examined the relative positions of the two mice closest to the estimated sound source .", "For all vocal signals , two distances were calculated: ( 1 ) the distance between the estimated sound source position and the closest mouse and ( 2 ) the distance between the estimated sound source position and the next closest mouse .", "These two distances were plotted in relation to each other to create two distance distribution matrices , one for assigned and one for unassigned vocal signals .", "Next , a difference distribution was calculated ( assigned-unassigned ) .", "The results revealed that assigned vocalizations occurred predominantly when the estimated sound source is close to one mouse , whereas unassigned vocalizations occur when two mice are equidistant from the estimated sound source ( Figure 4C ) .", "This indicates that the major reason for excluding vocalizations was ambiguity in the source of the signal and highlights the effort taken to avoid incorrect assignments .", "With this system , we were able to follow the vocal and physical interactions of mice in large , mixed sex groups and precisely distinguish sex-specific contributions during social interactions . 10 . 7554/eLife . 06203 . 006Figure 4 . Potential sources of bias in vocalization assignment .", "( A ) Assigned ( red ) and unassigned ( gray ) vocalizations are plotted as a function of time .", "In all groups , assigned and unassigned vocalizations occur throughout the experiment .", "( B ) The estimated sound source position of assigned ( red ) and unassigned ( gray ) vocalizations is displayed .", "Both assigned and unassigned vocalizations are less likely to be detected in the corners , but there is no difference in the spatial distribution for the two categories .", "( C ) The relative distance between sound source and the closest mouse ( x-axis ) or the second closest mouse ( y-axis ) for all vocalizations .", "Relative distances were calculated separately for both assigned and unassigned vocalizations , a 2-dimensional distance matrix was determined for each category and normalized by peak count , and then the difference between the distance distribution matrix for assigned and unassigned vocalizations was plotted .", "Red represents a higher proportion of assigned vocalizations , whereas blue denotes a higher proportion of unassigned vocalizations .", "Vocal signals were unlikely to be assigned when the closest and second closest mouse were equidistant from the estimated sound source ( blue diagonal line ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 006 Since both sexes vocalize in mixed sex groups , we examined the timing between male and female vocalizations .", "To explore the temporal relationship between male and female vocalizations , we plotted the vocalizations of both males within +/− 30 s of each female vocalization ( Figure 5B ) .", "Figure 5C shows a similar analysis where the vocalizations of both females were plotted relative to each male vocalization .", "A striking temporal correlation was revealed—when male vocalizations occurred , the probability of female vocalizations peaked .", "Similarly , when female vocalizations occurred , the probability of male vocalizations peaked .", "We quantified this effect by calculating the average rate of vocalizations triggered by either sex ( Figure 5D–E; peak male vocalization rate was 0 . 78 Hz; one-way ANOVA , F59 , 360 = 2 . 04 , p < 5 × 10−5; peak female vocalization rate was 0 . 19 Hz; one-way ANOVA , F59 , 360 = 5 . 2 , p < 10−22 ) .", "On average , both sexes showed an increase in vocalization rate that was significantly higher than expected by chance ( random permutation test , p < 0 . 006 and p < 0 . 004 ) .", "Overall , we found that 24% of male vocalizations occurred within 1 s of a female vocalization , while 61% of female vocalizations occurred within 1 s of a male vocalization .", "This coordination of vocal behavior may be indicative of an exchange of social information between males and females . 10 . 7554/eLife . 06203 . 007Figure 5 . Male and female mice vocalize together .", "( A ) Example spectrogram of male and female USVs .", "( B ) Raster plots of male USVs plotted relative to female vocalizations ( each row is associated with a single female vocalization with onset at t = 0 s; female vocalizations = 6832; rows sorted by male vocalization rate ) .", "( C ) Female USVs plotted relative to male vocalizations ( male vocalizations = 30 , 395; rows sorted by female vocalization rate ) .", "( D–E )", "Plots of female-vocalization-triggered average male USV rate ( D ) and male-vocalization-triggered average female USV rate ( E ) .", "Colored lines represent group averages ( n = 7 ) with SEM ( shaded patch ) .", "Black lines represent group averages ( n = 1000 ) for randomly generated trigger times .", "Light gray patches show the range of the randomly generated group averages . DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 007 To determine whether this temporal coordination in vocalization was associated with a particular social behavior we identified instances in which a vocalization from a single male was followed within 1 s by one or more vocalizations from a single female .", "This pattern was called a vocal sequence and the vocalizing male and female were called the vocal pair .", "Because four mice were present in the cage , the other mice were called the non-vocal pair .", "For each pair , we measured the speed and distance between the male and female during a 60 s window centered on vocal sequence onset ( see Figure 6A for example trajectories ) .", "The speed of the vocal pair was significantly faster at the time of the vocal sequence than either before or after the event ( Figure 6B; before: 1–30 s , after: 2–30 s; one-way ANOVA , F1740 , 10 , 446 = 11 . 9 , p < 10−6 ) .", "Despite a slight peak , the speed of the non-vocal pair did not change significantly over time ( one-way ANOVA , F1740 , 10 , 446 = 1 . 0 , p > 0 . 9 ) .", "We believe this peak occurs because all four mice are in the same cage , and therefore the behavior of the vocal pair may affect the behavior of the non-vocal pair .", "Moreover , the members of a vocal pair were significantly closer to each other at the onset of the vocal sequence than either before or after the event ( Figure 6C; before: 30–3 . 2 s , after: 5–30 s; one-way ANOVA , F1740 , 10 , 446 = 15 . 5 , p < 10−6 ) .", "In contrast , the distance between non-vocal mice did not change significantly over time ( one-way ANOVA , F1740 , 10 , 446 = 0 . 4 , p > 0 . 99 ) .", "For the vocal pair , we examined the relative positions of the mice before , during , and after the vocal sequence ( −25 , 0 , and 25 s , respectively ) .", "At the time of the vocal sequence , the male positions were significantly more clustered behind the females than either before or after the event ( Figure 6D; female-centered: χ0 . 05 , 22 = 220 . 9 , p < 10−5 , Tukey-type multiple comparison , before , q = 15 . 1 , p < 0 . 001 , after , q = 16 . 9 , p < 0 . 001; male-centered: χ0 . 05 , 22 = 198 . 9 , p < 10−5 , Tukey-type multiple comparison , before , q = 15 . 1 , p < 0 . 001 , after , q = 14 . 4 , p < 0 . 001 ) .", "These analyses show that vocal interaction between a male and female can occur when the mice are in close proximity to each other , moving quickly , with the male positioned behind the female .", "This behavior strongly resembles a chase , a fundamental component of mouse courtship ( Van Oortmerssen , 1971 ) . 10 . 7554/eLife . 06203 . 008Figure 6 . Mice participating in a vocal sequence are close to each other .", "( A ) Example trajectories of vocal and non-vocal pairs of mice .", "( B ) Vocal-sequence-triggered averages show that vocal ( v ) mice are faster at the time of the initial vocalization than either before or after the event ( p < 10−6 ) .", "The speed of the non-vocal ( nv ) mice did not change significantly over time ( p > 0 . 99 ) .", "For each event , the instantaneous speeds ( ±30 s from trigger start time ) were averaged for the vocal or non-vocal pair of mice .", "Colored and black lines represent the average speed between vocal and non-vocal pairs , respectively .", "( C ) The vocal pair was significantly closer at vocal sequence onset than either before or after the event ( p < 10−6 ) .", "Average distance between non-vocal mice did not change significantly ( p > 0 . 99 ) .", "Red arrows denote the periods in panel D . For B and C , shaded patch indicates SEM across groups ( n = 7 ) and dashed vertical lines denote the time of initial trigger vocalizations .", "( D ) Heat maps of relative position of female ( top row ) and male ( bottom row ) mice preceding ( pre ) , during ( 0 ) , and following ( post ) a vocal sequence .", "Males are significantly clustered behind the females at vocal sequence onset compared to before or after ( p < 0 . 001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 008 Our previous behavioral analyses were based on epochs when the initial trigger vocalization was from a male; however , the behavior of the vocally interacting animals may be different when the female starts the sequence .", "To investigate this possibility , additional analyses were performed for vocal sequences initiated by females , revealing similar results .", "Vocally interacting animals moved the fastest at the time of the vocal sequence ( before: 30–0 . 9 s , after: 2 . 5–30 s; one-way ANOVA , F1740 , 10 , 446 = 14 . 3 , p < 10−6 ) .", "For the non-vocal pair , the speed remained relatively constant ( one-way ANOVA , F1740 , 10 , 446 = 0 . 7 , p > 0 . 99 ) .", "Furthermore , as in the male-initiated vocal sequences , the vocal pair was significantly closer to each other at the vocal sequence onset than either before or after the vocal exchange ( before: 30–3 . 2 s , after: 3 . 6–30 s; ANOVA , F1740 , 10 , 446 = 16 . 4 , p < 10−6 ) .", "On average , non-vocal mice remained 35 cm apart ( ANOVA , F1740 , 10 , 446 = 0 . 3 , p > 0 . 99 ) .", "The positions of the males were significantly more clustered behind the females at the time of the vocal sequence compared to before or after the event ( female-centered: χ0 . 05 , 22 = 135 . 0 , p < 10−5 , Tukey-type multiple comparison , 25 s before , q = 9 . 9 , p < 0 . 001 , 25 s after , q = 14 . 4 , p < 0 . 001; male-centered: χ0 . 05 , 22 = 178 . 1 , p < 10−5 , Tukey-type multiple comparison , 25 s before , q = 15 . 1 , p < 0 . 001 , 25 s after , q = 11 . 9 , p < 0 . 001 ) .", "These results indicate that both male and female initiated vocal sequences occur during similar behaviors .", "To identify when males were chasing females , we manually annotated a subset of chases and used this annotation to train an automatic classifier ( Kabra et al . ( 2013 ) ; see Materials and methods ) .", "In every cage , each male chased each female ( median = 113; interquartile range ( IQR ) = 162 . 5–35 ) .", "In addition , each male vocally interacted with each female ( median = 111; IQR = 166–77 . 5 ) .", "The relationship between chases and vocal interactions was then examined for all male and female pairs ( n = 28; Figure 7A ) .", "The proportion of chases performed by each male varied across groups .", "In some cases , only one of the males did the majority of the chasing ( e . g . , group 4 , red symbols ) .", "In other cases , both males chased equally ( e . g . , group 6 , pink symbols ) .", "Similarly , the amount each of the females was chased varied .", "Most of the time , one of the females in the group was chased more than the other ( e . g . , groups 1 , 3 , 4 , 6 , and 7; black , green , red , pink , and cyan symbols ) .", "In contrast , females in groups 2 and 5 were chased for similar amounts of time ( blue and gray symbols ) .", "Despite this variation , the number of male-initiated vocal interactions was strongly correlated with both total chase time ( Figure 7A , r = 0 . 74 , p < 10−5 ) and number of chases ( r = 0 . 55 , p < 0 . 003 ) .", "Given this correlation we asked whether there was a consistent temporal relationship between vocal interactions and chases .", "Vocal interactions that occurred within 30 s of a chase ( 1743/3075 ) were plotted in relation to the onset of the nearest chase ( Figure 7B ) .", "The rate of vocal interactions occurring inside a chase was significantly higher than outside the chase ( inside: median = 0 . 18 Hz , IQR = 0 . 61–0; outside: median = 0 . 02 Hz , IQR = 0 . 04–0; Mann–Whitney U-test , z = 9 . 74 , p < 10−21 ) , with 47 . 2% of these vocal interactions occurring during a chase ( 823/1743 ) .", "Analyses of female-initiated vocal interactions showed a similar pattern ( total chase time–vocal interaction correlation: n = 28; r = 0 . 80 , p < 10−6; vocal interaction and chase timing: inside: median = 0 . 16 , IQR = 0 . 50–0; outside: median = 0 . 02 , IQR = 0 . 02–0; Mann–Whitney U-test , z = 9 . 77 , p < 10−21 ) .", "Our observation that the vocal interaction rate increased during chases suggests that both males and females actively participate in courtship displays . 10 . 7554/eLife . 06203 . 009Figure 7 . Vocal interactions are associated with courtship .", "( A ) The number of male-vocalization-initiated vocal interactions and total chase time for a given male-female pair was strongly correlated ( n = 28; r = 0 . 74 , p < 10−5 ) .", "Trend line ( red line ) was calculated using linear regression .", "Circles and triangles represent male 1 and male 2 , respectively .", "Open and filled depict female 1 and female 2 , respectively .", "Colors indicate group numbers ( black = 1 , blue = 2 , green = 3 , red = 4 , gray = 5 , pink = 6 , and cyan = 7 ) .", "( B ) Plots of male and female USVs as a function of time relative to chase .", "Vocal interaction rate is higher during chases than outside chases for male-initiated vocal interactions ( p < 10−21 ) .", "( C ) Mouse speeds at the time of the male vocalization during chases with and without vocal interactions .", "Top , male speed ( p > 0 . 25 ) .", "Bottom , female speed ( p < 10−10 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 009 Chase-related vocal interactions occurred when animals were in close proximity .", "Therefore , a possibility exists that the female's vocal contribution to the interaction was actually a male vocalization incorrectly assigned to a female .", "To control for this possibility , we used the data from the single mouse recordings and simulated a close proximity social interaction by randomly generating a second virtual mouse .", "To determine the possible positions of the virtual mouse that reflected the relative positions of vocally interacting mice in the multiple mouse experiment , we calculated the distance between each vocally interacting male and female at the time of the female vocalization as well as the orientation of the female relative to the male .", "The distance and orientation distributions were then sampled with replacement to generate the position of the virtual mouse relative to the real mouse in the single mouse experiments .", "In this control simulation , vocal signals were assigned to the real mouse 93 . 0% of the time , which indicates a 7% error rate .", "A series of analytical calculations were performed to determine the number of expected female vocalizations and vocal interactions .", "In the 1743 vocal interactions that occurred within 30 s of a chase , there were a total of 3486 vocalizations emitted .", "If male mice were the only animals vocalizing and all female vocalizations were incorrectly assigned based on a 7% error rate , we would have only expected a total of 244 female vocalizations .", "Instead , 1743 vocalizations were assigned to the female , which is greater than 7 times the number of expected incorrectly assigned male vocalizations .", "This analysis not only suggests that female mice vocalize in the presence of males , but moreover , they are vocally interacting with the males during courtship .", "Because female mice vocally participate in courtship chases , we investigated the specific role of the female's vocalization during vocal interactions .", "To isolate the effect of the female vocalization , we compared chases in which both the male and female vocalized ( vocal interaction chases ) with those in which only the male vocalized ( male only chases ) .", "We used speed at the time of the male vocalization as a proxy for female receptivity ( Kowalski et al . , 2004 ) .", "When examining the speed of the males , we found no difference between the two chase types ( Figure 7C , top panel; vocal interaction: median = 28 cm/s , IQR = 0 . 45–0 . 17; non-vocal interaction: median = 31 cm/s , IQR = 0 . 45–0 . 20; Mann–Whitney U-test , z = 1 . 13 , p > 0 . 25 ) .", "In contrast , female speed was significantly slower during chases with vocal interactions than without vocal interactions ( Figure 7C , bottom panel; vocal interaction: median = 28 cm/s , IQR = 0 . 49–0 . 12; non-vocal interaction: median = 51 cm/s , IQR = 0 . 73–0 . 32; Mann–Whitney U-test , z = 6 . 73 , p < 10−10 ) .", "In addition , vocal interaction chases were on average longer in duration than non-vocal interaction chases ( vocal interaction: median = 3 . 28 s , IQR = 5 . 02–2 . 07; non-vocal interaction: median = 2 . 38 s , IQR = 3 . 72–1 . 22; Mann–Whitney U-test , z = 4 . 45 , p < 10−5 ) .", "Our results show that the female drives the overall reduction in speed during a chase; thus , female vocal interaction may function as a signal of receptivity ." ], [ "In this study , we used a novel microphone array system to reveal an unprecedented level of vocal exchange between male and female mice during courtship .", "In the discussion , we consider and expand upon the most salient components of social vocalizations that emerged from our investigations , focusing on the potential impact of the tool , the role of female vocalizations , the synchrony of male and female vocalizations , and the function of vocal interactions during courtship .", "Mice produce ultrasonic vocalizations in many social contexts including establishment of territory , resident–intruder interactions , pup care , juvenile play , social reunion , and mating ( Sales and Pye , 1974 ) .", "In all of these social contexts , the presence of multiple vocalizing mice has hindered our ability to determine the specific role vocalizations play in social behaviors .", "Assigning a vocalization to a particular mouse in a social group is difficult for two reasons .", "First , there is no obvious visual sign that a mouse is producing a USV ( Chabout et al . , 2012 ) .", "Second , although mice appear to produce characteristic vocal patterns or songs ( Holy and Guo , 2005; Sugimoto et al . , 2011; Hoffmann et al . , 2012 ) , individual ultrasonic vocalizations are similar across animals , both within and between sexes ( Hammerschmidt et al . , 2012a ) .", "Therefore , a vocalization's acoustic features are likely inadequate for identifying which mouse in a social group emitted the sound .", "Previous studies have addressed the difficulty in determining the source by assuming a single mouse produced the majority of the observed vocalizations ( Barthelemy et al . , 2004; Choi et al . , 2011; Hanson and Hurley , 2012 ) , or by experimentally preventing one mouse from vocalizing ( Whitney et al . , 1973; Warburton et al . , 1989 ) .", "Both conditions overlook how social interactions may be impacted by the vocal contribution of multiple individuals .", "More importantly , these conditions cannot detect vocal exchanges between individuals .", "Consequently , the ability to track the vocal behavior of each animal is crucial in determining the function of mouse USVs .", "To examine the vocal contribution of every mouse during a social interaction , we developed and employed a microphone array system .", "In the past , microphone arrays have been used to study the acoustic features of many species ( Payne et al . , 2003; Mennill et al . , 2006; Chiu et al . , 2010; Collier et al . , 2010; Kalcounis-Rueppell et al . , 2010 ) .", "Other systems capitalized on the power of an array to determine the number of animals hidden in dense terrain ( Celis-Murillo et al . , 2009; Blumstein et al . , 2011 ) or map the boundaries of an animal’s territory ( Kirschel et al . , 2011 ) , whereas our system was specifically designed to link , with high resolution and accuracy , the vocal and social behaviors of mice recorded in a laboratory environment .", "Group size influences the behavior of individual animals ( Fitzsimmons and Bertram , 2013; Shen et al . , 2014 ) .", "For example , in a variety of species , the full range of a male’s vocal repertoire only occurs when multiple males are present ( Rand and Ryan , 1981; Martinez-Rivera and Gerhardt , 2008; Charlton and Reby , 2011 ) .", "Moreover , recent work has shown that mouse vocal behavior ( Portfors , 2007 ) and patterns of social interactions ( Shemesh et al . , 2013; Weissbrod et al . , 2013 ) are significantly different when mice are housed in more complex social groups than in dyads .", "These studies emphasize the importance of capturing the full behavioral repertoire .", "In the present study using a more dynamic social environment of two males and two females , we show that all mice vocalize and vocal exchanges occur between all possible male-female pairs .", "These findings illustrate the utility of the microphone array for clarifying the function of vocalizations in mouse social behavior .", "One of the most remarkable discoveries from the study was that male and female mice coordinate their ultrasonic vocalizations during courtship .", "These results are surprising because most previous studies concluded that only male mice produce courtship USVs ( Whitney et al . , 1973; Warburton et al . , 1989; Barthelemy et al . , 2004 ) .", "However , male and female mice emit USVs with practically identical acoustic structure ( Hammerschmidt et al . , 2012a ) , leaving open the possibility that some courtship USVs attributed to males are actually produced by females .", "Moreover , it is known that female mice possess the neural circuitry necessary to emit USVs during interactions with males because surgical or genetic lesions to the vomeronasal organ unmask this behavior ( Kimchi et al . , 2007 ) .", "Our findings expand on this observation and show that the behavior can be triggered in unaltered females .", "Our observation that the majority of a female's vocalizations occur during vocal exchanges with males suggests that female vocal behavior may depend on the presence of a vocally competent partner .", "This in turn , may explain the absence of ultrasonic vocalizations when female mice are exposed to male odor ( Maggio and Whitney , 1985 ) or paired with an anesthetized ( Whitney et al . , 1973 ) or devocalized ( Warburton et al . , 1989 ) male .", "Taken together , these results suggest that information from multiple modalities is necessary to activate these social neural networks .", "For courtship displays , the behavior of both partners is critical .", "Female responsiveness modulates male mating behavior throughout the animal kingdom ( Coleman et al . , 2004; Higham et al . , 2009; Akre and Ryan , 2011 ) .", "For example , in Drosophila melanogaster , males modulate their song patterning in response to variation in sensory cues about the female's position and motion ( Coen et al . , 2014 ) .", "Without accounting for the behavior of the female , the male's song pattern appears random .", "A similar interdependence of behavior between partners occurs in the early stages of human courtship , where a female's non-verbal displays modulate a male's approach behavior ( Moore , 1985 ) .", "Interactions between males and females during courtship also occur in the vocal domain ( Janson , 1984; Tobias et al . , 1998; Garamszegi et al . , 2007 ) .", "Most studies have found that vocal behavior is either important for joint territorial defense or maintaining affiliation , particularly between mated pairs ( Hall , 2004 ) .", "The association between vocal interactions and courtship chases in this study implies that vocal exchanges between mice may be involved in affiliation .", "As for the role of female vocalizations in courtship , many believe a female's participation may serve as an indication of her receptivity ( Janson , 1984 ) or may encourage competition between males ( Montgomerie and Thornhill , 1989 ) .", "In rats ( Thomas and Barfield , 1985 ) and hamsters ( Floody et al . , 1977 ) , female vocal participation in courtship is modulated by estrous state and has been proposed to signal female receptivity to nearby males , also consistent with a role in affiliation .", "Our results strengthen the argument that female vocalizations are associated with receptivity in rodents , since female speed during a chase is significantly slower when she subsequently vocalized than when she was silent .", "This is akin to other species; receptive females slow down permitting the courting male to approach and mate ( McGill , 1962; Beach , 1976; Hall , 1994; Kowalski et al . , 2004; Szykman et al . , 2007 ) .", "Based on the behaviors of the animals that co-occur during these vocal exchanges , we believe that the female's vocal contribution may facilitate this bond by indicating her receptivity .", "In many species , social interactions are essential for reproductive success and survival ( Bradbury and Vehrencamp , 1998 ) .", "These interactions are supported by information exchange through a variety of sensory modalities ( Adolphs , 2010 ) .", "Transfer of social information occurs over a wide range of timescales , from short-term information about an individual's current motivational state ( Moles and D'Amato F , 2000 ) to longer-term information about dominance status ( Ficken et al . , 1987; Grosenick et al . , 2007 ) or fertility ( Leong et al . , 2003 ) .", "Capturing the details of this information exchange is critical to developing a mechanistic understanding of how social information supports motivated behavior , and also in illuminating deficits of social interaction , such as autism .", "The microphone array system described here allows unprecedented access to the details of social interactions in groups of freely behaving mice , revealing temporally precise and behaviorally meaningful vocal communication between males and females ." ], [ "Singly housed male mice ( C57Bl/6J; n = 3; 3–5 months ) were used to test the sound source localization system .", "For multiple mouse studies , two male and two female mice were used in each experiment ( C57Bl/6J; n = 28; 6-12 weeks ) .", "Mice were isolate housed for at least two weeks prior to the start of the recordings and maintained on a 12/12 dark–light cycle with ad libitum access to food and water .", "At least one week prior to the start of the recordings , mice were marked with distinctive patterns for identification by applying harmless hair bleach to the fur ( Ohayon et al . , 2013 ) .", "The patterns were two vertical lines , two horizontal lines , one diagonal slash , or five dots .", "Each of the four subjects randomly received one of the four patterns .", "In rodents , reports indicate that USVs are important for female receptivity during estrus ( Floody et al . , 1977; McIntosh et al . , 1978 ) and , therefore two hours prior to the anticipated start of the experiment , non-invasive lavage and cytological assessment of vaginal cells were performed .", "Briefly , cells were collected by washing with 20 μl of sterile saline , placed on a slide , stained with Wright Stain , and examined under a light microscope .", "As described by Karim et al . ( 2003 ) , estrous stage was calculated based on the proportion of cell types observed .", "Proestrus consisted of mostly nucleated basal epithelial cells .", "In estrus , most cells were cornified squamous epithelial cells that lacked a nucleus .", "During metestrus , cells were a mixture of neutrophils and cornified squamous epithelial cells .", "Diestrus consisted of mostly neutrophils .", "If both females were in late proestrus/early estrus recordings were conducted .", "Otherwise recordings were postponed and the procedure for determining estrus was repeated again the following day .", "Male bedding was introduced into the female cage one day before experiment onset to ensure that females were cycling .", "Experiments began at dark cycle onset and lasted for five hours .", "Each mouse was individually recorded for three minutes following the experiment .", "HHMI Janelia Research Campus Institutional Animal Care and Use Committee approved all experimental protocols .", "Audio data were captured with a 4-channel microphone array ( Avisoft-Bioacoustics , Glienicke , Germany; CM16/CMPA40-5V ) , amplified ( 40 dB ) , low-passed filtered ( 200 kHz; Krohn-Hite , Brockton , MA; Model 3384 ) , and digitized at 450450 Hz with a National Instruments board ( Austin , TX; PXIe-1073 , PXIe-6356 , BNC-2110 ) .", "Externally triggered video data were acquired at 29 Hz with a camera ( Basler , Ahrensburg , Germany; A622f ) and stored on a PC ( Dell , Round Rock , TX; T7500 ) with StreamPix software ( Norpix , Montreal , Canada; StreamPix 5 ) .", "To synchronize the audio and video data , a 29 Hz square wave pulse was emitted from a function generator ( Agilent Technologies , Santa Clara , CA; Model 33522A-002 ) .", "A BNC splitter was used to simultaneously send the output signal from the function generator to the camera and the National Instruments equipment .", "This signal was time stamped at the sampling rate of the audio recordings and triggered the camera .", "Custom software was used to control and synchronize the data and video acquisition equipment .", "Recordings were made in a mesh-walled ( McMaster-Carr , Robbinsville , NJ; Nylon ) cage , with a frame of extruded aluminum ( 8020 , Inc . ; width = 66 cm , length = 66 cm , height = 66 cm ) , and surrounded with Sonex foam ( Pinta Acoustic , Inc . , Minneapolis , MN; VLW-35 ) .", "The cage was illuminated with infrared lights ( Reytec imaging , East Setauket , NY; part # IR-LT30 ) and filled to a depth of ∼7 . 5 cm with alpha-dri bedding ( Shepherd Specialty Papers , Richland , MI ) .", "A ring of LEDs , which was visible through the mesh walls , surrounded each microphone and helped to determine the microphone positions .", "The position of each mouse was automatically tracked using the Motr tracker program ( Motr; Ohayon et al . ( 2013 ) ; http://motr . janelia . org ) .", "Motr fits an ellipse around each mouse and reports the x and y position of the ellipse centroid , the length of the ellipse's major and minor axis , and the heading direction of each mouse for every frame in the video .", "Vocal signals were automatically extracted from the four channels of auditory recording using multi-taper spectral analysis .", "After removing signals below 30 kHz , overlapping segments in time were Fourier transformed using multiple discrete prolate spheroidal sequences as windowing functions ( K = 43 , NW = 22 ) .", "An F-test ( Percival and Waldan , 1993 ) was used to infer whether each time-frequency point was significantly above noise based on these independent estimates of intensity ( p < 0 . 05 ) .", "This procedure was performed for multiple segment lengths on each microphone channel to capture data at different temporal and spectral scales ( NFFTs = 128 , 256 , 512 ) .", "The data were combined in a single spectrogram whose pixel size corresponded to the time resolution of the shortest segment and frequency resolution of the longest .", "This image was then convolved with a square box ( 15 pixels in time X 7 pixels in frequency ) to fill in small gaps before the locations of contiguous regions , which exceeded a minimum pixel number ( 1500 ) , were characterized .", "Because one or more animals could produce discontinuous vocal signals , each discontinuous signal was extracted separately unless harmonics were present .", "Overlapping signals were considered harmonics when the overlapped length exceeded 90% of the shortest signal , and the frequencies of 90% or more of the overlap were within 10% of a factor of two or three of each other .", "Each vocal signal was preprocessed to run in the mouse sound source estimation program .", "The preprocessing steps involved cutting each extracted signal into time-frequency ‘snippets’—filtered pieces of the signal 5 ms long and 2 kHz wide .", "Because video data were collected at 29 Hz ( roughly 35 ms ) , each frame was associated with 7 time bins of audio data .", "Each of time bins used the same mouse position , making the localization sampling rate dependent upon the video sampling rate , as opposed to a per snippet sampling rate of 200 Hz .", "To determine the optimal duration of snippets , localization error as a function of snippet duration was plotted ( Figure 8A ) .", "Localization error increased dramatically for snippet durations under approximately 4 ms . Consequently , we selected 5 ms for snippet durations to improve the accuracy of the system .", "To select a frequency bandwidth , we examined ‘hot pixels’—pixels in the combined spectrogram that pass the multi-taper F-test for being significantly louder than noise .", "Figure 8B shows that localization error exponentially decays as the numbers of hot pixels within a snippet increases .", "After applying a cutoff criterion of 11 hot pixels , a bandwidth of 2 kHz was selected because it produced more than three snippets for most vocalizations .", "Therefore , localization was restricted to snippets that were 5 ms long and contained at least 11 hot pixels .", "This allowed us to use multiple estimates to determine the source location .", "Cage boundaries and microphone positions were manually annotated using the images from the video recordings . 10 . 7554/eLife . 06203 . 010Figure 8 . Effects of acoustic structure on localization fidelity .", "( A ) Localization error as a function of sound duration .", "The heat map shows that signals shorter than 5 ms ( red vertical line ) produce large errors between the estimated sound source location and the true position of the mouse .", "( B ) Localization error as a function of hot pixel count ( pixels in the sound's spectrogram that are significantly above background ) .", "Localization error is variable when the hot pixel count is low , and becomes more accurate above 11 hot pixels ( red vertical line ) .", "Inset ( dashed magenta box ) shows zoomed in region of heat map highlighting the increase in localization error for snippets with fewer than 11 hot pixels .", "Heat maps were normalized by peak counts . DOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 010 Mouse sound source estimation was performed using a procedure similar to that of Zhang et al . ( 2008 ) .", "Vocal signals were recorded on an array of four microphones , and a single location was found that best explained the different time delays observed between the six possible combinations of microphone pairs .", "A grid of points was generated that covered the floor of the arena with a spacing of 0 . 25 mm , and then the likelihood that the source of a vocal signal could be at a given point on the grid ( rg; ‘g’ for guess ) was determined .", "At each point , we computed the distance ( dk ) from rg to microphone k , ( 1 ) dk=‖rg−rk‖ , where rk is the position of microphone k .", "The signal recorded at microphone k was assumed to be proportional to the true signal , but delayed by a time τk given by: ( 2 ) τk=dk/c , where c is the speed of sound in air at the ambient temperature and standard atmospheric pressure ( 760 mm Hg ) .", "We then calculated the steered response power ( SRP ) , given by: ( 3 ) SRP=∑i=1K∑k=1Krik ( ( τi−τk ) /Δt ) , where rik ( ⋅ ) is the cross-correlation between signals recorded at microphone i and microphone k , given by: ( 4 ) rik ( p ) =∑n=0N−1Vi[m]Vk⋆[m]exp ( j2πmp/N ) , where Vi[m] is the discrete Fourier transform of the voltage recorded at microphone i .", "The SRP is the sum of the cross-correlations between each ordered pair of microphones ( including self-pairs ) , each evaluated at the time difference that would occur if the source was at the hypothesized location .", "However , the self-terms in the SRP ( where i = k ) do not vary with position rg , and because , ( 5 ) rik ( ( τi−τk ) /Δt ) =rki ( ( τk−τi ) /Δt ) , it suffices to calculate the reduced SRP , or RSRP , given by: ( 6 ) RSRP=∑i=1K∑k<iKrik ( ( τi−τk ) /Δt ) .", "That is , the RSRP includes exactly one term per unordered pair , not including self-pairs .", "The RSRP is a function of position , because the hypothesized delays τi and τk depend upon the hypothesized position rg .", "We therefore evaluate the RSRP at each grid point rg , and our estimate of the source location is given by the rg that maximizes RSRP .", "We sped up the computation by pre-computing each rik ( p ) for the range of possible p values , and computing rik ( p ) at each rg by linear interpolation into these pre-computed arrays .", "The fine spacing of the grid ( 0 . 25 mm ) was used to enable accurate localization of the maximum of RSRP .", "Ultrasonic vocal signals have power up to about 130 kHz and at this frequency the wavelength of the sound waves is ∼2 . 6 mm; thus our spacing was approximately 10 times finer than the shortest wavelengths present in the signals .", "Mouse Ultrasonic Source Estimation software is available for download at https://github . com/JaneliaSciComp/Muse .", "To assign vocal signals to specific mice , we used the estimated source position in combination with the location of the mice at the time of the vocal signal and a confidence measure in the estimated source position .", "Location outliers were identified and removed , and the covariance matrix of the remaining snippets was calculated using the method of Peña and Prieto ( 2001 ) .", "The x and y coordinates of the non-outlier snippets were then averaged to determined the estimated x and y coordinates of the vocal signal ( i . e . , estimated source position ) .", "The covariance matrix and estimated source position were then used to generate a probability density function over the cage for the vocal signal .", "The density at each mouse was then calculated from the probability density function .", "Densities ( D ) were used to calculate a mouse probability index ( MPI ) for each mouse using the following formula:MPIn=Dn∑i=1MDi , where n = mouse index and M = total number of mice .", "Vocal signals were only assigned to a mouse if the MPI was greater than 0 . 95 and the density was greater than 1 m−2 .", "By setting the MPI threshold to 0 . 95 , vocal signals were not assigned to a mouse when multiple mice were in close proximity to the estimated source .", "A density threshold of 1 m−2 was used to prevent a poorly localized vocal signal from being assigned to a mouse far from the estimated source location .", "Because of the presence of multiple mice in the cage , overlapping vocalizations were observed .", "The majority of the time these overlapping vocalizations were unassigned ( either the mice were equidistant to estimated source or the snippets from the overlapping vocalizations did not tightly cluster ) .", "These vocalizations were excluded from analysis .", "Table 1 lists the vocal signals detected , localized and assigned for each data set . 10 . 7554/eLife . 06203 . 011Table 1 . Vocal Signals detected , localized , and assigned for each data setDOI: http://dx . doi . org/10 . 7554/eLife . 06203 . 011SessionSignalsLocalizedAssignedIndividual male 1Male 2Female 1Female 2Proportion assigned*123 , 29217 , 444211510214653642650 . 12225 , 94219 , 531311819035053563540 . 16326 , 39519 , 889369023457693592170 . 19431 , 37125 , 147420129574154973320 . 17535 , 27428 , 0756337442810544713840 . 23652 , 59740 , 4836416274722796507400 . 16760 , 52548 , 71911 , 4845608403810847540 . 24*Proportion assigned is based on number of localized signal .", "The temporal relationship between male and female vocalizations was examined by calculating the average male vocalization rate centered on the time of each female vocalization .", "For every female vocalization , all male vocalizations within 30 s were partitioned into 1-s bins ( 60 bins total ) .", "Each bin was summed and normalized by the number of female vocalizations .", "The combined response of each recording session was then averaged .", "Note that sex triggered vocalization averages cover a 60 s window around the trigger vocalizations .", "We excluded potential trigger vocalizations that occurred within 30 s from the beginning or end of the data record ( 141 male and 3 female vocalizations ) .", "To determine whether the male vocal rate was significantly higher than expected by chance , a comparison data set was generated for every recording session using a random permutation test .", "The times of the female vocalizations were randomly shuffled and used to calculate a shuffle-dependent male vocalization rate .", "This procedure removes any temporal correlation between the two sets of vocalizations while preserving both the temporal structure of the male vocalizations as well as the total number of vocalizations .", "The shuffle-dependent male vocalization rate for each group was then averaged to produce a mean shuffle-dependent vocalization rate .", "The random permutation process was repeated 1000 times to generate a distribution of average shuffle-dependent vocalization rates , which were used to determine the probability that the male vocalization rate was higher than expected by chance .", "For each of the 60 bins , the number of times that the actual data were above the shuffled data was calculated .", "These proportions were used with a binomial parameter estimate to determine significance .", "The actual data were considered significant at a conservative alpha <0 . 006 if at most one of the average shuffle-dependent vocalization rates was greater than the average from the actual data .", "To examine the average female vocalization rate , the previously described analyses were also performed using male vocalizations as the trigger .", "To determine whether the vocalization rate changed significantly over time , a 1-way ANOVA was performed .", "Tukey multiple comparison tests were used to determine when the vocalization rate was significantly different from the other means .", "We called a sequence of vocalizations from a single male-female pair a vocal sequence if the male and female vocalized within 1 s of each other .", "The male and female mice participating in the vocal sequence were the vocal pair , while the other two mice were considered the non-vocal pair .", "This analysis was repeated for female-male vocal sequences .", "The behavior of the vocalizing and non-vocalizing mice was then examined separately for both male- and female-vocalization initiated events .", "We excluded vocal sequences that occurred within 30 s of the beginning or end of the data record , which removed 40 vocal sequences initiated by male vocalizations and 17 vocal sequences initiated by female vocalizations from the analysis .", "All vocal sequences were used when calculating the correlation between number of vocal sequences and number or length of chases ( see details below ) .", "For a 60 s window surrounding the onset of each vocal sequence , the position of the four mice was extracted from the tracked video files .", "At each frame , the Euclidean distance was calculated between the vocally active mice .", "This procedure was repeated for the non-vocal mice in the same set of video frames .", "After determining the distances between the pair of vocal mice as well as the non-vocal mice for each vocal sequence , we calculated the average inter-mouse-distance for every recording session .", "The cumulative average for both the vocal and non-vocal mice was determined from the average for each of the seven groups .", "To determine whether the distance between the vocal pair of mice or the non-vocal pair of mice changed significantly over time , a 1-way ANOVA was performed .", "Tukey multiple comparison tests were used to determine when the distance was significantly different from the other means .", "The relative position of the mice during the vocal sequence was examined twenty-five seconds before and after and at the start of each sequence ( pre , post and during ) .", "For each time point , the position and orientation of either the male or female mouse were translated and rotated such that the midpoint of the mouse’s body was centered and the mouse was facing upwards .", "The position and orientation of the other mouse in the pair was translated and rotated by the same amount as its vocal partner .", "A 2D histogram was generated with the distribution of the relative positions of the vocal partner .", "The bin size for the x- and y-axes was 0 . 01 cm .", "A region of interest surrounding the midpoint of the centered mouse ( circle with radius of 10 cm ) was used to determine whether the distribution of relative positions differed between the anterior and posterior regions at each time point .", "For both the male- and female-centered relative positions , a chi-square analysis was performed on the proportion of anterior counts to total counts at the pre , during , and post time periods .", "Post-hoc Tukey-type multiple comparison tests were used to compare the three proportions for the male- or female-centered data .", "For a 60-s window surrounding the onset of each vocal sequence , the instantaneous speed of the four mice was calculated between subsequent frames .", "The speed of each of the vocalizing mice was averaged to determine the mean instantaneous speed , which was then averaged across a recording session for all vocal sequences .", "This was repeated for non-vocal animals .", "The group average for both the vocal and non-vocal mice was determined from the average for each of the seven recording sessions .", "To determine whether the speed of the vocal pair of mice or the non-vocal pair of mice changed significantly over time , a 1-way ANOVA was performed .", "Tukey multiple comparison tests were used to determine when the speed was significantly different form the other means .", "An automated classifier ( Janelia Automatic Animal Behavior Annotator; JAABA; Kabra et al . ( 2013 ) , http://jaaba . sourceforge . net ) was used for identifying events when a female mouse was being followed .", "The classifier was trained on 5228 frames of video ( 2341 positive examples and 2887 negative examples ) , using the mouse position data extracted by Motr .", "A subset of non-training frames ( 4040 ) was manually scored and compared to the output of the classifier to determine the accuracy of the system .", "The chase classifier had a false negative rate of 2 . 7% and a false positive rate of 7 . 5% .", "To determine which of the two males was following the identified female , we used a set of heuristics .", "First , the trajectories of the potential male participant had to overlap with that of the chased female by at least 20% .", "Second , the distance between the two mice at the start of the chase had to be within 20 cm .", "Consecutive chases involving the same participants were merged when the two events were separated by ∼0 . 3 s or less .", "All chases needed to exceed ∼0 . 17 s .", "Vocal interactions that occurred during a chase were assigned to that chase if the vocalizing animals were participating in the chase .", "If the vocal interactions occurred within 30 s after the end of the previous chase , it was assigned to the previous chase .", "If the vocal interactions occurred within 30 s from the onset of the chase following the vocal interactions , then it was assigned to the following chase .", "If the vocal interactions were not within 30 s before or after the flanking chases , then it was not assigned to a chase .", "Vocal interaction rate during a chase was determined by dividing the number of vocal interactions in a chase by the duration of the chase .", "Vocal interaction rate outside a chase was determined by dividing the number vocal interactions outside a chase by the difference between the time of the chase and the one-minute window surrounding the onset of the chase .", "To determine whether the vocal interaction rate during a chase was significantly different from the rate outside the chase , a Mann–Whitney U-test was performed .", "Central tendency and variability were reported with the median and interquartile range , respectively .", "The interquartile range was shown as the 75th–25th percentiles of the data .", "Male vocalizations not followed by other vocalizations within 1 s were extracted and assigned to a chase if the vocalizing male was participating in the chase .", "These were classified as chases without vocal interactions .", "During chases , there were 107 male vocalizations that were not followed within a second by a female or unassigned vocalization and 823 vocal interactions .", "The average speed of the two vocally interacting animals at the time of the male vocalization was calculated as well as the speed of both the male and female .", "To determine whether the speed of the animals at the time of a male vocalization was significantly different between the two conditions , a Mann–Whitney U-test was performed ." ] ]
[ "During courtship males attract females with elaborate behaviors .", "In mice , these displays include ultrasonic vocalizations .", "Ultrasonic courtship vocalizations were previously attributed to the courting male , despite evidence that both sexes produce virtually indistinguishable vocalizations .", "Because of this similarity , and the difficulty of assigning vocalizations to individuals , the vocal contribution of each individual during courtship is unknown .", "To address this question , we developed a microphone array system to localize vocalizations from socially interacting , individual adult mice .", "With this system , we show that female mice vocally interact with males during courtship .", "Males and females jointly increased their vocalization rates during chases .", "Furthermore , a female's participation in these vocal interactions may function as a signal that indicates a state of increased receptivity .", "Our results reveal a novel form of vocal communication during mouse courtship , and lay the groundwork for a mechanistic dissection of communication during social behavior ." ]
[ "Male songbirds are famous for using singing to attract mates , and many other animals also make noises as part of their mating rituals .", "Male mice , for example , are known to make high-pitched noises as they court females—but these noises are beyond the range of human hearing .", "The scent of female mice is enough to cause male mice to start ‘singing’ , and female mice are attracted to the male's song .", "Many studies have examined how these songs affect the mating behavior of the mice .", "But it was not always easy for a researcher to tell which mouse in the pair was making the sounds , because the mice make no obvious movements when they sing .", "Neunuebel et al . have now used many microphones arranged closely together to accurately pinpoint the singing individuals in a cage full of mice .", "Unexpectedly , this approach revealed that female mice join the males in song when courting .", "This challenges the long held belief that only male mice sing to communicate their courtship interests , and suggests that previous studies may have incorrectly attributed sounds from female mice as coming from males instead .", "Neunuebel et al . showed that as a male mouse pursues a female , both may sing .", "When females sing , they slow down; this makes it easier for the male to catch them .", "Females who don't respond to the male's song , however , keep up the pace .", "This suggests the song may be a way for the female mouse to indicate that she is interested in mating with her suitor .", "The next step will be to measure whether the noises that a female makes change depending on the identity of the suitor .", "Previous research has shown that female mice have more and healthier pups when they are paired with a male that they choose as opposed to a random male .", "The same microphone array could now be used to see whether female mice ‘say’ different things when paired with a male they like versus a male they don’t ." ]
2015